Analytical Profiles of

Volume 5
Edited b y

Klaus Florey
The Squibb Institute for Medical Research New Brunswick, New Jersey Contributing Editors

Norman W. Atwater Glenn A. Brewer, Jr. Lester Chafetz

Boen T. Kho Gerald J. Papariello Bernard 2. Senkowski

Compiled under the auspices of the Pharmaceutical Analysis and Control Section Academy of Pharmaceutical Sciences

Academic Press New York San Francisco London

A Subsidiary of Harcourt Brace Jovanovich, Publishers

1976

EDITORIAL BOARD
Norman W. Atwater Jerome I. Bodin Glenn A. Brewer, Jr. Lester Chafetz Edward M. Cohen Jack P. Comer Klaus Florey Salvatore A. Fusari Erik H. Jensen Boen T. Kho Arthur F. Michaelis Gerald .J.Papariello Bruce C. Rudy Bernard Z. Senkowski Frederick Tishler

Academic Press Rapid Manuscript Reproduction

COPYRIGHT 0 1976, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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York, New' York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD.

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Library of Congress Cataloging in Publication Data Main entry under title: Analytical profiles of drug substances. Includes bibliographical references. Compiled under the auspices of the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences. 1. Drugs-Analysis-Collected works. 2. Chemistry, I. Florey, Medical and pharmaceutical-Collected works. 11. Brewer, Glenn A. 111. Academy of Klaus, ed. Pharmaceutical Sciences. Pharmaceutical Analysis and 1. Drugs-Analysis-Yearbooks. Control Section. [DNLM: QV740 AA1 A551 RM300.A56 616l.1 70-187259 ISBN 0-12-260805-4 (v. 5)

PRINTED IN THE UNITED STATES OF AMERICA

AFFILIATIONS OF EDITORS AND CONTRIBUTORS

H. Y. Aboul-Enein, USV Pharmaceuticals, Tuckahoe, New York, now: Riyadh
University, Riyadh, Saudi Arabia

G. D. Anthony, Searle and Company, Chicago, Illinois

N. W.Atwuter, Searle and Company, Chicago, Illinois

J. I. Bodin, Carter-Wallace Inc., Cranbury, New Jersey

G.A. Brewer, The Squibb Institute for Medical Research, New Brunswick, New
Jersey

D. E. Cudwulluder,School of Pharmacy, University of Georgia, Athens, Georgia L. Chufefz,Warner-Lambert Research Institute, Morris Plains, New Jersey A. R. Chamberlin, Stanford Research Institute, Menlo Park, California

2 L. Chung,Searle and Company, Chicago, Illinois .

A. P. K . Chatng, Stanford Research Institute, Menlo Park, California E. M.Cohen, Merck, Sharp and Dohme, West Point, Pennsylvania
J. P. Comer, Ei Ully and Company, Indianapolis, Indiana l

R. D. Duley, Ayerst Laboratories, Rouses Point, New York K. Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey
S A. Fusuri, Parke, Davis and Company, Detroit, Michigan .

vii

AFFILIATIONS OF EDITORS A N D CONTRIBUTORS

G G. Gallo, Gruppo LePetit, Milan, Italy .

R. Gomez, Hoffmann-LaRoche, Inc., Nutley, New Jersey R. B. Hagel, Hoffmann-LaRoche, Inc., Nutley, New Jersey

D.D.Hong, The Sterling-Winthrop Research Institute, Rensselaer, New York

E. H. Jensen, The Upjohn Company, Kalamazoo, Michigan
J , H. Johnson, Hoffmann-LaRoche, Inc., Nutley, New Jersey

H. W. Jun, School of Pharmacy, University of Georgia, Athens, Georgia

B. T. Kho, Ayerst Laboratories, Rouses Point, New York

P. Lim, Stanford Research Institute, Menlo Park, California

J. P. McDonnell, Salsbury Laboratories, Charles City, Iowa

E. A. MacMuZlan, Hoffmann-LaRoche, Inc., Nutley, New Jersey

A. F. Michaelis, Sandoz Pharmaceuticals, East Hanover, New Jersey

S. M.Miller, Wyeth Laboratories, Philadelphia, Pennsylvania

N. G.Nash, Ayerst Laboratories, Rouses Point, New York

C E. Onech, Ayerst Laboratories, Rouses Point, New York

G J. Papanello, Wyeth Laboratories, Philadelphia, Pennsylvania .
A. Post, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

P Radaelli, Gruppo LePetit, Milan, Italy .

J,

A. Raihle, Abbott Laboratories, North Chicago, Illinois

R, J. Rucki, Hoffmann-LaRoche, Inc., Nutley, New Jersey B. C Rudy, Schering-Plough,Corp., Bloomfield, New Jersey .

viii

AFFILIATIONS OF EDITORS A N D CONTRIBUTORS

F. Russo-Alesi, The Squibb Institute for Medical Research, New Brunswick,

New Jersey

B. Z. Senkowski, Hoffmann-LaRoche, Inc., Nutley, New Jersey

C.M. Shearer, Wyeth Laboratories, Philadelphia, Pennsylvania

F. Tishler, Ciba-Geigy, Summit, New Jersey

R J. Warren, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

E. H. Waysek,Hoffmann-LaRoche, Inc., Nutley, New Jersey L. L. Wearley, Searle and Company, Chicago, Illinois

ix

PREFACE
Although the official compendia list tests and limits for drug substances related to identity, purity, and strength, they normally do not provide other physical or chemical data, nor do they list methods of synthesis or pathways of physical or biological degredation and metabolism. For drug substances important enough to be accorded monographs in the official compendia such supplemental information should also be made readily available. To this end the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences, has undertaken a cooperative venture to compile and publish Analytical Profiles of Drug Substances in a series of volumes of which this is the fifth. The concept of Analytical Profiles is taking hold not only for cornpendial drugs but, increasingly, in the industrial research laboratories. Analytical Profiles are being prepared and periodically updated to provide physico-chemical and analytical information of new drug substances during the consecutive stages of research and development. Hopefully, then, in the not too distant future, the publication of an Analytical Profile will require a minimum of effort whenever a new drug substance is selected for compendial status. The cooperative spirit of our contributors had made this venture possible. All those who have found the profiles useful are earnestly requested to contribute a monograph of their own. The editors stand ready to receive such contributions.

Klaus Florey

xi

BENDROFLUMETHIAZIDE

Klaus FIorey and Frank M.Russo-Alesi

KLAUS FLOREY A N D FRANK M . RUSSO-ALES)

CONTENTS
1.

Description 1.1 History 1.2 Name, Formula, Molecuiar Weight 1.3 Appearance, Color,Odor Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Melting Range 2.6 Differential Thermal Analysis 2.7 Solubility 2.8 Crystal Properties 2.9 pKa Synthesis Stability and Degradation Drug Metabolic Products

2.

3.

4. 5. 6.

- Pharmacokinetics

Methods of Analysis 6.1 Elemental Analysis 6.2 Spectrophotometric Analysis 6.3 Colorimetric Analysis 6.4 Nonaqueous Titration 6.5 Chromatographic Analysis 6.51 Paper 6.52 Thin-Layer Identification and Determination in Biological Fluids Miscellaneous References

7.
8. 9.

BENDROFLUMETH IAZIDE

Description 1.1 Histor d l u m e t h i a z i d e belongs to the class of thiazide diuretics. Its synthesis was first reported by Holdrege, Babel and Cheneyl in 1959 and its diuretic activity was first described in 960 almost simultaneously by Lund and Kobing and by Kennedy, Buchanan and Cunningham

1.

s.

Name, Formula, Molecular Weight Bendroflumethiazide (also bendrofluazide, benydroflumethiazide, and benzylhydroflumethiazide) is 2H-1,2,4-Benzothiadiazine-7-sulfonamide, 3,4dihydro-3-(phenyl methyl)-6-trifluoromethyl-l,ldioxide or 3-Benzyl-3,4-dihydro-6-(trifluoromethyl)-2H-1,2,4-benzothiad~az~ne-7-sulfonam~de1,l dioxide [73-48-31, H > ' ( NH SO' @
0 0

1.2

HC-CH2
CF 3

H Mol. Wt. 421.41

1 SH1qF 3N304 2 '

1.3

Appearance, Color, Odor White or sliqhtly off-white, uniform free flowing crystalline powder i slight floral odor. Physical Properties 2.1 Infrared Spectrum The infrared spectrum f bendroflumethiazide is given in Figure 1
2.

P.

Nuclear Magnetic Resonance The 60 MHz proton magnetic resonance spectrum in d4-methanol containing tetramethylsilane as an intern93 reference (Figure 2) is assigned as follows :

2.2

Figure 1 .
rl

Infrared Spectrum of Bendroflumethiazide Instrument: Perkin-Elmer 621.

wrn G G

(batch #15); KBr, MeOH.

=w=

H H

c u

rl

cl fd

al

fdk kC\

al

..

Figure 2.

NMR Spectrum of Bendroflumethiazide (batch 15) in deuterated methanol (Instrument: Perkin Elmer R12B)

KLAUS FLOREY A N D FRANK M. RUSSO-ALES1

H ~1.67(s)

not assigned T2.7

I n d6-DMSO ( F i g u r e 3 1 , t h e NH p r o t o n r e s o n a n c e s were observed near ~ 1 . 7 5( s i n g l e t , superimposed

w i t h a r y l p r o t o n ) , ~2 ( b r o a d ) , 2 . 4 5 ( s i n g l e t ) and 2.57 ( s i n g l e t ) ( F i g u r e 3) i n a d d i t i o n t o t h e o t h e r p r o t o n s a s s i g n e d from t h e dq-methanol spectrum. The h i g h e r f i e l d s i n g l e t s a t ~ 2 . 4 5and 2.57 are t e n t a t i v e l y a s s i g n e d t o t h e sulfonamide -NH2 p r o t o n s . N o p r e f e r e n c e of assignment i s made of t h e o t h e r two -NH p r o t o n s . On a d d i t i o n of D 2 0 , t h e -NH p r o t o n s a r e r a p i d l y exchanged f o r d e u t e r i u m , r e s u l t i n g i n t h e d i s a p p e a r a n c e of t h e NH p r o t o n s and sharpening of t h e p r o t o n r e s o n a n c e n e a r ~5 which now a p p e a r s l i k e t h a t of t h e dq-methanol spectrum

U l t r a v i o l e t Spectrum Squibb House S t a n d a r d ( b a t c h #15) i n methanol f x h i b i t e d t h r e e maxima a t 208 mu (El 7 4 5 ) , 1 273 mp (E 565) and 326 mu (Ei 9 6 ) 1 3 . T h i s ag i s w i t h measurements by P i l s b u and JacksonfF and by Kracmar and L a s t o v k o v a f i who d i s c u s s t h e U . V . spectrophotometry of b e n z o t h i a d i a z i n e s and p r e s e n t s p e c t r a . Mass Spectrum The l o w r e s o l u t i o n mass spectrum ( F i g u r e 4 ) shows o n l y a v e r y weak m o l e c u l a r i o n (M+) of m / e 4 2 1 and a b a s e peak o f m / e 319 t h a t could a r i s e by a double p r o t o n r e a r r a n g e m e n t , which i s n o t a common f r a g m e n t a t i o n pathway. The assignment of some of t h e fragment i o n s i s shown below:
2.4

2.3

in

Figure 3.

NMR Spectrum of Bendroflumethiazide (batch 15) in deuterated DMSO (Instrument: Perkin-Elmer R12B)

3753 SO15497 BFI#15

12-FEB-76 I00 90.. 80.MR0587
I

70.60.-

5 0.40-

ie
MRSS/CHRRGE

INTENSITY SUM = 1 8 3 3 9

BRSE PERK Z = 8 . 3 2

Figure 4.

Low Resolution Mass Spectrum of Bendroflumethiazide (Instrument: AEI MS9)

BENDROFLUMETHIAZIDE

319

H
2"H2

330191

m/e 402 6 M+ m/e 4 2 1

77

-so2NH2

m/e 303 m/e 302 (-HI

m/e 239 \< m/e 255

-so2
H

m/e 319-

m/e 302 -so2NH2, m/e 2 2 2

m/e 174 m/e 175 (+HI m/e 176 (+2H)

m/e 158 m/e 159 (+HI

Although t h e s e a p p e a r t o be r e a s o n a b l e a s s i g n m e n t s , t h e y should be c o n s i d e r e d t e n t a t i v e s i n c e t h e a s s i nments a r e n o t confirmed by h i g h - r e s o l u t i o n d a t a 3.

Melting Range The m e l t i n g p o i n t d o e s n o t o c c u r s h a r p l y and depends on t h e r a t e of h e a t i n g . The m e l t i n g p o i n t of t h e Squibb House S t a n d a r d ( b a t c h 15) w a s r e p o r t e d a t 223.20 - 226.20 C. L i t e r a t u r e v a l u e s r a n g e from 2 0 2 ' - 28. 2'

2.5

2.6 2.7

D i f f e r e n t i a l Thermal A n a l y s i s Melting endotherm: 2210 C. (17). Solubilit d i n water and c h l o r o f o r m .

KLAUS FLOREY AND FRANK M. RUSSO-ALES1

S o l u b l e i n a l k a l i , a c e t o n e and a l c o h o l . S o l u b l e i n one e q u i v a l e n t of 0 . 1 N NaOH. S l i g h t l y s o l u b l e i n e t h e r . I n s o l u b l e i n a c i d , benzene, l i g r o i n and petroleum e t h e r l 7 . S t a b l e c o n c e n t r a t e d s o l u t i o n i n p o l y e t h y l e n e g l y c o l , water and dimethyla c e t a n i l i d e o r N-methyl-2-pyrolidinone have been claimed (18). Crystal Properties The powder x-ray d i f f r a c t i o n p a t t e r n i s p r e s e n t e d i n Table I and F i g u r e 517.
2.8

TABLE I
d (A) *
17.23 13.27 11.63 8.94 8.11 7.64 7.36 6.70 6.34 5.91 5.48 5.31 4.82 4.67 4.53 4.46 4.31 4.19 3.96 3.84 3.75 3.69 3.63 3.54 3.44 3.40 3.19 3.11 2.99
0

I/I0**
0.528 0.356 0.103 0*246 *d = i n t e r p l a n a r d i s t a n c e 0.148 - 1 0.118 11 n 0.100 Z@ 0.199 0.124 X = 1.539 A 0.341 0*141 Radiation: K a l and 0.194 0.162 Ka2 Copper 0.545 l'ooo i t i 0.206 ** R e l a t i v e hn g e n s t t y based on i h e s 0.175 i n t e n s i t y of 1 . 0 0 0.516 0.213 0.357 0.185 0.248 0.289 0.205 0.140 0.145 0.166 0.145 0.157

10

Figure 5.

Powder X-Ray Diffraction Pattern of Bendroflumethiazide (Instrument: Norelco)

KLAUS FLOREY AND FRANK M. RUSSO-ALES1

A pKa of 8.5320.05 was determined b using the solubility variation with pH method4<

2.9

pKa

3.

Synthesis (see Figure 6) The synthesis of bendroflumethiazide (I) by cyclization of 4-amino-6-trifluoromethyl-mbenzenedisulfonamide (11) with phenylacetaldehydel (111) was e c 1 e by Holdrege, Babel and Cheney and others Alternate approaches are condensation of 4-amino-6-trifluoromethyl-mbenzenedisulfonyl chloride (IV) with phenylacetaldehyde (111) in the presence of ammonia8,9 , condensation of I11 with phenylacetimine (V)10, reaction of 4-amino-6-trifluoromethyl-m-benzenedisulfonamide (11) with cetoxystyrene (VI) in the presence of acetic acid" and by hydrogenation of 3-benzyl-6-trifl~oromethyl-7-sulfamoyl-l,2~4benzothiadiazine 1,l-dioxide (VII) with lithium aluminum hydridel2.

4~'~r;'t6.

Stability and Degradation Bendroflumethiazide, as a solid, appears to be very stable. The solid, exposed to.606-C. for a period of two weeks showed no decomposition as measured by I.R. and modified Bratton-Marshal reaction. In ethanol (1 mg/ml), it showed significant changes (22% decomposition) at the end of two weeks at 60 C. In aqueous suspension, almost complete breakdown to the disulfonamide (11, Figurfg6) occurred at the end of one week at 6OOC.

4.

In alkaline solution (pH 12) bendroflumethiazide undergoes complete hydro1 sis disulfonamide (11) in one hour at 351: C. 2@2the 21 It is also unstable under certain acid conditions Drug Metabolic Products Pharmacokinetics No drug metabolites have been identified so far. The Pharmacokinetics ha55 been studied in In human beings, the rat24 and in human beings orally or intravenously administered doses of S35-labeled bendroflumethiazide a excreted almost quantitatively in 24 hourss5. The stability of the trifluoromethyl group was
5.

12

H2NSOZ

+
CF3

@
0
H2NS0
4

H=CHOCOCH I1

@H2-CH0

0
LiAlH4

v1

Y '
NH2
NaO/

" m0 '- ! C
J"/'
I11
0 0 +S4
-CH2

13

x-

C F'3
VII
H H

Q
v
a
c a, m
4J

c H -cH=NH

CF 3

Figure 6.

IV S y n t h e t i c Pathways t o B e n d r o f l u m e t h i a z i d e

SO'C1

+
I11

NH

l-l

0 k

I n

KLAUS FLOREY AND FRANK M. RUSSO-ALES1

studied in ratsz6. There was no detectable fluoride uptake in the teeth of rats on a carious diet. Bendrof umethiazide was found to be 94% protein bound4

i! .

6.

Methods of Analysis 6.1 Elemental Analysis Found : Calc. % (Squibb House Standard Batch 15) 42.63 C 42.75 3.27 H 3.35 13.83 F 13.53 9.92 N 9.97 0 15.21 S 15.22 15.60

Spectrophotometric Analysis The ultraviolet absorption maximum at 273 mp (Ei 565) can be used for the quantitation of bendro lumethiazide in dosage forms23I 27. Colorimetric Analysis Bendroflumethiazide can be quantitatively assayed in dosage forms by alkaline hydrolysis tothe disulfonamide (11, Figure 6) diazotization, coupling with N-(1-naphthyl) ethylenediamine and determ'nation of the absorption maximum at 515 n 2 r ' m83. Other coupling agents have been used29r31. This assay can also be used to determine the presence2pS2&he disulfonamide (11) in bendroflumethiazide Non-Aqueous Titration An assay based on titration with sodium methoxide in dimethylformamide using p-nitrobenzene-azo-resorcinol as indicator has been developed32. Pyridine as solvent and azo-violet as indicator can also be used2*. Chromatographic Analysis 6.51 Paper Chromatographic Bendroflumethiazide (Rf 0.76) can be separated from the disulfonamide (11, Figure 6) using methylisobutylketone saturated with formamide as mobile phase and 30% formamide
14

6.2

6.3

6.4

6.5

in methanol as stationary phase. For quantitation, the spots are eluted with methanol and the concentration is determined spectrophotometrically (see 6.2)33. Identification and separation from other thiazide diuretics by paper chromatography has also been reportedl5. 6.52 following table. Absorbent 250 Silica Gel G Thin-Layer Chromatography Thin layer chromatographic systems are compiled in the Solvent System Benzene-Ethyl Acetate (2:8) and Ethyl Acetate-Methanol Ammonium Hydroxide (85:10 :5) Propanol-2/12N Ammonia (8:2) Propanol-l/12N Ammonia (8:2) Butanol-l/12N Ammonia (8 :2) Pentanol-l/12N Ammonia (8:2) Ethyl Acetate/l2N Ammonia (8:2) Chloroformflethanol (8:2) Cyclohexane/Glacial Acetic Acid/ Acetone (4:1:5) Toluene/xylene/l-4-Dioxane/Isopropanol/ 25% Ammonia (50:10 :30 :10) Ethanol/Chloroform/Heptane (1:l:l) (1st) Ethanol: (2nd) Chloroform/ Bu tanol Ethanol containing 1.5% Water Ethanol Ref. 34 34 34 34 34 34 34 34 35
36 37

Rf 0.91 0.88 0.93 0.70 0.54 0.98 0.52 0.98

250 Silica Gel G n 11 G 11 II " G 11 II G 11 n G 1 1 I1 " G I1 11 " G

'I

Silica Gel Silica Gel Alumina GF-254 (Two Dimensional) Silica Gel Alumina G

0.98

37 38

0.71

Solvent System Ethanol/Benzene (80:20) Ethyl Acetate/Benzene (8:2) It I1 Benzene/Ethyl Acetate/25% Ammonia/ Methanol (20:80 :1:10) I1 I1 Ethyl Acetate/Benzene/25% Ammonia/ Methanol (60:4 0 :20) Ethyl Acetate/Benzene/Ammonia/ Silica Gel 25% Methanol (50:40:2:10) n-Hexane/Acetone/Ethylamine (60:30:10) Silica Gel n-Hexane/Acetone/Diethylamine (40:40:20) Chloroform/Methanol/Diethylamine Silica Gel (80:15:5) Benzene/Ethyl Acetate (2:8) Silica Gel G I1 Ethyl Acetate/Methanol/Ammonium " G Hydroxide (85:10:5 11 Ethyl Acetate/Benzene (8:2) G Identification of oral hypoglycemic and diuretic drugs by TLC has been described42. Absorbent Silica Gel G Silica Gel 7. Identification and Determination in Biological Fluids In plasma: Colorimetrically12 In urine: Calorimetrically 24 Radioactivity25 TLC39 r 40 t 41

Ref. 38 39 39 39 39 39 39 39 40 40 41

Rf 0.70 0.75
0.40

0.68
0.10 0.46

0.75

0.82 0.82
0.98

using metal ions

8.

Miscellaneous 43,44 Pharmaceutical preparation of bendroflumethiazide have been patented

BEN DROFLUMETH IAZIDE

9.

References

1. C. T. Holdrege, R. B. Babel and L. C. Cheney, J. Am. Chem. SOC. 81, 4807 (1959) . 2. A. C. Kennedy, K. b Buchanan and C. Cunningham, Lancet 1960-1, 1267. . 3. F. Lund, W. 0 Godtfredsen, Brit. Pat. 863,474 (1961); C.A. 55, 19,971d (1961) also U . S . Pat. 3,392,168 n968). 4. J. G. Topliss, M. H. Sherlock, F. H. Clarke, M. C. Daly, B. W. Pettersen, J. Lipski and N. Sperber, J. Org. Chem. 26, 3842 (1961). 5. J. Klosa and H. Voigt, J. =act. Chem. 16, 264 (1962), also Ger. (East) Pat. 3 1 , 4 8 r (1964); C.A. 63, 14,887g (1965), Brit. Pat. 1,049,322 (1960) C.A. 66, 65,5343 (1967) and Belg. Pat. 631,232 (1963); C.A. - 14,528b 60, (1964). 6. K. Abildgaard, Fr. Pat. 1,586,602 (1970) C.A. 74, 100,112J (1971). 7. E. Schoenfeldt and H. Thorsteinson, Brit. Pat. 879,592 (1961); C.A. 57, 844f (1962). 8. L. C. Cheney and C. T. Holdrege, Fr. Pat. 1,368,708 (1964); C . A . 62, 9157c (1965). 9. J. Klosa, Brit. Pat. 1,063,102 (1967); C.A. 67, 11,514e (1967). 57 10. Brit. Pat. 898,109 (1962); C.A. - 12,516a (1962). 11. Fr, Pat. 1,388,607 (1965); C.A. 63, 7,024b (1965). 12. Gl Hurka, Austrian Pat. 253,513 (1967); C.A. 67, 11,512~(1967). 13. J. D ux a m , The Squibb Institute, Personal Communjcation. V V 25, 14. J. Kracmar and M. Lastovkovi, Pharmazie 464 (1970); Cesk. Farm. 20, 287 (1971); C.A. 76, 144,8783 (92. 17) 15. V. B.Pilsbury and J. V. Jackson, J. Pharm. Pharmac. 18, 713 (1966). 16. F. J. Lunrand W. Kobinger, Acta Pharmacol. et Toxicol. 16, 297 (1960). 17. H. Jacobson, The Squibb Institute, Personal Communication. 18. J. Ueda, Japan Pat. 25,692 (1963); C.A. 60, 9,107f (1964). 19. M. Everhard, The Squibb Institute, Private Communication.

17

KLAUS FLOREY AND FRANK M. RUSSO-ALES1

20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
31.

32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42.

K. Matsushima and K. Kiyota, Iryo 23, 1561 (1969); C.A. 73, 112,897m (1970). J. C. Turner, A. W. Nichols and J. E. Sloman, The Pharmaceutical J. 1970, 622. J. Dobrecky and B. G. Gonzalez, Rev. Farm. 114, 34 (1972); C.A. 78, 7,794f (1973). A. I. Cohen, The S q u i s Institute, Personal Communication. J. J. Piala, J. W. Poutsiaka, C. J. Smith, J. C. Burke and B N. Graves, J. Pharmacol. . 134, Expt. Therapeutics - 273 (1961). H. R. Brettell, J. G. Smith and J, K. Aikawa, Arch. Internal. Med. 113, 373 (1964). G. Hasselmann and K. m o l d , J. Pharm. Pharmacol. 15, 339 (1963). A. M. Leal and M. B. S. Ramos Lopez, Rev. 59, Port. Farm. 2, 48(1963); C.A. - 11,188e (1964). National Formulary XIV, p. 61ff (1975). J. Bermejo, Galenica Acta - 255 (1961); 14, C.A. 56, 10,286e (1962). M. Ghxardoni and M. Fedi, Boll. Chim. Farm. - 26 (1962); C.A. 57, 9582 (1962). 101, J. F. Magalhaes and. M .G Piros, Rev. Farm. Bioquh. Univ. Sao Paulo - 273 (1971); 8, C.A. 75, 121,466~ (1971). H. C.Chiang, J. Pharm. Sci. 5 0 , 885 (1961). H. Roberts, The Squibb Institute, Personal Communication. P. J. Smith and T. S. Herman, Anal. Biochem. - 134 (1968). 22, K. C. Guven and S. Cobanlar, Ecsacilik Bul. 9, 98 (1967); C.A. 67, 102,839f (1967). S. Gecgil, Ecsacmik Bul. - 100 (1965); 7, C.A. 64, 14,032d (1966). M. Duzene and L. Lapiere, J. Pharm. Belg. - 275 (1965); C.A. 64, 7,970d (1966). 20, R. Adam and C. L. Lapsre, J. Pharm. Belg. 19, 79 (1964); C.A. 61, 8,134 (1964). R. Maes, M. Gijbxs and L. Larvelle, J. Chromatogr. 53, 408 (1970). D. Sohn, J. Simon, H. Moheeb, G. Shali and R. Tolba, J. Chromatogr. 87, 570 (1973). B. G. Osborne, J. ChromatGr. 70, 190 (1972). S. Agarwal, M. Walash, and M. I. Blake, 35, Indian J. Pharm. - 181 (1973).

18

BENDROFLUMETHIZAIDE

43. 44. 45.

M. G o l d b e r g , U.S.

Pat. 3,265,573 (1960); C.A. 6 5 , 1 3 , 4 5 9 ~ ( 1 9 6 6 ) . C . RifSirin and M. G o l d b e r g , U . S . P a t . 3 , 4 2 6 , 1 3 0 ( 1 9 6 9 ) ; C.A. 7 1 , 33,41513 ( 1 9 6 9 ) . A. xgren and T. Bzck, A z a . Pharm. S u e c i n a 10, 2 2 3 ( 1 9 7 3 ) .

L i t e r a t u r e surveyed through June 1 9 7 4 .

19

CEPHRADINE

Klaus Florey

KLAUS FLOREY

TABLE OF CONTENTS Description 1.1 Name 1.2 Definition 1.3 Formula and Molecular Weight 1.4 Appearance, Color, Odor 2. Physical Properties 2.1 Infrared Spectra 2.2 NMR Spectra 2.3 Mass Spectrum 2.4 Ultraviolet Spectrum 2.5 Optical Rotation 2.6 Melting Range 2.7 Differential Thermal Analysis 2.8 Thermogravimetric Analysis 2.9 Ionization Constant, pK 2.10 Solubility 2.11 Crystal Properties 3 . Synthesis 4 . Stability-Degradation 4.1 Bulk Stability 4.2 Solution Stability Drug Metabolism, Pharmacokinetics 5. 6. Methods of Analysis 6.1 Elemental Analysis 6.2 Microbiological Analysis 6.3 Iodometric Analysis 6.4 Spectrophotometric Analysis 6.5 Fluorometric Analysis 6.6 Colorimetric Analysis 6.7 Chromatographic Analysis 6.71 Paper 6.72 Thin-layer 6.73 Column 7. Determination in Body Fluids and Tissues 8. Determination in Pharmaceutical Preparations 9. References
1.

22

CEPHRADINE

Cephradine is @ , 7 ( - ) -2-amino- (1,4 cyclohexadien-l-yl)acetamido-3-rnethyl-8-0~0-5thia-l-azabicyclo-oct-2-ene-2-carboxylic acid; also 7-p-amino-2-(1,4 cyclohexadienyl) acetamido7-desacetyl-cephalosporanic acid. 1.2 -Definition Cephradine, unless specified otherwise, is defined as a hydrated form containing 3-6% of water (for further discussion see Section 2.11). 1.3 Formula,Molecular Weiqht

1. Description 1.1 Names

/ +
C

IH
NH2

- 8-

!jT~++-cH3

CO2H anhydrous monohydra te dihydrate odorless to

C16H19N304S Molecular Weights: 349.41 367.43 385.45 1.4 Appearance,Color,Odor White crystalline powder, slightly sulphurous. 2.

Physical Properties 2.1 Infrared Spectra Spectra of cephradine (Batch #NN005NC) (Figure l), cephradine dihydrate(house standard batch #NNOO5NB) (Figure 2), cephradine monohydrate recrystallized from acetonitrile-water (sample MSA 38719) (Figure 3 ) and cephradine monohydrate recrystallized from methanol (sample MSA 38680) (Figure 4) are presented'.

23

FREQUENCY

(a')

WAVELENGTH (MICRONS)

Figure 1.

Infrared Spectrum of Cephradine Batch #NN005NC, K r P e l l e t , Instrument, Perkin E l m e r 21. B

F i g u r e 2.

I n f r a r e d Spectrum of Cephradine Dihydrate(House S t a n d a r d Batch # NN005NB) K r P e l l e t , I n s t r u m e n t , P e r k i n E l m e r 621. B

Figure 3. Infrared Spectrum of Cephradine Monohydrate Recrystallized From Acetonitrile (Water Sample MSA 38719) KBr Pellet, Instrument, Perkin Elmer 621.

Figure 4 .

Infrared Spectrum of Cephradine Monohydrate, Recrystallized From Methanol (Sample MSA 38680) KBr Pellet, Instrument, Perkin Elmer 621.

KLAUS FLOREY

2.2

NMR S p e c t r a NMR s p e c t r a i n CF3COOH ( F i g u r e 5 ) and

D20 (Figure 6) a r e presented2. I n t r i f l u o r o a c e t i c a c i d (TFA), t h e compound e x i s t s i n p r o t o n a t e d form w i t h the NMR s h i f t o f the NH+ a t Z 2 . 4 4 w i t h r e f e r e n c e t o i n t e r n a l t e t r a m e t h y l s i l a n e ( T M S ) . The i m i d e NH p r o t o n a p p e a r i n g a s a d o u b l e t (J = 9.0 H z ) a t Z 2 . 0 0 i s c o u p l e d t o o n e o f B-lactam r i n g p r o t o n s , which a p p e a r a s a q u a r t e t (J = 9 . 0 , 4 . 0 Hz) a t C4.20. The s e c o n d @ - l a c t a m p r o t o n r e s o n a n c e i s a d o u b l e t (J = 4 . 0 H z ) a t % = 4.74. The S-CH2 g r o u p p r o t o n s a p p e a r a s AB q u a r t e t (J = 1 8 . 0 Hz) a t C 6 . 3 6 and 6.54 w h i l e t h e m e t h y l g r o u p a p p e a r s a t C7.61. I n t h e dihydrophenyl r i n g , t h e o l e f i n i c p r o t o n s a p p e a r a t f 3.62 (1 H ) and 4 . 2 1 ( 2 H ) . T h e r e s o n a n c e a t Z 7 . 1 3 i s a s s i g n e d t o f o u r p r o t o n s of the dihydro ring. F i n a l l y t h e m e t h i n e p r o t o n of the CHNHf g r o u p a p p e a r s a s a m u l t i p l e t a t f 5 . 0 0 . The s p e c t r u m o b t a i n e d i n d e u t e r i u m o x i d e c o n t a i n i n g a d r o p of sodium d e u t e r i u m oxide was recorded using 3-(trimethyl sily1)-1-propansulf o n i c a c i d sodium s a l t a s an i n t e r n a l r e f e r e n c e . The amine a n d NH g r o u p p r o t o n s a r e exchanged w i t h D20. I n t h e dihydrophenyl r i n g , t h e chemical s h i f t s a r e t w o o l e f i n i c hydrogens a t C 4 . 2 5 , one o l e f i n i c p r o t o n a t X 4 . 1 5 and t h e o t h e r f o u r hydrogens a t Z 7 . 3 1 . The @ - l a c t a m r i n g p r o t o n s a r e a p a i r o f d o u b l e t s (J = 4 . 0 H z ) a t t 4 . 4 2 and 4 . 9 4 The S - m 2 g r o u p p r o t o n s a p p e a r a s a AB q u a r t e t (J = 1 8 . 0 ) a t C 6 . 8 1 and 6 . 4 3 . The m e t h y l r e s o n a n c e i s a d o u b l e t a t C 8 . 2 1 and may be a r e s u l t o f p a r t i a l i s o m e r i z a t i o n of the d o u b l e bond i n t h e b a s i c s o l u t i o n . F i n a l l y , t h e m e t h i n e (CHNHd p r o t o n h a s a c h e m i c a l s h i f t o f 6.00C 2 NMR c a n a l s o be u s e d t o d e t e r m i n e t h e amount o f r e s i d u a l c e p h a l e x i n by comparing t h e a r e a u n d e r t h e p h e n y l r o o n s ( . C 2 . 5 0 ) and o l e f i n i c p r o t o n s ( z 3. E4)

5.

28

F i g u r e 5. N M R S p e c t r u m of C e p h r a d i n e B a t c h NN005NC i n CF3COOH I n s t r u m e n t : v a r i a n XL-100.

n I:

.,

F i g u r e 6.

NMR S p e c t r u m of C e p h r a d i n e B a t c h NNO05NC i n D20

I n s t r u m e n t : V a r i a n XL-100.

CEPHRADINE

Mass Spectrum B e c a u s e o f t h e low v o l a t i l i t y of cephr a d i n e , a t r i m e t h y l s i l y l d e r i v a t i v e was made u s i n g t h e r e a g e n t BSA (N,O-Bis- ( T r i m e t h y l s i l y l ) a c e t a m i d e ) . The low r e s o l u t i o n mass s p e c t r u m ( F i g u r e 7 ) i n d i c a t e s t h a t t w o and t h r e e t r i m e t h y l s i l y l g r o u p s were added. The compound h a v i n g t w o t r i m e t h y l s i l y l g r o u p s was t h e p r e d o m i n a n t p r o d u c t w i t h m o l e c u l a r i o n a t m / e 493. The loss of CH3 from t h e m o l e c u l a r i o n ( t y p i c a l o f t r i methyl s i l y l g r o u p s ) y i e l d e d an i o n a t m / e 4783. The d a t a s u p p o r t s t r u c t u r e I.

2.3

An i n t e n s e i o n a t m/e 180 (see s p e c t r u m ) c o r r e s p o n d s t o 11,

I1

a n o t h e r i n t e r v a l i o n a t m/e
1 1 1 .

230 c o r r e s p o n d s t o

H
C

II

0 Si(CH3I3
I11

which i s a t y p i c a l f r a g m e n t o b t a i n e d from 8-lactam r i n g fragmentations3.

31

1FIB

96l

85 1

t.
f.n
k-

>

75 1

/
MASS/CHARGE

Z
lL

68
56l
95 1

Z
Id

$ 3 ;

2
K

35 1

zh:
161

F i g u r e 7. Low R e s o l u t i o n Mass Spectrum of T r i m e t h y l S i l y l D e r i v a t i v e of Cephradine. I n s t r u m e n t : A E I MS-902.

2.4

U l t r a v i o l e t Spectrum Cephradine e x h i b i t s a s i n g l e a b s o r b a n c e peak a t 262 n m = 220 f o r b a t c h ~ N 0 0 5 N c ) ~ .

Optical Rotation The s p e c i f i c r o t a t i o n o f t h e h o u s e s t a n d a r d ( d i h y d r a t e , b a t c h #NN005NB) i n a pH 8 b u f f e r was found t o be + 8 8 . 3 O ( a s i s ) 5. The a v e r a g e s p e c i f i c r o t a t i o n of s e v e n b a t c h e s of c e p h r a d i n e a t a c o n c e n t r a t i o n o f 0.5% i n 0.1M a c e t a t e b u f f e r (pH 4 . 6 ) and c a l c u l a t e d on an anhydrous b a s i s was found t o be + 91.6O w i t h a r a n g e from + 89.22O t o 92.90. The e f f e c t o f pH on r o t a t i o n was found t o be s l i g h t , a s c a n be s e e n from Table 15. 27O Table 1 conc. Approx.

2.5

L 3- D 7
pH 4.01 5.00 6.03 7.00 8.20 Initial 77.450 78.250 -t 78.32O + 80.47O + 87.24O

1 %
5 Hours 78.45O 77.65O 77.83O + 76.68O + 85.74O

+ +

1 Hour 79.45O 78.75O + 79.10 + 78.37O + 86.04O

+ +

3 Hours 76.58O + 77.550 + 78. loo + 77.78O + 85.05O

+ + +

+ + + +

24 Hours 75.380 75.86O 77.770 76.95O + 84.32O

KLAUS FLOREY

M e l t i n q Range Cephradine m e l t s w i t h decomposition. The m e l t i n g range (USP) of cephradine d i h y d r a t e house s t a n d a r d (Batch NN005NB) was 183-185O. The m e l t i n g range of t y p i c a l b a t c h e s of cephrad i n e h a s v a r i e d from 175-192O. 2 . 7 D i f f e r e n t i a l Thermal A n a l y s i s The thermogram of cephradine e x h i b i t s a s i n g l e exotherm a t approximately 200-203O, depending on h e a t i n g r a t e . T h i s exotherm i n d i c a t e s o x i d a t i v e decomposition accompanied by melting6. O t h e o t h e r hand t h e thermogram of n c e p h r a d i n e d i h y d r a t e e x h i b i t s two endothermic peaks a t a b o u t 90 and a t a b o u t 102O which a r e r e l a t e d t o t h e h y d r a t i o n of t h e compound. A decomposition exotherm i s observed a t about 20OoC. The d i f f e r e n c e i n t e m p e r a t u r e between t h e two endothermic peaks i s a measure of t h e stress ( g r i n d i n g , h e a t i n g ) l e a d i n g t o d e h y d r a t i o n , to which t h e sample i s s u b j e c t e d 6 .

2.6

Thermograms of c e p h r a d i n e d i h y d r a t e , cephradine, c e p h a l e x i n7 , and cephradine monoh y d r a t e ( r e c r y s t a l l i z e d from a c e t o n i t r i l e - w a t e r ) ' a r e p r e s e n t e d i n F i g u r e a6. The absence Of an endotherm f o r cephradine s u g g e s t s t h a t cephradine i s n o t a t r u e h y d r a t e and t h e w a t e r i s n o t s t r u c t u r a l l y bound i n t h e c r y s t a l - l a t t i c e ( s e e a l s o s e c t i o n 2.11). On t h e o t h e r hand t h e d i h y d r a t e , t h e monohydrates o b t a i n e d by r e c r y s t a l l i z a t i o n from a c e t o n i t r i l e - w a t e r 7 ( s h a r p endotherm a t 150-152O) and methanol8 (Endotherm a t 152O)as w e l l a s cephalexin7 a r e t r u e h y d r a t e s ?
DTA h a s a l s o been used a s a s c r e e n i n g technique t o s t u d y t h e i n t e r a c t i o n o f c e p h r a d i n e with p o t e n t i a l adjuvants t o a parenteral formula t ion9

34

CEPHR AD IN E

Figure 8. D i f f e r e n t i a l The rmog rams

35

KLAUS FLOREY

Thermogravimetric A n a l y s i s By t h e r m o g r a v i m e t r i c a n a l y s i s (TGA) o f t y p i c a l batches o f c e p h r a d i n e from 3 t o 6% v o l a t i l e s ( w a t e r ) , w e r e found ( t h e o r y f o r monohydrate w a t e r 4 . 9 3 % ) . The d i h y d r a t e ( b a t c h B49988D)by TGA g a v e 9.8% v o l a t i l e s ( t h e o r y f o r d i h y d r a t e w a t e r 9 . 3 5 % ) . VPC was u s e d t o c o n f i r m the v o l a t i l e compound a s w a t e r 4 8 . The 13% loss shown b y TGA a t a b o u t 200-210(decomposition p o i n t ) i s assumed t o be due t o d e c a r b o x y l a t i o n 6 . 2 . 9 I o n i z a t i o n C o n s t a n t , pK By t i t r a t i o n w i t h sodium h y d r o x i d e a pK1 of 2 . 6 3 and pK2 o f 7 . 2 7 w a s found6. 2.10 S o l u b i l i t y T h e r e i s no d i f f e r e n c e i n s o l u b i l i t y of t h e v a r i o u s c r y s t a l forms. The s o l u b i l i t y o f c e p h r a d i n e i n b u f f e r s a t d i f f e r e n t pH v a l u e s i s reported i n Table 2. Table 2 S o l u b i l i t y of Cephradine a t D i f f e r e n t pH Values pH of S a t u r a t e d Solubility pH of b u f f e r Solution mq/ml
2.8

4.00 water 60% s u c r o s e 5.00 6.03 7.20 8. 20 9. i a

4.02 4.91 5.0 5.04 5.90 6.12 7.09 7.41

35.8 21.3 17.1 21.1 20.5 28.2 36.7 49.6

Cephradine i s p r a c t i c a l l y i n s o l u b l e i n e t h y l e t h e r , c h l o r o f o r m , b e n z e n e and h e x a n e . The a n t i b i o t i c i s very s l i g h t l y soluble i n acetone and a b s o l u t e e t h a n o l . I t i s f r e e l y soluble i n propylene glycol. The s o l u b i l i t y terminology u s e d i s t a k e n from U S P X V I I I . The i n t r i n s i c d i s s o l u t i o n r a t e s of c e p h r a d i n e and i t s d i h y d r a t e a s w e l l as
36

CEPHRADINE

c e p h a l e x i n were found i d e n t i c a l a t an a g i t a t i o n i n t e n s i t y of 100 rpm 10 The observed i n t r i n s i c d i s s o l u t i o n r a t e s (mg/ml/min/cm2) a r e a s f o l l o w s : Intrinsic Compound Temperature D i s s o l u t i o n Rate 2 2oc Cephradine 0.08 0.1 37oc Cephradine dihydrate 2 2oc 0.08 37OC 0.09

2 2% 0.08 3 7% 0.09 2 . 1 1 Crystal Properties There i s c o n s i d e r a b l e evidence f o r polymorphism and f o u r polymorphs o r r a t h e r pseudopolymorphs have been c h a r a c t e r i z e d s o f a r . 1. ) Cephradine, h y d r a t e d . Although t h e w a t e r c o n t e n t v a r i e s from 3-6% i t i s n o t a s t o i c h i o m e t r i c hydrate s i n c e t h e water apparently moves f r e e l y i n t h e c r y s t a l l a t t i c e ( s e e s e c t i o n 2 . 7 ) . I t i s o b t a i n e d from aqueous s o l u t i o n . Anhydrous cephradine h a s been p r e p a r e d and was found t o be v e r y s t a b l e and r e s i s t a n t t o o x i d a t i o n t o c e p h a l e x i n , when k e p t a n h y d r o u s ( s e e s e c t i o n 4 . 1 ) . However, i t cannot be p r o p e r l y c h a r a c t e r i z e d , s i n c e i t immediately h y d r a t e s on exposure t o t h e atmosphere. 2 . ) Cephradine d i h y d r a t e . T h i s compound which c r y s t a l l i z e s from aqueous s o l u t i o n under c o n t r o l l e d c o n d i t i o n s l l , i s a t r u e dihydrate (see section 2 . 7 ) . I t i s v e r y s t a b l e and r e s i s t a n t t o o x i d a t i o n t o c e p h a l e x i n . However, on d e h y d r a t i o n ( l o s s of c r y s t a l s t r u c t u r e ) t h e d i h y d r a t e becomes v e r y u n s t a b l e ( s e e s e c t i o n 4 . 1 ) . The s t r u c t u r e of c e p h r a d i n e d i h y d r a t e was determined by s i n g l e c r y s t a l x-ray d i f f r a c t i o n i n o r d e r t o i n v e s t i g a t e t h e p o s i t i o n of t h e w a t e r molecules i n t h e c r y s t a l and t h e hydrogen

Cephalexin

37

KLAUS FLOREY

bonding of t h e w a t e r t o v a r i o u s s i t e s i n t h e cephradine molecule50. The hydrogen bonding p a t t e r n i s i n d i c a t e d i n f i g u r e 9 . There are t w o molecules i n t h e u n i t c e l l . The p r o j e c t i o n of one i s shown. The second i s symmetry r e l a t e d by 180 r o t a t i o n i n t h e p l a n e of p r o j e c t i o n , and t r a n s l a t i o n of 1 / 2 of t h e b r e p e a t , Atoms of t h e second molecule i n v o l v e d i n hydrogen bonding a r e i n d i c a t e d w i t h a prime n o t a t i o n . The bonding may b e summarized a s follows: Water oxygens a r e numbered 69 and 71. Water oxygen #69 bonds w i t h t h e @-lactam carbonyl of molecule 1, t h e amide n i t r o g e n 13, of molecule 2 , and w i t h t h e second w a t e r molecule # 7 1 . Water oxygen # 7 1 Ponds w i t h one of t h e carboxyl oxygens, 64 , of molecule 2 , and w i t h w a t e r molecule #69. The amino n i t r o g e n , N5 of molecule 2 bonds w i t h b o t h carboxyl oxygens. 3 . ) Cephradine monohydrate r e c r y s t a l l i z e d from a c e t o n i t r i l e water8. T h i s i s a t r u e h y d r a t e (see s e c t i o n 2 . 7 ) . 4 . ) Cephradine r e c r y s t a l l i z e d from anhydrous methanol8 a l s o a p p e a r s t o b e a t r u e monoh y d r a t e , a l t h o u g h a n o t h e r one h a l f mole of unbound w a t e r was a l s o p r e s e n t . N o r e s i d u a l methanol was found. Cephradine, g e n e r a l l y , seems t o have a tendency t o form s o l v a t e s s i n c e a c e t o n i t r i l e and e t h y l e n e g l y c o l s o l v a t e s have been observed 1 2 Powder x-ray p a t t e r n s of t h e f o u r c r y s t a l forms a r e p r e s e n t e d i n Tables 3-613. The u n i t c e l l dimension, s p a c e group and c e l l c o n t e n t d e t e r m i n a t i o n s of t h e f o u r c r y s t a l forms w e r e made by s i n g l e x-ray c r y s t a l l ographyl and a r e p r e s e n t e d i n Table 7. For f u r t h e r d i s c u s s i o n on t h e s e c r y s t a l forms, s e e s e c t i o n s on I R ( 2 . 1 ) , DTA(2.7) and Bulk S t a b i l i t y ( 4 . 1 ) .
38

I a oc

F i g u r e 9.

D i s t r i b u t i o n of water molecules i n cephradine dihydr at e .

KLAUS FLOREY

Table 3 Powder X-Ray P a t t e r n of Cephradine,Hydrated Batch NN005NC d 1/10 d 1/10

15.80 11.90 8.04 5.98 5.61 5.34 4.92 4.66 4.48 4.32 4.20 4.00 3.93

14.1 47.4 15.4 19.2 24.4 100.0 21.8 11.5 21.8 57.7 26.9 30.8 14.1

3.76 3.61 3.47 3.33 3.24 3.18 3.08 2.90 2.80 2.74 2.67 2.57 2.43

14.1 35.9 12.8 17.9 12.8 11.5 25.6 12.8 11.5 10.3 14.1 9.0 11.5

Table 4 Powder X-Ray P a t t e r n of Cephradine Dihydrate Batch NN005NB (House S t a n d a r d ) d d 1/10 1/10

11.60 10.50 8.75 7.05 6.20 6.00 5.80 5.61 5.23 5.10 4.68 4.55 4.45 4.25 3.94 3.87 3.80

35.4 15,6 10.4 19.8 37.5 17.7 24.0 56.3 20.8 37.5 7.3 9.4 26.0 17.7 9.4 12.5 28.1

3.76 3.67 3.55 3.45 3.42 3.19 3.08 2.92 2.80 2.68 2.61 2.57 2.49 2.46 2.39 2.31

22.9 26.0 100.0 43.8 46.9 28.1 30.2 54.2 10.4 15.6 15.6 13.5 14.6 17.7 7.3 12.5

CEPHRADINE

Table 5 Powder X-Ray Pattern of Cephradine Monohydrate Recrystallized from Acetonitrile-Water Sample #38720 d 1/10 d 1/10
9.92 8.83 6.96 6.45 5.64 5.43 4.92 4.84 4.74 4.39 4.23 4.00 3.91 3.64 3.41 3.36 3.22 3.10
100.0 94.0 82.0 24.0 80.0 35.0 15.0 22.0 91.0 45.0 100.0 45.0 35.0 21.0 32.0 29.0 24.0 61.0

3.05 3.02 2.96 2.90 2.80 2.77 2.75 2.70 2.65 2.60 2.47 2.42 2.38 2.35 2.24 2.21 2.14

17.0 18.0 25.0 14.0 17.0 35.0 14.0 13.0 18.0 26.0 11.0 26.0 23.0 31.0 25.0 17.0 25.0

41

KLAUS FLOREY

Table 6 Powder X-Ray Pattern of Cephradine Monohydrate Recrystallized from Methanol Sample # 38708 d
11.20 8.83 8.18 7.83 6.80 6.23 5.82 5.64 5.21 4.86 4.74 4.59 4.34 4.19 4.13 4.00 3.89
1/10 100.0 17.0

d
3.78 3.63 3.51 3.30 3.17 3.08 2.94 2.82 2.74 2.65 2.62 2.54 2.45 2.42 2.36 2.30

19.0 10.0 18.0 19.0 25.0 13.0 14.0 37.0 45.0 43.0 32.0 34.0 25.0 26.0 32.0

14.0 26.0 33.0 24.0 15.0 16.0 26.0 18.0 21.0 17.0 18.0 16.0 14.0 14.0 17.0 14.0

1/10

42

P 0

Table 7

CELL coNsTAms->

SPACE

MEASURED C E L L COJTI'ENTS

12 cephradine

4 cephradine Acetonitrile Monohydra t e ' Recrystallized from 17.58 M e th an o 1

I
9.4

21.6

90

3568

P212121

1.33

8 cephradine 8 water

Synthesis Cephradine (111, Figure 10) is synthesized by coupling 7-aminodesacetoxycephalosporanic acid (7-ADCA) (11) with a protected derivative of dihydrophenylglycine (I), Figure 10 Synthetic Pathway to Cephradine
3.

0hz
CH
I

COOH

Intermediate

+ H2N

COOH

0 " :
CO - JJ < m 0

m2

CH3

COOH
I11

such as the tert.-butoxylcarbonyl derivative which can be converted to a mixed anhydride with ethylchloroformate and reacted with 7-ADCA14. Cephradine can also be made by forming the methyl acetoacetic ester enamine derivative of dihydrophenylglycine-which is converted to a mixed

CEPHRADINE

anhydride w i t h benzoyl c h l o r i d e p r i o r t o coupling Cephradine i s t h e n cr s t a l l i z e d w i t h 7-ADCAI2. It also from a b i p h a s i c MIBK-aqueous s o l u t i o nY2 can be c r y s t a l l i z e d from w a t e r a l o n e , a s w e l l a s from o t h e r s o l v e n t s ( s e e s e c t i o n 2 . 1 1 ) .

S t a b i 1 ty-Degrada t i o n i 4 . 1 Bulk S t a b i l i t y C e p h r a d i n q w h e n k e p t u n d e r d r y and c o o l s t o r a g e c o n d i t i o n s , h a s shown r e a s o n a b l e b u l k s t a b i l i t y 1 2 . Like o t h e r 1,4 cyclohexadienes such a s 2,5 d i h y d r ~ p h e n y l a l a n i n e ~ ~ p h r a d i n e i s ce , prone t o a slow r a t e of o x i d a t i o n of t h e c y c l o hexadiene r i n g t o t h e benzenoid r i n g . The e x a c t mechanism of t h i s r e a c t i o n i s n o t known, b u t i n a d d i t i o n t o oxygen and w a t e r , t r a c e m e t a l s s u c h a s i r o n seem t o have an a c c e l e r a t i n g e f f e c t . The o x i d a t i o n t o c e p h a l e x i n a s w e l l a s d e g r a d a t i o n (loss of b i o p o t e n c y ) can be p r e v e n t e d o r s i g n i f i c a n t l y r e d u c e d b y s t o r a g e a t low t e m p e r a t u r e and e x c l u s i o n o f oxygen, a s w e l l a s removal of w a t e r . T h i s l a s t method, however, i s i m p r a c t i c a l due t o t h e e x t r e m e l y h y g r o s c o p i c n a t u r e o f anhydrous c e p h r a d i n e 1 2 . On t h e o t h e r hand c e p h r a d i n e d i h y d r a t e 11 b e i n g a t r u e s o l v a t e (see s e c t i o n 2 , l l ) w h e r e water c a n n o t move f r e e l y i n t h e c r y s t a l l a t t i c e e x h i b i t s remarkable r e s i s t e n c e t o c e p h a l e x i n f o r m a t i o n , l o s s o f b i o p o t e n c y and c o l o r d e v e l o p m e n t d u r i n g e x t e n d e d s t o r a g e u n d e r a i r l 2 . The d i f f e r e n c e b e t w e e n c e p h r a d i n e and i t s d i h y d r a t e i s i l l u s t r a t e d i n Table 812. However upon p a r t i a l o r f u l l d e h y d r a t i o n of c e p h r a d i n e d i h y d r a t e u n d e r a v a r i e t y of conditions, drying a t higher temperatures o r c e r t a i n kinds of m i l l i n g t h i s e x c e l l e n t s t a b i l i t y i s not maintainedlz.
4.

45

KLAUS FLOREY

Table 8 Average Bulk S t a b i l i t y Data for Three Batches of Cephradine and f o r Three Batches of Cephradine Dihydrate A f t e r 9 Months of S t o r a g e Under A i r Cephradine ( " a s i s " ) Initial Bioassay, mcg/ml 949 2.7 Cephalexin, % Bioassay, mcg/ml Cepha l e x i n , % 5Oc 947
3.0

RT

942 3.5

4OoC 924 4.6

5OoC 849 7.0

Cephradine Dihydra t e ( " a s i s " ) 870 897 906 870 909 2.3 2.3 2.3 2.2 2.4

Cephradine i s moderately l i g h t s e n s i t i v e . When exposed t o U.V. l i g h t , t h e s o l i d t u r n s yellow on t h s u r f a c e b u t no loss of b i o The s e n s i t i v i t y of a c t i v i t y w a s noted f 2 c e p h a l o s p o r i n C t o l i g h t h a s been p r e v i o u s l y no t e d l 6 . Other than c e p h a l e x i n , no d e g r a d a t i o n p r o d u c t s of s o l i d cephradine have been i d e n t i f i e d TLC examination of degraded (loss of biopotency) samples r e v e a l e d a m u l t i p l i c i t y of U.V. a b s o r b i n g and f l u o r e s c e n t s p o t s 1 7 . 4.2 S t a b i l i t y i n Solution I n aqueous s o l u t i o n c e p h r a d i n e t e n d s t o be q u i t e s t a b l e a t p 4.0 and below and much l e s s H s t a b l e a t higher p u n i t s . A pH-stability profile H i s p r e s e n t e d i n Table 96 Cephradine was found to be f u l l y p o t e n t for at l e a s t e i g h t h o u r s i n a v a r i e t y of p a r e n t e r a l i n f u s i o n s o l u t i o n s 2 3 . Although cephradine, l i k e o t h e r cephalo s p o r i n s i s much more r e s i s t a n t t o opening of t h e B-lactam r i n g by a l k a l i t h a n p e n i c i l l i n s , t h e r i n g does open up w i t h subsequent f u r t h e r d e g r a d a t i o n , Ring opening a l s o l e a d s t h e loss of U.V. absorbance a t 262 nm.

46

Table 9 S t a b i l i t y of C e p h r a d i n e i n Phosphate B u f f e r a t Room Temperature

p H of S o l u t i o n
4.0 6.0 8.0

2 days
95.9 73.0 67.1 64.0

P e r c e n t of Remaining B i o a c t i v i t y 4 days 7 days 10 days

105.1 86.9 43.1 41.1 99.3 69.5 24.5 25.2 99.3

14 days
95.2 18. 5 10.9 11.7

30.2
13.4 13. 3

10.0

*
5

ph of samples a t s t a r t of s t u d y . The pH o f t h e h i g h pH samples d r i f t e d toward lower pH v a l u e s a s t h e s t u d y p r o g r e s s e d .

One of t h e a l k a l i n e d e g r a d a t i o n p r o d u c t s p r e c i p i t a t e s from t h e s o l u t i o n and h a s been i d e n t i f i e d a s 2-[6-(1,4 cyclohexadien-l-yl)-2, 5-dioxo-3-piperazinyl~-5,6-dihydro-5-methyl-2H-l,3-thiazine-4-carboxylic a c i d , sodium s a l tI8.

KLAUS FLOREY

The B-lactam r i n g o f c e p h r a d i n e i s q u i t e res i s t a n t t o p e n i c i l l i n a s e , b u t opens r e a d i l y w i t h cephalosporinasel9. On t h e a c i d s i d e NMR s t a b i l i t y s t u d i e s w i t h a 2% s o l u t i o n a t pH 1 . 6 , h e l d a t 60C, d e m o n s t r a t e d t h a t t h e r e i s no 0-lactam r i n g opening. However p o s s i b l e s h i f t i n g o f t h e d o u b l e bond from c a r b o n s 2-3 t o 3-4 was o b s e r v e d b y NMR and c o n f i r m e d by loss of U.V. a b s o r b a n c y a t 262 nm. A f t e r 93 h o u r s NMR i n d i c a t e d a 50% d o u b l e bond i s o m e r i z a t i o n 2 . Very v i g o r o u s treatment with strong a c i d w i l l lead a t l e a s t i n p a r t t o a s p l i t o f t h e amide l i n k a g e t o form 7-ADCA and d i h y d r o p h e n y l g l y c i n e 1 2 . This cleavage can a l s o be a c h i e v e d e n z y m a t i c a l l y w i t h p e n i c i l l i n acylase19. I n a phosphate b u f f e r a t pH 6 v i g o r o u s a e r a t i o n o r e x p o s u r e t o room l i g h t However 10 d i d n o t a c c e l e r a t e degradation2'. h o u r s of e x p o s u r e t o a Hanovia U.V. lamp of an aqueous s o l u t i o n (pH 5 ) of c e p h r a d i n e more t h a n doubled t h e c e p h a l e x i n c o n t e n t w i t h l i t t l e Cephradine was found t o change i n b i o p o t e n c y 1 2 . be s t a b l e f o r a t l e a s t t h i r t y days i n f r o z e n (-S0C) human s e r u m and u r i n e . No significant l o s s was d e t e c t e d by r e p e a t e d thawing and On t h e o t h e r hand when i n c u b a t e d refreezing2'. w i t h s er u m a t 37OC f o r 6 h o u r s t h e r e was a 20% loss of a c t i v i t y . I n c u b a t i o n w i t h whole b l o o d under t h e same c o n d i t i o n s c a u s e d l i t t l e o r no loss of b i o p o t e n c y 2 2 . I t was found5I, t h a t t h e a c t i v i t y of c e p h a l o s p o r i n s c o n t a i n i n g a p h e n y l g l y c i n e m o i e t y ( c e p h a l o g l y c i n and t o a l e s s e r d e g r e e c e p h a l e x i n and c e p h r a d i n e ) i s p r o g r e s s i v e l y 10s t i n t h e p r e s e n c e of c u p r i c i o n . T h i s d e g r a d i n g e f f e c t of c u p r i c i o n can be i n h i b i t e d b y d-penicillamine.

40

CEPHRADINE

Druq Metabolism Cephradine-3H 250 m d r y f i l l e d c a p s u l e s w e r e g a d m i n i s t e r e d t o e i g h t normal male s u b j e c t s a s a s i n g l e o r a l dose i n a b i o a v a i l a b i l i t y study24. Serum and u r i n e s a m p l e s were a s s a y e d i n a d o u b l e b l i n d f a s h i o n f o r b i o l o g i c a l a c t i v i t y and i n a n open f a s h i o n f o r r a d i o c h e m i c a l a c t i v i t y . The mean p e a k c e p h r a d i n e s e r u m c o n c e n t r a t i o n (7.0 2 1 . 0 kgm/ml SE by b i o a s s a y a n d 7 . 8 f 0 . 9 pgm/ml - SE b y r a d i o a s s a y ) o c c u r r e d 55 m i n u t e s a f t e r d o s i n g and d e c r e a s e d w i t h a b i o l o g i c a l h a l f - t i m e o f 40 m i n u t e s . The c u m u l a t i v e a r e a s u n d e r t h e c u r v e s f o r c e p h r a d i n e were 1 6 1 . 5 f 29 and 1 7 7 . 3 f 34.7 min. pgm/ml f o r b i o a s s a y and radioassay curves respectively. C e p h r a d i n e was r a p i d l y e x c r e t e d . A p p r o x i m a t e l y 77% of c e p h r a d i n e was e x c r e t e d w i t h i n t h e t h r e e h o u r p e r i o d f o l l o w i n g d r u g a d m i n i s t r a t i o n . A f t e r 24 h o u r s a p p r o x i m a t e l y 87% o f t h e a d m i n i s t e r e d d o s e of c e p h r a d i n e was r e c o v e r e d i n t h e u r i n e a s m e a s u r e d b y both b i o and r a d i o c h e m i c a l a s s a y25 Four h e a l t h y f e m a l e s u b j e c t s r e c e i v e d a s i n g l e i n t r a m u s c u l a r i n j e c t i o n of 1 gram o f The ~ e p h r a d i n e - ~ H ~ ~ . mean p e a k c o n c e n t r a t i o n o f 1 0 f 1 . 7 ug/ml SE i n plasma w a s r e a c h e d a t 2 h o u r s a f t e r a d m i n i s t r a t i o n and t h e n d e c r e a s e d with a b i o l o g i c a l h a l f - l i f e of 2 hours. Binding of c e p h r a d i n e t o plasma p r o t e i n was f o u n d t o be 6% o v e r t h e r a n g e of c o n c e n t r a t i o n s found i n plasma d u r i n g t h e a b s o r p t i v e a n d e x c r e t o r y phases. A b s o r p t i o n , b a s e d on e x c r e t i o n o f r a d i o a c t i v i t y i n u r i n e o v e r t h e 24 h o u r p e r i o d + 6.6% + SE f o r f o l l o w i n g a d m i n i s t r a t i o n , w a s 92 t h e f o u r s u b j e c t s . A n o t h e r 1% was e x c r e t e d i n f e c e s w i t h i n t h e 72-hr p e r i o d o f t h e e x p e r i m e n t . T h e r e w a s no s i g n i f i c a n t d i f f e r e n c e i n e x c r e t i o n b a s e d on t h e r e c o v e r y o f r a d i o a c t i v i t y o r antimicrobial a c t i v i t y i n urine. E x c r e t i o n was 66 f 6.9% f SE a t t h e e n d o f 6 h r and 8 5 f 6.5%
5.

49

Recovery of antimicrobial a c t i v i t y i n u r i n e and t h e a r e a under t h e c u r v e f o r c o n c e n t r a t i o n o f a n t i b i o t i c i n plasma were i n e x c e l l e n t agreement w i t h t h o s e found when l a b e l l e d c e p h r a d i n e w a s administered o r a l l y . Cephradine a p p e a r s t o be s l o w l y r e l e a s e d from t h e s i t e o f i n j e c t i o n t o g i v e l e v e l s of a n t i b i o t i c i n plasma which, though r e a c h i n g a maximum a t 1/3 t h o s e found a f t e r o r a l a d m i n i s t r a t i o n , p r o v i d e t h e same amount o f b i o a v a i l a b l e a n t i b i o t i c o v e r a l o n g e r p e r i o d of t i m e 2 4 N o c a n i n e o r human drug m e t a b o l i t e s w e r e found so f a r e x c e p t t r a c e amounts of c e p h a l e x i n , a s i d e n t i f i e d by m a s s spectrometry26. The above i n f o r m a t i o n was p u b l i s h e d ( r e f e r e n c e s 27-31).
6.

5 S E a t t h e end of 1 2 hours.

Methods o f A n a l y s i s 6 . 1 Elemental A n a l y s i s Calc. f o r anhydrous Calc. f o r monohydrate Found f o r c e p h r a d i n e b a t c h NN057NC Calc. f o r d i h y d r a t e Found f o r c e p h r a d i n e dihydrate batch N O 5 B N ON

55.01 52.30 51.96 49.85 49.77

5.48 5.76 5.83 6.01

12.03 11.44 11.16

10.90

9.16 8.73
9.00

8.31 8.39

6.06

10.95

CEPHRADINE

M i c r o b i o l o g i c a l Assay For b u l k and f o r m u l a t e d p r o d u c t s a t u r b i d i m e t r i c method u s i n g S t r e p t o c o c c u s f a e c a l i s A.T. C. C. 1 0 , 5 4 1 i s c o n v e n i e n t . A l t e r n a t i v e l y a g a r p l a t e methods u s i n g S a r c i n a l u t e a A . T . C. C. 9341, B a c i l l u s pumilus A . T . C. C. 14,884 o r S t a p h y l o c o c c u s a u r e u s , A.T.C. C. 6538P = F A 209P, D a r e a l s o employed. F o r b l o o d and body f l u i d samples an a g a r p l a t e method i s u s e d employing S a r c i n a l u t e a a s t e s t o r anism b e c a u s e of i t s g r e a t s e n s i t i v i t y3 2 , 4 6 , 4 7 , The minimum i n h i b i t o r y c o n c e n t r a t i o n (MIC) of cephradine f o r t h e f o u r t e s t c u l t u r e s i s a s follows: mcq/ml Sarcina lutea 0.04 Staphylococcus aureus 0.40 0.40 Bacillus pumilus Streptococcus f a e c a l i s 50.0 Cephradine i s s l i g h t l y more b i o a c t i v e a g a i n s t S t r e p t o c o c c u s f a e c a l i s and S a r c i n a l u t e a than cephalexin, while the reverse hold t r u e f o r Staphylococcus a ~ r e u s ~ ~ . 6.3 Iodometric Analysis Cephradine can be d e t e r m i n e d by t h e i o d o m e t r i c a s s a y . The B-lactam r i n g i s opened w i t h a l k a l i o r c e p h a l o s p o r i n a s e f o l l o w e d by i o d i n a t i o n a t an a c i d pH (pH 4 . 5 p h o s p h a t e 34 b u f f e r ) . About 4-5 moles o f i o d i n e a r e c o n s u m e d , I t i s i n t e r e s t i n g t o n o t e t h a t p e n i c i l l i n s under t h e same c o n d i t i o n consume 8-9 moles o f i o d i n e . The p r e c i s i o n of t h e i o d o m e t r i c a s s a y f o r c e p h r a d i n e i s n o t a s good a s t h a t f o r p e n i c i l l i n s when a l k a l i i s u s e d f o r i n a c t i v a t i o n . The p r e c i s i o n can be c o n s i d e r a b l y improved by u s i n g cephalos o r i n a s e i n s t e a d of a l k a l i f o r r i n g opening A l s o , w i t h t h e l a t t e r method much b e t t e r agreement was o b t a i n e d w i t h t h e m i c r o b i o -

6.2

16.

51

logical assay (see Table 10) even with severely degraded bulk samples. It therefore can be considered stability indicating. Table 10 Comparison of the cephradinase-iodometric, alkali-iodometric * and bioassay methods for the determination of cephradine in bulk powders Cephradine potency = mcg/mg Sample NN054ND NNO 5 9ND NN061ND NN054N.D NNO59ND NNO61ND Bioassay 812 854 778 578 424 405 Cephalosporinaseiodometric assay 813 043 846 578 4 6 6 464 Alkali-iodometric assay
979 991
980

744 643 639

* Results

are the means of determinations on two consecutive days. The powders were stored at 5OoC two years in bottles with varying volumes of head space.

CEPHRADINE

The p r e s e n c e of 7-ADCA d o e s n o t i n t e r f e r e w i t h t h e i o d o m e t r i c a s s a y 19 I t was found t h a t when i o d i n a t i o n was c a r r i e d o u t a t an a l k a l i n e r a t h e r t h a n a c i d pH, 1 3 e q u i v a l e n t s o f i o d i n e were consumed 34 6.4 Spectrophotometric Analysis The u l t r a v i o l e t a b s o r b a n c e peak of c e p h r a d i n e a t 262 n m ( s e e s e c t i o n 2 . 4 ) can be u s e d a s an i d e n t i f y a n d homogeneity a s s a y i n formulations35. Opening of t h e B-lactam r i n g w i t h a l k a l i o r preferably, with cephalosporinase a b o l i s h e s t h e U.V. a b s o r b a n c e a t 260nm. This h a s been made t h e p r i n c i p a e o f a q u a n t i t a t i v e a s s a y which a p p e a r s t o be s t a b i l i t y i n d i c a t i n g i n t h e " p r a c t i c a l " r a n g e ( l e s s t h a n 20% loss o f b i o a c t i v i t y ) 19. 6.5 Fluorometric Analysis Cephradine can be a s s a y e d q u a n t i t a t i v e l y i n a l k a l i n e medium b y f l u o r i m e t r y , ( e x c i t a t i o n wave l e n g t h 350 nm, e m i s s i o n wave l e n g t h 495 nm). T h i s method h a s been used f o r b l o o d l e v e l s t u d i e s b u t i s n o t recommended f o r t h e d e t e r m i n a t i o n i n u r i n e because of e r r a t i c r e s u l t s . 6.6 Colorimetric Analysis The w e l l known hydroxylamine method f o r p e n i c i l l i n h a s been a d a p t e d b y F A t o D cephradine a s a batching assay. It i s not s t a b i l i t y indicating. Strongly alkaline reaction c o n d i t i o n s and f e r r i c n i t r a t e w e r e used 5 When f e r r i c ammonium s u l f a t e was u s e d a t pH 7 o n l y 15% o f t h e r e s p o n s e normal f o r p e n i c i l l i n s was o b t a i n e d 3 7. A c o l o r i m e t r i c method u s i n g 5 , 5 ' d i t h i o b i s ( 2 - n i t r o b e n z o i c a c i d ) a t pH 9.2 p r o d u c e s a y e l l o w c o l o r which can be q u a n t i t a t e d b y measuring t h e peak a b s o r b a n c e a t 412 nm38.

53

KLAUS FLOREY

Chromatoqraphic A n a l y s i s 6 . 7 1 Paper Cephalexin (slower moving component) can be s e p a r a t e d from c e p h r a d i n e w i t h a n-butanol-t-amyl alcohol-~ater(7:l:4)system and q u a n t i t a t e d b y b i o a u t o g r a p h y u s i n g S t r e p t o c o c c u s a u r e u s 209P a s t h e a s s a y organisn?? Dihydrophenlyglycine(faster moving component) can be s e p a r a t e d from c e p h r a d i n e w i t h a t e r t . amyl a l c o h o l - s e c . - b u t a n o l - w a t e r (4:4: 1) s y s t e m and q u a n t i t a t e d w i t h a n i n h y d r i n - c o p p e r complex 40

6.7

Thin-Layer T h e r e a r e two TLC methods b y which c e p h a l e x i n and o t h e r i m p u r i t i e s can be s e p a r a t e d from c e p h r a d i n e a n d b o t h c e p h a l e x i n a n d c e p h r a d i n e can be q u a n t i t a t e d . Both methods 40 g i v e comparable r e s u l t s . I n t h e f i r s t method , s i l i c a g e l p l a t e s a r e impregnated w i t h s i l i c o n e f l u i d and d e v e l o p e d f o r 2-1/2 h o u r s i n a pH 4 . 1 M c I l v a i n e b u f f e r and a c e t o n e ( 1 0 0 : l . S ) . T h e z o n e s a r e l o c a t e d w i t h U.V. l i g h t , e l u t e d a n d q u a n t i t a t e d s p e c t r o p h o t o m e t r i c a l l y a t 260 nm. The s e p a r a t i o n scheme of t h e known components is a s follows :
A t 22OC

6.72

compound Dihydrocephradine Cephradine Cephalexin 7-ADCA 7 -ACA Dihydrophenylglycine Phenylglycine

Rf 0.31 0.43 0.51 0.65 0.65 0.74 0.80

Cephradine 0.72 1.00 1.20 1.51 1.51 1.72 1.88

To d e t e r m i n e r e s i d u a l 7-ADCA q u a n t i t a t i v e l y , i t i s a d v a n t a g e o u s t o change t o a s o l v e n t s y s t e m o f pH 6.5 M c I l v a i n e s b u f f e r acetone(50:l). The 7-ADCA zone i s e l u t e d w i t h 0.24 M sodium b i c a r b o n a t e a n d t h e a b s o r b a n c e a t

54

CEPHRADINE

260 n i s measured41. m The s e c o n d method42, a m o d i f i c a t i o n of t h e f i r s t i s p r e f e r r e d because of g r e a t e r e a s e of performance. The p l a t e s a r e i m p r e g n a t e d w i t h t e t r a d e c a n e i n s t e a d of s i l i c o n e and s i n c e t h i s i n t e r f e r e s w i t h t h e u. v. a b s o r b a n c e , q u a n t i t a t i o n is c a r r i e d o u t with ninhydrin. T h i s method can a l s o b e u s e d t o d e t e r m i n e d i h y d r o p h e n l y g l y c i n e and 7-ADCA semiq u a n t i t a t i v e l y . Approximate Rf v a l u e s a r e a s follows: C ep h r a d i n e 0.2 Cephalexin 0.3 7 -ADCA 0.6 Dihydrophenyl- 0.7 glycine 6 . 7 3 Column Chromatoqraphy High p r e s s u r e l i q u i d chromotog r a p h y (HPLC) h a s b e e n u s e d t o q u a n t i t a t e c e p h r a d i n e a n d r e s i d u a l c e p h a l e x i n i n c e p h r a d i n e . The mobile p h a s e i s a pH 4 . 3 g l a c i a l a c e t i c a n h y d r o u s sodium s u l f a t e s y s t e m , a Dupont s t r o n g c a t i o n exchange r e s i n , a t ambient t e m p e r a t u r e and a p r e s s u r e o f 1000 p s i g . Sulfamethazine is u s e d as i n t e r n a l s t a n d a r d and t h e o r d e r of elut i o n i s s u l f a m e t h a z i n e , c e p h a l e x i n and ~ e p h r a d i n e ~ A .m o d i f i c a t i o n , u s e s a n a c e t a t e ~ 0.17M sodium s u l f a t e b u f f e r pH 4 . 7 , Dupont Zipax c a t i o n exchange r e s i n , n- (4-methoxy-methyl6 - m e t h y l - 2 - p y r i m i d i n y l s u l f a n i l a m i d e as i n t e r n a l s t a n d a r d and a p r e s s u r e o f 300 p s i g . The o r d e r of e l u t i o n is cephalexin, cephradine, i n t e r n a l s t a n d a r d . 7-ADCA, when p r e s e n t , w i l l a p p e a r i n When a d j u s t i n g t h e p H o f t h e v o i d volume44. t h e mobile p h a s e t o 3.70 a n d r a i s i n g t h e p r e s s u r e t o 1800 p s i g , t h e s y s t e m was a b l e t o s e p a r a t e c e p h r a d i n e ( d - i s o m e r ) from t h e s y n t h e t i c a l l y p r e p a r e d l-isomer which p e a k s between c e p h a l e x i n and c e p h r a d i n e . N o l-isomer was
55

KLAUS FLOREY

A d e t e c t e d i n r e g u l a r c e p h r a d i n e samples4. r e v e r s e phase system, u s e f u l f o r q u a n t i t a t i o n i n formulation h a s a l s o been d e s c r i b e d 4 9 . A 1 mm x 2.1 mm i . d . column, ODS-Sil-X-I1 packing and 7 % methanol, 93% 0.05M ammonium c a r b o n a t e a s mobile phase were used. P r e s s u r e , 1 0 0 0 p i g ; f l o w 0 . 6 ml/min; d e t e c t o r W (254 nm); s e n s i t i v i t y , 0.08 AUFS.

Determination i n Body F l u i d s and T i s s u e s Cephradine h a s been determined microbiol o g i c a l l y (see s e c t i o n 6.2) i n human serum, i n human u r i n e , human lung t i s s u e , human eye t i s s u e and i n s p i n a l f l u i d . I t has been determined f l u o r o m e t r i c a l l y (see s e c t i o n 6.5) i n dog serum.
7.

Determination i n Pharmaceutical P r e p a r a t i o n I n pharmaceutical p r e p a r a t i o n s ( c a p s u l e s , o r a l suspensions and i n j e c t a b l e s ) i n f r a r e d has been used f o r i d e n t i t y t e s t s , t h e hydroxylamine and iodometric a s s a y f o r b a t c h i n g , t h e microb i o l o g i c a l and cephalosporinase-iodometric a s s a y s f o r s t a b i l i t y and chromatography f o r d e t e c t i o n of impurities.
8.

56

CEPHRADI N E

9.

References
B. T o e p l i t z , The S q u i b b I n s t i t u t e , P e r s o n a l communi c a t i o n . M. S. P u a r , The S q u i b b I n s t i t u t e , P e r s o n a l communication. P. T. Funke, The S q u i b b I n s t i t u t e , P e r s o n a l communication. J. Dunham, The S q u i b b I n s t i t u t e , P e r s o n a l communication. F. M. R u s s o - A l e s i , The S q u i b b I n s t i t u t e , P e r s o n a l communication. H. J a c o b s o n , The S q u i b b I n s t i t u t e , P e r s o n a l communication. L. P. M a r e l l i , A n a l y t i c a l P r o f i l e s o f Drug S u b s t a n c e s , Vol. 4 , p 21, A c a d e m i c Press, 1974. M. S. A t w a l , The S q u i b b I n s t i t u t e , P e r s o n a l communication. H. J a c o b s o n and I. G i b b s , J. Pharm. S c i . 62, 1543 ( 1 9 7 3 ) . D. E. A u s l a n d e r , The S q u i b b I n s t i t u t e , P e r s o n a l communication, F. D u r s c h , The S q u i b b I n s t i t u t e , U . S . P a t e n t 3,829,620, June 25, 1 9 7 4 . F. D u r s c h , The S q u i b b I n s t i t u t e , P e r s o n a l communication. Q. Ochs, T h e S q u i b b I n s t i t u t e , P e r s o n a l communication. J. E. D o l f i n i , H. E. A p p l e g a t e , G. Bach, H. Basch, J. B e r n s t e i n , J. S c h w a r t z a n d F. L. Weisenborn, J. Med. Chem. 1 4 , 117 (1971) : U . S . P a t e n t 3 , 4 8 5 , 8 1 9 ( 1 9 6 9 ) . M. L. Snow, C. L a u i n g e r and C. R e s s l e r , J. Org. Chem. 3 3 , 1 7 7 4 ( 1 9 6 8 ) . L. Demain, N a t u r e 2 1 0 , 4 2 6 ( 1 9 6 6 ) H. R. Roberts, The S q u i b b I n s t i t u t e , P e r s o n a l communication.

1.
2.

3. 4. 5. 6. 7.

8. 9.

10.

11.
12.

13. 14.

15. 16. 17.

57

KLAUS FLOREY

18.

19. 20.

A. I. Cohen, P. T. Funke and M. S. Puar, J. Pharm. S c i , 6 2 , 1559 ( 1 9 7 3 ) . B. M. F r a n t z , The Squibb I n s t i t u t e , P e r s o n a l communication; J. Pharm. S c i . , i n p r e s s , R. V a l e n t i and H. J a c o b s o n , The Squibb

21.
22. 23. 24. 25.

26. 27.

28.

29.

30.

31.

32.

33.

I n s t i t u t e , P e r s o n a l communication. M. A. L e i t z , The S q u i b b I n s t i t u t e , P e r s o n a l commun i c a ti o n , K. J. K r i p a l a n i and A. V. Dean, The S q u i b b I n s t i t u t e , P e r s o n a l communication. D. Adam, Munch. Med. WSchr. 1 6 1 9 4 5 ( 1 9 7 4 ) . 1, I. Weliky and R. Vukovich, The S q u i b b I n s t i t u t e , P e r s o n a l communication. A. V a h i d i , R. Vukovich, E. S. N e i s s and E. S c h r e i b e r , The S q u i b b I n s t i t u t e , P e r s o n a l communication. P. T. Funke, The Squibb I n s t i t u t e , P e r s o n a l communication. Weliky, I . , and Z a k i , A . , S e l e c t e d P r o c e e d i n g s from t h e 8 t h I n t e r n a t i o n a l Congress o f Chemotherapy, S e p t . 8-14,1973, A t h e n s , Greece, pp. 1-5. Vukovich, R . , M a r t i n e z , M . , and N e i s s , E . S . , S e l e c t e d P r o c e e d i n g s from t h e 8 t h I n t e r n a t i o n a l Congress o f Chemotherapy, S e p t . 8-14, 1973, A t h e n s , Greece, pp. 30-34. Z a k i , A. , S c h r e i b e r , E . C . , Weliky, I . , K n i l l , J . R . , and Hubsher, J . A . , J. C l i n . Pharmacol, New Drugs 14: 118-126 ( 1 9 7 4 ) . I. Weliky, H. H. Gadebusch, K. K r i p i l a n i , P. Arnow and E. C. S c h r e i b e r , A n t i m i c r o b i a l Agents and Chemotherapy 2 4 9 ( 1 9 7 4 ) . , G. R e n z i n i , G. Ravagnan, B. O l i v a , E. S a l v e t t i , and R. A u r i t i , Q u a d e r n i di A n t i b i o t i c a ; 1972, 17. T. B. P l a t t , The S q u i b b I n s t i t u t e , P e r s o n a l communication. S. Wind, The Squibb I n s t i t u t e , P e r s o n a l c o m u n i c at i o n .
58

CEPHRADINE

34.
35.

36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.
51.

J. Alicino, The Squibb Institute, Personal communication. H. Lerner and S. Willis, The Squibb Institute, Personal communication. A. F. Heald, C. E. Ita and E. Solney, The Squibb Institute, Personal communication. C. Sherman, The Squibb Institute, Personal communication. J. Kirschbaum, J. Pharm. Sci., 63 923 (1974). A. Vahidi and H. R. Roberts, The Squibb Institute, Personal communication. H. R. Roberts, The Squibb Institute, Personal communication. F. P. Targos, The Squibb Institute,Personal communication. I. R Salmon, The Squibb Institute,Personal . communica tion. A. Peterson and D. Guttman, Smith Kline & French Laboratories, Personal communication. J. Kirschbaum, The Squibb Institute, Personal communication. H. H. Pu and R. B. Poet, The Squibb Institute, Personal communication. D. M. Isaacson, The Squibb Institute, Personal communication. J. R. Ster, H. Weisblatt and J. D. Levin, The Squibb Institute, Personal communication. A. N. Niedermayer, The Squibb Institute, Persona1 communication. E. R. White, M. A . Carroll, J.E. Zarembo and A. D. Bender, J. Antibiotics - 205 (1975). 28, J. Z. Gougoutas and B. K. Toeplitz, Univ.of Minnesota and Squibb Institute, Personal communication. J. V Uri, P. Actor, L. Phillips and . J. A. Weisbach, Experientia &54(1975).
59

CHMROQUINE PHOSPHATE

Donald D,Hong

DONALD D. HONG

CONTENTS Analytical Profile

Chloroquine Phosphate

1.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor Physical Properties 2.1 Ultraviolet Spectrum rescence Spectrum 22 . 2.3 N A ear Magnetic Resonance Spectrum Spectrum 2.4 M a & % 2 5 ~ p cal Rotation . t i morphism and Melting Range 2.6 2.7 Dissociation Constant

2.

2.8 DH 2 9 seezing Point Depression .

2.10 Solubility 2.11 Differential Scanning Calorimetry

3.

Synthesis
Drug Metabolic Products

4.
5.

4.1 Biotransformation Products 4.2 Distribution in Human Tissues

Methods of Analysis 5.1 Phase Solubility Analysis 5.2 Identification by Spot Tests 5.3 Non-Aqueous Titration 5.4 Spectrophotometric Analysis 5.5 Fluorometric Analysis 5.6 Gravimetric Analysis 5.7 Chromatographic Analysis 5.71 Paper 5.72 Thin-Layer 5.73 Gas 5.8 Miscellaneous Methods Referencea

6 .

62

CHLOROQUINE PHOSPHATE

1.

Description 1.1
Name, Formula, Molecular Weight

The chemical name of chloroquine phosphate i n Chemical Abstracts is found under t h e heading Quinoline and designated as 7-Chloro- [4- (4-diethylamino-1-methylbutylamino) Iquinoline diphosphate. The hydrochloride and s u l f a t e e a l t e are a l s o a v a i l a b l e .

2H2PO4-

C@&CN3

2H3Po4

Molecular Weight:

515.87

1 . 2 Appearance, Color, Odor Chloroquine phosphate i s a w h i t e , o d o r l e s s , c r y s t a l l i n e powder having a b i t t e r taste: it d i s c o l o r e gradually on exposure t o l i g h t . 2. Physical P r o p e r t i e s 2 . 1 U l t r a v i o l e t Spectrum
A 10 y/ml s o l u t i o n of chloroquine phosphate i n 0.01 N H C 1 when scanned between 360 and 210 n e x h i b i t s m t h r e e maxima, three minima and s e v e r a l shoulders i n t h e region from 270 t o 225 nm, as shown i n Figure 1. The maxima are located a t 343 n ( a = 36.1), 328 n ( a = 32.6) and m m 222 n ( a = 59.9). The r a t i o of m /%% i s 1.11. Minima

were observed a t 335 nm, 280 n a342h3 nm. m 2.2 Fluorescence Spectrum

Figure 2 shows t h e fluorescence spectrum of chloroquine phosphate obtained on a s o l u t i o n of 0.2 mg/ml pH 7.9 phosphate b u f f e r using an Aminco-Bowman spectrophotofluorometer. Excitation a t e i t h e r 320 n o r 370 nm m produced emission s p e c t r a with a maximum a t 400 nm, t h e
63

si

.- . . . . . . . . .
........
. . . . .

...~. . . . . . . . .

... . . . . . . . .

...

...
......

... . . .

.
. . I

'

1.! .
,.
j
.

..
...
....
1

. .

,
i (

+
. . I

"I

i
, . .
!.

3I

i
I

64

CHLOR OQUlNE PHOSPHATE

Fluorescence
Figure2.

70

F luorescence Spectrum of Chloroquine Phosphate i n pH 7.9 Phosphote Buffer (Sterling-Winthrop House Reference Standard Lot N-087- JF)

WAVE L ENGTH-MILL IMICRONS

65

DONALD D. HONG

l a t t e r e x c i t a t i o n wavelength providing a higher emission response.

2.3

Nuclear Magnetic Resonance Spectrum

(Ml NR

The spectrum i n Figure 3 was obtained w i t h a Varian A60 NMR Spectrometer using a 20$ s o l u t i o n i n D20 containing TMS as an e x t e r n a l standard. The s p e c t r a l 1. assignments are summarized below ( )

c1
Protons No. Protons Derived from Integration
a Chemical Shi

Multiplic doublet doublet broad s i n g l e t quintet broad s i n g l e t sharp s i n g l e t doublet multiplet singlet doublet doublet

CH3 -CH2
CH -CH CH2 CH2-N CH-N

6
3

Cgc

1-68-1 -80 1.90-1.9

4 6
1 exchanged
1 1 1 1

NH

2.35 3 -55-3 a 9 1 4.38-4.65 5 -39 7.15-7 28 7 58-7 -75 7.75 8-33-8.40 8 48-8-55

b a
C

2.4

Mass Spectrum

The mass spectrum is shown i n Figure 4 and was obtained using a J o e l JMS-01SC mass spectrometer with an i o n i z i n g energy of 75 eV. The h i g h e s t mass observed a t m/e 319 i s a thermal breakdown product where two phosphoric acid moieties were l o s t from t h e parent compound. The base peak a t m/e 86 i s due t o t h e N,N,N-diethYlmethylene fragment (1).

66

A/-+-

L
I . .
, . I . ' . . I . ,

. . , . . . .I
. I
71
, '

. , .

I 1

I
40

I I
10

'

I
00

I
(0

Y I 4

an

I
$8

I
8

Figure 3.

NMR Spectrum of Chloroquine Phosphate.

Instrument:

Varian A60

DONALD D. HONG

Intensity

100

F
W

t z W
v)

>
80
319

5 60
40

+ a
W

L
A

BACKGROUND

a 20
0
20

.
40

.
60 ' 80

100 I20 140160 I80

Fig.4 Mass Spectrum of Chloroquine Phosphate (Sterling-Winthrop House Reference Standard Lot N-087-JF)

m/e

250

300

60

CHLOROQU IN E PHOSPHATE

2.5

Optical Rotation

Chloroquine phosphate e x h i b i t s e s s e n t i a l l y no o p t i c a l a c t i v i t y , e x i s t i n g as a racemic mixture. Riegel and Sherwood ( 2 ) have shown t h a t n e i t h e r of t h e o p t i c a l l y a c t i v e enantiomorphs showed any s i g n i f i c a n t d i f f e r e n c e s i n a n t i m a l a r i a l a c t i v i t y i n birds and f o r t o x i c i t y i n dogs. 2.6 Polymorphism and Melting Range

Chloroquine phosphate e x i s t s i n two polymorphic forms giving rise t o two melting ranges. USP x ( 3 ) r e p o r t s n one melting between 193" and 195" and t h e o t h e r between 210" and 215". Mixtures of t h e forms melt between 193" and 215". It i s possible t o obtain one form s e l e c t i v e l y by r e g u l a t i n g t h e rate of c r y s t a l l i z a t i o n ( 4 ) . 2.7 Dissociation Constant

The pKa's f o r chloroquine phosphate by t h e t i t r i m e t r i c method were found t o be 8.10 and 9.94 ( 5 ) . 2.8
@
A 18 aqueous s o l u t i o n has a pH of about 4.2.

2.9

Freezing Point Depression

Cryoscopic measurements were made on (w/v) solutions of t h e drug. Freezing Point Depression Chloroquine phosphate 0.73 "

lo$ and 2o$

Calculated

**
69

7.oe

Isotonic

DONALD 0.HONG

The value f o r "calculated i s o t o n i c " s o l u t i o n was obtained by graphic i n t e r p o l a t i o n t o FPD of 0.550", representing 0 . d sodium chloride s o l u t i o n ( 5 ) .
2.10 S o l u b i l i t y

Chloroquine phosphate i s f r e e l y soluble i n water: p r a c t i c a l l y insoluble i n alcohol, i n chloroform and i n

ether (3).

2.11 D i f f e r e n t i a l ScanninR Calorimetry (DSC)

Two polymorphic forms of chloroquine phosphate a r e exhibited by DSC. A mixture of t h e two c r y s t a l forms may be demonstrated a l s o by t h e t r a n s i t i o n temperatures ( 6 ) . The D C thermogram of a chloroquine phosphate s t a n d a r d shown S i n Figure 5 was obtained on a Perkin-Elmer DSC-lB d i f f e r e n t i a l scanning calorimeter a t a heating rate of 10C per minute under nitrogen. T h i s i s an example of t h e low melting form. Figure 6 shows another sample of chloroquine phosphate containing a mixture of t h e low and high melting forms.
Both t h e low and high melting polymorphs may be obtained from t h e same aqueous s o l u t i o n of chloroquine phosphate by s e l e c t i v e c r y s t a l l i z a t i o n . The high melting form usually occurs as small c r y s t a l s while t h e low melting polymorph c r y s t a l l i z e s as s i g n i f i c a n t l y l a r g e r c r y s t a l s . The two forms e x h i b i t s l i g h t d i f f e r e n c e s i n t h e i r I R curves from a KBr matrix.

3.

Synthesis

E l d e r f i e l d (7) and Kenyon i s collaboration w i t h Wiesner and K w a r t l e r (8) and more r e c e n t l y Basu, e t a1 ( 9 ) have summarized t h e American e f f o r t t o develop an a n t i malarial drug necessitated by World W a r 11. T h i s need was compounded when Japan seized c o n t r o l of t h e East Indies, e f f e c t i v e l y c u t t i n g o f f t h e n a t u r a l sources of quinine, which was t h e drug of choice f o r malaria a t t h e time. Chloroquine was one of t h e f r u i t s of t h i s concerted e f f o r t . The synthesis of chloroquine was f i r s t reported by t h e German chemists Andersag, Breitner and Jung (10, 11). Since t h e p a t e n t l i t e r a t u r e lacked t h e d e t a i l information required t o prepare t h e necessary intermediates, a research program w a s s t a r t e d a t Winthrop Chemical Company r e s u l t i n g i n a method o f synthesis for chloroquine by Surrey and
70

CHLOROQUINE PHOSPHATE

Figure 5. OSC Thermogram of Chloroquine Phosphate (Sterling-Winthrop House Reference Standard Lot N - 0 8 7 - J F ) Low Melting Form
189-202.5-207.5
I c
OC

(Corr.)
-4

t
1

m
I

* t
0
I

0
0

0
(D

0 I

t
I

r-

*
I

0
OD
I

0
Q,

t I

TEMPERATURE

O K

71

DONALD D. HONG

Figure 6 DSC Thermogram of Chloroquine Phosphate (Lot An-K-67) Mixture of Low and High Melting Forms

1-

189-202-1 -207.5 OC (Corrl

2 5-2231 2 2 6 O C (Corr)
ENDO

EX0

t 1

0 m

e
1

O
I

0
c

0
d I
00

Q
I

*
I
O K

Q,

0 0
In

TEMPERATURE

72

CH LOROQUINE PHOSPHATE

K m e (12). a mr
The two key intermediates required i n t h e synthes is a r e 4 7-d i ch l o r oquinoline and 4-d i e t h y lami no 1 methylbutylamine ( novol diamine). The s y n t h e t i c scheme i s shown i n Figure 7.

--

4.

Drug Metabolic Products

4.1

Biotransformation Products

Several metabolites in a d d i t i o n t o t h e unchanged drug have been i s o l a t e d from human t i s s u e s and urine. Titus, e t a1 (13) used counter-current d i s t r i b u t i o n t o i s o l a t e t h e d e s e t h y l compound ( A ) from t h e u r i n e of human volunteers. Kuroda (14) i s o l a t e d four metabolites of chloroquine from human t i s s u e s using a combination of paper chromatography and W spectroscopy. I n a d d i t i o n t o t h e desethyl compound ( A ) of T i t u s , they were t h e b i s d e s e t h y l (B), t h e c a r b i n o l (C) and t h e ~-amino-7-chloroquinoline( D ) d e r i v a t i v e s .

--

Similar observations were seen from u r i n e of human s u b j e c t s b u t no t r a c e of t h e 4-hydroxy-7-chloroquinoline w a s found. The unchanged drug was always found t o be t h e major compound. McChesney, e t a1 (15, 16) using a fluorescence technique confirmed t h e above findings and i n a d d i t i o n found t r a c e s of t h e hl-aldehyde (E) and t h e b-carboxy (F) d e r i v a t i v e s . They l i s t e d t h e amount of determinable excretory products as TO$ chloroquine, 23s desethylchloroquine, 1-2$ bisdesethylchloroquine and t h e o t h e r s as t r a c e degradation products. I n a d d i t i o n they reported t h a t about one-third of t h e administered chloroquine was unaccounted and remains obscure i n t h i s very complex blotransformation of t h e drug.

73

DONALD D. HONG

Figure 7: Synthesis of Chloroquine Phosphate

m-Chloroaniline

Ethyl oxalacetate

Ethyl(m-chloropheny1imino)succinate

(1) NaOH heat

c1 COOH
(2) HC1

k
1

and isomer separation OH

co0c.$l5

7-Chloro-4-hydroxyquinoline-2-carboxylic acid

Ethyl-7-chloro-4-hydroxyquinoline-2-carboxylate

-COOH

-.

Cl

c1

a >
I ! H 2

CH3CHCH2CH2CH9 (CpHg )2

novol diamine

., 1

7-Chloro - 4hydroxyquinoline

4,7-Dichloroquinoline

Chloroquine diphosphate

- H3P04 <
CL Chloroquine.base

74

CHLOROQUIN E PHOSPHATE

Compound Chloroquine

R
-CHCH$H2CH$I (C2H5 )2

CH3
A

-CHCH2CH$H$IHC2Hrj

CH3

I
I

-CHCH$H$H$H2

CH3
C

-CHCH2CH2CH20H

D E

4
H

-FHO

-rHC82CH2CwH
CH3

CH3

4.2

D i s t r i b u t i o n in Human T i s s u e s

Chloroquine i s predominantly l o c a l i z e d i n t h e l i v e r and t o lesser d e g r e e s i n t h e s p l e e n , h e a r t , kidney, lung, b r a i n , l e u c o c y t e s and s k i n . Chloroquine was o r i g i n a l l y used t o treat malaria. Subsequently it was found t o be e f f e c t i v e a g a i n s t o t h e r p a r a s i t i c d i s o r d e r s . According t o Rubin, e t a1 (17) t r a c e s o f t h e d r u g and its m e t a b o l i t e s were found i n t h e blood and u r i n e of s u b j e c t s up t o f i v e y e a r s after d i s c o n t i n u i n g long-term t h e r a p y .

75

DONALD D. HONG

5.

Methods of Analysis

5 . 1 Phase S o l u b i l i t y Analysis (PSA)

PSA i s probably n o t promising f o r c h l o r o q u i n e phosphate because t h e m u l t i p l e pKa's would allow s u b s t a n t i a l d i s p r o p o r t i o n a t i o n (18).
5.2

I d e n t i f i c a t i o n of Chloroquine Phosphate by Spot Tests

I n t h e approximate period of two decades f o l l o w i n g World W r I1 where t h e r e was i n c r e a s i n g use of c h l o r o q u i n e a phosphate f o r mass p r o p h y l a x i s of malaria it became n e c e s s a r y t o have a r e l a t i v e l y s i m p l e method t o i n s u r e t h a t t h e d r u g was t a k e n r e g u l a r l y . T e s t s of t h i s t y p e u s u a l l y measure t h e d r u g i n u r i n e . Other tests u t i l i z e v a r i o u s r e a g e n t s t o r e a c t e i t h e r w i t h t h e pure d r u g o r t h e d r u g i n dosage form. Some of t h e s e t e s t s are summarized i n Table 1.

5.3 Non-Aqueous T i t r a t i o n
Chloroquine phosphate can be t i t r a t e d w i t h a c e t o u s 0.1 N p e r c h l o r i c a c i d . The t i t r a t i o n may be c a r r i e d o u t manually w i t h c r y s t a l v i o l e t as i n d i c a t o r o r determined p o t e n t l o m e t r i c a l l y . The t i t r a t i o n is r a p i d a l t h o u g h n o n - s e l e c t i v e . T h i s n o n - s p e c i f i c i t y i s no drawback, however, so long as good i d e n t i f i c a t i o n tests are a l s o adopted ( 3 0 ) . Wu, e t a1 (31) have r e p o r t e d t h e determina t i o n of e l e v e n a n t i m a l a r i a l d r u g s u s i n g t h i s t i t r i m e t r i c procedure. Each ml of 0.1 N HClO4 i s e q u i v a l e n t t o 25.79 mg of chloroquine phosphate.

5.4

S p e c t r o p h o t o m e t r i c Assay

The W s p e c t r a of c h l o r o q u i n e base and phosphate s a l t are s i m i l a r i n 0.01 N HC1. Absorption maxima a r e observed a t 343, 328, 256 and 222 nm. Measurements are most f a v o r a b l y made a t 343 n where a b s o r p t i o n i s most m i n t e n s e and least a f f e c t e d by i n t e r f e r i n g s u b s t a n c e s i n t h e b i o l o g i c a l sample. The c h l o r o q u i n e b a s e is o b t a i n e d by e t h e r o r chloroform e x t r a c t i o n of an a l k a l i n e homogenate of t h e b i o l o g i c a l sample. After s e p a r a t i o n of i n t e r f e r i n g
76

CH LOROQUIN E PHOSPHATE

Table 1
Test

Spot T e s t s Color P a l e green Violet Yellow Sensitivity

NA NA NA

Form Tablet Tablet Tablet

Ref.
19

1. Complex w i t h Copper
2. Complex w i t h Cobalt

19 .
20

3 . Dimethylaminobenzaldehyde ( E h r l i c h ' s reagent ) 4. Styphnic a c i d

Pure drug Pure drug

Rosettes of p l a t e s
NA

0.5~

21 22

5. N i t r o p r u s s i d e and
P i p e r a z i n e (Lewin's r e a g e n t )*

SOY

6. Eosin yellowish
( D i l l & Glazko)

Urine Urine

Yellow t o v i o l e t -red

NA 2.5-9- 5~ per rnl urine

2 mg/liter

23

7. Mercuric iodide/KI
( W e r -Tanre t ' s reagent )

**
Yellow

24

8. Methyl orange 9. Complex w i t h

HClO 4/AuClj
10. Aconitic a c i d /

Urine

25

Pure drug Rosettes o r b i o l . and d e n d r i t e s extract Pure drug Red

0.4~

26

57

27

a c e t i c anhydride/ e t h y l e n e di c h l o ri de

11. H2SO4/KClOj 12. H C ~ / K C ~ O ~

Biol. extract Biol. extract

Red - v i o l e t

5Y
10Y

20 28

Yellow Yellow Blue-violet to b l u e -green

15. 25$ H2SOl+/Chlorinated Biol. line extract

10Y 0.8 mg$

28
29

1 4 . BPB/boric a c i d

Free b a s e

**

S p e c i f i c f o r N-ethyl group T u r b i d i t y i s measured

77

DONALD D. HONG

materials, t h e base i s i n t u r n extracted i n t o a s o l u t i o n of 0.1 or 0.01 N H C 1 and q u a n t i t a t i v e l y determined by measuring i t s W absorption. A l t e r n a t i v e l y t h e acid s o l u t i o n can be made a l k a l i n e and t h e base extracted w i t h e t h e r o r chloroform. The organic phase i s evaporated t o dryness and t h e residue further examined by I R and paper o r TLC.
A b r i e f summary of t h e spectrophotometric procedures f o r t h e q u a n t i t a t i v e determination of chloroquine and metabolites i s summarized i n Table 2.

Table 2

Spectrophotometric Methods I s o l a t i o n from Human Tissues Blood Tissues Tissues+ Urine Reference

Method of Analysis

UV, I R , TLC, GLC uv, I R , PC

uv uv

32

33

34
35

uv

14

Includes i d e n t i f i c a t i o n of chloroquine metabolites a l s o

5.5

Fluorometric Analysis

Fluorometric procedures have been extensively u t i l i z e d f o r t h e q u a n t i t a t i v e determination of chloroquine i n b i o l o g i c a l materials. I n t h e e a r l y days of fluorescence when instrumentation was n o t s u f f i c i e n t l y advanced, an a d d i t i o n a l i r r a d i a t i o n s t e p was required t o convert t h e chloroquine t o a more i n t e n s e fluorophore ( 3 6 ) . With t h e advent during t h e mid 1950's of t h e highly s e n s i t i v e spectrophotofluorometers u t i l i z i n g t h e xenon a r c source and monochromators, it i s now possible t o measure t h e chloroquine fluorescence d i r e c t l y (15, 16, 37, 3 8 ) . Brodie, e t a1 (36) found t h a t t h e fluorescence of chloroquine i n pH 9.5 b o r a t e b u f f e r after i s o l a t i o n from b i o l o g i c a l specimen may be s t a b i l i z e d by t h e a d d i t i o n of cysteine. The sample i s then irradiated with UV l i g h t and t h e measurement made using a s u i t a b l e fluorometer. The s e n s i t i v i t y of t h e procedure i s about 0.lmcg.

5.6 Gravimetric Analysis

Parikh and Mukherji ( 3 9 ) reported t h e q u a n t i t a t i v e formation of chloroquine-silicatungatate CSi0278

CH LOROQUI N E PHOSPHATE

1803 -2(C18H2@3Cl). 2H20] p r e c i p i t a t e from chloroquine phosphate and s i l i c a t u n g s t i c a c i d . By use o f t h e approp r i a t e g r a v i m e t r i c f a c t o r t h e v a r i o u s s a l t s o f chloroquine could be determined. USP XVI (10) a l s o contained a g r a v i m e t r i c method whereby t h e chloroquine base is measured.

5.7

Chromatographic Analysis

5.71 Paper Chromatosaphic Analysis

A number o f paper chromatographic systems for chloroquine phosphate and i t s base are summarized i n Table 3. Goldbaum and Kazyak ( 4 1 ) use i o d o p l a t i n a t e r e a g e n t t o v i s u a l i z e t h e chloroquine which i n t u r n may be e l u t e d o f f t h e paper and t h e s p o t q u a n t i t a t e d by o t h e r means.

5.72

Thin-Layer Chromatographic Analysis

The f o l l o w i n g TU: systems (Table 4 ) are u s e f u l

as an i d e n t i t y t e s t and i n t h e e v a l u a t i o n of t h e p u r i t y o f

t h e drug substance. The n a t u r e of t h e i m p u r i t i e s p r e s e n t is a l s o h e l p f u l i n t h a t it t e l l s i n d i r e c t l y , f o r example, s i m i l a r i t y o r d i e s i m i l a r i t y o f t h e manufacturing process.

A l l of t h e systems u t i l i z e precoatcd s i l i c a g e l containing a fluorescent indicator.

5.73 Gas-Liquid Chromatographic Analysis (GLC)

The f o l l o w i n g procedures have been demonstrated t o be a p p l i c a b l e t o t h e GLC examination of chloroquine base. Vanden Heuvel, - - ( 4 6 ) Viala, e t a1 (47) and Kazyak and e t a1 Knoblock (48) have r e p o r t e d t h e d e t e c t i o n of t h e drug i n microgram amounts from b i o l o g i c a l samples after s o l v e n t e x t r a c t i o n . Holtzman (49) has shown t h a t a8 l i t t l e as 5 nanogram q u a n t i t i e s of chloroquine base could be d e t e c t e d u s i n g e l e c t r o n c a p t u r e . The c o n d i t i o n s r e p o r t e d , however, are f o r pure drug and m a y be o f p o t e n t i a l value i n t h e a n a l y s i s o f t h e substance i n b i o l o g i c a l m a t e r i a l e . The following c o n d i t i o n s have been used f o r t h e
CIA: d e t e r m i n a t i o n of chloroquine base (50).

Column: 3.8s S i l i c o n e Gum SE 30, 4 f t , g l a s e Support : D i a t o p o r t S Detection: FID

79

Table 3 S o l v e n t System
1. n-Butanol s a t ' d
w i t h buffer
2.

Paper Chromatographic Systems Paper Detection

Species

Rf - Reference
0.15
0.16 0.26
0.89 0.36

base
base

Whatman No. 2 s a t ' d w i t h pH 3 .O M a c I l v a i n e ' s buffer Whatman No. 2 sat 'd w i t h pH 5 .O MacIlvaine ' s buffer Whatman No. 2 sat 'd w i t h pH 6.5 Sorensen's b u f f e r Whatman No. 2 sat 'd w i t h pH 7.5 S o r e n s e n ' s buffer Whatman N o . 1 s a t ' d w i t h petroleum (195-220' f r a c t i o n ) as above

uv uv
W

41
41 41

n-Butanol sat 'd w i t h buffer n-Butanol s a t ' d w i t h buffer n-Butanol sat 'd
w i t h buffer

3.

base
base
base
base base

4.
0 W

uv

41
42
42 42

5.

Ethanol-Water-Conc ammonia (35-63-2)

AS

w w w
uv
W
W

6.

NO. 5 (45-53-2)

0.60
0.79

7 - AS NO. 5 (55-43-2) 8. 9.
10. AS NO. AS

as above
as above as above

5 (65-33-2)

base

0.87 0.88 0.89

0.89

42

NO. 5 (75-23-2)

base

42
42 42

As No.

5 (85-13-2)

base
base

as above as above

1 . As No. 5 1

(95-3-2)

uv

CHLOROQUINE PHOSPHATE

Table System Methanolwater-conc a m o n i a (72:25:3) Benzene-methanolieopropylamine (87:10:3) Chlorof orm-aethanolisopropylamine (94:3 :3)

- TLC Systems
Rf x 100
Reference

S p o t t i n g Soln Chloroform Chloroform' MeOH-H 0

2%

43 43 43 43 43 43 44 44 44 44

41 40
15
28

(737
Chloroform2 H20/MeOH/CHC13 / i sopropylaaine

Chloroform-isopropylamine

(97:3 1

E t h e r -hexane i sopropylm i n e (90:~:~) n-Butanol-conc mmonia-

water

(85:k:ll)

Water
Water

59
60
28

Ethylacetate-conc ammoniae b s . a l c o h o l (5:2:2)

25$ ammonia-benzene-dioxanee t h a n o l (1:10:8 :1)

Water
Water
Water

Chloroform-cyclohexanediethylamine ( 5 :4:1)

40

25% ammonia-methanol
(3:200) Acetone -water -ammonia (90 :40 :1 )

20
15

abs. a l c o h o l S

45

Spotting solution 1. The drug i s e x t r a c t e d i n t o chloroform a f t e r b a s i f y i n g w i t h 10% Na2C03. 2. S i m i l a r t o above b u t from human plasma. 3 . Spotted as t h e b a s e . Detection
A

B
C D E

- W-254 - I o d i n e vapor/20$ H2S04 - Dragendorff's r e a g e n t - w-360 - Iodoplatinate reagent

81

DONALD D. HONG

Temperature :

I n j port 275 "C Column 240 O C Detector 250 "C Flaw Rate: 30 ml/min helium Retention Time: 7.0 min

5.8

Miscellaneous Methods of Analysis

Roushdi and Shafik (51) reported t h e determinat i o n of chloroquine phosphate by t i t r a t i o n with an anionic s u r f a c t a n t , d i o c t y l sodium sulfosuccinate (Aerosol O.T. ), using dlmethyl yellow as i n d i c a t o r .
A complexometric method using bismuth complexonate t o p r e c i p i t a t e t h e chloroquine base and t i t r a t i n g t h e liberated EWIA w i t h zinc s u l f a t e was also reported by t h e s e authors (52).

Various quinine salts and chloroquine phosphate have been determined using ammonium reinckate (53). For chloroquine phosphate t h e insoluble r e i n c k a t e s a l t I s formed a t pH 1, removed, and t h e amount of excess reagent I n t h e f i l t r a t e i s measured colorirnetrlcally. The d i f f e r e n c e i n absorbance between t h e sample and blank and t h e standard and blank r e p r e s e n t t h e absorbances of t h e sample and standard s o l u t i o n s , r e s p e c t i v e l y .

82

CH LOROQU IN E PHOSPHATE

6.

References
1. S. Clemans, Sterling-Winthrop Research I n s t i t u t e ,

Personal Communication. B. Riegel and L. T. Sherwood , Jr. , JACS, 71, 1129 (1949)3- United S t a t e s Pharmacopeia X M , p. 82 (1975). 4. R. L. Kenyon, J. A. Wiesner and C. E. K w a r t l e r , Ind. and Ehg. Chem. , 659 (1949). 5. P. Dederick, Sterling-Winthrop Research I n s t i t u t e , Unpublished Data. 6. W. Houghtaling, Sterling-Winthrop Research I n s t i t u t e , Personal Communication. 2598 (1946). 7. R . C. E l d e r f i e l d , Chem. Eng. News, 8. R . L. Kenyon, J. A. Wiesner and C. E. K w a r t l e r , Ind. and Eng. Chem. , 654 (1949). 9. U. P. Basu, A. Raychaudhuri and R. De, Res. and Ind., 2.

41,

6 ,

2,

B r e i t n e r and II. Jung, German Patent 683,692 (1939). 1 . Ibid , U . S. P a t e n t 2,233,970 ( t o Winthrop Chemical 1 Company) (1941). 12. A. R. Surrey and H. F. Hammer, JACS, 68, 113 (1946). 13 E. 0. T i t u s , L. C. Craig, C. Golumbic, H. R. Mighton, I. M. Wampen and R. C. E l d e r f i e l d , J. Org. Chem.,
10.

H. Andersag, S.

10,

165 (1965).

-.

14.
15.

UJ39 (194a)*

K. Kuroda, J. Pharmacol. Exp. Ther., 156 (1962). E. W . McChesney, W. D. Conway, W. F. Banks, Jr. , J. E. Rogers, and J. M. Shekosky, fbid., 1 1 482 5,

u,

( 1966 16. E. W . McChesney, M. J. Fasco and W. F. Banks, Jr., 17 *

Ibid., 323-(1967). M. Rubin. H. N . Bernstein and N. J. Z v a i f l e r , Arch. Ophthalmil. , 70, 474 (1963 ) L. T. Grady, Drug Standards Laboratory, Personal Communication. P. Cooper, Pharmaceutical J., 177, 53 (1956)I b i d . , p. 495. E. G. C. Clarke, J. Pharm. and Pharmacol., l0, 194 (1958 * E. S i l v a , Anal. Chem., Proc. I n t . Symp. i n Honor of F r i t z F e i g l , Birmingham, Engl., A p r i l 1962. Elsevier, 1963 , p. 78. J. L e l i j v e l d and H. Kortmann, Bull. WHO, 42, 477

- 158,
-

18.

19

20. 21. 22. 23 * 24. 25

G. Fuhrmann, 22, 663 (1960). W. T. Haskins, Am. J. Trop. Med. Hyg.,

83

( 1970

w.,

7, 199

(1958).

DONALD D. HONG

26

A. F. Fartushnyi, Farmatsiya, l8, 77 (1969); CA 71: 47882 v (1969). , 27. R. S t r u f e , Clin. Chlm. Acta, 2 753 (1960). 28. A. F. Fartusknyl, Sudebro-Med. Ekspertiza. Mln. Zdravokhl SSR, lo, 45 (1967); CA 66:114281 k (1967). 0. Fuhrmann and K. Werrbach, 2. Tropenmed. 29 P a r a s i t o l . , l, 269 (1965): CA 65:12725 b (1966). 6 . M. E. Auerbach, Sterling-Winthrop Research I n s t i t u t e , Unpublished Data. T. S. Wu, T. 0 . Sun and T. H. Tang, Yao Hsueh Hsueh Pao, 253 (1958); CA 53~20691 (1959). d 32. R. W. Prouty and K. Kuroda, J. Lab. Clin. Med., 2,

6,

33
34.

V. Waaret, Archiv. For Pharmacl. Og. Cheml.,

477 (1958)-

71, 116

37

A. E. Robinson, A. I. Coffer and F. E. Camps, J. Pharm. and Pharmacol., 22, 700 (1970). J. Stepan and B. Kakac, Med. Exp., ll, 352 (1964). B. B. Brodie, S. Udenfrlend, W. D i l l and T. Chenkin, J. Biol. Chem., 319 (1947). E. M. Ensor, Trans. Roy. SOC. Trop. Med. Hyg., 60,

(1964)

x,

75 (1966). 38- E. W. McChesney, 39

40. 41.
42.

W. F. Banks and J. P. McAullff, A n t i b i o t i c s and Chemotherapy, 12, 583 (1962). P. M. Parlkh and S. P. Mukherji, Ind. J. Pharm., 5, 269 (1.963). United S t a t e s Pharmacopeia XVI, p. 151 (1960). L. R. Goldbaum and L. Kazyak, Anal. Chem., 28, 1289

J. Vecerkova, J. Solc and K. Kacl, J. Chromat., l0,

(1956).

43

44.

45
46.
47
48.

R. Crain, Sterling-Winthrop Research I n s t i t u t e , Unpublished Data. L. T. Grady, Drug Standards Laboratory, APhA, Unpublished Data. R. Castagnou and M. Sylvestre, Bull. SOC. Pharm. Bordeaux, 1 5 121 (1966); CA 6 7 ~ 6 2 8 2 1 0, k. W. J. A. VandenHeuvel, B. 0. A. Kaahti and E. C. Horning, C l t n . Chem., g, 351 (1962). A. Viala, J. P. C a n 0 and A. Durand, J. Chromatog., 1 1 299 (1975). 1, L. KaWsk and B. C. Knoblock, A n a l . Chem., 1448
J. L. Holtzman, Anal. Biochem., 66 (1965). J. Grego, Sterling-Wlnthrop Research I n s t i t u t e , Unpublished Data.

479 (l963).

(1963)

s,

49
50

u,

84

CHLOROOU IN E PHOSPHATE

51.
52.

53.

I. M. Roushdi and R . M. Shafik, J. Pharm. Sci. U.A.R., 2, 65 (1968). Ibid., p . 57. S-K. Sheng, C - I . Hsieh and S-H. Peng, Yao Hsueh Hsueh Pao, l , 662 (1965); CA 64:7967 c (1966). 2

Acknowledgments The writer wishes t o thank M r . E. L. P r a t t and D r . R . S. Browning f o r reviewing t h e m n u a c r i p t , D r . S. D. Clemans f o r t h e NMR and M.S. data, Mr. W. W. Houghtaling f o r t h e D C data, Dr. L. T. Grady (USP Laboratories) f o r S t h e TLC data, Mrs. E. V. Miller f o r t h e drawings and Miss

S. F. Casey f o r typing t h e p r o f i l e .

85

DAFSONE

Chester E. Orzech, Norris G. Nash, and Raymond D. Daley

CHESTER E. ORZECH ma/.

CONTENTS 1 . DESCRIPTION 1 1 Name, Formula, Molecular Weight . 1 2 Appearance, Color, Odor . PHYSICAL PROPERTIES 2 1 Infrared Spectra . 22 Nuclear Magnetic Resonance Spectrum . 23 Ultraviolet Spectra . 2 4 Mass Spectrum . 2 5 Differential Thermal Analysis (DTA) . 2 6 Differential Scanning Calorimetry (DSC) . 2 7 Crystal Properties . 2 8 Solubility . 2 9 Fluorescence Spectra . 2.10 Melting Point SYNTHESIS STABILITY-DEGRADATION DRUG METABOLIC PRODUCTS

2 .

3.

4.

5 .

6. METHODS OF ANALYSIS 61 Identification Tests . 6.2 Elemental Analysis 6 3 Colorimetric Methods . 6 4 Titration Methods . 6 5 Fluorometric Methods . 6.6 Paper Chromatography 6 7 High Pressure Liquid Chromatography . 6.8 Gas Chromatography 6.9 Thin Layer Chromatography 7 . ACKNOWLEDGMENTS

8 REFERENCES .

88

DAPSONE

1.

DESCRIPTION

1.1 Name, Formula, Molecular Weight Dapsone i s 4,4'-diaminodiphenyl sulfone, a l s o known as p i p '-sulf onyldianiline and b i s (4-aminophenyl) sulfone. Chemical Abstracts indexes dapsone a s benzenamine, 4,4'-sulfonylbis-, s t a r t i n g w i t h Volume 76; previously the index name was a n i l i n e , 4,4'-sulfonyldi-. The C S Registry Number is [80-08-0]. A
0

H2N

H 2$ * c
0

Mol. W t . :

248.31

1.2 Appearance, Color, Odor Dapsone i s a white or creamy white odorless cryst a l l i n e powder. 2.

PHYSICAL PROPERTIES

2.1 Infrared Spectra Infrared s p e c t r a of two c r y s t a l forms of dapsone (designated Form 1-and Form 11) are shown i n Figures 1 and 2. The samples were prepared a s mineral o i l m u l l s between potassium bromide p l a t e s . A Beckman Model I R - 1 2 spectrophotometer was used. The spectrum of Form I i s similar t o those published by Pouchert (1) and by Hayden e t a 1 (2). The spectrum of a hydrate of dapsone has a l s o been observed; s i n c e i t i s d i f f e r e n t from the s p e c t r a of the anhydrous forms, samples should be dried a t 105OC before obtaining spectra. Some of the absorption bands may be assigned a s follaws (3):

89

( D

Figure 1 .

Infrared Spectrum of Dapsone (Form I ) , Mineral Oil Mull

Figure 2.

Infrared Spectrum of Dapsone (Form II), Mineral O i l Mull

CHESTER E. ORZECH H a l .

Band frequency, c m 3200-3500 3000-3 100 2800-3000 1635

Assignment
N-H S t r e t c h

Aromatic C-H S t r e t c h Mineral O i l

N-H Deformation

1590, 1500, 1440 1460, 1380 1300 Region 1150 830-840 550

Aromatic C=C S t r e t c h Mineral O i l Asymmetric -SO2- S t r e t c h Synunetric -SO2- S t r e t c h

2 Adjacent H on aromatic ring

-SOz- Scissoring

2.2 Nuclear Magnetic Resonance Spectrum The NMR spectrum shown i n Figure 3 was obtained by dissolving USP reference standard dapsone i n acetoned containing tetramethylsilane as i n t e r n a l reference. Tke spectrum was produced using a Varian EM-360 NMR spectrometer. The s e r i e s of peaks centered a t 7.2 ppm is an A2Bp p a t t e r n t h a t is t y p i c a l of para s u b s t i t u t i o n and the broad s i n g l e t a t 5.4 ppm is due t o the amine protons. The peaks a t 2 ppm and 3 ppm a r e due t o the solvent. These values c o r r e l a t e well with those previously reported
(4)

2.3 U l t r a v i o l e t Spectra Figure 4 i s t h e u l t r a v i o l e t absorption spectrum of dapsone i n methanol s o l u t i o n , run on a Cary Model 14 spectrophotometer. The s o l u t i o n contained 8.0 mg of dapsone per l i t e r of methanol, and was r u n a g a i n s t methanol ( 1 c c e l l s ) . The spectrum e x h i b i t s peaks a t m 295 n and 260 nmwith a b s o r p t i v i t i e s of 30,100 and m 18,300 l./mole c m respectively. The u l t r a v i o l e t i d e n t i t y t e s t of the B r i t i s h Pharmacopoeia (5) s p e c i f i e s peaks a t
92

I---

1 0

Figure 3.

Nuclear Magnetic Resonance Spectrum of Dapsone

. . .

v
94
(D

z
a

2
% m
a

210

250 WAVELENGTH
I 0
W

300 IN NANOMETERS
in Methanol, 1 cm Cells

I L

350

In

Figure 4 .

Ultraviolet Spectrum of Dapsone, 8 mg/l.,

z -

CHESTER E. ORZECH eta/.

295 and 260 n with a b s o r p t i v i t i e s of about 30,000 and m 18,100 l./mole cm respectively. A s i m i l a r spectrum i s obtained i n 95% ethanol s o l u t i o n , except t h a t the a b s o r p t i v i t i e s a r e apparently lower. Baliah and Ramakrishnan ( 6 ) r e p o r t peaks a t 295 and 261 n w i t h a b s o r p t i v i t i e s of 27,000 and 17,800 1./ m mole cm. Hayden e t a 1 (2) r e p o r t peaks a t 295 and 260 nm. Maschka e t a 1 (7) reported u l t r a v i o l e t s p e c t r a of dapsone a t various hydrogen ion concentrations; the d a t a a r e a s follows: Condition Wave length of Maxima, nm 291.0 258.5 291.0 259.0 289.5 273.5 264.5 234.5 Molar Absorptivity, l./mole cm 19,820 12,190 17,950 10,810 13,490 1,850 2,000 10,790

1.

pH 11.0

2.
3. 4.

p 6.8 H p 1.8 H

2 2

HCl

2.4 Mass Spectrum Figure 5 shows the low r e s o l u t i o n mass spectrum of dapsone. The d a t a w a s obtained w i t h an LKB 9000s mass spectrometer, with an i o n i z a t i o n voltage of 70 eV, source Some of the peaks may be assigned a s temperature 250'C. follows (8):

m/e
248 140 108

Assignment

M+

92

95

96

50
MASS/ CHARGE
Figure 5.
v1

R AT10
a
0)

M s Spectrum of Dapsone as

DAPSONE

2.5 D i f f e r e n t i a l Thermal Analysis (DTA) The DTA curve i n Figure 6 was obtained with a DuPont Model 900 instrument. The curve shows a sharp endothermic s o l i d - s o l i d phase t r a n s i t i o n a t 84OC and a melting endotherm a t 178OC.
2.6 D i f f e r e n t i a l Scanning Calorimetry (DSC) The DSC melting thermogram of dapsone c r y s t a l l i z e d from methanol and water i s shown i n Figure 7 , 'The thermogram was obtained with a Perkin-Elmer DSC-1B d i f f e r e n t i a l scanning calorimeter a t a heating r a t e of 1.25C/minute i n a nitrogen atmosphere. The p u r i t y of samples of t h i s compound can be determined by a n a l y s i s of the melting thermogram (9).

2.7 C r y s t a l Properties B u t t (10) reported t h a t dapsone can be obtained i n a t l e a s t two c r y s t a l forms, with d i f f e r e n t melting points. Infrared s p e c t r a (see Section 2.1) and x-ray powder d i f f r a c t i o n p a t t e r n s a l s o i n d i c a t e t h a t dapsone occurs i n a t l e a s t two c r y s t a l forms, end t h a t it forms a c r y s t a l l i n e hydrate. The powder d i f f r a c t i o n p a t t e r n s a r e given i n Table 1 . These p a t t e r n s were obtained with a Norelco d i f fractometer, using n i c k e l - f i l t e r e d copper Kcr r a d i a t i o n . The c r y s t a l and molecular s t r u c t u r e of dapsone has been determined by Alleaume and Decap (ll), and by Dickinson e t a 1 (12, 13). Both groups r e p o r t 4 molecules i n an orthorhombic u n i t c e l l , symmetry P212121, with s i m i l a r dimensions (I):
a=25.57 a=8.065

+ 0.01, b=8.07 + 0.01, c=5.77 5 0.01 (11) + 0.005, b=25.57 + 0.02, c=5.760 2 0.001 ( 1 2 )
c

97

Figure 6 .

Differential Thermal Analysis Curve of Dapsone

to

20

21

OAPSONE hHF ~4.90 KCAL.

Tf

a,

00 Irz

25

30

PURITY = 99.99 %

II

99

(0 (0

\
I

I-

Figure 7 .

Differential Scanning Calorimeter Curve of Dapsone

CHESTER E. ORZECH e t a / .

TABLE 1 DAPSONE X-RAY POWDER DIFFRACTION PATTERNS F o r m I1

c

Hydrate
d -

1/10 4 8 32 53 4 9 60 34 17 100 49 62 1 48 22 44 2 1 10 2 10 4 46 2 9 4 6 4 4 7 3 2 5 5 3 2 5 3 4 8

d 13.07 10.78 9.21 7.41 6.85 6.66 6.57 6.40 5.80 5.68 5.45 5.35 5.01 4.66 4.57 4.37 4.27 3.99 3.86 3.82 3.52 3.50 3.42 3.37 3.33 3.28 3.20 3.13 3.09 3.02 2.89 2.86 2.73 2.68 2.52 2.43 2.40 2.34 2.28 2.21

1/10 5 2 1 7 8 89

1/10 1 4 3 16 29 13 47 28 1 7 7

12.79 7.74 6.86 6.42 5.88 5.66 5.28 5.03 4.71 4.64 4.42 4.30 4.12 4.00 3.84 3.78 3.65 3.46 3.42 3.32 3.21 3.16 3.09 2.98 2.94 2.88 2.80 2.78 2.74 2.64 2.56 2.51 2.44 2.40 2.35 2.31 2.25 2.20 2.17 2.13

18
18 3 3 27 54 23 97 20 96 9 8 21 100 23 25 8 9 17 2 8 22 14 26 9 17 3 3 6 2 2 5 2 2

12.55 12.25 11.13 8.54 7.73 7.62 7.54 7.45 6 -82 6.39 6.25 5.98 5.74 5.68 5.62 5.53 5.45 5.32 5.12 5.00 4.93 4.86 4.79 4.68 4.59 4.55 4.45 4.39 4.31 4.27 4.22 4.18 4.09 4.05 4.01 3.95 3.86 3.83 3.77 3.72

4
28 35 19 17 11 4 3 21 58 9 2 100 39 75 6 27 13 54 31 12 87 19 13 4 15 19 4 3

100

DAPSONE

2.08 2.06 1.96

5 1 2

2.17 2.10

3.65 3.58 3.46 3.42 3.37 3.35 3.30 3.28 3.23 3.20 3.14 3.09 3.02 2.96 2.87 2.84 2.82 2.79 2.72 2.69 2.65 2.62 2.58 2.50 2.45 2 -42 2.27 2.15 2.11 2.10 2.07 1.96

1 14 1 1 9 13 6 15 8 30 10 21 14 8 6 5 3 3 6 6 5

7 7 1 3 3 2 6 5 2 2 3 3

2.8 S o l u b i l i t y The s o l u b i l i t y a t room temperature is a s f o l l o w s : Solvent Methanol Ethanol (95%) 2-Propanol Ethyl Acetate Ethyl Ether Chloroform Benzene Water 2,2,4-trimethylpentane
101

Approximate S o l u b i l i t y , mg/ml

52 28 6 34 0.9 3 0.5 0.2 (14) c0.2

CHESTER E. ORZECH eta/.

Dapsone is very soluble i n acetone and a c e t o n i t r i l e ; one gram of dapsone dissolves i n 1.8 m l of acetone or 5 m l of a c e t o n i t r i l e . It is a l s o soluble i n d i l u t e mineral a c i d s ; one gram dissolves i n about 10 m l of 1E hydrochloric acid.
2.9 Fluorescence Spectra Peters e t a 1 (15) found the e x c i t a t i o n maximum a t 285 n and the fluorescence maximum a t 350 nm, i n ethylene m dichloride s o l u t i o n , Ellard and Gammon (16) reported an e x c i t a t i o n maximum a t 298 n and the fluorescence maximum m a t 345 nm, i n e t h y l a c e t a t e . Glazko e t a 1 (73) and Cucinell m e t a 1 (17) reported the e x c i t a t i o n maximum a t 297 n and the fluorescence maximum a t 340 nm, i n e t h y l a c e t a t e ,

2.10 Melting Point A range of melting points has been reported for dapsone :
172'C 172-173OC 172 174'C 174OC 174- 176'C 175OC 175-176'C 176OC 176.3-177.5OC 178-179OC 1 7 9- 1 8 O o C

Bu t (10) reported t h a t dapsone can -e obtained ,n two forms, one melting a t about 178.5OC, the other a t about 180.5'C.
3.

SYNTHESIS

Dapsone has been prepared by various procedures, s t a r t ing with 4,4'-dinitrodiphenyl s u l f i d e ( 2 1 , 2 2 , 31, 32) prepared from p-chloronitrobenzene (33), or by oxidation of 4,4'-diacetylaminodiphenyl s u l f i d e (19, 23, 25) prepared from 4-nitro-4'-aminodiphenyl s u l f i d e (19, 23, 24) or 4,4'diaminodiphenyl s u l f i d e (25). It has a l s o been made from a s u l f i n a t e and a halonitrobenzene (24, 26, 2 9 , 3 5 ) ; from 4acetylaminobenzenesulfonyl chloride and a c e t a n i l i d e (20, 36, 3 7 , 38); from a c e t a n i l i d e and thionyl chloride ( 2 7 , 39, 40); from 4,4'-dichlorodiphenyl sulfone (18, 28, 30, 41, 42, 43); from the diphthaloyl derivative of 4,4'-diaminodi102

f n

0 "
0 rn
9)

cu

rnx

D U

F
cw
I4 0

r
cn
U

m d a

G 0

cn

0 "
0
U

2
d 4

0 $1
m

0
x

0 "

V=O U
. r

x z
a
X m z

co

.
I4

aJ

2 "

'4

ii

rnx

"

8
X " X

I
4

x m 0 rn

rfl

0 "

103

CHESTER E. ORZECH era/.

phenyl sulfide (44, 45); and from 4,4'-dimethyldiphenyl sulfone by oxidation to the dicarboxylic acid and Hofmann degradation of the diamide (46). The 4,4'-diacetylaminodiphenyl sulfone obtained by any of these methods can easily be hydrolyzed to the diamino compound ( 3 . 2) Sanghavi (47) has reviewed the various methods of synthesis. A few of these procedures are outlined in Figure 8.

4 STAB1LITY-DEGRADATION .
No reports of stability studies or degradation of dapsone were found.
5, DRUG METABOLIC PRODUCTS Dapsone metabolic products have been reported as follows: Me tabo1i te Dapsone glucuronide Species Man Monkey Rabbit Man Rat Man Dog Man Monkey Rabbit Dapsone monohydroxylamine su 1f amate Dapsone monohydroxylamine glucuronide Monoace tyl dapsone sulfamate
Man

References
(48,49,50) (51,521 (48,51,53, 54,55)

Dapsone sulfamate Dapsone monohydroxylamine Monoace tyl dapsone

(48,501 (51,521 (56,57,58) (56,581 (15,51,57, 59,60,61) (51,52,62) (51,61,62) (58) (581 (50) (51,52)

Man Man Monkey

104

0 0

x x

Dapsone sulfamate (DDS-S)

ru

Dapsone glucuronide (DDS-G)

rl

5
cd

cn

al

a a

..

ocv)+o

8
I

( I

z ,

hl

Dap s one (DDS )

Dapsone monohydroxylamine (DDS-NOH)

Az ox y -d aps one (AZmy-DDS )

82 2-

105

o=u0

ot;;:

X V

Monoace t y l dapsone ( AD MDS

X z

p: z

II

Monoace t y l dapsone hydroxylamine (MADDS-NOH)

Diace t y l azoxy-dapsone
rl

o=u 5:
Some Dapsone Metabolites

X m V

Figure 9.

CHESTER E. ORZECH era/.

Monoacetyl dapsone glucuronide

Man Monkey Rabbit Rat Man Man Rat Man

(50,511 (52) (51) (51) (57) (571 (57) (57)

Monoacet y l dapsone hydroxylamine

Az my-da p s one

Diace t y l azoxy-dapsone

The s t r u c t u r e s of some of these metabolites, a r e shown i n Figure 9. 6. M T O S OF ANALYSIS EH D

6.1 I d e n t i f i c a t i o n Tests Dapsone is e a s i l y i d e n t i f i e d by the physical prope r t i e s described i n s e c t i o n 2 above. Where i d e n t i f i c a t i o n of dapsone i n formulations i s necessary, i t can be e x t r a c t e d by various organic solvents such a s methanol, chloroform, ethylene d i c h l o r i d e , e t c . I f the i d e n t i f i c a t i o n of very small amounts of dapsone i s necessary, the colorimetric method of P e t e r s e t a1 (63), the c o l o r i m e t r i c t e s t s described i n s e c t i o n 6.3, or the fluorometric t e s t s described i n s e c t i o n 6.5 may be useful. 6.2 Elemental Analysis The elemental composition of dapsone i s as follows: Element Carbon Hydrogen Nitrogen Su If u r Oxygen %Theory

58.04 4.87 11.28 12.91 12.89

6.3 Colorimetric Methods There a r e two b a s i c colorimetric methods f o r dapsone. The f i r s t and most widely u s e d is the method of Bratton and Marshall (64). Dapsone is diazotized, then coupled with N-(1-naphthy1)-ethylenediamine t o form a n 820 dye. Other i n v e s t i g a t o r s have used t h i s b a s i c method but have used d i f f e r e n t coupling reagents: N i t t i e t a 1 (651, dimethyl- 0 -naphthylamine; Rose and Bevan (66), N-@-sulfa106

DAPSONE

toethyl-m-toluidine; Schoog (67), l-sulfomethylaminonaphthalene-8-sulfonic a c i d ; Merland (68), N-naphthyl-N' ,N'-die thy 1 py lene d iamine pro The second type of method, reported by Levy and Higgins (69), i s based on the formation of a Schiff base between dapsone and 4-dimethylaminobenzaldehyde. This method is reported t o be 2.4 times more s e n s i t i v e than the Bratton-Marshall technique and, i n a d d i t i o n , i t i s reported t o be s p e c i f i c f o r dapsone i n the presence of i t s metaboli tes.

6.4 T i t r a t i o n Methods Tomicek (70) t i t r a t e d dapsone potentiometrically with perchloric acid i n g l a c i a l a c e t i c acid. Wojahn brominated dapsone with bromide-bromate and b a c k - t i t r a t e d the excess bromate with sodium t h i o s u l f a t e s o l u t i o n ( 7 1 ) . The USP method of assay i s t i t r a t i o n of the cooled, s t r o n g l y acid sample s o l u t i o n with sodium n i t r i t e s o l u t i o n t o a n electrometric endpoint (72). 6.5 Fluorometric Methods Glazko (73) reported a fluorometric method f o r dapsone i n plasma and u r i n e samples. Complete d e t a i l s of sample preparation a r e given, The fluorescence of the e t h y l a c e t a t e e x t r a c t is determined by a c t i v a t i n g a t 297 nm and measuring the emission a t 340 nm. Ellard and Gammon (16) developed an e x t r a c t i o n scheme t o determine dapsone and two possible metabolites, MADDS and DADDS, fluorom e t r i c a l l y i n plasma and urine samples. Peters e t a1 (15) modified and expanded the above procedure. They extracted with ethylene dichloride instead of e t h y l a c e t a t e and thus eliminated the problem of quenching caused by small quant i t i e s of water i n the e t h y l a c e t a t e e x t r a c t .
6.6 Paper Chromatography Longenecker (74) reported procedures f o r p a r t i t i o n chromatography of dapsone by both ascending and descending chromatography on paper and s t r i n g . Bushby and Woiwood (54, 55) u s e d paper chromatography t o i s o l a t e and i d e n t i f y dapsone and some d i a z o t i z a b l e metabolites by paper chromatography and electrophoresis. Wadia e t a 1 (75) reported Rf values f o r dapsone and twenty f i v e other diaminodiphenyl s u l f i d e s , sulfoxides, and sulfones using four d i f f e r e n t solvent systems. J a r d i n and S t o l l (76), Khosla e t a 1 (77), and T s u t s u m i (48) a l l used paper chromatography i n t h e i r s t u d i e s on the metabolism of dapsone.

107

CHESTER E. ORZECH eta/.

6.7 High Pressure Liquid Chromatography Gordon and Peters (78) separated dapsone, monoa c e t y l dapsone (MADDS), and d i a c e t y l dapsone (DADDS) a t the 1 t o 20 ,,g l e v e l on a s i l i c a g e l column with e t h y l a c e t a t e as solvent. The e f f l u e n t was monitored a t 280 nm. Murray e t a1 (79) used a fluorometric d e t e c t o r and increased the s e n s i t i v i t y of the determination t o the 10 ng level. This procedure was modified by Murray e t a 1 (80) t o increase the s e n s i t i v i t y t o 0.1 ng i n 0.5 m l . Ribi e t a 1 (81) used pressure accelerated chromatography through microparticulate s i l i c a g e l columns, packed by c e n t r i f u g a t i o n , t o separate dapsone, MADDS, and DADDS, with chloroform-methanol (97:3) as solvent and u l t r a v i o l e t absorption a t 254 n f o r detecm tion. Gordon e t a1 (82) used a s i l i c a g e l column with chloroform-carbon t e t r a c h l o r i d e (7:3) solvent t o examine dapsone f o r impurities. Column e f f l u e n t was monitored by the absorption a t 280 nm. Fractions were c o l l e c t e d and evaporated, and the residues were dissolved i n ethylene d i chloride. The fluorescence of the re-dissolved f r a c t i o n s was measured. Impurities determined were 2,4'-diaminodiphenyl sulfone and 4-aminodiphenyl sulfone Krol and Mannan (83) developed a method f o r the a n a l y s i s af dapsone using a commercially a v a i l a b l e s i l i c a g e l column 6- P o r a s i P ) , a solvent containing isopropyl a l cohol, a c e t o n i t r i l e , e t h y l a c e t a t e , and pentane (1:1:1:7), and d e t e c t i o n a t 254 m o A 30 c by 4 mm I.D. column was m s u i t a b l e e i t h e r f o r assay f o r dapsone i n t a b l e t s or f o r analyzing f o r impurities i n dapsone. The following r e l a t e d compounds can be separated from dapsone: (a) 2,4'-diaminodiphenyl sulfone; (b) 4-aminodiphenyl sulfone; (c) 4-amino4'-chlorod iphenyl s u l f one ; (d ) 4,4'-d iace tamidod iphenyl sulfone (DADDS); (e) 4-amino-4I-ace tamidodiphenyl s u l f one (MADDS); ( f ) 4-amino-4'-hydroxydiphenyl sulfone; ( g ) 4,4'diace tamidodiphenyl sulfoxide; 4,4'-diaminodiphenyl s u l f i d e (90).

6.8 Gas Chromatography Burchfield e t a 1 (84, 85) reported t h a t dapsone can be chromatographed d i r e c t l y . Because t h e i r l a t e r work r e quired g r e a t e r s e n s i t i v i t y , and, i n a d d i t i o n , the determination of MADDS and DADDS, the iodo d e r i v a t i v e s were prepared by d i a z o t i z a t i o n and an e l e c t r o n capture d e t e c t o r w a s used. Using a 4 f o o t by 0.25 inch O D g l a s s U-tube packed .. with 3% Poly-A-103 on 100-120 mesh Gas Chrom Q a t 285'C with nitrogen, 280 pg of iodo d e r i v a t i v e was e a s i l y d e t e c t able.

108

DAPSONE

Chang e t a 1 (52) investigated the metabolism of dapsone by chromatographing t r i m e t h y l s i l y l d e r i v a t i v e s of the metabolic products on 3% OV-17 (Gas Chrom Q) a t 275OC. 6.9 Thin Layer Chromatography Thin layer chromatography systems have been compiled i n Table 2 .

7.

AK O LD MNS C N WE G E T

The w r i t e r s wish t o thank D r . B. T. Kho f o r h i s review of the manuscript, D r . G. S c h i l l i n g of Ayerst Research Laboratories f o r h i s mass s p e c t r a l data and i n t e r p r e t a t i o n , the l i b r a r y s t a f f f o r t h e i r l i t e r a t u r e search, and Mrs. B. Juneau f o r typing the p r o f i l e .

109

TABLE 2 Adsorbent Silica gel Silica gel DF-5 Silica gel G

11

SoLvent Sys tem

! %
0.62
03 .0

Ref. 86 87

1 1) Chloroform-Butanol-Pe troleum Ether (1: :

Toluene-Ethyl acetate (1: 1) Chlorofom-Hep tane-E thanol (1: 1 1) : Ace tone-Chloroform (1:9) Chloroform-Ethyl Ether (85: 15) Toluene-Ethyl acetate (1:l) Chloroform-Methanol (95:5) Chloroform-Methanol (9: 1) Chloroform-Ethanol (9:l) Chlorof orm-Ace tone-Die thanolamine (5 : :1) 4

0.60 0.20 0.11 0.27 0.47

88
89
11
11

11

11

11

11

11

04 .8
0.65 0.69 07 .8

11

11

11

I1

11

I1
11

Me thano1-Ace tone-Die thanolamine (50: :. ) 50 15

Ace tone -Chlorofo m (9: ) 1 Isopropyl alcohol-cyclohexane-259. Axunonia (65:25:10) Dime thy1 formamide-Die thylamine-EthanolEthyl acetate (1:1:6:12)

11

08 .4 08 .5 08 .8

11

11

11

11

11

DAPSONE

8 .
1.

REFERENCES
C. J. Pouchert, "The Aldrich Library of Infrared Spectra", Aldrich Chemical Company, Inc., Milwaukee, Wisconsin, 1970, spectrum 843F. A. L. Hayden, 0. R. Samul, G. B. Selzer, and J. Carol, J. Ass. Off. Agr. Chem. 4 , 797-900 (19451, spectrum 5

2 . 3 .

4 .
5 .
6 . 7 . 8 . 9 .
1. 0 11.

12. 13. 14. 15. 16. 17.

18

19. 20. 21. 22.

.-

29. L. G. Bellamy, "The Infra-red Spectra of Complex Molecules", 3rd ed., John Wiley and Sons, Inc., New York, N. Y, 1975. . J. G. G r a s s e l l i (ed.), "Atlas of S p e c t r a l Data and Physical Constants f o r Organic Compounds", Chemical Rubber Company, Cleveland, Ohio, 1973, p. B-918. " B r i t i s h Pharmacopoeia 1973", Her Majesty's S t a t i o n e r y Office, London, England, 1973, p. 140. V. Baliah and V. Ramakrishnan, J. Indian Chem. SOC. 35, 151-6 (1958); C.A. 53, 10954. A. Maschka, M S t e i n , and W. Traoer, Monatsh. 85, 168. 81 (1954); C.A. 48, 11191a. G. S c h i l l i n g , Ayerst Research Laboratories, Montreal, P.Q., Canada, private comunication. "Thermal Analysis Newsletter", Nos. 5 and 6, The PerkinE l m e r Corporation, Norwalk, Conn. L. T. B u t t , J. Pharm. Pharmacol. 5, 568 (1953). M. Alleaume and J. Decap, Compt. rend. 261 (7) (Groupe 8), 1693-4 (1965). C. Dickinson, J. M. Stewart, and H. L. Annnon, J. Chem. SOC. D 1970, 920-1. C. W. Dickinson, Diss. Abstr. I n t . B 33 (5), 2009-10 (1972). C. U. Linderstrom-Lang and R. F Naylor, Biochem. J . . 83, 417-24 (1962). J. H. Peters, G. R. Gordon, and W. T. Colwell, J. Lab. Clfn. Med. 76, 338-47 (1970). G. A. ELlard and P. T. Gammon, I n t . J. Leprosy 37, 398405 (1969). S A. Cucinell, Z. H. I s r a i l i , and P. G. Dayton, A e. . mr J. Trop. Med. Hyg. 2, 322-31 (1972). B. Ciocca and L. Canonica, Chimica e i n d u s t r i a ( I t a l y ) 26, 7-9 (1944); C.A. 40, 3104. V. A. Zasosov and M. I. Galchenko, J. Applied Chem. (USSR) 19, 580-4 (1946); C.A. 4l, 2702f. K Ramaz M Raghavan, and P. C. Guha, J. Indian Chem. . . SOC. 30, 723-4 (1953); C.A. 49, 2347e. E. F & r and J. Wittmann, Ber. 2, 2264-73 (1908). R. E Buckles, J. Chem. Educ. 2, . 36-7 (1954).

111

CHESTER E. ORZECH eta/.

23. 24. 25. 26.

28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48.

G. W. R a i z i s s , L. W. Clemence, M. Severac, and J. C. Moetsch, J. Am. Chem. SOC. 2763-5 (1939). R. 0. Roblin, J. H. Williams, and G. W. Anderson, J. Am. Chem. SOC. 63, 1930-4 (1941). A, M. VanArendonk and E. C. K l e i d e r e r , J. Am. Chem. SOC. 62, 3521-2 (1940). "Organic Syntheses", Vol. 2 2 , John Wiley and Sons, Inc., New York, N. Y., 1942, pp. 31-4. S. Sugasawa and K. S a k u r a i , J. Pharm. Sac. Japan 6 0 , 22-4 (1940); C.A. 34, 3704. H. Heymann and L. F. F i e s e r , J. Am. Chem. Sac. 67, 1979-86 (1945). H. Dorn and G o H i l g e t a g , Ger. ( E a s t ) P a t e n t 33,094; C.A. 64, PC65Og. F r e n c h P a t e n t , 829,926; C.A. 33, 1760. H. G. Biswas, S c i . C u l t . ( C a l c u t t a ) 28, 586-8 (1962); C.A. 2, 3806e. Yu-Li Chi and Jo-Yung Moh, K'e Hsueh Tung Pao 2, 50-1 (1957); C.A. 53, 18896i. N i e t z k i and Bothof, Ber. 27, 3261 (1894). Hodgson and Rosenberg, J. Chem. SOC. 180 (1930). A. M. Morales, Anales fac. farm. y bioquim., Univ. n a c l . mayor San Marcos (Lima, Peru) 2 607-14 (1950); , C.A. 49, 210b. K e r e s z y and Wolf, Hung. P a t e n t 120,021; C.A. 33, 4600. I n d i a n P a t e n t 47,585; C.A. 49, 1101c. S w i s s P a t e n t 243,601; C.A. 43, 7960e. S P. Raman and S. C Niyogy, Ann. Biochem. and E x p t l . . . Med. ( I n d i a ) 2, 207-10 (1955); C.A. 50, 1 2 8 8 0 ~ . W. Braun, Ger. P a t e n t 964,593; C.A. 53, P12240g. Brit. P a t e n t 506,227; C.A. 33, 9328. A. G i r a r d , French P a t e n t 844,220; C.A. 34, 7543. Ya. Ya. Makarov-Zemiyanskii, Nauch. T r u d y Moskov. Tekhnol. I n s t . Legkoi Prom., Sbornik 1957, No. 8 , 315-19; C.A. 54, 1 5 9 7 5 ~ . V. A. Zasosov, E. I. Metel'kova, and M. I. Galchenko, Med. Prom. S.S.S.R. 13, No. 2 , 18-20 (1959); C.A. 53, 19944a. S. Radulova and E. Topalova, Vet. Med. Nauki ( S o f i a ) 2 , 873-7 (1965); C.A. 64, 17460g. P.P.T. Sah, S. A. PeopGs, S. T. Kwan, and H. J. Sah, Arzneim.-Forsh. 17, 425-31 (1967); C.A. 6 7 , 424772. N. M. Sanghavi, I n d i a n Chem. J. 6 (11, 179-81 (1971). S. T s u t s u m i , Chem. and Pharm. B u i l . (Tokyo) 2, 432-6 (1961); C.A. 56, 2850h.

c,

112

DAPSONE

49. 50.

J. H P e t e r s , G. R. Gordon, D. C. Ghoul, J. G. . Tolentino, G. P. Walsh, and L. Levy, Am. J. Trop. Med. Hyg. 2, 450-7 (1972). 51. A. J. Glazko, T. Chang, J. Baukema, S. F. Chang, A. Savory, and W. A. D i l l , Int. J. Leprosy 37, 462-3 (1969). . . 52 T Chang, S F. Chang, J. Baukema, A. Savory, W. A. D i l l , and A. J. Glazko, Fed. Proc. 28, 289 (1969). . 53. G A. E l l a r d , B r i t . J. Pharmacol. 26, 212-7 (1966). . 54 S R. M. Bushby and A. J. Woiwood, Biochem. J. 63, 406-8 (1956). . 55. S R. M. Bushby and A. J. Woiwood, Amer. Rev. Tuberc. 72, 123-5 (1955). . 91 56. S Tabare111 and H. Uehleke, Xenobiotica 1 7 , 501-2. . . 57. 2 H. I s r a i l i , S. A. Cucinell, J. Vaught, E Davis, J. M, Lesser, and P. G. Dayton, J Pharmacol. Exp. . Ther. 187, 138-51 (1973). 58. H. U e h m e and S. T a b a r e l l i , Naunyn-Schmiedeberg's Arch. Pharmacol. 278, 55-68 (1973). 59. J. H. P e t e r s , G. R. Gordon, L. Levy, M. A. Storkan, R. R. Jacobson, C. D. Enna, and W. F. Kirchheimer, Am. J. 'hop. Med. Hyg. 2, 222-30 (1974). 60. G. A. E l l a r d and P, T. Gammon, I n t . J. Leprosy 37, 398-405 (1969). . 61. J. H. P e t e r s , G. R. Gordon, R. G e l b e r , and L Levy, Int. J. Leprosy 37, 463 (1969). 62 0. R. Gordon, J. H. Peters, R. G e l b e r , and L. Levy, Proc. West. Pharmacol. SOC. 13, 17-24 (1970). . . 63. J H. P e t e r s , S C. Lin, and L. Levy, I n t . J. Leprosy 36, 46-51 (1969). . 64. A. C Bratton and E. K. Marshall, J. Biol. Chem. 537-50 (1939), 65. F. N i t t i , D. Bovet, and V. Hamon, Compt. rend. soc. b i o l . 128,26-8 (1938); C.A. 32, 6332. . 66. F. L Rose and H. G. L. Bevan, Biochem. J. 38, 116 (1944). 67. M. Schoog, Arzneim.-Forsch. 2, 512-4 (1952); C.A. 47, 3392b. 68. R. Merland, Ann. b i o l . clin. ( P a r i s ) 13, 21-32 (1955); C.A. 2, 8359c. 69. L. Levy and L. J. Higgins, I n t . J. Leprosy 34, 411-4 (1966). . 116-30 70 0 Tomicek, C o l l . Czech. Chem. Comuns. 2, (1948); C.A. 42, 8108g.

- 620-31 (1970). 83,

J. O'D. Alexander, E. Young, T. McFadyen, N. G. F r a s e r , W. P. Duguid, and E. M. Meredith, B r i t . J. Dermatol.

128,

113

CHESTER E. ORZECH et 81.

71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83

84. 85. 86. 87. 88. 89. 90.

H. Wojahn, Suddeut. Apoth.-Ztg. 8 8 , 395-6 ( 1 9 4 8 ) ; C.A. 43, 1908f. T n i t e d S t a t e s Pharmacopeia XIX", Mack Publishing Co. Easton, Pa., 1975, pp. 118-19 and 626. A. J. Glazko, W. A. D i l l , R. G. Montalbo, and E. L. Holmes, Amer. J. Trop. Med. Hyg. 465-73 (1968). W. H. Longenecker, Anal. Chem. 3, 1402-5 ( 1 9 4 9 ) . P. S Wadia, M. C. Khosla, and N Anand, J. S c i . Ind. . . Research (India) E , 148-52 ( 1 9 5 5 ) ; C.A. 2, 12195d. C. J a r d i n and A Stoll, Semaine hop. Therap. 34, 611. 16 ( 1 9 5 8 ) ; C.A. 20627d. M. C. Khosla, J. D. Kohli, and N. Anand, J. Sci. Ind. Research (India) E, 51-6 ( 1 9 5 9 ) ; C.A. 19131d. G. R. Gordon and J. H. Peters, J. Chromatogr. 47, 2697 1 (1970). J. F. Murray, G. R. Gordon, and J. H. Peters, J. Lab. Clin. Med. 78, 464-71 (1971). J. F. MurraE G. R. Gordon, C. C. Gulledge, and J. H. Peters, J. Chromatogr. 107, 67-72 (1975). E. Ribi, S C. Harris, J. Matsumoto, R. Parker, R. F. . Smith, and S. M. S t r a i n , J. Chrom. S c i . l, 708-11 0 . (1972). G. R. Gordon, D. C. Ghoul, and J. H. P e t e r s , J. Pharm. Sci. 6 4 , 1205-7 (1975). a. J.?rol and C. V. Mannan, Ayeret Laboratories, Inc., Rouses Point, N Y, private communication. . . H. P. Burchfield, R. J. Wheeler, and E. E. S t o r r s , Int. J. Leprosy 37, 462 (1969). H. P. Burchfield, E. E. S t o r r s , R. J. Wheeler, V. K. Bhat, and L. L. Green, Anal. Chem. 4 5 , 916-20 (1973). E. Sawicki, . IJohnson, and K. K o s i z k i , Microchem. J. 10 ( 1 - 4 ) , 72-102 ( 1 9 6 6 ) . L. Fishbein and J. Fawkes, J. Chromatogr. 22, 323-9 (1966). T. S Gloria, Rev. Fac. Farm. Bioquim. Univ. Sao Paulo . 4 , 391-400 ( 1 9 6 6 ) ; C.A. 67, 8 4 9 1 7 ~ . L. M S Armasescu and M. Manda, Farmacia (Bucharest) . . 22 ( 3 ) , 165-70 ( 1 9 7 4 ) ; C.A. 8 l , 158721t. Compounds ( a ) , (b), and ( c ) were kindly provided by G. R. Gordon, Stanford Research I n s t i t u t e , Menlo Park, Ca.

17,

52,

53,

L i t e r a t u r e surveyed through June, 1975.

114

FLUCYTOSINE

Edward H. Waysek and James H. Johnson

EDWARD H. WAYSEK AND JAMES H. JOHNSON

INDEX
Analytical P r o f i l e

Flucytosine

1.

Description

1.1
1.2 2.

Name, Formula, M o l e c u l a r Weight Appearance, Color, Odor

Physical Properties 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 I n f r a r e d Spectrum N u c l e a r M a g n e t i c Resonance Spectrum U 1 t r a v i o l e t Spectrum F l u o r e s c e n c e Spectrum Mass Spectrum Opt i ca 1 Rota t i o n M e l t i n g Range D i f f e r e n t i a l Scanni ng C a l o r i m e t r y Thermogravirnetric Analysis Solubility X-Ray C r y s t a l P r o p e r t i e s D i s s o c i a t i on Constant

3.
4. 5.

Synthesis Stability Drug M e t a b o l i c P r o d u c t s

5.1 6.

Toxicology

Methods o f A n a l y s i s

6.1
6.2

6.3 6.4 6.5 6.6 6.7

Elemental A n a l y s i s Phase S o l u b i l i t y A n a l y s i s Th i n-Layer Chromatograph i c Ana 1 ys i s Non-Aqueous T i t r a t i o n Fluorometric Analysis D i r e c t Spect rophotornetr i c A n a l y s i s Fluorine Analysis

6.71
6.72

Free F l u o r i d e Analysis O r g a n i c a l l y Bound F l u o r i n e A n a l y s i s

116

FLUCYTOSIN E

6.8
7.
8.

Biological Assays

Acknowledgements References

117

EDWARD

H. WAYSEK AND JAMES H. JOHNSON

1.

Description

1.1

Name, Formula, Molecular Weight F 1 ucytos ine i s 4-ami no-5-f 1uoro-2 ( 1 H) -pyr i m i done.

C4H4FN30 1.2

Molecular Weight: Appearance, Color, Odor

129.1

F l u c y t o s i n e i s a white, odorless, c r y s t a l l i n e powder. 2. Physical P r o p e r t i e s 2.1 I n f r a r e d Spectrum ( I R ) The i n f r a r e d spectrum o f f l u c y t o s i n e i s present e d i n F i g u r e 1. The spectrum was recorded w i t h a Perkin-Elmer Model 621 G r a t i n g I n f r a r e d Spectrophotometer as a Fluorolube suspension from 4000 t o 1350 cml and as a m i n e r a l o i l The assignsuspension from 1350 t o 400 cm. ments f o r t h e c h a r a c t e r i s t i c bands i n t h e I R spectrum a r e l i s t e d i n Table 1 ( I ) .

118

FIGURE 1 I n f r a r e d Spectrum o f Flucytosine

W AVEL ENGTH

2.5
10 0

MICROMETERS 7 8 9 1 0

1 1 2 4

1822 35 50 10 0 80
60 40

8 80
W

260

540
CtT t-

I -

20
0

20
0 200

4000 3500 3000 2500 2000

1700

1400

10 10

800

500

FREQUENCY (CM-')

EDWARD H. WAYSEK AND JAMES H. JOHNSON

Table I

I n f r a r e d Ass ignmen t s For F I ucytos ine

Frequency (cm-I) Characteristic O f Bonded NH Enol i c OH (associated) CO stretch = N-C=H (amidine) C C (extended conjugation) =

3374, 3138
2800-2200 (broad)

1684, 1672 1647 1554

2.2

Nuclear Magnetic Resonance Spectrum (NMR) The NMR spectrum o f f l u c y t o s i n e i n F i g u r e 2 was determined on a JEOL C-60 HL Spectrometer a t ambient temperature (ca. 25"). The sample was d i s s o l v e d i n 0.1N deuterium c h l o r i d e c o n t a i n i n g sodium 2,2-dimethyl-2-silapentane s u l f o n a t e as an i n t e r n a l reference (MeSi = 0 ppm). The C-6 = 5.5 Hz) a t p r o t o n e x h i b i t e d a doublet ( J h- f 8.03 ppm.

2.3

U l t r a v i o l e t Spectrum The u l t r a v i o l e t spectrum o f f l u c y t o s i n e i n 0.1N h y d r o c h l o r i c a c i d i n the r e g i o n 340 t o 210 nm e x h i b i t s a maximum a t 283-287 nm (E = 9.2 x 103 ) and a minimum a t 243-247 nm. The spectrum p r e sented i n F i g u r e 3 was obtained from a reference standard s o l u t i o n o f f l u c y t o s i n e a t a concent r a t i o n o f 0.85 m per 100 m l o f 0.1N H C I ( 2 ) . g

2.4

Fluorescence Spectrum F i g u r e 4 shows the e x c i t a t i o n and emission spectra o f a s o l u t i o n o f reference standard f l u c y t o s i n e from 250 t o 650 nm. The s p e c t r a measured i n a methanol s o l u t i o n o f f l u c y t o s i n e (0.025 mg/ml u s i n g a Farrand MK-1 spectrof luorometer, showed one e x c i t a t i o n maximum a t 290 n and one emission maximum a t 366 nm. m

120

FLUCYTOSINE

FI GURE 2
NMR Spectrum of Flucytosine

121

EDWARD H. WAYSEK AND JAMES H. JOHNSON

FIGURE 3
Ultraviolet Spectrum of Flucytosine

.7

.6

.5

w.4
0

f 8
v)

9.3

*t
.I

_I n- _ 300210 250 I 35 NANOMETERS

122

FIGURE

4:

Fluorescence Spectra

of Flucytosine
I I

Excitation Spectrum

Spectrum

250

300

350

400 450

500

NANOMETERS

550

600 650

EDWARD H. WAYSEK AND JAMES H. JOHNSON

2.5

Mass Spectrum The mass spectrum o f f l u c y t o s i n e was o b t a i n e d u s i n g a CEC 21-110 mass spectrometer w i t h an i o n i z i n g energy o f 70 eV. The o u t p u t from the mass spectrometer was analyzed and presented i n t h e form o f the bar graph, shown i n F i g u r e 5, by a Varian 100 MS dedicated computer system

(3)
The spectrum shows a s t r o n g molecular ion peak a t m/e 129. The peak a t m/e 101 i s due t o t h e loss o f CO from the p a r e n t mass and t h e f r a g ment o f m/e 86 i s due t o t h e loss o f HNCO, a l s o from t h e molecular i o n . 2.6 Optical Rotation F l u c y t o s i n e e x h i b i t s no o p t i c a l a c t i v i t y .

2.7

M e l t i n q Range A sharp m e l t i n g p o i n t i s n o t observed w i t h f l u c y t o s i n e . The m e l t i n g range depends on t h e r a t e o f h e a t i n g . When the USP Class l a procedure (4) i s used, the m e l t i n g p o i n t l i e s between 292" and 298C. M e l t i n g i s accompanied by m e l t decomposition.

2.8

D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC) When scanned a t a programmed r a t e o f 10C per minute using n i t r o g e n as the i n e r t gas, decomp o s i t i o n occurs b e f o r e any observed t r a n s i t i o n , t h e r e f o r e , DSC i s n o t u s e f u l f o r c h a r a c t e r i z a t i o n o f t h i s compound ( 5 ) .

2.9

Thermogravimetric A n a l y s i s (TGA)

No weight loss was observed by TGA between

ambient temperature and 210C a t a h e a t i n g r a t e o f 10C per minute. A weight loss began a t about 210C and amounted t o about 64% o f t h e sample weight a t 5OO0C ( 6 ) .

124

FIGURE

5:
v\

Mass Spectrum of F l u c y t o s i n e

..

125

EDWARD H. WAYSEK AND JAMES H. JOHNSON

2.10

Solubi 1 i t y The s o l u b i l i t y data f o r f l u c y t o s i n e obtained a t 25'C i s g i v e n i n Table I1 ( 7 ) . The s o l u b i l i t i e s were measured a f t e r an e q u i l i b r a t i o n p e r i o d of t h r e e hours. Table I 1 Flucytosine S o l u b i l i t y

So 1 ven t

Solubi 1 i t y (mg/ml)

3A a l c o h o l Absolute ethanol Ace tone Benzene Chloroform Diethyl ether Ethyl acetate Hexane 2 p ropa no 1 Met hano 1 USP a l c o h o l Water

1.3
0.5 <o. 1 <o. 1 <o. 1 <o. 1 <o. 1 <o. 1 1.1 5.0

1.7
14.2

2.11

X-Ray C r y s t a l P r o p e r t i e s The x-ray powder d i f f r a c t i o n p a t t e r n o f f l u c y t o s i n e i s presented i n Table I l l ( 8 ) . The instrumental c o n d i t i o n s are g i v e n below, Instrumental Conditions General E l e c t r i c Model XRD-6 Spectrogoniometer Genera t o r Tube Target Radiation Optics 50 KV, 12.5 mA Copper 0 Cu (Cu Ka = 1.5418 A) 0.1' Detector s l i t M.R. S o l l e r s l i t 3" Beam s l i t 0.0007" N i f i l t e r 4' take o f f angle

126

FLUCYTOSINE

Goniometer

Recorder Sample

Scan a t 0.2"/minute 20 A m p l i f i e r g a i n - 16 coarse 8.17 f i n e Sealed p r o p o r t i o n a l c o u n t e r tube and D.C. v o l t a g e a t p l a t e a u , p u l s e h e i g h t s e l e c t o r EL; 8.0 v o l t s , Eu; o u t Rate meter T.C. 4, 2000 c/s f u l l scale Chart speed 1" p e r 5 min. Prepared by g r i n d i n g a t room temperature Table I l l

X-Ray Powder D i f f r a c t i o n P a t t e r n o f F l u c y t o s i n e 20 d(i)

13.74 14.76 15.14

17.26

18.76
20.18 21.81 22.75 23.94

6.45 6 .OO 5.85 5.14 4.73

4.40 4.08

0.4 * 39 .76
.09 .22

25.94 26.44
29.24

3.91 3.72 3.44 3.37

3.06 2.99 2.92 2.80 2.68 2.39 2.26 2.22 2.12

1 .oo
.72

.81 .98 .16

.29

.67
.14 .15 .55
.22 .22

29.94
30.65;: 32.01*

33.48 37.57 39.98

40.70 42.74

.07
.09

51.75
*Broad Peaks

1.77

.15 .15

127

EDWARD H. WAYSEK AND JAMES H. JOHNSON

(1) d - ( i n t e r p l a n a r d i s t a n c e )

nA 2 sin 0

(2) [ / I 1= r e l a t i v e i n t e n s i t y 2.12 D i s s o c i a t i o n Constant The apparent pKa values f o r f l u c y t o s i n e have been determined s p e c t r o p h o t o m e t r i c a l l y t o be 2.90 2 0.05 and 10.71 2 0.05 ( 9 ) . The apparent d i s s o c i a t i o n constants were c a l c u l a t e d from t h e UV s p e c t r a l data i n water a t v a r i o u s pH values. pKal (2.90) represents the p r o t o n a t i o n o f N-3 and pKa (10.71) represents d e p r o t o n a t i o n a t the ami8e moiety. These values a r e i n good agreement w i t h t h e pKa's reported by Ueda and Fox f o r 3-methylcytosine (10).

3.

Synthesis F l u c y t o s i n e may be prepared by the r e a c t i o n scheme shown i n F i g u r e 6. 5 - F l u o r o u r a c i l i s r e f l u x e d w i t h d i m e t h y l a n l l i n e and phosphorous o x y c h l o r i d e . The 2,4-di ch l o r o - 5 - f 1 uoro-pyr i m i d i n e formed i s reacted w i t h ammonia and ethanol t o g i v e 2-chloro-4-amino5-f l u o r o p y r i m i d i n e which y i e l d s f l u c y t o s i n e hydroc h l o r i d e upon being t r e a t e d w i t h concentrated hydroc h l o r i c acid. F l u c y t o s i n e h y d r o c h l o r i d e i s neutral i z e d w i t h ammonium hydroxide forming 5 - f l u o r o c y t o s i n e (11).

4.

Stability F l u c y t o s i n e s t o r e d a t room temperature f o r f i v e years was s t a b l e when t e s t e d by u l t r a v i o l e t a b s o r p t i o n and t h i n - l a y e r chromatography u s i n g e t h y l a c e t a t e : methano1:conc. NHhOH (50:50:2) as t h e s o l v e n t system (12). The s t a b i l i t y o f 0.14; b u f f e r e d s o l u t i o n s i s shown i n Table I V (13).

128

FIGURE 6:
0

Synthesis of Flucytosine

CI
LL

H 5-Fluorouracil
129

= z

-4 o

2.4-Dichloro5-f luoropyrimidine

2-Chloro-4-amino5-f luoropyrimidine

y 2
NH40H

fi
Lt
H
Flucytosine hydrochloride

HCI

H Flucytosine

EDWARD H. WAYSEK AND JAMES H. JOHNSON

Table I V Stabi 1 i t y o f 0.1% B u f f e r e d S o l u t i o n

~~

Before Heat g H z T n i ng

A f t e r 1 Hour a t 100C pH APHA Color 0-10 0-10 0-10 0-10 Clarity Clear
I1 I1 I1

p H
5.50
3.10

APHA Color
0-10 0-10 0-10 0-10

Clarity Clear
I1
I1
I1

% Recover:
92.9

3.15
5.60 6.90 9.40

6.90 9.40

95.9
99.0

98.5

Method Used:

U l t r a v i o l e t a b s o r p t i o n and t h i n - l a y e r chromatography.

The s t a b i l i t y o f f l u c y t o s i n e has a l s o been s t u d i e d i n i t s dosage forms (14). I n ampuls f l u c y t o s i n e was s t a b l e t o the e x t e n t o f 2% o r less breakdown a t room temperature f o r 36 months. No degradation products, urea, u r a c i 1, and 5 - f l u o r o u r a c i 1 , were detected i n capsules a f t e r s i x months a t 37C and twelve months a t 25C.

5.

Drug M e t a b o l i c Products
I t has been r e p o r t e d t h a t f l u c y t o s i n e d i d n o t undergo s i g n i f i c a n t b i o t r a n s f o r m a t i o n i n humans a f t e r o r a l dosage and 90% o f the f l u c y t o s i n e administered was e x c r e t e d unchanged i n t h e u r i n e (15). Koechl i n , e t a l . have s t u d i e d t h e metabolism o f f l u c y t o s i n e i n t h e r a t and i n man u s i n g drug l a b e l e d i n the 2p o s i t i o n w i t h carbon-14. Rats given a p a r e n t e r a l dosage d i d n o t metabolize f l u c y t o s i n e and the dosage was recovered q u a n t i t a t i v e l y , mostly i n t h e u r i n e . F l u c y t o s i n e was deaminated t o 5 - f l u o r o u r a c i l t o t h e e x t e n t o f 20-30% a f t e r o r a l a d m i n i s t r a t i o n t o r a t s , which was i n t u r n metabolized f u r t h e r t o a - f l u o r o 6 - u r e i d o - p r o p i o n i c a c i d , urea, and carbon d i o x i d e .

N m e t a b o l i c degradation o f f l u c y t o s i n e i n humans was o

130

F LUCY TOSl N E

found a f t e r a s i n g l e o r a l 2 g dose (16). Polak and Scholer (17, 18) using r a d i o l a b e l e d f l u c y t o s i n e i n Candida a l b i c a n s , determined t h a t f l u c y t o s i n e e n t e r s the c e l l by means o f the enzyme c y t o s i n e permease and i s deaminated t o 5 - f l u o r o u r a c i 1 by c y t o s i n e deaminase. The 5 - f l u o r o u r a c i 1 thus formed i s i n c o r p o r a t e d i n t o t h e RNA i n place o f u r a c i l by means o f t h e f o l l o w i n g pathway ( 1 9 ) .

5- f 1 uorocy tos in e 5 - f 1 uorourac i l+5- f 1 uorour id i n e

monophosphat e -+5-f1uorouridine diphosphate+5-fluorouridine triphosphate +RNA (up t o 50% o f u r a c i 1 replaced by 5-f luorouraci 1)

5.1

Toxicology The f o l l o w i n g e x c e r p t on t h e t o x i c o l o g y o f f l u c y t o s i n e was taken from Drug E v a l u a t i o n Data (15): "The LD f o r f l u c y t o s i n e has been s t u d i e d i n r a t s a d o m i c e . The LD i n r a t s i s r e p o r t e d as 8 g/kg o r a l l y and 100 $&kg i n t r a v e n o u s l y . The LD f o r mice i s r e p o r t e d t o be 2 g/kg o r a l l y anaOsubcutaneously, 1.2 g/kg i n t r a p e r i toneal ly and 500 mg/kg in t ravenous 1 y I '

6.

Methods o f A n a l y s i s

6.1

Elemental A n a l y s i s The r e s u l t s from t h e elemental a n a l y s i s a r e l i s t e d i n Table V (20).

131

EDWARD H. WAYSEK A N D JAMES H. JOHNSON

Table V Elemental A n a l y s i s o f F l u c y t o s i n e

E 1emen t
C

% Theory
37.22

% Found
37.24

H N
F 6.2 Phase S o l u b i l i t y

3.12
32.55 14.72

3.13
32.40 14.60

Phase s o l u b i l i t y a n a l y s i s o f f l u c y t o s i n e may be c a r r i e d o u t u s i n g methanol as t h e s o l v e n t . F i g u r e 7 shows a t y p i c a l example and l i s t s t h e c o n d i t i o n s o f t h e a n a l y s i s (21).

6.3

Thin-Layer Chromatographic A n a l y s i s (TLC) The t h i n - l a y e r chromatography o f f l u c y t o s i n e and o t h e r f l u o r o p y r i m i d i n e s has been e x t e n s i v e l y s t u d i e d by Hawrylyshyn, Senkowski, and W o l l i s h (22). The R f values presented i n Table V I were o b t a i n e d u s i n g v a r i o u s s o l v e n t systems w i t h 10 c g o f sample s p o t t e d on a s i l i c a g e l p l a t e 1 and developed f o r a t l e a s t 10 cm. The p l a t e s were a i r d r i e d and viewed under shortwave u l t r a v i o l e t radiation. One and two-dimensional TLC systems a r e g i v e n by Koechlin, e t a l . (16) f o r the s e p a r a t i o n o f f l u c y t o s i n e , a-fluoro-8-ureido-propionic a c i d , 5 - f l u o r o u r a c i l , and urea i n u r i n e . The s o l v e n t systems used were e t h y l a c e t a t e : f o r m i c a c i d : water (60:5:35), 2-propanol :ammonia (20: l ) , and methanol :water (85: 15). The f o l l o w i n g TLC system i s used f o r the detect i o n o f f l u o r o u r a c i l i n f l u c y t o s i n e drug substance (23). Twenty p1 o f a 25 mg/ml s o l u t i o n o f f ucytosine i n a mixture o f g l a c i a l acetic a c i d and w a t e r (8:2) i s s p o t t e d o n a s i 1 i c a gel GF P a t e and developed by ascending chromato-

132

FIGURE

PHASE SOLUBILITY ANALYSIS

SAMPLE 8 FLUCYTOSINE SOLVENT METHANOL SLOPE: O.57t0.2% EQUILIBRATION: 20 HRS at 25. EXTRAPOLATED SOLUBILITY 7.01t0.1 mq

wr

q of SOLVENT

25 50 75 SYSTEM COMPOSITION rng of SAMPLE per g of SOLVENT

EDWARD H. WAYSEK AND JAMES H. JOHNSON

graphy i n a m i x t u r e o f c h 1 o r o f o r m : g l a c i a l a c e t i c a c i d (65:35). A f t e r development for a t l e a s t 14 cm, t h e a i r - d r i e d p l a t e i s viewed u n d e r shortwave u l t r a v i o l e t r a d i a t i o n . Flucytosine has an a p p r o x i m a t e R v a l u e o f 0.3 w i t h t h i s f system and f l u o r o u r a c i I 0.45 (24). Table V 1 R f Values f o r F l u c y t o s i n e i n V a r i o u s S o l v e n t Systems (22) S o l v e n t System

Value

1.
2.

6. 7.
8. 9.
11.
12. 10.

3. 4. 5.

a c e t a t e : acetone: w a t e r (70:40: 10) E t h y l acetate:methanol:conc. ammonium h y d r o x i d e (75:25: 1 ) Acetone Ether Ethyl acetate Methanol 2-propanol E t h y 1 a c e t a t e :methano 1 (80 :20) E t h y l a c e t a t e : m e t h a n o l (75:25) E t h y l a c e t a t e : w a t e r (100: I ) E t h y l a c e t a t e : w a t e r (100:3) E t h y 1 acetate:me t h a n o l :a c e t i c a c i d (75:25: 1) Non-Aqueous T i t r a t i o n

Ethy 1

0.1

0.3 0.02 0.0 0.0 0.6 0.1 0.2 0.2 0.0 0.0

0.4

6.4

F l u c y t o s i n e may be t i t r a t e d i n a m i x t u r e of g l a c i a l a c e t i c a c i d : a c e t i c a n h y d r i d e (2: 1) using acetous p e r c h l o r i c a c i d w i t h a glass/ calomel e l e c t r o d e system ( 2 3 ) .

6.5

F l u o r m e t r i c Analysis

A s p e c t r o f l u o r o m e t r i c method o f a s s a y i n g f l u c y t o s i n e i n b i o l o g i c a l f l u i d s has been r e p o r t e d b y Wade and Sudlow ( 2 5 ) . Flucytosine i s isol a t e d from p r o t e i n - f r e e b i o o g i c a l f l u i d s by TLC and e l u t e d f r o m t h e s i l ca g e l w i t h w a t e r . A f t e r making t h e s o l u t i o n a k a l i n e , t h e

134

FLUCYTOSINE

fluorescence i s measured. E x c i t a t i o n was c a r r i e d o u t a t a wavelength o f 300 nm and t h e emission was measured a t 365 nm.

6.6

D i r e c t Spect rophotomet r i c Ana l y s i s

D i r e c t spectrophotometric a n a l y s i s can be used t o o b t a i n a q u a n t i t a t i v e assay o f f l u c y t o s i n e i n capsules (23). I n d i l u t e hydrochloric acid ( 1 i n 100) the r e p o r t e d maximum i s 283-287 w i t h = 9.2 x 103.

6.7

Fluorine Analysis

6.71

Free F l u o r i d e A n a l y s i s The d e t e r m i n a t i o n o f f r e e f l u o r i d e i n f l u c y t o s i n e drug substance can be c a r r i e d o u t by d i r e c t measurement w i t h a f l u o r i d e s p e c i f i c i o n e l e c t r o d e (23). Electrode p o t e n t i a l measurements a r e made i n a c i t r a t e containing acetate b u f f e r solution The p o t e n t i a l (pH between 5.0 and 5 . 5 ) . measurements a r e converted t o f r e e f l u o r i d e c o n c e n t r a t i o n by r e f e r e n c e t o a c a l i b r a t i o n curve.

6.72

O r g a n i c a l l y Bound F l u o r i n e A n a l y s i s Bound f l u o r i n e may be determined by SchHniger combustion f o l l o w e d by measurement w i t h a f l u o r i d e - s p e c i f i c i o n e l e c t r o d e (26).

6.8

B i o l o q i c a l Assays Shadomy, e t a l . (27) i n d i c a t e d t h a t t h e a c t i v i t y o f f l u c y t o s i n e should be s t u d i e d i n a completely s y n t h e t i c medium. I n l a t e r s t u d i e s (28) Shadomy described a c y l i n d e r p l a t e bioassay f o r t h e d e t e r m i n a t i o n o f f l u c y t o s i n e i n sera, u r i n e s , cerebrosp i na f l u i d s , and t ssue homogena tes The i n d i c a t o organ ism used was & c e r e v i s i a e ATCC 9763 on yeas t n i t rogen base agar supple-

135-

EDWARD H. WAYSEK AND JAMES H. JOHNSON

mented w i t h L-asparagine and d e x t r o s e . For s e r a and o t h e r b i o l o g i c a l f l u i d s t h e lower s e n s i t i v i t y 1 i m i t was 0.4 t o 0 . 5 pg f l u c y t o s i n e / m l . Block and Bennett (29) r e p o r t e d a c y l i n d e r p l a t e b i o assay t h a t p e r m i t t e d t h e d e t e r m i n a t i o n o f f l u c y t o s i n e i n b i o l o g i c a l f l u i d s i n t h e presence o f a m p h o t e r i c i n B which o t h e r w i s e would i n t e r f e r e . B l a k e r and D o u t t (30) d e s c r i b e d a d i s c d i f f u s i o n method f o r a s s a y i n g f l u c y t o s i n e i n serum and o t h e r body f l u i d s u s i n g a s t r a i n o f Saccharomyces c e r e v i s i a e as t h e t e s t organism on y e a s t n i t r a t e base agar. The method was usef u l i n t h e c o n c e n t r a t i o n range 2 t o 6 pg/ml. Holt and Newman (31) a l s o r e p o r t e d a method f o r t h e r o u t i n e assay o f f l u c y t o s i n e i n b i o l o g i c a l f l u i d s u s i n g C . a l b i c a n s ( C a r s h a l t o n 2606) as t h e i n d i c a t o r o r g a n i s m . The method r e a d i l y d e t e c t e d 0.1 pg f l u c y t o s i n e / m l . M ks and a E i c k h o f f (32) s t u d i e d t h e a n t i f u n g a a c t i v i t y o f f l u c y t o s i n e using b r o t h - d i l u t i o n microt i t e r d i l u t i o n , a g a r - d i l u t i o n , and d i s c s u s c e p t i b i l i t y tests. Their invest gation indicated t h a t the disc s u s c e p t i b i l t y t e s t may be a p p l i c a b l e i n d i a g n o s t i c m i c r o b i o l o g y l a b o r a t o r i e s f o r y e a s t s such as Candida and T o r u lops i s .

7.

Acknow 1edgemen t s The a u t h o r s w i s h t o acknowledge M.V. Go, D r . K. B l e s s e l , E . Kohler, and t h e S c i e n t i f i c L i t e r a t u r e Department o f Hoffmann-La Roche I n c . for t h e i r assistance i n the preparation o f t h i s a n a l y t i c a l p r o f i le.

136

FLUCYTOSINE

8.

References

1. 2.

3.
4.

S . Traiman, Hoffmann-La Roche Inc., Personal Communication. R. Gomez, Hoffmann-La Roche Inc., Personal Communication. W. Benz, Hoffmann-La Roche Inc., Personal Comnun i c a t ion.

Pharmacopeia of t h e h i t e d S t a t e s X I X , 652 (1975).

R.J. Rucki, Hoffmann-La Roche Inc., Personal Communication. R.J. Rucki, Hoffmann-La Roche Inc., Personal Commun i c a t ion. E . Bass, Hoffmann-La Roche Inc., Personal Communi c a t i o n . A.M. Chiu and W. McSharry, Hoffmann-La Roche I n c . , Personal Communication. V . Toome, Hoffmann-La Roche l n c . , Unpublished Data. T. Ueda and J.J. Fox, J . Am. Chem. Soc., 4024 (1963). L.A. Dolan, Hoffmann-La Roche Inc., Unpublished Data R. Gomez, Hoffmann-La Roche Inc., Unpublished Data. J. Guastella, Hoffmann-La Roche Inc., Unpubl i s h e d Data. J.B. Johnson, Hoffrnann-La Roche Inc., Unpubl i s h e d Data.

5.
6. 7.
8. 9.
10.

85,

11.
12.

13. 14.
15.

Drug I n t e l l i g e n c e and ClinicaZ Pharmacy,

1,75

16.

17.
18. 19.
20.

15,

(1973). B. Koechlin, F. Rubio, S. Palmer, T. G a b r i e l , and R. Duschinsky, Biochemical PhamcoZogy,

A . Polak and H. Scholer, Path. Microbiol., 2, 148-159 (1973). A. Polak and H. Scholer, Path. MicrobioZ., 39, 334-347 ( 1973) . A . Polak and H. Scholer, Hoffmann-La Roche Inc., Basle, Switzerland, Unpublished Data. F. S c h e i d l , Hoffmann-La Roche I n c . , Unpublished Data

435 (1966).

137

EDWARD H. WAYSEK AND JAMES H. JOHNSON

21. 22. 23. 24.

E. Bass, Hoffmann-La Roche Inc., Personal Commun i c a t i o n . M. Hawrylyshyn, B.Z. Senkowski, and E.G. W o l l i s h MicrochemicaZ Journal, 8, 15-22 (1 964) . Pharmcopeia of the UniTed States, X I X , 199 (1975). B.C. Rudy and B.Z. Senkowski, AnaZyticaZ

Profiles of Drug Substances, Vol. 2, 1973,

25. 26. 27. 28. 29.

30.

31.
32.

Academic Press, p . 239. D. Wade and H . Sudlow, J . Pharm. Sci., 828 (1973). B . C . Jones, J.E. Heveran, and B.Z. Senkowski, J . Pharm. S c i . , &, I036 (1971). S. Shadomy, H.J. Shadomy, J.A. McCay, and J P U t z , Antimicrobial Agents and Chemotherapy, 452 (1968). S Shadomy, AppZied Microbiology, (6) , 871-877 (1969). . . . . .. E.R. B l o c k and J.E. Bennett, AntimkrobiaZ Agents and Chemotherapy, (61, 476-482 ( 1 972) R.G. B l a k e r and B.J. D o u t t , AntimicrobiaZ Agents and Chemotherapy, 2, (6), 502-503 (1972). R.J. H o l t and R.L. Newman, J . Clin. Path., 26, 167-174 (1973). M . I . Marks and T.C. E i c k h o f f , AntimicrobiaZ Agents and Chemotherapy, 49 1 (1970)

62,

..

17,

1,

138

GLUTETHIMIDE

Hassan Y. Aboul-Enein

HASSAN Y. ABOUL-ENEIN

CONTENTS Analytical Profile - Glutethimide

1.

2.

3. 4. 5. 6.

Description 1.1 Nomenc lature 1.11 Chemical Names 1.12 Generic Name 1.13 Trade Names 1.2 Formulae 1.21 Empirical 1.22 Structural 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Odor Physical Properties 2.1 Crystal Properties 2.11 Crystallinity 2.12 X-Ray Diffraction 2.13 Melting Range 2.14 Differential Scanning Calorimetry 2.2 Solubility 2.3 Optical Activity 2.4 Molecular Orbital Calculations & Dipole Moment 2.5 Acid Dissociation Constant 2.6 Identification 2.7 Spectral Properties 2.71 Ultraviolet 2.72 Infrared 2.73 Nuclear Magnetic Resonance 2.74 Mass Spectrum Synthesis Stability and Decomposition Products Metabolism Method of Analysis 6.1 Titrimetric Methods 6.11 Aqueous 6.12 Non-Aqueous 6.2 Colorimetric 6.21 Qualitative 6.22 Quantitative 6.3 Polarographic Analysis 6.4 Ultraviolet Spectrophotometric 6.5 Fluorometric Analysis
140

GLUTETHIMIDE

CONTENTS (cont'd) 6.6 Chromatographic Analysis 6.61 Paper 6.62 Thin Layer 6.63 Gas Chromatography High Voltage Electrophoresis Biological Assay Nuclear Magnetic Resonance

6.7 6.8 6.9

141

HASSAN Y, ABOUL-ENEIN

1.

Description 1.1 Nomenclature 1.11 Chemical Names: a. (+) 2-Ethyl-2-phenylglutarib. O(-ethyl-O(-phenylglutarimide. c. 3-ethyl-3-phenyl-2,6-piperid. e. 3-ethyl-3-phenyl-2,6-dioxo3-ethyl-3-phenyl-2,6-diketo-

mide

dinedione. piperidine. piperidine. 1.12 1.13 1.2

Generic Name: Glutethimide Trade Names: Doriden, Noxyron, Elrodorm.

Formulae: 1.21 1.22 Empirical : C13H15N02 Structural :

1.3 1.4

Molecular Weight: 217.26 Elemental Composition: C, 71.86; H, 6.96; N, 6.45; 0, 14.73

142

GLUTETH lMl DE

1.5

Appearance, Color, Odor: Glutethimide is a colorless crystalline white solid, odorless, has a bitter taste

2.

Physical Properties: 2.1 Crystal Properties:

2.11 Crystallinity Glutethimide is a crystalline solid. A typical photomicrograph of glutethimide obtained by sublimation, recrystallization from 50% aqueous ethanol and recrystallization from concentrated ammonia is shown in Fig. (1). These crystalline structures can be used as a microscopic means of identification of glutethimide (1). The behavior of glutethimide crystals extracted from the tablet formulations was discussed by Penprose and Biles (1). 2.12 X-Ray Diffraction: Analytical x-ray diffraction data for glutethimide, its anhydrous, hydrous forms and optical active antipodes were studied in detail by Bonamico et al. (2) along with other analogs. The elementarcrystal structure for these forms are shown in Table I. Further information regarding conformation of the chemical structure of glutethimide through analysis of x-ray diffraction patterns are given by Bonamico e t &. (2). The optical crystallographic properties for glutethimide crystals are as follows ( 3 ) : q1.572; p 1.585, x 1 . 5 9 0 Optic Sign: Negative 2V. large Extinction: parallel and inclined Elongation: positive System: 6-sided rods and plates

143

FA.

1 -

Photomicrographs showing variation in crystal structure of glutethimide (1). A-sublimation, B-from 50% EtOH, C-from conc. NH40H.

X-Ray
0

Table I D i f f r a c t i o n Data f o r G l u t e t h i m i d e
0
0
_ C ,A .

Form
A

aA ,
7.5320.02 20.40+0.06

b,A
30.50+0.09

z
8

v
2487E3
0

B -

Special Groz

G l u t e t h i m i d e hydrous rhombic prism G l u t e t h i m i d e anhydrous m o n o c l i n i c prisms

10.83+0.03

0.

11.3920.03

2 0 . 6 0 2 0 . 0 6 16

4791A3

92 40 220

D;5
P

?bca

c&

2COr

Pc
0

- c;-

G l u t e t h i m i d e (+)or (-1 antipode rhombic p r i s m s

6.8720.02

25.7550.07

6.9020.02

1221A3

HASSAN Y. ABOUL-ENEIN

2.12

X-ray D i f f r a c t i o n ( c o n t i n u e d )

The c h a r a c t e r i s t i c d i f f r a c t i o n p a t t e r n s were o b t a i n e d by s u b j e c t i n g g l u t e t h i mide powder t o C u K ) I - r a d i a t i o n from x-ray spectrometer and r e c o r d i n g t h e d i f f r a c t e d r a d i a t i o n on a c h a r t , u s i n g a modified GM-tube w i t h a rec o r d i n g p o t e n t i o m e t e r (1). The D-distances were c a l c u l a t e d as shown i n T a b l e 11. Table I1 10.3 7.23 6.22a 6.01 5. lla 4.59 3.78a 3.52 3.27 3.20

a
2.13

Strong

M e l t i n g Range The N a t i o n a l Formulary X I V ( 4 ) s p e c i f i e s a m e l t i n g r a n g e f o r g l u t e t h i m i d e between 8 6 0 and 89O a s a c r i t e r i a of a c c e p t a b i l i t y . Furthermore, it w a s r e p o r t e d t h a t g l u t e t h i m i d e showed a m.p. range of 85O-880 w i t h r e s i d u a l c r y s t a l s growing s l u g g i s h l y o n l y below 80 i n t o g r a i n s and prisms ( 5 ) . The m e l t s o l i d i f i e s t o a g l a s s . The g l a s s y powder showed nD 1.5403. The e u t e c t i c t e m p e r a t u r e of g l u t e t h i m i d e w i t h azobenzene and w i t h b e n z i l w a s r e p o r t e d t o be 53O and 61, r e s p e c t i v e l y .

of g l u t e t h i m i d e r e p o r t e d i n t h e l i t e r a t u r e .

Table I11 shows t h e m e l t i n g range

146

X-Ray

Table I D i f f r a c t i o n Data f o r G l u t e t h i m i d e
0 0
C ,A -

* F
Glutethirnide h y d r o u s rhombic prism
Glutethimide a n h y d r o u s m o n o c l i n i c prisms

a,A
7.5320.02 20.40+_0.06

b,A
30.50fp.09 11.39+_0.03

2
8 16

! !
0

S p e c i a l Group

10.83+0.03 20.60+0.06

2 4 8 7 ~ ~
0

o* 4 7 9 1 ~ ~ 2 40 5 2 0 9

D215
P

bca

- Cjh
2COr

Pc 6.8720.02 25.7550.07 6.9020.02 4 1 2 d 3

- c;

Glutethimide (+)or ( - ) antipode rhombic prisms

P212121

HASSAN Y . ABOUL-ENEIN

Table I11 m.p., C O 91-92 0 85-87, bo 3168 82-83 ( is6propanal) 86-88 anhydrous glutethimide 80-85 (EtOAc-pet. ether) 83-85 68.5.70.5 (ail EtOH) 2.14 Reference 1 6 7 8 9
10 11

Differential Scanning Calorimetry Reubke and Mollica (12) reported the application of the differential scanning calorimetry in the quantitative estimation of purity of glutethimide and the detection of polymorphism. They reportedAH value for glutethimide to be 28.7+1 cal/gm. at 86.80 - 87O. Solubilit : d i n water (1.0 mg/ml) , 1 in 5 of ethanol, 1 in 12 of ether and than 1 of chloroform, dichloromethane. in acetone, ethyl acetate. A saturated in water is acidic to litmus.
2.2

soluble 1 in less Soluble solution

Recently, it was reported that the tetramethyl-substituted amides of pimelamide, suberamide, azelamide and sebacamide markedly enhance the solubility of glutethimide in aqueous solution (13). The solubility of glutethimide was increased significantly above the critical concentrations and from the nature of the solubility curves, a micellar type of solubilization appears to be dominant. Optical Activity Glutethimide is marketed as the racemic Keberle mixture of 2-ethyl-2-phenylglutarimide. et al. (14) describes a procedure for resolution the racemic mixture to the pure optical antipodes as shown in Scheme 1 The sedative. hypnotic activity of the (+) isomer is 2-3 times more potent than the ( - 1 isomer (15).
148

2.3

GLUTETH IMI DE

c 6H 5

-: 2 -co

H (CH2)2C02H

Pi""
It

+,

( 2 ) -e thy l-2 -pheny lg lu t a r i c -2

acid

r e s o l v e d w i t h (+) and (-1 d -phenethylamine

Jy3

H (-)-isomer
Scheme 1
149

phn
H (+)-isomer

\D

rl

m
rl
rl rl W
IW

* rl
rl

*
0 rl

*
rl I Pl 0 rl
0
rl

0 ui
N 0 rl

Pl 0 rl

I
0 rl

*
0 rl

*
m
0
N

I
I
0
4

0 rl

Pl

0
X

rl

0
X

v
Y

w
W
N

s
w
V
II
+ ,

X
rl

8
X

:
2

rl

I 1
N N

+I
+I
W
W

W
I-

* W
rl

+I
m 4
rl

rl
rl

ui
NO
U U

-k

I
ui

I
N O n

ND

N O
u

N O

II
a,

T
Ll

Ll

8
.r(

a,

I
h Y

150

GLUTETHIMIDE

2.3 Optical Activity (continued) The specific rotations, melting points for the (+) and (-1 isomers are given in Table IV as recorded'in the literature. 2.4 Molecular Orbital Calculations and Dipole Moment The molecular orbital calculations of glutethimide was discussed by Andrews (19) using two methods, extended Huckel theory (EHT) and complete neglect of differential overlap CND0/2. Calculated dipole moment (D) of glutethimide was found to be 8.67 using EHT, while the experimental dipole moment (D) was reported by Lee and Kumler (20) to be 2.83. Andrews showed that CNDO/2 method is more suitable for assessing net atomic charges than the EHT.

H 2.83D Furthermore, Lee and Kumler (20) concluded from their study that of the dipole moments of several six-membered cyclic imides the imide groups in these compounds are in a cis-cis conformation. 2.5 Acid Dissociation Constant Doornbos and De-Zeeuw (21) measured the "proton lost" dissociation constant K H and the "proton gained" K3H dissociation conszant for glutethimide by a previously described potentioK2H and K metric titration method at 2 0. for glutethimide at ionic strength of p=O.?O was found to be 4.518.
151

HASSAN Y . ABOUL-ENEIN

Identification The f o l l o w i n a i d e n t i f i c a t i o n t e s t s a r e p u b l i s h e d i n B . P . 1973 ( 2 2 ) a s a p a r t of t h e i d e n t i f i c a t i o n of g l u t e t h i m i d e .

2.6

1. The l i g h t a b s o r p t i o n , i n t h e r a n g e 230 t o 350 nm, o f a 2-cm l a y e r o f a 0.03 p e r c e n t w/v s o l u t i o n i n d e h y d r a t e d a l c o h o l e x h i b i t s t h r e e maxima, a t 252 nm, 258 nm, and 2 6 4 nm; e x t i n c t i o n a t 252 nm, a b o u t 1.0, a t 258 nm, a b o u t 1.1, a n d a t 2 6 4 nm, a b o u t 0 . 8 6 .
2. Heat 1 g w i t h 5 ml of sodium hydroxi d e s o l u t i o n and 1 5 m l o f water on a w a t e r - b a t h f o r t h i r t y m i n u t e s , cool and a c i d i f y t o l i t m u s paper w i t h d i l u t e h y d r o c h l o r i c acid; f i l t e r ; m e l t i n g p o i n t of t h e p r e c i p i t a t e , a f t e r washing w i t h water and d r y i n g a t l o o o , a b o u t 159'.

F u r t h e r m o r e , Imaoka and Ogura ( 2 3 ) publ i s h e d a p r o c e d u r e f o r i d e n t i f i c a t i o n of g l u t e t h i m i d e i n t a b l e t s by s p o t t e s t . A powdered sample (2-3 mg) on Whatman's T e s t P a p e r i s w e t t e d c w i t h 2 d r o p s a c e t o n e , 1 d r o p 1% o p p e r acetate o r 1% o b a l t acetate s o l u t i o n , k e p t f o r 30 s e c o n d s c then 1 drop 1 0 % isopropylamine s o l u t i o n i n a c e t o n e w a s a d d e d , g l u t e t h i m i d e g i v e s a clear v i o l e t colored spot.

Most b u l k i n g a g e n t s , e . g . l a c t o s e , s t a r c h , s u c r o s e , gun a r a b i c , g l u c o s e o r t a l c w i t h t h e e x c e p t i o n of a l g i n i c a c i d d o e s n o t i n t e r f e r e w i t h t h e test.

2.7

Spectral Properties 2.71

Ultraviolet Glutethimide i n s o l u t i o n absorbs u l t r a v i o l e t r a d i a t i o n over a b r o a d range t o prod u c e a s p e c t r u m w i t h maximum a t 257 251, 263 nm = 19 for ( t y p i c a l spectrum, F i g . 2 1 , w i t h A t h e wave l e n g t h 257 n ( 2 4 - 2 6 ) . m

!&,

152

OL g0.40
0

0 20

000

220

260 300 340 WAVE L NGT t4, rni i1i m ic rons

I
153

L
I

Fig.

2-

U l t r a v i o l e t spectrum of g l u t e t h i m i d e i n methanol ( 2 4 ) .

HASSAN Y . AEOUL-ENEIN

I n f r a r e d Spectrum The i n f r a r e d s p e c t r u m of g l u t e t h i mide i s shown i n F i g . 3. The s p e c t r u m w a s obt a i n e d on a P e r k i n - E l m e r 621 s p e c t r o p h o t o m e t e r from a K B r p e l l e t .

2. 72

The s t r u c t u r a l a s s i g n m e n t s h a v e b e e n c o r r e l a t e d w i t h t h e f o l l o w i n g band f r e quencies: Frequency ( c m - l ) 319 0-3100 1710 1680 1200 760-700 Assignment
NH s t r e t c h i n g i m i d e C=O i m i d e c a r b o n y l C=O i m i d e c a r b o n y l n e x t

t o t h e d -p h en y l group stretching Monosubstituted phenyl

C-0

Further information with regard t o t h e i n f r a r e d s p e c t r a of g l u t e t h i m i d e i s g i v e n b y s e v e r a l authors (27-29). 2.73 N u c l e a r M a g n e t i c Resonance S p e c t r u m

A t y p i c a l NMR s p e c t r u m of g l u t e t h i mide i s shown i n F i g . ( 4 ) . The sample w a s

d i s s o l v e d i n c a r b o n t e t r a c h l o r i d e . The s p e c t r u m w a s d e t e r m i n e d on a V a r i a n T-60 NMR S p e c t r o m e t e r w i t h TMS as the i n t e r n a l s t a n d a r d . The f o l l o w i n g s t r u c t u r a l a s s i g n ments h a v e b e e n made f o r F i g . ( 4 ) . C h e m i c a l S h i f t (dl T r i o l e t 0.80 M u l k p l e t 1 . 8 t o 1.97 S i n g l e t 6.9 7 Broad s i n g l e t 8.27 Assignment
-CH
9

-CH2-CH3 a n d t w o -C>of glutarimide ring A r o m a t i c phenyl protons NH i m i d e , e x c h a n g e a b l e with D20.

-CH --J

Further information concernins t h e i n t e r p r e t a t i o n of t h e NMR s p e c t r u m of g l u t e t h i m i d e and r e l a t e d c y c l i c imides c a n b e o b t a i n e d

154

FA.3

I n f r a r e d spectrum of g l u t e t h i m i d e , K B r p e l l e t .

Glutithimide (1)

F i g- 4 - .

NMR spectrum of g l u t e t h i m i d e i n CC14 c o n t a i n i n g TMS a s i n t e r n a l s t a n d a r d .

GLUTETH IMlDE

from C a s i n i and S a l v i ' s ( 2 9 ) s t u d y of t h e NMR o f a s e r i e s of c y c l i c i m i d e s and a l s o by Defay and Dorlet (30).

Mass S p e c t r a The mass s p e c t r u m of g l u t e t h i m i d e o b t a i n e d by c o n v e n t i o n a l e l e c t r o n impact i o n i z a t i o n shows a m o l e c u l a r i o n M+ a t m/e 2 1 7 . The M+ i o n p e a k i s o n l y 5 % ( F i g . 5 ) . The b a s e p e a k i s a t m / e 1 1 7 . The mass s p e c t r a l f r a g m e n t a t i o n mechanism w a s d i s c u s s e d by Rucker ( 3 1 ) a s shown Mass s p e c t r o m e t r y w a s a l s o u s e d as i n Scheme 2 . an analytical tool f o r rapid, accurate identif i c a t i o n o f and d i a g n o s i s o f g l u t e t h i m i d e o v e r d o s e s and p o i s o n i n g cases ( 3 2 - 3 3 ) .
2.74 The c h e m i c a l i o n i z a t i o n ( C I ) mass s p e c t r o m e t r y o f g l u t e t h i m i d e w a s r e c e n t l y rep o r t e d by S a f e r s t e i n and Chao ( 3 4 ) . The i o n i z a t i o n w a s a c c o m p l i s h e d by t h e r e a c t i o n of t h e d r u g w i t h e i t h e r i o n i z e d g a s e o u s methane or i s o b u t a n e . The r e s u l t i n g s p e c t r u m w a s l e s s complex t h a n t h a t p r o d u c e d by c o n v e n t i o n a l E I i o n i z a t i o n and u s u a l l y r e s u l t s i n t h e f o r m a t i o n of a molecular i o n p l u s o n e (M+ + 1) i . e . , 218 f o r glutethimide.

3.

Synthesis
X ( C H 2 ) 2-C-CN

CH2CN N a N H 2 C2H5C:

basic catalyst
X=-CN

or

roac
H2S04
E t

Scheme 3
157

SRMPLE

NUMBER

73Z

h i

G L uT ET H1 M ID E

'001
'09 '0h

C D I I T I 2ED 3 G
117

YH3d 35HB

'0R

i 1
I

132
115 1 GO

X 3EltllN3Xf3d I

158

'3 02 ' 0

5.m.
M/E

am.

!in.

- -

Fig. 5

Mass Spectrum of Glutethimide (EI).

w
() I

k c,

a,

GLUTETHIMIDE

rnlr =

189

m/c

146

m/e = 137

m/e = 117

Scheme 2

Mass spectral fragmentation mechanism of glutethimide (31).

159

HASSAN Y . ABOUL-ENEIN

Glutethimide is synthesized as shown in Scheme 3. Starting with benzylcyanide which is treated with ethyl chloride or bromide in the presence of sodium amide to yieldd-ethyl benzylcyanide. This is then allowed to react with methyl acrylate or acrylonitrile (Michael condensation) under the catalytic influence of piperidine or another suitable basic catalyst, thus, forming methyl 4-cyano-4-phenylhexanoate or its corresponding dinitrile derivative, respectively. The latter intermediate was purified by distillation and cyclized in the presence of acetic acid and 9 0 % sulfuric acid to yield glutethimide (6, 35-37). Another synthetic route for glutethimide is described by Iwase - - ( 9 ) et al. shown in Scheme 4. A mixture of l-phenyl-lethylpropane-1,3-dicarboxylic acid and p-nitrosoaniline was treated at 180-185O under nitrogen atmosphere for 5 hrs., refluxed with 2% sodium hydroxide for 3 hrs., acidified, washed with ether, neutralized with sodium carbonate, extracted with chloroform to yield glutethimide.
NO

Et

NH2

Scheme 4

160

GLUTETHIMIDE

13.

O t h e r r o u t e (38) f o r g l u t e t h i mide s y n t h e s i s i s shown i n Scheme 5.

CH 2 =CHCN

HC 1 9 C1CH2CH2C02CH (CH3) isopropanol

i s o p r o p y l /3-chlarnproDionate

dimethyl p i p e r i d i n e sulfate

gH5

C gHg$HCN C2H5

Scheme 5

161

HASSAN Y. ABOUL-ENEIN

Stability & Decomposition Products Several reports showed that glutethimide is hydrolyzed to h-ethyl-4-phenylglutaramic acid in alkaline solution (36, 39). Yamaha and Mitzukami (40) reported that this decomposition occurs in sodium hydroxide and sodium carbonate solutions by second order reaction but not in sodium bicarbonate solution. The degradation rate constants at 20, 30 and 400 in 0.01N sodium hydroxide 1.67 x 10-1 and 3.56 x 10-1 were 7.66 x a?d those in 0.01M sodium carbonate were 3.56 x 10- , respectively. The activation energies in 0.01N sodium hydroxide and 0.01M sodium carbonate were 14.3 and 14.6 K cal., respectively. Furthermore, Wesolowski et al. (41) studied the kinetics of degradationoflutethimide in buffered aqueous solutions in the pH range 1.5-8.0. They supported the previous reports that the degradation of glutethimide is a base-catalyzed reaction since the contribution of ionized species to the reaction rate in the pH range studied is very small. The rate is first order with respect to the concentration of glutethimide. The apparent energy of activation Ea, for the degradation of glutethimide at pH8 is found to be in the order of 25.86 K cal./mole. However, the energy of activation corrected for heat of ionization of water at SOo, 60, and 650 was found to be 13.47 K cal., 13.92 K cal., and 14.15 K cal./mole, respectively. The mechanism proposed for the glutethimide degradation is shown in Fig. 6 and involves direct attack by a hydroxyl ion on the unhindered carbonyl of the glutarimide ring followed by the cleavage of the ring to 4-ethyl-4-phenyl-glutaramic acid.

4.

162

GLUTETHIMIDE

r
8 Ph OH,
0
N
0 Slow
7

HO
H

c =o

Fig. 6 The Mechanism of G l u t e t h i m i d e Degradation ( 4 1 ) .

A p l o t of l o g t+ v e r s u s p ( F i g . 7) shows t h a t H below pH5, g l u t e t h i m i d e i s v e r y s t a b l e s i n c e a t p 1.0 and 5.0 t h e r a t e i s independent of hydroH gen i o n c o n c e n t r a t i o n .

163

24 2.2
20 1.8 1.6
CI

t-

r
-J

5 C

v)

I4

1.2

i?

k\
5.0 6.O

Q8C
0.6

7.0
PH
t3c

04-

0.2

0.0 40
0

Fig. 7 - -

Log t4, the half-life of the reaction in months against pH at 25 (41).

164

GLUTETH l MI DE

Metabolism The distribution and metabolism of glutethimide in the dog and rat were extensively studied and summarized by Keberle - al. (42). The et metabolic fate in man was studied by Bitikofer et - al. (43). Both of these studies indicated that the (+)-isomers are metabolized via two different Eiochemical routes. The ( r e m o s i ) + was hydroxylated on the glutarimide ring at the 4position to yield 4-hydroxy-2-ethyl-2-phenylglutarimide, a small percent of which undergoes dehydration to give 2-ethyl-2-phenylglutaconimide. The (-)-isomer predominantly hydroxylated on the ethyl side chain to form 2-(l-hydroxyethyl)-2phenylglutarimide, of which a small percent lose a moleculae of acetaldehyde to form 2-phenyl--. glutarimide, Fig. ( 8 ) . In 1969, Post and Schutz (44) identified a phenolic metabolite which they assumed to be 2-(4-hydroxyphenyl) glutarimide.
5.

Recently Stillwell - - (45) identified et al. more polar metabolites present in the urine of rats and guinea pigs, (Fig. 9). They characterized these metabolites by combined GC/MS as follows: a. 4-hydroxy-2-ethyl-2-phenylglutarimide b. 3-hydroxy-2-ethyl-2-phenylglutarimide C. 2-(l-hydroxyethyl)-2-phenylglutarimide d. 2-ethyl-2-(4-hydroxyphenyl)glutarimide e. 2-ethyl-2-(3,4-dihydroxyphenyl)glutarimide f. 2-ethyl-2-(3,4-dihydroxy-l,5-~yclohexadien- l-yl)glutarimide g. 2 - e t h y l - 2 - p h e n y l g l u t a c o n i m i d e h. 2-phenylglutarimide They demonstrated that the hydroxylation of the aromatic ring in the rat and guinea pig occurs V J an epoxide pathway. These authors also supported the fact that the rat and guinea pig, like the dog and man, metabolize the (-)-isomer of glutethimide by hydroxylation of the ethyl side chain to form 2- (1-hydroxyethyl) -2-phenylglutarimide. However, the metabolism of the ( + I 165

(+)-isomer Dextrorotatory

i
0

.
0

~
0

G 1 u - o ~ o C 6 H 5
0 H
45%

2%

glucuronidation dog man rat guinea pig

do9 man rat guinea pig

Glu = glucuronic acid

H
(+)

Glutethimide

(-)-isomer Levorotatory

0 0 :
-CH3CHO,
0
h -

C6H5
0

-N-

0
n

4%

45%

man rat guinea pig and dog Fig. 8 Metabolism of Glutethimide

do9 man r a t and guinea pig

OH

man

rat guinea p i g

Man (minor)

OH
0

N
u

N
H rat guinea p i g (minor)
H

rat guinea p i g

rat guinea p i g

F i g . 9 P o l a r M e t a b o l i t e s of G l u t e t h i m i d e

HASSAN Y . ABOUL-ENEIN

isomer seems to be species dependent. In contrast to man and dog, the rat apparently metabolized the (+)-isomer of glutethimide by hydroxylating the aromatic as opposed to the heterocyclic ring to form 2-ethyl-2-(4-hydroxyphenyl)glutarimide, however, some hydroxylation of the heterocyclic ring occurs. The guinea pig hydroxylates both the aromatic and the heterocyclic rings and these metabolites are presumably formed from the (+)-isomer. Further studies on the metabolism of the ( - ) and (+) isomers by the rat and guinea pig are needed to validate these conclusions. Ambre and Fischer (46) showed that monohydroxylated metabolite of glutethimide accumulated in the plasma of humans intoxicated with glutethimide overdose. This metabolite was subsequently isolated from urine of dogs given large doses of glutethimide and was chemically identified as 4-hydroxyglutethimide (47). This metabolite was found to possess twice the sedative-hypnotic activity of glutethimide (47). Recently, it was shown that the concentration of 4-hydroxyglutethimide is highest when the respiratory status of the patient was near its lowest (48, 49). This led to the conclusion that 4-hydroxyglutethimide accumulation plays an important role in acute glutethimide poisoning. 4-Hydroxyglutethimide and other potential glutethimide metabolites were chemically synthesized and pharmacologically tested by Aboul-Enein - et al.
(50).

Among other metabolites previously identified in animals and man, Andreson _ et al. (51) recently reported the identification and chemical characterization of 2-ethyl-2-(4-hydroxyphenyl) glutarimide in human urine from patients overdosed with glutethimide as a major metabolite. They also characterized 2-ethyl-2-(3-methoxy-4hydroxypheny1)-glutarimide as a new metabolite of glutethimide.
168

GLUTETHIMIDE

The toxic effects possibly caused by the metabolites of glutethimide has interested many research laboratories to continue investigating this problem. 6.
6.1

Methods of Analysis Titrimetric Methods 6.11

Aqueous A titrimetric method was developed for analysis of glutethimide. The procedure involves the alkaline hydrolysis of glutethimide with standard alcoholic potassium hydroxide and subsequent back titratation of the unconsumed alkali with standard hydrochloric acid using phenolphthalein as indicator (521, a method adapted by the British Pharmacopeia (22). Non-Aqueous Ellert et - (53) described a - al. . . method for determination of glutethimide by nonaqueous titration with 0.1N sodium methoxide in methanol-benzene in a solution of ethylenediamines (against 0-nitroaniline) or in pyridine (against Azo Violet). Glutethimide can also be quantitated in tablet formulation by non-aqueous titration in dimethyl formamide and titrating with propanol-benzene 0.1N potassium hydroxide (using metanil yellow as indicator) (54). 6.2 Colorimetric Qualitative The following color reaction is published in the B.P. 1973 (22) as part of an identification scheme. Shake 10 mg with 2 ml of water, 0.1 g of hydroxylammonium chloride and 1 ml of sodium hydroxide solution, allow to stand for ten minutes and add 2 ml of dilute hydrochloric acid and 1 ml of ferric chloride test-solution; a deep brownish-red color is produced.
169

6.12

6.21

HASSAN Y . ABOUL-ENEIN

Quantitative Sheppard ( 3 9 ) and a s s o c i a t e s u t i l i z e d a method based on t h e f o r m a t i o n of t h e c o l o r e d complex between f e r r i c i o n and t h e hydroxamate r e s u l t i n g from t h e r e a c t i o n of g l u t e t h i m i d e w i t h a l k a l i n e hydroxylamine f o r d e t e r m i n a t i o n of t h e drug i n u r i n e . The c o l o r w a s r e a d w i t h i n f i v e minutes a t 5 1 0 nm. The p r e s e n c e of l a c t o s e and a n i o n s which complex w i t h f e r r i c i o n o r s a l t s which form p r e c i p i t a t e s w i t h f e r r i c i o n i n t e r f e r e w i t h t h i s method. Phang e t - ( 5 5 ) i n t r o d u c e d a m o d i f i c a t i o n t o Shep. -r a l' s procedure so t h e f a t t y i m p u r i t i e s do n o t pa d i n t e r f e r e w i t h t h e q u a n t i t a t i o n of g l u t e t h i m i d e i n vomitus m a t e r i a l .
6.22

Belova and Zinakova (56) rep o r t e d a s i m i l a r c o l o r i m e t r i c method i n which t h e c o l o r developed was measured a t 4 9 0 n w i t h m a no. 5 l i g h t f i l t e r and claimed t h a t t h e p r e s e n c e of l a c t o s e does n o t i n t e r f e r e w i t h t h e r e s u l t of g l u t e t h i m i d e d e t e r m i n a t i o n . T h i s p r o c e d u r e , however, i s n o t v e r y s e n s i t i v e o r a p p l i c a b l e t o blood specimens and i s r e l a t i v e l y n o n - s p e c i f i c ( 5 7 ) . Polarographic Analysis G l u t e t h i m i d e f a i l e d t o have p o l a r o g r a p h i c wave on a . c . and d . c . polarograms u s i n g 0.1M l i t h i u m c h l o r i d e , o r tetramethylammonium c h l o r i d e as s u p p o r t i n g e l e c t r o l y t e (58).
6.3

R e c e n t l y , a d e t a i l e d s t u d y of a n i n d i r e c t p o l a r o g r a p h i c d e t e r m i n a t i o n of g l u t e t h i mide a f t e r n i t r a t i o n was r e p o r t e d by Lauermann ( 5 9 ) . The method c a n be used t o q u a n t i t a t e g l u t e t h i m i d e i n post-mortum t i s s u e s and b i o l o g i c a l f l u i d s , w i t h s e n s i t i v i t y of 0 . 8 mg/100g of m a t e r i a l . The c o n v e r s i o n of t h e phenyl group t o n i t r o p h e n y l group a f t e r n i t r a t i o n makes it possible t o u t i l i z e t h e polarographic technique i n a n a l y s i s of g l u t e t h i m i d e .

170

GLUTETHlMl DE

Ultraviolet Spectrophotometric The ultraviolet absorption of glutethi257 nm (60) is-used as mide in methanol at a sensitive criteria for its analysis. This method is sensitive to a concentration range of 50-500 pg/ml (61).
6.4

Ultraviolet spectroscopy has been extensively applied to glutethimide determination in biological fluids such as (62-64) serum and urine and pharmaceutical preparations (54, 65) with high degree of sensitivity. Fluorometric Analysis A fluorometric procedure was described for the quantitative determination of glutethimide in table formulations (66). The method is based on the fluorogen resulting from the reaction of glutethimide with conc. sulfuric acid containing formaldehyde. The fluorescent intensity is a straight line function of concentration over a wide range. The excitation maxima were observed at 280 and 365 nm and the corresponding maximum fluorescent emission occurred at 450 nm. The procedure is applicable for the determination of glutethimide in the presence of its degradation product 4-ethyl-4phenylglutaramic acid, since the latter yields only 1/10 the fluorescence of glutethimide. The fluorescent reaction does not occur with compounds lacking a phenyl group or containing a substituted phenyl ring and there is no interference with the tablet excipients.
6.5 6.6

Chromatographic Analysis
6.61

Paper The chromatographic behavior of qlutethimide and related analogs was established 6y Davies and Nicholls (67) without interference from the chemically related barbiturates by ascending paper chromatography. Whatman No. 1 paper was used and some paper was impregnated with liquid paraffin ( 4 % in hexane), olive oil
171

HASSAN Y. ABOUL-ENEIN

(20% in acetone) or tributyrin (10% in acetone). All compounds were applied in amounts of 100 ) g I by the window technique. Several reagents were used to detect glutethimide after chromatography. These include the hypochlorite reagent by Creig and Leaback (681, alkaline, hydroxylamine spray of Sheppard - - (39), and 1% HgN03 solution (69). Some of et al. the most useful solvent systems are shown in Table V. Table V Solvent System 10:5:4 PhMe-HOAC-H 0 CC1 HOACmH20 1:2:1 1 0 % w/v aq. 4iaC1 0.066M Na3P04(pH 7.3 on tributyrin paper Reference 67 67 67 67

A method for rapid detection of glutethimide and other hypnotic was described (70) using pigment impregnated paper, and a combination of 2 dimensional chromatographs. Solvent system used in this technique were piperidine-petroleum ether (1:9) followed by cyclohexane, CHC13 (1:3) or solvent system piperidine-petroleum ether (1:9) followed by benzene: CHC13 (1: 5 ) . The applicability of pigment impregnated paper for the resolution of hypnotics in cadaveric material was also discussed. Kloecking (69) described the use of wedge-strip procedure to achieve better separation of glutethimide and other barbiturates in chemical-toxicological analysis than the usual ascending descending chromatograms. A good separation was obtained by using ascending chromatograms with dioxane-xylene-toulene-isopropanol-258 NH (1:2:1:4:2) upper phase on Schleicher and Zchnell 2045 Bgl paper for 24
172

GLUTETHlMl DE

h o u r s a t 20. Other p e r t i n e n t i n f o r m a t i o n i n t h i s r e g a r d i s t o b e found i n references (71-74). Thin Layer S e v e r a l t h i n l a y e r chromatog r a p h i c s y s t e m s have been d e v e l o p e d t o s t u d y v a r i o u s a s p e c t s of g l u t e t h i m i d e . S e v e r a l p r o cedures w e r e described f o r its detection, identification i n biological fluids, forensic and t o x i c o l o g i c a l a n a l y s i s , (82-90). S e v e r a l s o l v e n t s y s t e m s u s e d f o r i t s i d e n t i f i c a t i o n and s e m i q u a n t i t a t i o n of g l u t e t h i m i d e a r e shown i n Table V I .
G a s Chromatography Many i n v e s t i g a t o r s h a v e u s e d g a s chromatography t o a n a l y z e g l u t e t h i m i d e . A l t h o u g h most of t h e p u b l i s h e d w o r k i s c o n c e r n e d almost e n t i r e l y w i t h a n a l y s i s of g l u t e t h i m i d e and i t s m e t a b o l i t e s i n b i o l o g i c a l f l u i d s and t i s s u e s , many of t h e methods c o u l d b e m o d i f i e d e a s i l y f o r a n a l y s i s of g l u t e t h i m i d e i n pharmac e u t i c a l dosage forms.

6.62

6.63

Berry ( 9 1 ) conducted i n - d e p t h s t u d y f o r t h e g a s c h r o m a t o g r a p h i c beh a v i o r of g l u t e t h i m i d e i n p r e s e n c e of s e v e r a l b a r b i t u r a t e s on 1 2 GC columns. OV-225 and CDMS were t h e most u s e f u l of t h e columns t e s t e d f o r a n a l y s i s of t h e b a r b i t u r a t e s , g l u t e t h i m i d e and i n t e r f e r i n g d r u g s a t t h e r a p e u t i c and o v e r d o s e l e v e l s i n p l a s m a . The method w a s s e n s i t i v e , r a p i d a n d s u i t a b l e f o r emergency t o x i c o l o g i c a l cases.
B l o o m e r e t - (92) d e v e l o p e d a - al. r a p i d method f o r d i a g n o s i s of s e v e r a l a c i d i c and n e u t r a l s e d a t i v e - h y p n o t i c s u s i n g GC i n i n t o x i c a t e d p a t i e n t s . The p r o c e d u r e p e r m i t s s i m u l t a n e o u s i d e n t i f i c a t i o n and measurements of a

GC are l i s t e d i n T a b l e VII.

A number o f t h e s y s t e m s u s e d f o r

173

Table VI Solvent Svstem CHC13:Et20 85:15 CHC13 :EtOH 90:10 Developer KI-benzidine KI-o-tolidine HC1 (after treatment with gaseous C12)
Ag

Rf 06 .6 0.96

Reference 75 75

EtOAcaeOH:NH385:5:2.5 CHCI3:Me2CO 9: 1
2

0.1% KMnO4 and 1% NO3 1% HgS04

0.89 Rf were reptd. on various commerc. avail. silica gel tlc, plates, sheets, films

76 77 77 77 78

EtOAc:MeOH:NH40H 85:lO : 5 Cyc10hexane:CgHg :Et2NH 15 :: 32 CHC13 :Me2C0 9 :1 0.2% aq. solution CuSO with pyridine (50:4,-grey color developed.

IsoPrOH : CHCl : 5%NH4OH 2 45: 45:lO

0.84

79

Table VI (cont'd) Solvent System Et0Ac:cyclohexane-dioxane: MeOH:H20:NH40H 50:50:10:10: 1.5:0.5 50:50:10:10:0.5:1.5 EtOAc-cyclohexane:MeOH :NH40H 56:40:0.8:0.4 70:15:10:5 Developer

Reference 79

0.1% diphenyl carba0.89 zone (orange pinkspot):1% AgOAc (bluepurple spot: HgS04 (purple fades away).

79

EtOAc:cyclohexane:NH~0H 50: 40 :O .1

79 79 1% diphenyl carbazone;Hg (NO3)

W at 254 nm C12

EtOAc :cyclohexane: NH40H : MeOH:H20 70:15;2:8:0.5

CHC13:BuOH:HC02H 70:40:3.5 (chamber saturated with NH40H) EtOAc (redisti1led) Dioxane: CH2C1 :H20 l:2:1 CHC13 :Me2C0 9 :

80

and starch KI solution

0.68 0.96 0.55

81

Table VII Gas Chromatographic Systems Used for Glutethimide Analysis All systems listed used flame ionization detectors.
3% OV-17 on Supelcoport

Column

(100-120 mesh) (derivatized with dimethylformanide dimethyl acetal)

Carrier Gas 55 ml/min He

Column Temp, Co 160-2200 at 7"/min

Ref. 95

3% OV-1 on 100-120 mesh Gas Chrom Q

A

30 ml/min He

120-2200 at 1o0/min 183 183 220 220

96 97 97 98 98

o ,

4 % OV-17 on 80-100 mesh Chromosorb WHPAW-DMSC

15 psi N2 25 psi N2 N2 N2 50-60 ml/min N2

3.8% SE-30 on 80-100 mesh Chromosorb WHPAW-DMSC 10% Dexsil 300 on 800-100 mesh Chromosorb WHP

17% Dexsil 300 on 80-100 mesh Chromosorb WHP

1% Carbowax 20M*

205

91

on 80-100 mesh Chromosorb W, HP.

T a b l e V I I (cont'd) Column 3% P o l y A - 1 0 3 * Carrier Gas 50-60 m l / m i n N2 50-60 50-60

Column T e m p , Co 2 15
230 230 200 1 90 220-240

Ref. 91 91 91 91 91 91 91 91 91 91 91 99

3% NGA+0.07% t r i m e r acid*

m l / m i n N2

1% SP-1000*

m l / m i n N2
N2

3% PPE-20*
1 0 % A p i e z o n L*
2

50-60 m l / m i n 50-60 50-60 50-60 50-60 50-60 50-60

m l / m i n N2

4 % CDMS*

m l / m i n N2

I J

3 % OV-l*

m l / m i n N2
ml/min N2

155
200 1 65 160 205 200

5% OV-17*
3% OV-25*
4% ov-210* 4 % OV-225*

m l / m i n N2
ml/min N2

50-60 m l / m i n N2 4 0 m l / m i n N2

3% OV-225 o n 8 0 - 1 0 0 mesh Chromosorb W

Table VII (cont'd) Column 8% X F 1112 on 60-80 mesh Chromosorb WHMDS 7% DC-200 on G a s Chrom Q (80-100 mesh) derivatized as N methyl derivative Carrier G a s 25 ml/min N, .4
50 ml/min He

Column Temp, Co 19 5 19 0

Ref. 100 10 1

5% SE-30 on 70-80 mesh AW Chromosorb W

4

60 ml/min N2

19 5

10 2

l
Q)

on 80-100 mesh chromosorb, W, HP.

GLUTETHIMIDE

variety of sedative agents. By virtue of acidbase extraction with chloroform, the acidic and neutral agents are separated. The chloroform extracts were dried, evaporated and the residue dissolved in isopropanol. Column used was 3% SE-30 on 80-100 mesh Chromosorb W. Gel filteration using Sephadex LH-20 was used to remove impurities from chloroform extracts of acidified blood, urine and homogenized tissue sample. Quantitative determination was made by GC using 15% SE-30 in Chromosorb W at column temperature of 15Oo-25O0
(93).

Fischer and Ambre ( 9 4 ) showed that analysis of urine from patients intoxicated with glutethimide on columns containing SE-30, OV-1 and OV-17 lead to an overestimation of the unchanged drug in urine. These columns were considered non-selective. However, 3% OV225 on 80-100 mesh Supelcoport and 2% Carbowax 20M on 100-120 mesh Supelcoport were selective to separate the drug from its potentially interfering metabolites and thus, eliminates the possible overestimation of glutethimide in biological samples. Several gas chromatographic methods for measurement of glutethimide in biological fluids are reported employing a variety of extraction procedures and gas chromatographic conditions (103-123). High Voltage Electrophoresis Hoffman - - (124) described a method et al. for the separation, detection and semiquantitation of glutethimide and other barbiturates from urine and other biological fluids using high voltage electrophoresis. It was reported that glutethimide migrates at the cathode side at a distance of 0.22 relative to crotylbarbital.

6.7

179

HASSAN Y . ABOUL-ENEIN

B i o l o g i c a l Assay G l u t e t h i m i d e c a n b e d e t e c t e d by hemagg l u t i n a t i o n i n h i b i t i o n method d e s c r i b e d by A n t i s e r a from r a b b i t s Valentour e t a l . (125). i n j e c t e d w i t h g l u t e t h i m i d e - b o v i n e serum a l b u m i n ( 1 0 mg/ml) w e r e s e n s i t i v e enough t o d e t e c t 50 n g g l u t e t h i m i d e /0.1 m l plasma o r u r i n e .
6.8

The method w a s u s e d t o a n a l y z e u r i n e and plasma p a t i e n t s s u s p e c t e d of g l u t e t h i m i d e intoxication. N u c l e a r M a g n e t i c Resonance Aboul-Enein (126) h a s r e p o r t e d a method f o r q u a n t i t a t i v e d e t e r m i n a t i o n of- g l u t e t h i m i d e by NMR b o t h i n powder and t a b l e t f o r m u l a t i o n s . The method o f f e r s a r a p i d , a c c u r a t e p r o c e d u r e f o r a n a l y s i s of t h e d r u g . F u r t h e r m o r e , i t p r o v i d e s a confirmatory i d e n t i f i c a t i o n f o r glutethimide.
6.9

180

GLUTETHIMIDE

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18,

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98. 99.
100.

F.L.

F r i c k e , J . A s s o c . O f f . Anal. C h e m . , 55, 1162 (1972). K . C o h e n , R . T . Koda, J . C h e m . E d u c . , 51, L 133 (1974).

101. 102. 103. 104. 105. 106. 107. 108. 109.

110 *
E.

111.
112. 113. 114. 115. 116. 117. 118. 119. 120.

B r o c h m a n n - H a n s s e n and A. B a e r h e i m Svendsen, J . Pharm. S c i . , 51, 318 (1962). B . P . K o r z u n , S.M. B r o d y , P X . Keegan, R.C. Luders, and C.R. R e h m , J. L a b . C l i n . Med., - 333 (1966). 68, I. S u n s h i n e , R. Maes and R. Feracci, C l i n . C h e m . , 2, 595 (1968). P . G r i e v e s o n and J . S . G o r d o n , J . C h r o m a t o g r . , 44, 279 (1969). J . M a c G G , C l i n . C h e m . , 17,587 (1971). H . E . S i n e , M . J . McKenna, T.A. R e l e n t and M.H. Murray, C l i n . C h e m . , 16, 587 (1970). L.R. G o l d b a u m and J.J. D o m G s k i , J. F o r e n s i c , S c i . , 11, 233 (1966). A. W i n s t e n , and D. B r o d y , C l i n . C h e m . , 14, 589 (1967). K.D. P a r k e r , J . A . W r i g h t , A . F . H a l p e r n , and C.H. H i n e , J . Forensic. S c i . S O C . , 125, (1968). W.C. G r i f f i t h s , S.K. O l e k s y k , I . D i a m o n d , C l i n . B i o c h e m . , 6, 124 (1973). J . H . Kaufman, Am, J . Med. T e c h n o l . , 39,

A. P r e m e l - C a b i c ,
951 (1973).

338 (1973). R . J . Flagagan , G . W i t h e r s , J. C l i n . P a t h o l , 25, 899 (1972).

P.

Allain, Therapie,

28,

R.G. C o o p e r , M . S . G r e a v e s , G . O w e n , C l i n . Che . - m-18, 1343 (1972). E . Watson, S . M . K a l m a n , C l i n . C h i m . A c t a ,

K M . Solon, Anal. I n s t r u m . , 185 (1972). C . C a r d i n i , V. Quercia, A. C a l o , B o l l .

38, 33 (1972).

10,

186

GLUTETHIMIOE

Chim. Farm., - 300 (1968). 107, 121. M. Gold, E. Tassoni, M. Etzl, Clin. Chem.,
122.
123. 124. .

M Gold, E. .

Tassoni, M. Etzl and G. Mathew, Clin. Chem., 20, 195 (1974). K. h v y , T. Schwartz, Clin. Chim. Acta,
54, 19 (1974).

19, 158 (1973).

F Hoffmann, M. Buchner , and W. Hebecker , Pharmazie 29, 208 (1974). 125. J.C. Valentour, W.W. Harold, A.B. Stavitsky, G. Kananen, I. Sunshine, Clin. Chem. 43, Acta - 65 (1973). 1 126. H.Y. Aboul-Enein, Anal. Chim. Acta, 73,
399 (1974).

Acknowledgment The author expresses appreciation to Miss Marie Belmonte for typing the manuscript and Mr. S. Kobrin for his technical assistance in photographing the figures in this profile.

187

LEVODOPA

Ralph Gomez, Robert B. Hagei, and Edward A . MacMulian

RALPH GOMEZ eta/.

INDEX

Analytical P r o f i l e

Levodopa

1.

Description 1.1 Name, Formula, Mol ecul a r Weight 1.2 Appearance, Color, Odor Physical P r o p e r t i e s 2.1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 U l t r a v i o l e t Spectrum 2.4 Fluorescence Spectrum 2.5 Mass Spectrum 2.6 Optical Rotation 2.7 M e l t i n g Range 2.8 D i f f e r e n t i a l Scanning C a l o r i m e t r y 2.9 Thermogravi m e t r i c A n a l y s i s 2.10 S o l u b i l i t y 2.11 C r y s t a l P r o p e r t i e s 2.111 X-Ray D i f f r a c t i o n 2.1 12 C r y s t a l S t r u c t u r e 2.12 D i s s o c i a t i o n Constant Synthesis Separation o f Racemates Stabi 1 it y and Degradation Drug M e t a b o l i c Products Methods o f A n a l y s i s E 1ernent a 1- An a 1y s is 7.1 Phase S o l u b i l i t y A n a l y s i s 7.2 Chromatographic Methods 7.3 7.31 Thin-Layer Chromatography 7.32 Paper Chromatography 7.33 Gas-Liquid Chromatography 7.34 Column Chromatography 7.4 D i r e c t Spectrophotometric Analysis Col o r i met ri c Ana 1y s is 7.5 Non-Aqueous T i t r a t i on 7.6 Determination o f D-Dopa 7.7
190

2.

3.

4. 5.
6.

7.

LEVODOPA

8. 9.

Acknow 1 edgements References

191

RALPH GOMEZ et al.

1.

Descri p t i on 1.1 Name, Formula, Molecular Wei q h t Levodopa i s (-)-3-(3,4-Dihydroxyphenyl)-L-

alanine.

HO b C H 2 C H C O O H I
NH2

1.2

Appearance, Color, Odor

Levodopa i s an odorless w h i t e t o o f f - w h i t e c r y s t a l 1ine powder. 2. Physical P r o p e r t i e s 2.1 I n f r a r e d Spectrum

The i n f r a r e d spectrum o f levodopa is shown i n The spectrum was recorded on a PerkinFigure 1 ( 1 ) Elmer Model 621 G r a t i n g I n f r a r e d Spectrophotometer and was measured i n a K B r p e l l e t which contained 1 mg o f levodopa i n 300 mg o f KBr.

The f o l 1owi ng a b s o r p t i ons have been assigned f o r Figure 1 : a. OH s t r e t c h i n g (bonded): 3375 cm'l, 3210 cm-l b. NH +: 3070 cm-1, 2700-2300 cm-1 (broad) c. CO&: 1656 cm-1, 1569 cm-1 d. Aromatic CH o u t o f plane bending o f two a d j a c e n t 821 cm-1, 816 cm-1 free H's: 2.2 Nuclear Magnetic Resonance Spectrum (NMR)

The NMR spectrum o f levodopa, recorded on a JEOL C60HL spectrometer, i s shown i n F i g u r e 2 ( 2 ) . The spectrum was recorded u s i n g a s o l u t i o n o f 60 mg o f levodopa/0.4 m l D20 t 0.1 m l DCL. The s p e c t r a l assignments a r e l i s t e d i n Table I .
192

Id
0 -0

>
1

cc
0

5
c, V
Q)

Ln
-0 W

L + ET

Id

t-

lo-

rD

\,

co

0
(0

0 d-

8
3 3 N V l l l W S N V H l Yo

193

FIGURE 2 NMR Spectrum o f Levodopa

LEVODOPA

TABLE I
NMR Spectral Assignments f o r Levodopa

Proton

Chemical S h i f t (6) 3.22 4.44 6.80 6.92 7.05 ppm ppm ppm ppm ppm

Spectral S t r u c t u r e (J i n H z l Mu1 t i p l e t (7.5) T r i p le t Doublet (2 sets; 2.0, Doublet Doublet

8.0)

The remaining protons exchange i n t h e deuterated solvent used. 2.3 U l t r a v i o l e t Spectrum

The u l t r a v i o l e t spectrum o f levodopa ( 4 mg o f levodopa/100 m l o f 0.1N h y d r o c h l o r i c a c i d ) i n t h e r e g i o n o f 230 t o 370 nm e x h i b i t s one maximum a t 280 nm ( E = 2.8 x 103) and one minimum a t 250 nm. The spectrum i s shown i n Figure 3 ( 3 ) . 2.4 F1uorescence Spectrum

The e x c i t a t i o n and emission spectra o f levodopa are presented i n Figure 4 ( 4 ) . The sample was dissolved i n methanol a t a concentration o f 1.43 mg o f levodopa/ml and t h e spectra were recorded using a Farrand MK-1 recording spectrofluorometer. Levodopa e x h i b i t s e x c i t a t i o n maxima a t 236 nm and 286 nm and emission peaks a t 320 nm and 620 nm. 2.5 Mass Spectrum

The low r e s o l u t i o n mass spectrum o f levodopa i s shown i n Figure 5 ( 5 ) . The spectrum was obtained using a
195

RALPH GOMEZ etal.

FIGURE 3

U1t r a v i ol e t Spectrum o f Levodopa

0.6

0.f

0.4

2 a

O.!

8 m
02 .

0. I

C
I

250

300 NANOMETERS

350

196

-8 W
a
P
-0

-8
-8 In
0

>
I

cc
0

c,
V

5 L
-$

-8, K

a
P

-s.,

I O U

Ln

a
V

z a
0 2

a
V
v)

L
0
3
F

-8 m

LL

-8 m

-s:

(u

197

FIGURE 5 Mass Spectrum o f Levodopa

10 0 90
80

A l I S N 3 1 N I 3hllWl3tl

3 I 5

70

60 50

198

2 40 I a
I W

30

20
1 0 0

50

100

150

200

m/e

LEVOOOPA

Varian MAT spectrometer w i t h an ionizing voltage of 70 eV, which was interfaced w i t h a Varian data system 6201. The data system accepts the output of the spectrometer, calculates the masses, compares the i n t e n s i t i e s t o the base peak and plots this information as a s e r i e s of l i n e s whose heights are proportional t o the i n t e n s i t i e s . The molecular ion f o r levodopa was measured a t m/e = 197. Other c h a r a c t e r i s t i c masses were observed a t m/e = 179 corresponding t o the loss of H20 from the molecular ion, m/e = 152 which corresponds t o the loss of COOH from the parent mass, and m/e = 123, the base peak, which i s the 3,4 dihydrovphenyl moiety. A h i g h resolution scan confirmed the r e s u l t s o f the low resolution spectrum. Table I1 l i s t s the elemental compositions f o r the ions as determined by h i g h resolution mass spectrometry.

TABLE I 1
High Resolution Mass Spectrum of Levodopa

Found Mass 197.0678 179.0540 177.01 37 175.9964 165.0149 161.0472 152.0722 151.0633 139.0207 136.0537 134.0612 132.0446 1 30.0032 127.01 62 123.0415 2.6

Calcd. Mass

C -

H 11 9 5 2 5 7 10 9 5 8 10

N 1 1 0 1 0 1 1 1 1 0 0 0 0 0 0

0 4 3 4

1 79.0583 177.01 89 1 75.9984

165.01 88 161.0477 152.071 3 151.0634 139.0270 136.0525 134.0580 132.0423 1 30.0055 1 27.01 84 123.0447 Optical Rotation

197.0689

9 9 9 8 8 9 8 8 6 8 5 5 8 9 7

2 3 7

The s p e c i f i c rotation of an aluminum complex of levodopa i n an acetate buffer a t 25OC i s plotted versus
199

RALPH GOMEZ et a/.

wavelength i n Figure 6 ( 6 ) . The s p e c i f i c rotation observed a t 589 nm was approximately -41" while t h e rotation a t 365 nm was approximately -122". In 0.1N hydroc h l o r i c acid the s p e c i f i c rotation of levodopa a t 589 nm has been reported t o be -11" ( 7 ) . Chafetz and Chen (8) have reported the s p e c i f i c rotation of an acidified solution of levodopa containing methenamine t o be approximately -165" a t 589 nm. 2.7 (9). Differential Scanning Calorimetry An endotherm was obtained i n the 290"-300C region where me1 ti ng , accompanied by sample decomposi t i on, occurred. The temperatures observed f o r the decomposition t r a n s i t i o n s a r e dependent on instrumental conditions as well as sample size and cannot be considered c h a r a c t e r i s t i c f o r the compound (10).
2.8

Melting Range Levodopa me1 t s w i t h decomposition above 270C

Thermogravimetric Analysis (TGA) A TGA scan showed no loss o f weight as the temperature increased from 30" t o 270C a t a r a t e of 1O"C/ minute (10). Solubility The approximate s o l u b i l i t y data obtained f o r a sample of levodopa a t 25C i s l i s t e d i n Table I11 (11). The equilibration time was 20 hours f o r each system. TABLE I11
Sol ubi 1i t y Data f o r Levodopa

2.9

2.10

Sol vent Water 95% Ethanol 3 Alcohol A

200

Sol ubi 1i t y (mg/ml ) 3.0

0.3

0.15

FIGURE 6

Specific Rotati on Versus Wavelength for Levodopa

-50"

z 2
I201

2 0
a 0
LL

2 -100" a
v)

NOtlVlOtl 3M133dS

-150"

300

400 500 WAVELENGTH IN NANOMETERS

600

RALPH GOMEZ eta/.

TABLE I11 (cont.)

Sol ubi 1i t y Data f o r Levodopa

So 1 vent

Sol ubi 1 i t y (mg/ml ) 0.1 <0.01 0.1 <o. 01 <o * 01 <o. 01 <0.01 0.2 <0.01 0.03 0.07

M t h a no1 e 2-Propanol Chloroform D i ethyl Ether Petroleum Ether (3O"-6O0C) Benzene Dimethylacetami de Propylene Glycol Benzyl A1 coho1 Acetone Acetoni tri 1 e 2.11

Crystal Properties 2.111 X-Ray Diffraction The X-ray powder d i f f r a c t i o n data f o r levodopa a r e presented i n Table IV (12); instrumental conditions are gi ven be1 ow. Instrument and Operating Conditions GE Model XRD-6 Spectrogoniometer e I n s t ru m n t Generator 50 KV, 12.5 mA Copper (Cu Ka = 1.5418 A) Tube Target Optics 0.1" Detector s l i t M.R. S o l l e r s l i t 3" Beam s l i t 0.0007" Ni F i l t e r 4" Take o f f angle Scan a t 0.2" 20 per minute Goni ometer Amplifier gain-16 coarse; 8.7 f i n e . Detector Sealed proportional counter t u b e and DC voltage a t plateau. Pulse height selection El 5 v o l t s , Eu out. Rate meter T.C. 4, 2000 CIS full scale.
0

202

LEVODOPA

Recorder Samples

Chart speed 1 "/5 minutes. Prepared by g r i n d i n g a t room temperature. TABLE I V Levodopa Powder D i f f r a c t i o n Data

20 6.54 13.08 14.75 15.31 16.85 17.91 18.45 19.75 21.21 22.38 22.73 23.05 23.85 24.91 25.86 26.35 26.92 28.57 29.61 31.25 33.19 33.64 34.31 35.43 36.15 36.28 37.25 37.74 38.34 39.35 40.04

d (!)* 13.52 6.77 6.01 5.79 5.26 4.95 4.81 4.50 4.19 3.97 3.91 3.86 3.73 3.58 3.45 3.38 3.31 3.13 3.02 2.86 2.70 2.66 2.61 2.53 2.49 2.48 2.41 2.38 2.35 2.29 2.25
203

I/Io**
4 2 4 2 20 4 53 14 100 21 42 38 3 60 55 6 28 10 9 21 8 12 6 5 6 21 9 25 8 5 10

RALPH GOMEZ er a/.

TABLE IV (cont.)

Levodopa Powder Diffraction Data

0

20 -

d (A)*

40.80 41.24 41.82

*d

2.21 2.19 2.16

I/Io** 6 8 8

rn (interplanar distance)
percent r e l a t i v e intensity (based on maximum intensity o f 1 .OO) Crystal Structure Levodopa has been observed t o e x i s t as plates. Rapid recrystallization from water a t ures w i t h o u t agitation produced the needlehabit. The needles were found t o exist i n two forms (13).

n h

**I/Io

112

needles and low tempera 1i ke crysta crystalline

Mostad, e t a l . (14) have determined the structures of two monoclinic crystal forms of levodopa. A s t a b l e form was produced by the slow diffusion of absolute alcohol i n t o a half-saturated solution of levodopa in fgrmic acid, The c e l l dimensi ns are as follows: a = 13.629 A; b = 5.308 8; c = 6.049 ; and B = 97.53'.

A l e s s s t a b l e form of levodopa was obtained as t h i n needles by using ethyl ether as a precipitating agent. This form i s n o t s t a b l e when separated I t s cell dimensions are: from the p t h e r liquo a = 16.9 A; b = 5.88 c = 9.0 8; and B = 99'.

8;.

Additional data on crystal structures have been reported (15, 16). Dissociation Constants The dissociation constants f o r levodopa have been determined t i trimetri cal ly and are reported as fol lows (1 7) :

2.12

204

LEVODOPA

iH2

NHZ

3. Synthesis Yamada, e t a l . (18) report the synthesis of levodopa starting with three different compounds: 3-(3,4-methylenedioxypheny1)-L-alanine ( I ) ; N-acetyl-3-(3,4-methylenedioxypheny1)-L-alanine ( 1 1 ) ; or N-acetyl-3-(3,4-methylenedioxypheny1)-L-alanine Z-menthyl ester (111). The s t a r t i n g materi a1 , red phosphorous and a mixture of HI and acetic anhydride are refluxed for several hours t o give (-)-3-(3,4 di hydroxyphenyl )-L-a1 ani ne. The reaction scheme i s shown in Figure 7.

4. Separation of Racemates
Methods described in the l i t e r a t u r e for the separation of DL-3-( 3,4-dihydroxyphenyl )alanine into i t s opti cal isomers general ly i nvol ve the resol uti on of an i ntermedi a t e in the synthesis. The optically pure intermediate i s then reacted further t o give pure levodopa or D-Dopa (19-24).
A procedure has been described which was used t o resolve DL-3-(3,4-dihydroxyphenyl)alanine into i t s optically active antipodes ( 2 5 ) . A supersaturated solution of the racemic mixture was seeded with crystals of one pure enantiomorph which crystallized pure enantiomorph. The mother liquor was transferred t o a separatory vessel, heated and treated with fresh finely ground racemic mixture; the amount added was approximately twice the amount of pure enantiomorph obtained in the f i r s t recrystallized stage. A preferential solution of the deficient enantiomorph occurred which l e f t the antipode behind as a crystalline product. The method
205

FIGURE 7
Synthesis o f Levodopa

G !
I
I u 0
U J

CH2-

CH-COOH ?HZ

t
CH2 -CH-COOH

NH- COCH3

II

LEVODOPA

d n
0

CH2 - CH -COO

LEVODOPA

i s applicable only when the optically active antipodes form a racemic mixture and. not a compound.
Stabi 1 i t y and Degradati on Levodopa, i n the presence of moisture, i s rapidly oxidized by atmospheric oxygen and turns green (9). The oxidation of levodopa i n basic solution r e s u l t s i n the formati on of me1 ani n and re1 ated intermediates (26). The s t a b i l i t y of levodopa i n the bulk form was studied under conditions of elevated temperature by storing samples a t 105C f o r varying periods of time. The samples were examined f o r evidence of degradation by measuring the color o f a 10%w/v solution i n 10% hydrochloric acid and by t h i n layer chromatography. The color of the solutions showed some darkening a f t e r 24 hours, and increased w i t h increasing heating time. However, no degradation was detected i n these solutions by thin-layer chromatography (27).
5.

Drug Metabolic Products The major metabolites of levodopa i n humans have been reported t o be 3-methoyy-4-hydroxyphenylacetic acid, 3,4d i hydroxyphenylacetic acid, dopamine, and 3-methoxytyrosine (28-33). Barbeau (34) has reported t h a t patients given levodopa show an increase i n the excretion of methylated derivatives such as o-methyldopa, 3-methoxytyramine and 5hydroxyi ndol eaceti c acid. Wada and Fel lman (35) have presented evidence t h a t 2,4,5-trihydroxyphenylacetic acid i s a metabol i c product of 3,4-di hydroxyphenylpyruvate, a levodopa metabolite. Gjessing and Borud (36) and O'Gorman, e t a l . (30) have also studied the metabolic f a t e o f levodopa i n humans, Figure 8 shows the metabolism of levodopa as described by them. The chemical names o f the compounds i n Figure 8 are l i s t e d i n Table V.

6.

TABLE V

Metabolites of Levodopa Referred t o i n Figure 8

I,
11. 111.

IV.
V.

3,4-dihydroxyphenylalanine 3,4-dihydroxyphenylethylarnine 3,4-dihydroxyphenylpyruvic acid 3,4-di hydroyyphenyll a c t i c acid 3,4-dihydroxyphenylacetic acid

207

RALPH GOMEZ e t a / .

FIGURE 8

Metabolism o f Levodopa

208

LEVODOPA

TABLE

V (cont.)

M e t a b o l i t e s o f Levodopa R e f e r r e d t o i n F i g u r e 8

VI. VI I. VI I I IX. X. XI. XI I. XIII. XI V .

7. 7.1

3,4-dihydroxyphenylethanol
3,4-di hydroyyphenyl a c e t a l dehyde 3-methoxy-4-hydroxyphenyl a1 a n i ne 3-methoxy-4-hydroxyphenyl p y r u v i c a c i d 3-methoxy-4-hydroxyphenyll a c t i c a c i d 3-methoxy-4-hydroxyphenyl a c e t i c a c i d 3-methoxy-4-hydroxyphenylethanol

3-methoxy-4-hydroxyphenylacetaldehyde

3-methoxy- 4- hyd roxy pheny 1e t hy 1ami ne

Methods o f A n a l y s i s Elemental A n a l y s i s

A t y p i c a l elemental a n a l y s i s o f a sample o f levodopa i s presented i n Table V I (37).

TABLE

VI

Elemental A n a l y s i s o f Levodopa Element

C

% Theory
54.82 5.62 7.10 32.46

% Found

H N 0
7.2

54.82 5.74 7.10 32.34 (by d i f f e r e n c e )

Phase S o l u b i l i t y A n a l y s i s Phase s o l u b i 1it y a n a l y s i s has been c a r r i e d o u t f o r levodopa u s i n g 1 :1 methanol :water as t h e s o l v e n t . An example i s presented i n F i g u r e 9 along w i t h t h e c o n d i t i o n s under which t h e a n a l y s i s was performed ( 3 8 ) . 7.3 Chromatographic Methods 7.31 Thin-Layer Chromatography (TLC)

The f o l l o w i n g TLC procedure appears i n USP X I X and i s u s e f u l f o r s e p a r a t i n g levodopa from 3methoxytyrosine and 3,4,6-trihydroxyphenylalanine ( 6 ) . An A v i c e l p l a t e , p r e v i o u s l y predeveloped i n t h e d e v e l o p i n g
209

2.5 -

2 0.
1.5:

- - -

LEVOWPA PHASE SOLUBILITYANALYSIS

LEVODOPA

solvent, is spotted w i t h 0.1 mg of levodopa from 9:l acetone:4% hydrochloric acid. The p l a t e i s subjected t o ascending chromatography i n n-butanol :gl aci a1 a c e t i c acid: double d i s t i l l e d water:methanol (1 50: 75:75:15). After development of a t l e a s t 15 cm, t h e p l a t e i s a i r dried and sprayed w i t h 2:l 10%w/v f e r r i c chloride:5% w/v potassium ferricyanide. The approximate Rf values a r e summarized i n Table VII.
TABLE VII

Summary of TLC Data Compound 3,4,6 Trihydroyyphenylalanine Levodopa 3-Methoxytyrosi ne Approximate Rf 0.25 0.4 0.5

Additional TLC separations of levodopa from related compounds o r metabolites have been reported (39-42). A summary of these methods i s found i n Table VIII.
TABLE VIII

Thin-Layer Chromatographic Systems f o r Levodopa Rf Value Adsorbent Solvent of Levodopa Reference Cellulose MN 300 Awl Alcoho1:Formic 0.50 39 Acid: Water (40 :40 :
20 1

Cellulose MN 300 n-Propano :Water (65:25) Cellulose MN 300 n-Heptane Carbon Tetrachloride: Methanol ( 70 :40 :30) Polyami de Isobutanol :G1 aci a1 Acetic Acid:Cyclohexane (80:7:10)

0.39
0.02

39 39

0.18

40

211

RALPH GOMEZ et a/.

TABLE V I I I ( c o n t . ) Thin-Layer Chromatographi C Systems f o r Levodopa Adsorbent Cellulose Sol vent n-Butanol :61 a c i a1 A c e t i c A c i d :Water ( 5 : l :3) E t h y l Acetate: G1a c i a1 A c e t i c A c i d :Water (5:1.5:3) E t h y l Acetate:nButanol :G1 a c i a1 A c e t i c Acid: Water (3:2:1:3) Methyl E t h y l Ketone :Acetone: 2.5N G l a c i a l A c e t i c A c i d (40:20:20) 7.32 Paper Chromatography
Rf Value of Levodopa

Reference 41

0.28

C e l l u l ose

0.23

41

Cellulose

0.33

41

Cell ulose

0.16

42

PaDer chromatowaohi c s e o a r a t i ons o f levodopa have been used f o r i d e n t i f i c a t i o n as w e l l as f o r i t s s e p a r a t i o n from r e l a t e d compounds (41, 43, 44, 4 5 ) . Table I X i s a summary o f some o f t h e paper chromatographic methods employed f o r levodopa. Whatman number 1 paper was used i n each case. TABLE I X Paper Chromatographic Systems f o r Levodopa Sol vent n-Butanol :Gl a c i a1 A c e t i c Acid:Water (5:1:3) Development Descending
Rf Value o f Levodopa

Reference 41

0.27

212

LEVODOPA

TABLE IX (cont.) Paper Chromatographic Systems f o r Levodopa Sol vent Ethyl Acetate: Glacial Acetic Acid: Water (5:1.5:3) Methanol :Water: Quinoline (160: 40 :8) n-Butanol :Pyridine: 0.2N Sodium Acetate (1:l:l) n-Butanol :Pyridine: 1M Sodium Acetate (1 :1:2)
Rf Value Development o f Levodopa Des cendi n g 0.21

Reference 41

Des cendi n g

0.42

43

Descending

0.47

44

Descending

0.68

44

n-Butanol :Pyridine Descending (1 :1 ) saturated w i t h 1M Sodium Acetate n-Butanol :Pyridine: Water (1 :1:1) Benzene :Methanol : n-Butanol :Pyridine: Water (1 :2: 1 :1:1) Methanol :Water: Pyridine (20:5:1) Methanol :Water: Pyri d i ne (20:5: 1 ) Descending Descending

0.40

44

0.54 0.36

44 44

Descend i n g Ascending

0.46 0.37

44 44

213

RALPH GOMEZ etal.

TABLE IX (cont.)
Paper Chromatographic Systems f o r Levodopa

Sol vent
To1uene:Ethyl Acetate :Py ri d i ne : Water :Methanol (1:l:l:l:l) To1uene:Ethyl Acetate:Methanol : Water (1 :1:1 : l ) Aqueous Phase Water saturated w i t h methyl ethyl ketone Water saturated w i t h methyl ethyl ketone n-Butanol :Ethanol : Water ( 2 : l : l )

Rf Value Development Descending

o f Levodopa

Reference 44

0.59

Descending

0.62

44

Descending

0.05

44

Ascending

0.02

44

Descend

0.23 0.3

44 44

Methanol :n-Butanol : Descend Benzene: Water ( 2 :1 : 1:1) Methanol :n-Butanol : Descending Benzene:Water (4:3: 2:l) Methanol :n-Butanol : Ascending Benzene:Water (4: 3: 2:l)

0.2

44

0.20

44

To1 uene: Ethyl Acetate: Methanol : 0.1N HC1 ( 1 : l : l : l )

Descending

0.75

44

214

LEVODOPA

TABLE IX (cont.) Paper Chromatographic Systems for Levodopa Sol vent Methanol :Awl A coho1 :Benzene : 1 2N H 1 (37:17.5: C 35: 12.5) Development Descending
Rf Value of Levodopa Reference

0.51

44

n-Butanol saturated Descending w i t h 1N H 1 C n-Butanol saturated Ascending w i t h 1 HC1 N

$ e r t . -Butanol : Descending Acetone: Formic Acid: Water (160 :160: 1 :39)

0.19 0.18

44 44 44

0.08

t e r t . -8utanol: Ascending Acetone: Formic Acid: Water (160:160:1:39)

0.06

44

Ch1oroform:Glacial Acet i c Acid: Water (2:l:l) n-Butanol :G1 aci a1 Acetic Acid:Water (4:l:l)
t e r t . -Butanol : Acetone:Propionic Acid:Water (160: 160: 1 :39)

Descending

0.80

44

Descending

0.21

44

Descending

0.06

44

Benzene: Propionic Acid:Water ( 2 : l : l )

Descending

0.83

44

215

RALPH GOMEZ et al.

TABLE I X ( c o n t . ) Paper Chromatographic Systems f o r Levodopa Sol vent Development Ascending Rf Value o f Levodopa 0.42 Reference 45

t e r t . -Butanol :
Methyl E t h y l Ketone :Formi c Aci d :Water (40:30: 15: 15) n-Butanol : G a c i a1 A c e t i c 1 Acid: Water (50: 25 :25) Phenol-Water (Lower Phase) Water:sec. Butanol :t e r t . Butanol (48.4:43: 8.6 ) Upper Phase

Ascending

0.44

45

Ascending Ascending

0.38 0.33

45 45

2-propanol :Water: Concentrated HC1 (65:18.4: 16.6)

Ascending

0.40

45

see. -Butanol :
Water-Upper Phase Ethyl Acetate: Formi c Acid :Water ( 70 :20 :10) E t h y l Acetate: Water:Formic Acid (60: 35 :5)-Upper Phase

Ascending Ascending

0.17 0.30

45 45

Ascending

0.00

45

216

LEVODOPA

TABLE IX (cont.) Paper Chromatographic Systems f o r Levodopa Sol vent

t e r t . -Butanol :

Development

Rf Value of Levodopa

Reference 45

A cendi n g s

0.10

Methyl Ethyl Ketone: Water: Formic Acid: (44: 44: 11 :

0.26)

Chrom tography A gas-1 i q u i d chromatographic procedure f o r the determination of levodopa purity and detection of possible impurities has been developed (46).
7.33

- s-Liquid G

Gas-1 i q u i d chromatography permits the q u a l i t a t i v e identification of the impurities by r e l a t i n g the retention times r e l a t i v e t o an added internal standard. Deri vati z a t i on was carried out by the reaction o f 1evodopa and/or proposed impurities w i t h bis-trimethylsilylacetamide reagent (BSA). Because levodopa is v i r t u a l l y insoluble i n most non-acidic o r non-aqueous solvents, the conversion t o the trimethylsi lyl (TMS) derivative was accomplished without the use o f a solvent. Direct addition of the BSA reagent t o the compound followed by moderate heating was s u f f i c i e n t for complete derivative formati on. The TMS deri vati ves were successful ly chromatographed on a column packed w i t h 20% SE-30 on Gas Chrom Q under isothermal conditions and detected using a thermal conductivity detector. In order t o compensate for column c h a r a c t e r i s t i c s , instrumental variati ons , and sample introduction technique, an internal standard, docosane, was employed for r e l a t i v e retention time data and response data. Assay values calculated by area normalization yielded a precision o f f 0.5% f o r f i v e degrees of freedom.

217

RALPH GOMEZ et al.

An absolute determination u s i n g an internal standard yielded a precision of 2 1.2% f o r f i v e degrees of freedom.

Gehrke and S t a l l i n g (47) reported the quantitative gas-liquid chromatographic separation of the N-trifluoroacetyl-n-butyl e s t e r of levodopa from 14 other non-protei n amino acids The temperature programmed chromatography was carried out on columns of 60/80 mesh acid-washed Chromosorb W coated w i t h 5% w/w DC-550. + Yields of 98 - 3% were obtained.

Atkinson, Brown and Gelby (48) separated the trimethylsilyl derivatives of levodopa, tyrosine, phenylalanine, N-acetyl tyramine, tyramine, N-acetyldopamine and dopamine isothermally on various columns. B using the y column i n conjunction w i t h a flame ionization detector, levodopa was detected a t levels as low as 1-2 pg. Col u r n Chromatography A part of a study of the biosynthesis and s metabolism of catecholamines, Masuoka, e t a l . (49) developed a separation of the constituents of tissue extracts by column chromatography. The e x t r a c t s were separated i n t o three fractions on an alumina column by eluting successively w i t h 0.5M (pH 6.1) ammonium acetate buffer, 0.01M (pH 4.0) ammonium acetate buffer and 2 N a c e t i c acid. The t h i r d fraction was then passed through a Dowex 50 column which separated levodopa from the other constituents. The fractions were monitored by measuring the absorbance a t 279 nm. 7.34 Spiegel and Tonchen (50) described a method f o r the separation of levodopa from catecholamines found i n plasma. The sample was adsorbed on alumina, eluted w i t h 0.1N hydrochloric a c i d , then adsorbed on and eluted from an AG50W-X4 cation-exchange column.

A method f o r the separation of catechol derivatives, including levodopa, from guinea p i g brains by column chromatography wi t h Duo1 i t e C-25 was described by Nakajima (51).

218

LEVODOPA

Rolland, Lasry and Lissitzky (52) separated levodopa from L-tyrosine and other amino acids contained in protein or natural extracts on Dowex 50. The limit of detection of levodopa was reported t o be 50-200~. Direct Spectrophotometric Analysis Levodopa, i n tablets or capsules, can be assayed directly by an ultraviolet absorption procedure, The tablets are finely ground or the contents of the capsules are mixed. A portion of the powder i s weighed and quantitatively diluted w i t h 0.4N hydrochloric acid, The contents are mixed, f i l t e r e d and the absorbance of an appropriately diluted solution i s measured a t 280 nm. The amount of levodopa i n tablets or capsules i s determined by comparing the absorptivity of the sample a t 280 nm with the absorptivity of a solution of levodopa reference sample simi larly prepared and measured ( 6 ) . Colorimetric Analysis An automated method utilizing the Doty reaction has been successfully applied t o the uantitative determination o f levodopa in tablets 753). The method i s specific f o r the catechol confi gurati on and w 11 indicate i any decomposition due t o the oxidation of t h i s moiety. The tablets are dissolved i n 1N sulfuric acid, homogenized w i t h d i s t i l l e d water, and quickly combined w i t h sodium b i s u l f i t e solution t o prevent oxidation. A ferrous c i t r a t e solution i s introduced into the system followed by a strongly basic buffer which produces a stable purple color measureable a t 545 nm. The amount of levodopa i s calculated by comparison with a cal ibration curve prepared from pure levodopa, similarly treated (54). Non-Aqueous Titration A potenti ometri c t i t r a t i on w i t h perch1 oric acid i n glacial acetic acid is the method o f choice t o assay levodopa. The sample i s dissolved in formic acid, glacial acetic acid i s added, and the t i t r a t i o n i s carried o u t with l 0.1N perchloric acid i n glacial acetic acid. Each m of 0.100N perchloric acid i s equivalent t o 19.72 mg of levodopa ( 6 ) .
7.6

7.4

7.5

219

RALPH GOMEZ et a/.

7.7

Determi n a t i on o f G-Dopa

Coppi, V i d i and Bonardi (55) described a method f o r t h e determination o f D-Dopa i n levodopa. The method i s based on t h e q u a n t i t a t i v e r e a c t i o n o f a levodopa decarboxyl ase, present i n a Streptococcus frecaZis suspension, which converts 1evodopa t o dopami ne w i t h o u t a f f e c t i n g D-Dopa. D-Dopa was separated from dopamine by e l u t i n g a s o l u t i o n o f t h e m i x t u r e w i t h 0.05M pH 6.0 phosphate b u f f e r on an Amber1 it e I RC50 ion-exchange c o l urn. The e l u a t e was assayed f l u o r i m e t r i c a l l y f o r D-Dopa according t o Anton and Sayre (56). 8. Acknowledgements The authors wish t o acknowledge t h e assistance o f t h e S c i e n t i f i c L i t e r a t u r e Department and t h e Research Records O f f i c e o f Hoffmann-La Roche Inc. f o r t h e i r 1it e r a t u r e searches.

220

LEVODOPA

9.

References 1. 2. 3. 4. Waysek, E., Hoffmann-La Roche I n c . , Personal Communication. Johnson, J., Hoffmann-La Roche I n c . , Personal Communi c a t i o n . Corrade, C., Hoffmann-La Roche, Inc., Personal Communi c a t i o n . Boatman, J., Hoffmann-La Roche, Inc., Personal Communication. Benz, W., Hoffmann-La Roche Inc., Personal Communication. "The U n i t e d States Pharmacopei a X I X " , pp. 280, 281 (1975). Osborn, S., Hoffmann-La Roche Inc., Personal Communic a t ion. Chafetz, L. and Chen, T.M., J . Pharm. S c i . , 63, 807 (1974). T e r c k Index", E i g h t h Ed., p. 397. Rucki, R., Hoffmann-La Roche Inc., Personal Communication. MacMull an, E. , Hoffmann-La Roche I n c . , Personal Communication. Chiu, A. M., Hoffmann-La Roche I n c . , Personal Communication. Sci are1 10 , J and Sheridan, J , Hoffmann-La Roche I n c . , Personal Communi c a t i o n . Mostad, A,, Ottersen, T. and Romming, Chr., Acta Chem. Scand. , 24, 1864 (1970). Jandacek, R. J . and E a r l e , K. M., Acta C r y s t . , - 841 (1971). B27, Becker, J. W., Thathachari , Y. T. and Simpson, P. G., Biochem. Biophys. Res. C o m n . , 41, 444 (1970). Gorton, J. E. and Jameson, R. F., J . Chem. SOC. ( A ) , 2615 (1968). Yamada, S., F u j i i , T. and S h i o i r i , T., Gem. Pharm. BuZZ., 10, 693 (1962). H a r r i n g t o n , C . T . and Randall, S. S., Biochem. J . , 25, 1028 (1931). Vogler, K. and Baumgartner, H., HeZv. a i m . Acta, 35, 1776 (1952). Emada, S., F u j i i , T. and S h i o r i , T., Chern. Pham. BUZZ., 680 (1962).

5.
6.

7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

20.
21.

10,

221

RALPH GOMEZ er a/.

22 * 23. 24. 25. 26.

27.
28.

29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39 40.

Berenyi, E., Budar, Z . , Pallos, L., Magdanyi, L. and Benko, P . , Hung. Teljes 858, 28 Aug. 1970, Appl. 28 Oct. 1969; CA:74:64389u. Berenyi, E. Budai, Z. Pallos, L . , Benko P. and Magdanyi, L . , Hung. Teljes 859, 28 Aug. 1970, Appl 28 Oct 1969; CA: 64390n. Hever, I. and Villanyi, E., Hung. Teljes 383, 6 June 1970, Appl. 16 Nov. 1968; CA: 64391~. Merck and Co., Inc., Neth. Appl. 6,514, 950, May 18, 1966. Lerner, A. 6. and Fitzpatrick, T. B . , Physical Rev,, 30, 91 (1950). Burke, C. M . , Sokoloff, H . K. and Heveran, J . E . , Hoffmann-La Roche Inc., Personal Communication. Abrams, W. B . , Spiegel, H . E., Leon, A. S . , Pocelinko, R. M., and Solomon, H. M., Am. Med. Assoc. 119th Ann. Conv., Chicago, I l l i n o i s , June 21-25, 1970. Bronaugh, R. L . , McMurtry, R. J . , Hoehn, M. M., and Rutledge, C. O . , Pharmacologist, 15, 247 (1973). O'Gorman, L. P . , Borud, 0. Khan, I. A. and Gjessing, L. R., CZin. Chim. Acta, 29, 111 (1 970) Routh, J . I . , Bannow, R. E . , Fincham, R. W . , and S t o l l , J . L . , CZin. Chim., 1 867 (1971). , S i i r t o l a , T. and Hirviaho, L . , Scand. J. CZin. Lab. Invest., 3, ( S u p p l . 130), 14 (1973). Tyce, G. M., Muentner, M. D. and Owen, C. A., J r . , Mayo CZin. Proc., 45, 645 (1970). Barbeau, A , , and Mzowell, F. H . , "L-Dopa and Parkinsonism," F. A. Davis Co., P h i l a . , Pa., pp. 360-362, 1970. Wada, G. H. and Fel lman, J . H . , Biochemistry, - 5212 (1973). 12, Gjessing, L. R. and Borud, O . , S c m d . J . CZin. Lab. Invest., 17,80 (1965). Scheidl, F . , Hoffmann-La Roche Inc., Personal Communication. Bass, E . , Hoffmann-La Roche Inc., Personal Communication. O i ttmann J., J . Chromatogr., 32, 764 (1968). 134 ( 1969). Sapi r a , J D. , J , hromatogr.

,L T,

222

LEVODOPA

41. 42. 43. 44 * 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56.

Vahidi, A. and Siva Sankar, D. V., 43, 135 (1969). Baumann, P. , Scherer, B. , Kramer, W . and Matussek, N . , J . Chromatogr., 59, 463 (1971). Mattok, G. L. , J . Chromatogr.,T~, 254 (1964). McGeer, E. G. and Clark, W. H. , J . Chromatogr., 14, 107 (1964). --r F i n k , K . , Cline, R. E . and F i n k , R. M., Anal. Cham., 35, 389 (1963). Mahn, F. P . , a n i u s , G . , Venturella, V. S . and Senkowski, B. Z . , Hoffmann-La Roche Inc., Personal Communication. Gehrke, C. W . and S t a l l i n g , D. L . , S e p a r . Sci., - 101 (1967). 2, Atkinson, P. W . , Brown, W . V. and Gilby, A. R . , AnaZ. Biochem., 40, 236 (1971). Matsuoka, D. T . , Drell, W . , Schatt, H. F. , Alcaraz, A. F. and James, E . C . , AnaZ. Biochem., - 426 (1963). 5, Spiegel, H . E . and Tonchen, A. E . , CZin. Chem., 16, 763 (1970). Nakajima, K . , Osaka f/iagaku Igaku Zasshi, 72, 897 (1960). Rolland, M. Lasry, S . and Lissitzky, S . , BUZZ. SOC. Chim. BioZ., 42, 1065 (1960). Doty, J . R., Anal. &em., 20, 1166 (1948). Billmeyer, A . , Geller, M., Venturella, V. and Senkowski , B. , Hoffmann-La Roche Inc. , Personal Communication. Coppi, G., Vidi, A. and Bonardi, G . , J . Pharm. S c i . , 61, 1460 (1972). Anton,T. H. and Sayre, D. F. , J . PharmacoZ. Exp. Ther., 145,326 (1964).
J . Chromatogr.,

223

SODIUM LEVOTHYROXINE

Alex Post and Richard J. Warren

ALEX POST AND RICHARD J. WARREN

CONTENTS

I.

Description

1.1

Nomenclature

1.11
1.12 I . 13

Chemical Names G e n e r i c Names Trade Names

I .2

Formula, M o l e c u l a r Weight, S t r u c t u r e

I .21
1.22

E m p i r i c a l Formula, M o l e c u l a r Weight Structure C o l o r , Odor

I .3
2.

Appearance,

Physical Properties

2. I

Spectra I P r o p e r t i e s 2.11 U l t r a v i o l e t A b s o r p t i o n Spectra

2.12
2.13

I n f r a r e d Spectrum N u c l e a r Magnetic Resonance S p e c t r a 2.131 2. I 3 2 P r o t o n NMR Spectrum Carbon- I 3 NMR Spectrum

2.14

Mass Spectrum Specific Optical Rotation

2. I5 2.2 2.3

Solubility Crystal Properties 2.31 2.32 Crystallinity X-Ray D i f f r a c t i o n 226

SODIUM LEVOTHY ROX IN E

CONTENTS (continued) 2.4 pKa, Ionization Constant 2.5 Melting Range 2.6 Differential Thermal Analysis 2.7 Thermogravimetric Analysis 3. Synthesis 31 . Chemical Synthesis 3.11 L-Thyroxine, Monosodium Salt

3.12 D-Thyroxine, Monosodium Salt 3.2 Nonenzymic Synthesis of L-Thyroxine

3.3

Synthesis of Radiolabeled L-Thyroxine

4. Stability

5. Drug Metabol ism 5.1

5.2

Metabolic Products Biological Half-Life

6. Elemental Analysis 61 .

Determination of Organically Bound Iodine

6.11

Oxygen Flask Combustion

t

lodometric Titration

6.12 Ashing

N-Bromosuccinimide Titration

6.13 Oxygen Flask Combustion + Coulometric Titration

6.14 Specific Ion Electrode

6.2 Determination o f Water 6.21

USP X I X

227

ALEX POST AND RICHARD J. WARREN

CONTENTS ( c o n t i n u e d ) 6.22 6.3 Thermogravimetric Analysis

Chromatographic A n a l y s i s 6.3 I 6.32 6.33 Paper Chromatography T h i n Layer Chromatography Column Chromatography 6.331 6.332 6.333 6.334

Ion Exchange Chromatography

Gel F i l t r a t i o n Chromatography Gas L i q u i d Chromatography H i g h Performance L i q u i d Ckomatography

6.4 6.5 6.6 6.7 6.8 6.9

Neutron A c t i v a t i o n Analysis Po I arograph i c Ana I y s i s K i n e t i c Methods o f A n a l y s i s Double-Isotope D i l u t i o n A n a l y s i s Determination of Stereoisomeric P u r i t y Equilibrium Dialysis

7.
8.

Methods o f A n a l y s i s References

A Compilation

228

SODIUM LEVOTHYROXINE

I.

Description

Sodium l e v o t h y r o x i n e i s a p h y s i o l o g i c a l l y a c t i v e m a t e r l a l b e i n g t h e levo-isomer o f t h y r o x i n e . The d a t a p r e s e n t e d i n t h l s a n a l y t i c a l p r o f i l e , unless otherwise stated, w i l l r e f e r t o t h e levo-isomer. References t o d a t a o b t a i n e d f o r t h e dextro-isomer, sodium d e x t r o t h y r o x i n e , t h e DL-form, o r f o r t h e f r e e amino a c i d of e i t h e r s t e r e o i s o m e r w i l l be so des i gnated. I. I Nomenclature

1.11

Chemical Names

Several chemical names have been used t o d e s c r i b e sodium l e v o t h y r o x i n e and sodium d e x t r o t h y r o x i n e : ( a ) Sodium d e r i v a t i v e o f 3-[4-(4-hydroxy-3,5d i iodophenoxy)-3,5-di iodophenyll-L-a l a n i n e l ( b 1 L-3,3', sa I t, p e n t a h y d r a t e 2 5,5'-Tetra i o d o t h y r o n 1 ne, sod i um

( c ) !3-[(3,5-D i iodo-4-hydroxyphenoxy d i i o d o p h e n y l l - a l a n i n e , sodium s a l t , p e n t a h y d r a t e z ( d ) D-Tyrosine, 0-(4-hydroxy-3,5-di monosod i um sa I t, h y d r a t e d ( e l L-Tyrosine, 0-(4-hydroxy-3,5-di monosodium s a l t , h y d r a t e 4

-3,5-

odophenyll-

3,5-d

i odo-,
iodo-,

iodophenyll-

3,5-d

I . 12 G e n e r i c Names
Sodium d e x t r o t h y r o x i n e (D-T4, Na); sodium l e v o t h y r o x i n e (L-T4, Na); and L - t h y r o x i n e , sodium (L-T4, Na). I . 13 Trade Names

C h o l o x i n , S y n t h r o i d , L e t t e r , C y t o l e n , Levoid, E l t r o x i n , Laevoxin, Levaxin, Oroxine, and S y n t h r o i d Sodium.

1.2

Formula, M o l e c u l a r Weight, S t r u c t u r e

229

ALEX POST AND RICHARD J. WARREN

I .21

E m p i r i c a l Formula, M o l e c u l a r Weight (a) (b) Sodium S a l t , P e n t a h y d r a t e : C15H1014NNa04 5H20 Anhydrous Sodium S a l t :

cl l 01qNNa04 SH

888.96 798.86

(c)

Free A c i d :
15 11 4

776.93

I .22

Structure

I
Na '
-0

O--@-CH

2- YH-COOH

5H20

I '
I.3
Appearance,

NH2

C o l o r , Odor

The sodium s a l t , pentahydrate, i an o d o r l e s s , w h i t e t o p a l e b u f f powder o r c r y s t a l l i n e powder The anhydrous powder I s l i g h t ye1 low t o b u f f c o l o r e d and hygroscopic3.

s.

2.

Physical Properties 2. I S p e c t r a I P r o p e r t i es 2.11 U l t r a v i o l e t Absorption Spectra

Gemml I I s r e p o r t e d X max 325 ( E = 6207) I n 0.4 N KOH and A max 295 ( E = 4160) i n 0.4 N HCI. Evidence has beenp r e s e n t e d t h a t t h e s h l f t I n t h e maxima I s due t o t h e d i s s o c i a t i o n o f t h e p h e n o l i c h y d r o x y l group. Edelhoch6 a l s o r e p o r t e d X max 325 ( E = 6180) i n 0.1 - NaOH. N The u l t r a v i o l e t spectrum o f t h y r o x i n e i n a c i d i f i e d e t h a n o l (pH 2.017 i s shown i n F i g u r e I . A maximum a t 300 nm ( E = 4600) and a s h o u l d e r a t 290 nm a r e bands c h a r a c t e r i s t i c o f t h e c o n j u g a t e d a r o m a t i c system. F i u r e 2 i s t h e u l t r a v i o l e t spectrum i n a l k a l i n e e t h a n o l (pH 13.0) which produces t h e sodium s a l t o f t h e

230

:$
0.8 0.7 0.6

CONC: 2.004 x W5Y SOLVENT: Acidifird Ethrnd (pH 2.0) CELL: 5 an

r u

0.4 0.5

0.3

0.2 0.1

260

300

350

400

Figure 1

13.0)

-u)

-to

(v

260

300

0 - 0

350

400

0 - 0

232

Figure 2
Q)

SOD I UM LEVOTHY ROXl N E

p h e n o l i c h y d r o x y l on t h e a r o m a t i c system. The s p e c t r u m shows t h e expected s h i f t i n maximum t o 328 nm ( E = 6580) w l t h corresoonding increase i n i n t e n s i t y . 2.12 I n f r a r e d SDectrum

F i g u r e 3 i s t h e i n f r a r e d spectrum o f t h y r o x i n e , USP, t a k e n as a m i n e r a l o i l d i s p e r s i o n from 4000-625 c m - l on a Perkin-Elmer Model 457 i n f r a r e d s p e c t r o p h o t m e t e r . The f o l l o w i n g a b s o r p t i o n bands a r e a ~ s i g n e d : ~ Table I I n f r a r e d S p e c t r a l Assignments Wavelength (cm-l) 3600 3500-3200 1610 1415) I245 Assignment OH broad, NH2 + H20

cooR-0-R

The i n f r a r e d spectrum o f L - t h y r o x i ne has been r e p o r t e d by Hagen, e t a1.8 2.13 N u c l e a r Magnetic Resonance S p e c t r a 2.131 P r o t o n NMR Spectrum

The p r o t o n NMR spectrum ( F i g u r e 4 ) was o b t a i n e d I n a DMs0-d~ s o l u t i o n which c o n t a i n e d a b o u t 100 mg t h y r o x i n e / m l and t e t r a m e t h y l s i l a n e as t h e i n t e r n a l r e f e r e n c e . The spectrum was o b t a i n e d on a P e r k i n - E l m e r Model R32 90 MHz s p e c t r o m e t e r . The assignments a r e as f o l l o w s : Table 2 P r o t o n NMR S p e c t r a l Assignments

233

Figure 3

MATERIAL:

INSTRUMENT: P r L R E l m r R32.90 MHz

RUN IN: DMSO-dg

I
k

235
L
I
I I I
I

Figure

ALEX POST AND RICHARD J. WARREN

Table 2 (continued) Proton P o s i t i o n a S i g n a l Appearance Chemical S h i f t (ppm) 3.00 3.51 5.30 7.09 7.83

broad m u l t i p l e t
broad m u l t i p l e t broad, N : t H20 H singlet singlet 2.132 Carbon-13

b
c

d
e

NMR Spectrum

The carbon-13 NMR spectrum o f t h y r o x i n e was t a k e n i n DMSO-d, s o l u t i o n on a V a r i a n CFT-20 s p e c t r o m e t e r . The spectrum i s shown i n F i g u r e 5. The assignments a r e a s f o l lows:? Table 3 Carbon-13

NMR

S p e c t r a l Assignments

I
Carbon-13 P o s i t i o n

COOH

Chemical S h i f t (ppm) 169.30 151.27 149.80 140.90 139.06 125.00 91.50 87.59 54.78 o v e r l a p p e d by s o l v e n t

b c e

d
and

f
9

i
j
2.14

Mass Spectrum The f i e l d d e s o r p t i o n mass spectrum o f

236

MATERIAL Thyroxine INSTRUMENT: Varian CFT 20 RUN IN: DMSOd6

4000

3000

2000

1000

Figure 5

ALEX POST AND RICHARD J. WARREN

9 L-thyroxine was o b t a i n e d on a Varian MAT CH-5 spectrometer. The r e s u l t s a r e presented as a bar graph I n F i g u r e 6. The presence o f a t r a c e o f t r l iodothyronlne i s evidenced by f r a g ments a t m/e 577, 607 and 651. Mass s p e c t r o m e t r i c s t u d i e s o f t h y r o x i n e t r l m e t h y l s i l y l d e r i v a t i v e s have been reported.10
2.15 Specific Optical Rotation

As b o t h t h e lev0 and d e x t r o isomers a r e pharmacologically important substances, t h e i r r e s p e c t i v e s p e c i f i c r o t a t i o n values have been l i s t e d i n several compend i a as c r i t e r i a o f a c c e p t a b i l i t y . The f o l l o w i n g a r e severa I r e p o r t e d va I ues, t o g e t h e r w It h references ( R I ) :

I somer
Lev&
Lev4 Lev&

Concentration 0.66 g 0.66 g 3%

Solvent

*C X - -max 21 546 546

[a]

+ 13.03 g CZH50H

6.07 g 0.5 N NaOH

-3.2 I 1
-3.2

+ 13.03 g CTH50H

6.07 g 0.5 N NaOH

25

0.13 N NaOH i n 2o D 70% CFH5OH I N NaOH : C2H50H Levoa 2.2% 2o D (1-72) I N NaOH : C2H5OH Levoa 3.28% 2, D ( 172) 24 g 0.5 N NaOH Levoa 17,5 D 3.25% t 56 g C2A5OH I N HCI : C2H50H Levoa D 25 5% (172) 0.2 N NaOH I n Lev& D 20 3% 70% & H ~ O H I N HCIC: C ~ H S O H 20 Lev& D 2% ( 1-74] I N NaOH : C2HsOH Lev& 20 D 3% ( 1-72) I N HCI : C2H50H 2o Levoa D 2% ( 174) I N HCI : C2H50H 2o Dextro' D 2% ( 1741 I N NaOH i n Dextrob 25 D 3% C2HsOH aAnhydrous f r e e amino ac i d bAnhydrous sod iurn s a l t

-4.4 1 2 -5.7 1 2 -5.4 1 3 -4.6 1 3 +I5

14

-4.5 14 +I6 t o +20 -5 t o 4 -6 t i 9 . 1 25 -19.2 15

t5 t o

+6

238

Matsrial: Thyroxine
EmitbrWireCumnt: 23mA

win dippad m DMSO sdution

InmUm*n: Vrirn MAT CH-5 O f

A
Q

Soura T a n ~ e m re: 50C u

P.akr>m

El 'r

HI

++'

+ -

239

:l i
10
0

El

Figure 6

ALEX POST AND RICHARD J. WARREN

S p e c i f i c r o t a t i o n s o f L - t h y r o x i n e (C = 29, 0.2 N H C I - e t h a n o l ) a t s e v e r a l wave1 engths and t e m p e r a t u r e s have been r e p o r t e d : 1 5 Temp

Cal
589 nm +19.5 t19. I +18.9 +18.4 578 nm t20.3 +20. I +19.7 +19.3 546 nm +23.3 t23.2 +22.8 +22. I 436 nm +42.2 +42.6 +42.0 +41.5 364 nm +7 I .O +70. I +64.5 +62.7

10 20 30 40

(OC)

Equivalent s p e c i f i c r o t a t i o n values, b u t o f opposite sign, were a I so r e p o r t e d f o r D - t h y r o x i ne.I5 U s i n g t h e above data, F e l d e r , e t a l l 5 o b t a i n e d t h e o p t i c a l r o t a t o r y d i s p e r s i o n c u r v e s and e s t a b l ished t h e L- c o n f i g u r a t i o n o f t h e samp l e o f L - t h y r o x i n e .

2.2

Sol u b i I i t v
Table 4 L-Thyroxine Solubi I i t y So I v e n t
H20

g/lOO

ml

Ref

# 3 1 1 1 1 16

95% e t h a n o l a1 k a l i h y d r o x i d e s chI orofom ethyl ether pH 7.4 b u f f e r

0. 14 0.3, 0.4 so I ub 1 e almost l n s o l u b l e almost i n s o l u b l e 0.022-0.044

E v e r t 1 6 used a phosphate b u f f e r a t pH 7.2-7.8 a t 38OC and a range o f i o n i c s t r e n g t h from 0.032-0.162. He p o s t u l a t e d t h a t t h e i n c r e a s e i n i o n i c s t r e n g t h by t h e a d d i t i o n o f sodium c h l o r i d e produced a change i n t h e i o n i c atmosphere o f t h e c e n t r a l i o n r e s u l t i n g i n a ' s a l t i n g - i n and s a l t i n g - o u t ' e f f e c t . U l t r a v i o l e t a b s o r p t i o n a n a l y s i s was used t o e s t a b l i s h t h e s o l u b i l i t i e s . E v e r t has p r e s e n t e d s o l u b i l i t y c u r v e s which show t h e e f f e c t o f t e m p e r a t u r e (25' and 38OC) and o f i o n i c s t r e n g t h s on t h e s o l u b i l i t y o f L - t h y r o x i n e sodium. S o l u b i l i t y i s s i g n i f i c a n t l y increased above pH 7.4.
240

SODIUM LEVOTHYROXINE

2.3

Crysta I Properties 2.31 Crystallinity

C r y s t a l d a t a o n L - t h y r o x i n e , sodium s a l t , p e n t a h y d r a t e , have been r e p o r t e d by Cody, e t a l .I7 2.32 X-Ray D i f f r a c t i o n

The c r y s t a l and m o l e c u l a r s t r u c t u r e s o f L - t h y r o x i n e h y d r o c h l o r i d e , monohydrate have been determined b y X-ray c r y s t a l lography and r e p o r t e d i n t h e I i t e r a t u r e . 2 8 The c r y s t a l system i s m o n o c l i n i c , and t h e sample c r y s t a l l i z e d i n space group C 2 w i t h a = 17.23, b = 5.14, C = 25. 15 $ = 90.47'; Z = 4.

R;

2.4

pKa,

I o n i z a t i o n Constant

The a p p a r e n t pKa o f t h e phenol i c h y d r o x y l , c a r b o x y l and amino f u n c t i o n s has been r e p o r t e d : Funct i o n ca r b o x y I phenolic hydroxyl amino

pKa
2.2 6.7 10. I

Ref

Ref # 3.832 8.085 9.141

19 5
19

20 20
20

a l n 75% d l m e t h y l s u l f o x i d e - w a t e r and 0.1 M KNO3 T i t r a n t : p o t e n t i o m e t r i c w i t h sodium h y d r o x i d e 2.5 M e l t i n g Range

The fol l o w i n g m e l t i n g ranges have been r e p o r t e d f o r thyroxine:

I somer
L-T4 L-T4 D-T4 L-T4

M e l t i n g Range

(OC)

Reference # 12

233-235 ( d e c m p 1 235-236 (decomp 1 237 ( d e c m p ) 236 ( c o r r )

2
2 8

241

ALEX POST AND RICHARD J. WARREN

The m e l t i n g r a n g e o f L - t h y r o x l ne (SKBF r e f e r e n c e 88-2746-227) employing t h e USP X I X , C l a s s I procedure, was 235-236OC (decomp 1. 21 2.6 D i f f e r e n t i a l Thermal A n a l y s i s

A d i f f e r e n t i a l thermal a n a l y s i s curve o f L-thyroxine (SK&F r e f e r e n c e 88-2746-2271, o b t a i n e d on a DuPont Model 900 D i f f e r e n t i a l Thermal A n a l y z e r from r o o m t e m p e r a t u r e t o 25OOC a t a h e a t i n g r a t e o f 10C p e r m i n u t e under n i t r o g e n sweep, i s shown i n F i g u r e 7. A sharp e x o t h e r m i c change o c c u r r e d from 230-235OC, i n d l c a t l n decomposition w i t h no o b s e r v a b l e p r i o r m e l t i n g (endotherm). Be
2.7 Thermogravlmetrlc Analysis

The t h e r m o g r a v l m e t r l c a n a l y s i s o f L - t h y r o x i n e sodium, pentahydrate, was o b t a i n e d on a DuPont T h e r m o g r a v l m e t r i c A n a l y z e r ( s e e F i g u r e 8).z1 The compound was heated a t a r a t e o f 2OoC p e r m i n u t e under n i t r o g e n sweep t o 475OC. A w e i g h t loss of ~ 9 was observed. As expected, i n c e p t i o n o f r a p i d % decomposition appeared t o o c c u r a t a p p r o x i m a t e l y 20OoC. The r e s u l t o b t a i n e d by t h e loss on d r y i n g p r o c e d u r e o f t h e USP X I X 4 was 8.75%. 3. Synthesis 3.1 Chemical S y n t h e s i s 3. I I L-Thyroxl ne, Monosodi um Sa I t

The s y n t h e t i c r o u t e t o L - t h y r o x i n e , and subseq u e n t l y t o I t s monosodium s a l t , pentahydrate, was d e s c r i b e d by Chalmers, e t a l l 2 and i s p r e s e n t e d i n F i g u r e 9. Evidence was a l s o p r e s e n t e d showing t h e s t e r e o s p e c i f i c i t y o f t h i s s y n t h e s i s . The o v e r a l l y i e l d was 26%.
To a c o o l e d suspension o f L - t y r o s i n e ( 1 ) i n s u l f u r l c a c i d was added n i t r i c a c i d which on n e u t r a l i z a t i o n ( I I ) . An a l k a l i n e s o l u t i o n o f yielded 3,5-dinitro-L-tyrosine ( I I ) a c e t y l a t e d w i t h a c e t i c a n h y d r i d e y i e l d e d t h e amide ( I l l ) . E s t e r i f i c a t i o n o f ( I l l ) t o ( I V ) was e f f e c t e d u s i n g e t h y l a l c o h o l and p - t o l u e n e s u l f o n i c a c i d and t h e n a z e o t r o p i n g t h e w a t e r w i t h c h l o r o f o r m . The d l p h e n y l e t h e r ( V ) was p r e p a r e d from ( I V ) by t r e a t m e n t w i t h p - t o l u e n e s u l f o n y l c h l o r i d e and
242

h)

P 0

50

100

150

200

250

T, %(CORRECTED FOR CHROME1 ALUMEL THERMOCOUPLES)

Figure 7

1
0

50

100

150

200

250

300

I 350

400

I 450

I
500

T. 'C(CORRECTE0 FOR CHROMl AlUMEl THERMOCOUPLES)

Figure 8

t
I
0 0

8 1= o v
;

N r n I I 0 0

I I 0--z

1 1
0-2

I
I 0

IN

I 0

\ I m

."
I 0 0
0

z
I
0

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0

1 1 0--z

1 v=o

8
N

V -2
I

1 1

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245

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I Z
0 0

8
I 1
0-2
\ W

o=o I

I
0 0

0,

6
m
m

I ,
I
I

o
0

I 1
Q,

81
o o=o
I I
0 --z

IN

0, I o

l;9
\'

U .L

a ,

0,
I
0

246

0 0

u 0--z
I 1

r x
0--z
0

I N
I

I
I

cu

I
0

iu
cu

OH Jp q\H

0 I

0 +

(0

247

ALEX POST AND RICHARD J. WARREN

p-methoxyphenol. Reduction o f ( V ) t o t h e d i a m i n e ( V I ) was r e a d i l y o b t a i n e d by t r e a t i n g an a l c o h o l s o l u t i o n o f ( V ) w i t h Raney N i . Replacement o f t h e amino groups was b r o u g h t about v i a t e t r a z o t i s a t i o n and Sandmeyer procedures. T e t r a z o t i s a t i o n was c a r r i e d o u t by t h e slow a d d i t i o n of a s o l u t i o n o f ( V I ) i n a m i x t u r e o f a c e t i c and s u l f u r i c a c i d s t o one o f sodium n i t r i t e i n t h e m i x t u r e o f t h e same a c i d s . The d l a z o nium groups were removed by t h e a d d i t i o n o f a s o l u t i o n o f i o d i n e i n aqueous sodium i o d i d e .

A l I t h e p r o t e c t i v e groups p r e s e n t i n ( V I I ) were removed by t r e a t m e n t w i t h a m i x t u r e o f h y d r o i o d i c a c i d and g l a c i a l a c e t i c a c i d . lodination of (VIII) wlth a s o l u t i o n o f Iodine i n ethylamlne yielded L-thyroxine ( I X ) . The a d d i t i o n o f L - t h y r o x i n e t o a b o i l i n g 2 sodium c a r b o n a t e s o l u t i o n produced t h e d e s i r e d p r o d u c t ( X I .

3.12

D-Thyroxine,

Monosodium S a l t

E l k s and Wal l e r Z 3 prepared t h e D-isomer by ( I I-from Figure 9 ) f i r s t inverting the 3,5-dinitro-L-tyrosine t o t h e D-form ( X I I - F i g u r e 10) by t r e a t m e n t o f ( I 1 ) w l t h n i t r o s y l b r o m l d e which, a f t e r ammonolysis o f ( X I - F i g u r e l o ) , yielded t h e 3,5-dinitro-D-tyrosine (XI I - F i g u r e 10). The D - t h y r o x i n e was t h e n p r e p a r e d I n a s e r i e s of r e a c t i o n s e s s e n t i a l l y e q u i v a l e n t t o t h a t used b y Chalmers, e t a l l 2 [see ( I I I ) - ( X I i n F i g u r e 91. 3.2 Nonenzymic S y n t h e s i s of L-Thyroxine

I n an a t t e m p t t o e x p l a i n t h e b i o s y n t h e s i s o f t h y r o x i n e from 3,5-di i o d o - L - t y r o s l n e , Shi ba, e t a I z 4 proposed a nonenzymic model f o r t h i s pathway ( F i g u r e I I ) . A t n e u t r a l pH and a t room t e m p e r a t u r e and i n t h e presence o f sodium g l y o x y l a t e ( I l l , c u p r i c a c e t a t e , and oxygen, t h e r e a c t i o n proceeded r a p i d l y t h r o u g h a t r a n s a m i n a t i o n v i a a m e t a l c h e l a t e o f t h e S c h i f f base o f d l i o d o t y r o s i n e and g l y o x y l i c a c i d ( I I I ) . O x i d a t i v e coup1 i n g o f 4-hydroxy-3,5d i l o d o p h e n y l p y r u v i c a c i d ( I V ) w i t h t y r o s i n e y i e l d e d t h e Lthyroxine (V). The a n a l y t i c a l d a t a p r e s e n t e d e s t a b l i s h e d t h e s t e r e o s p e c i f i c i t y of t h i s s e r i e s o f r e a c f i o n s . The o v e r a l I y i e l d was 8%.

240

NO2 L-HO-I=)..-CH-COOH \ NO2

NH2 I

KBr, NaN02 >H2S04

V CH2 -CH - COOH Br I

n02

(I1

from F i g u r e 9)

(XI 1

aqueous NH3

H O O C H 2 - CH-COOH 1

\ I

NH2 NO2
(XI I )

F i g u r e 10

0 -0--0

z
I
N

"", YZ" ' , ,

-O

/ + I
+
0-0

2
I

+
Y Y

4
?

%Y&
P

+ +

r:

250

SODIUM LEVOTHYROXINE

3.3

Synthesis o f Radiolabeled L-Thyroxine

Weeke and O r s k o ~ ~ ~ developed a r a t h e r f a c i l e have rocedure f o r t h e s y n t h e s i s and p u r i f i c a t i o n o f monolabeled P251-LT4 w i t h a v e r y h i g h s p e c i f i c a c t i v i t y f o r use i n radiolmnune assay. F i v e mCI o f 1251 ( s p e c i f i c a c t i v i t y > 14 mCi/ug) were added t o 50 p1 o f 50 mmol/l phosphate b u f f e r , pH 7 . 5 , f o r b u f f e r i n g t h e a l k a l i n e 1251 l u t i o n . A d d i t i o n o f 2 pg so (20 pI 1 o f 3 , 5 , 3 ' - t r l i o d o - L - t h y r o n l n e and 90 pg (25 1.11) o f chloramine-T, m i x i n g f o r 15 seconds and h a l t i n g t h e r e a c t i o n w i t h 240 pg ( I 0 0 p l ) o f sodium m e t a b i s u l f l t e , y i e l d e d a 1251-LT4 o f very h i g h s p e c i f i c a c t i v i t y (~3000 mCl/mg T4). The 1 2 5 1 l a b e l i s presumably i n t h e 5' p o s i t i o n .

A more general and simple procedure f o r t h e syntheor s i s o f r a d i o a c t i v e L-thyroxine, c a r r y i n g t h e label I3lI 14C e i t h e r i n t h e phenol i c r i n g o r i n t h e non-phenol i c r i n g and t h e s i d e chain, was r e p o r t e d b y Shiba and Cahnmann.26 I t i s based on t h e c o u p l i n g o f 4-hydroxy-3,5-diiodophenyIp y r u v i c a c i d and d i i o d o t y r o s i n e . Labeled 4-hydroxy-3,Sd l l o d o p h e n y l p y r u v i c a c i d was prepared by t h e condensation of i o d i n a t e d p-hydroxybenzaldehyde w i t h a c e t y l g l y c i n e and t h e h y d r o l y s i s o f t h e azlactone. Depending on t h e s p e c i f i c labeled compound prepared, y i e l d s ranged from 12-209.
4. Stability ne and i g h t . On nk c o l o r . s stable in

The USP,4 NF,3 and B P I s t a t e t h a t levothyrox d e x t r o t h y r o x i n e sodium a r e t o be p r o t e c t e d from exposure to l i g h t these compounds may assume a p I n a d d i t i o n , t h e USP4 s t a t e s t h a t I e v o t h y r o x i n e dry a i r .

Several i n v e s t i g a t o r s have s t u d i e d t h e s t a b i l i t y o f t h y r o x i n e as a f u n c t i o n o f t h e d e l o d i n a t i o n process which y i e l d s f r e e i o d i n e and t r i iodothyronine. StanburyaP has reviewed t h e pathway and mechanism o f t h i s process by b o t h i n v i t r o and I n v i v o experimentation. He i n d i c a t e d t h a t t h e major f a c t o r I n t h e d e i o d i n a t i o n process i s i n v o l v e d w i t h t h e f a c t l e i o n l z a b l I i t y o f t h e hydroxyl group. Because o f t h i s , one can expect t h a t t h e lodlnes i n t h e 3' and 5' p o s i t i o n s a r e more l a b i l e than those i n t h e 3 and 5 p o s i t i o n s

251

ALEX

POST AND RICHARD J. WARREN

( r e f e r t o I ,221. The r e l e a s e d i o d i d e i s t h e n o x i d i z e d t o f r e e i o d i n e . T h a t t h i s appears t o be t h e i n v i v o mechanism has subsequently been borne o u t by o t h e r i n v e s t i g a t o r s . High energy g a m a r a d i a t i o n has been shown t o cause r a p i d d e i o d l n a t i o n and t h e f o r r n a t l o n o f o t h e r i o d l n a t e d o r g a n i c molecu 1 es .28 The r a t i o n a l e and e x p l a n a t i o n f o r t h e spontaneous d e i o d i n a t i o n o f 1311 l a b e l e d t h y r o x i n e Is s t i I I o f concern. TaurogBg has shown t h a t t h e s p o t t i n g o f d i l u t e aqueous b u f f e r s o l u t i o n s on f i l t e r p a p e r and d r y i n g f o r 10-20 minutes l e d t o a s i g n i f i c a n t and v a r i a b l e loss o f 1311 from t h e 1 3 1 1 - t h y r o x i n e . T h i s e f f e c t was a l s o observed when g l a s s paper and t h i n l a y e r s i I i c a g e l were used. R e d u c t i o n i n t h e amount o f d e i o d i n a t i o n o c c u r r e d when e t h a n o l or p r o p y l e n e g l y c o l was added t o t h e sample. Shortwave u l t r a v i o l e t l i g h t g r e a t l y enhanced t h e r a t e o f d e i o d i n a t i o n . These spontaneous d e i o d i n a t i o n e f f e c t s were a l s o s t u d i e d by J o l i n , e t a l . 3 0 They i n d i c a t e d t h a t t h i s e f f e c t a r i s e s when one o f t h e components o f t h e s o l u t i o n under s t u d y becomes an a c t i v e d e i o d i n a t i o n agent d u r i n g t h e a n a l y t i c a l procedure. Thus, a s t r i c t adherence t o p r o c e d u r e must b e f o l lowed. Reviczky and Nagy3l found t h a t under u l t r a v i o l e t r a d i a t i o n d e i o d i n a t i o n o c c u r r e d t o a g r e a t e r e x t e n t and more r a p i d l y i n aqueous s o l u t i o n s t h a n i n b u t a n o l , t h e r e l e a s e o f I o d i d e i s p r o p o r t i o n a l t o t h e decrease i n pH, and chromatography showed t h a t i n a d d i t i o n t o t h e presence o f f r e e i o d i d e t r i i o d o t h y r o n i n e was t h e p r i m e decomposition p r o d u c t .

5.

Drug Metabolism 5. I Biological Half-Life

I n a s t u d y s i m i l a r t o t h a t of S t e r l i n g , e t a1,32 Demeester-Mi r k i ne showed t h a t t h e ha I f - I i f e o f 1 3 1 1 - t h y r o x i ne i s 7 days i n serum and 0.4 days i n t i s s u e s . 3 3

5.2

Metabol i c P r o d u c t s

T h y r o x i n e i s c o n v e r t e d t o t r i i o d o t h y r o n i n e i n hypot h y r o i d human s u b j e c t s m a i n t a i n e d on s y n t h e t i c sodium L252

SODIUM LEVOTHYROXINE

t h y r o x i n e a d m i n i s t e r e d o r a l ly.34 T h i s was c o n f Inned by S t e r l i n g , e t al,32 who i n j e c t e d p u r i f i e d t h y r o x i n e l a b e l e d w i t h 14C i n r i n g A and i n t h e a l a n i n e s i d e c h a i n . Methods f o r d e t e r m i n i n g t h i s c o n v e r s i o n have been reviewed by Surks and Oppenheimer. 35

6.

Elemental A n a l v s i s

The elemental c o m p o s i t i o n o f L - t h y r o x i n e as t h e monosodium s a l t p e n t a h y d r a t e , t h e anhydrous monosodium s a l t , and t h e anhydrous f r e e a c i d i s as f o l l o w s :

%
Monosodium (Pentahydra t e ) Carbon Hyd r o g en N it rogen Oxygen Sod i urn Iodine

(Theory) Monosodium (Anhydrous) Amino A c i d (An hy d r o u s 1

20.27 2.27 I .58 16.20 2.59

57.10

22.55 I .26 1.75 8.01 2.88 63.54

23.19 I .43 I .80 8.24

65.34

The t h e o r e t i c a l percentage o f w a t e r i n t h e monosodium pentah y d r a t e form i s 10.13%.

6.1

D e t e r m i n a t i o n of O r g a n i c a l l y Bound l o d l n e

As t h e c r i t e r i o n o f a c c e p t a b i I i t y i n b o t h t h e USPd and t h e NF3 i s t h e assay v a l u e f o r i o d i n e , t h e methods o f a n a l y s i s employed t o d e t e r m i n e t h i s element have been c a r e f u l l y investigated.

6. I I

Oxygen F l a s k Combustion36 T i t r a t ion37

lodometric

This method i s d e s c r i b e d i n b o t h t h e USP X I X and NF X I V monographs f o r sodium l e v o t h y r o x i n e and sodium d e x t r o t h y r o x i ne.

Apparatus - The a p p a r a t u s c o n s i s t s o f a heavyw a l l e d c o n i c a l , d e e p l y l i p p e d o r cupped 500-ml f l a s k ( u n l e s s a l a r g e r f l a s k Is s p e c i f i e d ) , f i t t e d w i t h a ground-glass s t o p p e r t o which i s fused a sample c a r r i e r c o n s i s t i n g o f
253

ALEX POST AND RICHARD J. WARREN

heavy-gauge p l a t i n u m w i r e and a p i e c e o f welded p l a t i n u m gauze measuring about 1.5 x 2 cm. Procedure - Weigh a c c u r a t e l y about 25 mg o f samp e on a p i e c e o f h a l i d e - f r e e f i l t e r paper measuring about 4 cm square, and f o l d t h e paper t o e n c l o s e it. P l a c e t h e samp e, t o g e t h e r w i t h a f i l t e r paper f u s e - s t r i p , i n t h e p l a t num gauze sample h o l d e r . P l a c e t h e a b s o r b i n g l i q u i d , cons s t i n q of a m i x t u r e of 10 m i of sodium h y d r o x i d e s o l u i o n 71 i n 1001, i n t h e f l a s k and m o i s t e n t h e j o i n t o f t h e stoDoer w i t h w a t e r . F l u s h t h e a i r from t h e f l a s k w i t h a stream o f r a p l d l y f l o w i n g oxygen, s w i r l i n g t h e I i q u i d t o f a v o r i t s t a k i n g up oxygen. [NOTE--saturation o f t h e l i q u i d w i t h oxygen i s e s s e n t i a l f o r t h e s u c c e s s f u l performance o f t h e combustion procedure.] I g n i t e t h e f u s e - s t r i p by s u i t a b l e means. I f the s t r i p i s ignited outside the flask, invert the f l a s k so t h a t t h e a b s o r p t l o n s o l u t i o n makes a seal around t h e stopper, and h o l d t h e s t o p p e r f i r m l y i n p l a c e . When t h e combustion i s complete, p l a c e a few m l o f w a t e r around t h e s t o p p e r and a l l o w t h e f l a s k t o s t a n d f o r a b o u t 15 minutes. Loosen t h e s t o p p e r , and r i n s e t h e s t o p p e r , sample h o l d e r , and s i d e s o f t h e f l a s k w i t h about 20 m l o f water, added i n s m a l l p o r t i o n s . Add I m l o f an o x i d i z i n g , s o l u t i o n prepared by a d d i n g 5 m l of bromide t o 100 ml of a I i n 10 s o l u t i o n o f sodium a c e t a t e i n g l a c i a l a c e t i c a c i d . I n s e r t t h e stopper I n t h e f l a s k , and shake v i g o r o u s l y f o r I minute. Add 0.5 m l o f f o r m i c a c i d , r e p l a c e t h e s t o p p e r , and shake v i g o r o u s l y f o r I m i n u t e . Remove t h e s t o p p e r , and r i n s e t h e s t o p p e r , t h e sample h o l d e r , and t h e s i d e s o f t h e f l a s k w i t h s e v e r a l small p o r t i o n s o f water. Bubble n i t r o g e n t h r o u g h t h e f l a s k t o remove t h e oxygen and excess bromine, add 500 mg o f p o t a s s i u m i o d i d e , s w i r l t o d i s s o l v e , add 3 ml o f d i l u t e d s u l f u r i c a c i d , s w i r l t o mix, and a l l o w t o s t a n d f o r 2 minutes. T i t r a t e w i t h 0.02 N sodium t h i o s u l f a t e , adding 3 m l o f s t a r c h TS as t h e N endpoTnt i s approached. Each ml o f 0.02 - sodium t h i o s u l f a t e i s e q u i v a l e n t t o 0.1057 mg o f i o d i n e .
I ,

6.12

Ash i ng t N-Bromosucc i n i m i de T i t r a t i o n

Bakarat, e t al,38 ashed t h e sample i n t h e presence o f potassium c a r b o n a t e and, a f t e r d i l u t i o n w i t h water, t i t r a t e d t h e s o l u t i o n w i t h 0.02 N N-bromosuccinimide. Accuracy o f g r e a t e r t h a n 99% was o b t a i n z d f o r samples cont a i n i n g 5-10 mg o f t h y r o x i n e .

254

SODIUM LEVOTHYROXINE

6.13

Oxygen F l a s k Combustion T i t r a t i on

+ Coulometric

A modif i c a t i o n d g o f t h e oxygen f l a s k combustion method has been used p r i o r t o t h e c o u l o m e t r i c t i t r a t i o n 4 0 o f t h e iodide: To a 500-ml oxygen f l a s k f i l l e d w i t h oxygen I s added 10.00 m i o f 0.4 N potassium h y d r o x i d e and s e v e r a l m i l l i g r a m s o f h y d r a z i n e s u l f a t e . The f i l t e r paper t a b i s i g n i t e d , and a f t e r combustion i s c o m p l e t e t h e f l a s k i s c o o l e d f o r a few seconds i n a stream o f c o l d w a t e r and s e t a s i d e f o r a t l e a s t 30 minutes f o r complete a b s o r p t i o n o f t h e combustion p r o d u c t s . The a b s o r b i n g s o l u t i o n i s a c i d i f i e d w i t h 10.00 m l o f a s o l u t i o n o f 0.6 N n i t r i c a c i d i n 20% g l a c i a l a c e t i c a c i d . The f l a s k i s s w i r l e d t o remove some o f t h e carbon d i o x i d e evolved, stoppered and v i g o r o u s l y shaken. An a l i q u o t o f 4.00 m l i s t i t r a t e d c o u l o m e t r i c a l l y on a s u i t a b l e c o u l o m e t r i c t i t r a t o r , such as t h e Aminco-Cotlove Automat i c T i t r a t o r . 4 2 j 42

6. I4

S p e c i f i c Ion E l e c t r o d e

The s p e c i f i c i o n e l e c t r o d e s e n s i t i v e t o i o d i d e has been used by P a l e t t a and Pantenbeck43 f o r samples of L-T4. The i o d i n e i s s t r i p p e d from t h e compound w i t h a c t i v a t e d a l u minum f o i I a t pH I I a t 6OoC i n 10 minutes. A f t e r n e u t r a l l z a t i o n w i t h d i l u t e h y d r o c h l o r i c a c i d , t h e p o t e n t i a l is measu r e d u s i n g t h e s p e c i f i c i o n e l e c t r o d e 4 4 versus t h e calomel r e f e r e n c e e l e c t r o d e . The amount o f i o d i d e p r e s e n t i s d e t e r mined by comparing t h e p o t e n t i a l r e a d i n g w i t h t h o s e o b t a i n e d f o r a s e r i e s o f standard s o l u t i o n s c o n t a i n i n g t o lo- grams o f i o d i d e p e r m l . The assay r e s u l t s compared f a v o r a b l y w i t h t h o s e o b t a i n e d by t h e c o n v e n t i o n a l t i t r i m e t r i c p r o c e d u r e u s i n g a sodium t h i o s u l f a t e t i t r a n t .

6.2

D e t e r m i n a t i o n o f Water

L - t h y r o x i n e monosodium i s g e n e r a l l y p r e p a r e d as a h y d r a t e . Thus, i n o r d e r t o compare samples on an anhydrous b a s i s , t h e m o i s t u r e c o n t e n t i s determined.

6.21

G r a v i m e t r i c Method (USP X I X , Method I l l , p.668) From t h e monograph f o r l e v o t h y r o x i n e sodium

255

ALEX POST AND RICHARD J. WARREN

t h e d e t e r m i n a t i o n i s as f o l l o w s : ' D r y about 500 mg, a c c u r a t e l y weighed, o v e r phosporous p e n t o x i d e a t 6OoC and a t p r e s s u r e n o t exceeding 10 mm o f mercury f o r 4 hours. 6.22 Thermogravi m e t r i c Ana I y s i s (TGA) i n 2.7 has been found X I X procedure. A X I X p r o c e d u r e showed thermogravimetry I t

The procedure d e s c r i b e d t o y i e l d e q u i v a l e n t r e s u l t s t o t h e USP sample ( B a t c h #27) analyzed by t h e USP t h e presence y f 8.75% m o i s t u r e , and by showed 8.85%. 6.3 Chromatographic A n a l y s i s

As t h y r o x i n e i s o f t e n contaminated w i t h t r i i o d o t h y r o n l n e , and i n t h r y o i d powders w i t h a s e r i e s o f i o d i n a t e d t h y r o n i n e s a n d / o r t y r o s i n e s , and i n s e r a w i t h s i m i l a r compounds, it i s i m p o r t a n t t h a t t h e chromatographic system employed be c a p a b l e o f s e p a r a t i n g t h e s e r e l a t e d compounds. Thus, i n t h e s e v e r a l chromatographic t e c h n i q u e s used t o e v a l u a t e t h r y o x i n e and t h r y o x i n e - c o n t a i n i n g m a t e r i a l s , t h e R f , RT, e t c . o f t h e s e r e l a t e d compounds a r e a l s o r e p o r t e d when a v a i l a b l e . The f o l l o w i n g a b b r e v i a t i o n s o f t h r y o x i n e and r e l a t e d compounds w i l l be used: T4 T3 T2 TI T thyroxine 3,3', 5 - t r i io d o t h y r o n i ne 3,5-d i i o d o t h y r o n i ne 3-iodothyron i n e t h y r o n i ne i norgan ic iod i de t y r o s i ne 3-iodotyrosine 3,5-diiodotyrosine Paper Chromatography (PC)

I-

TY MIT DIT 6.31

A s i g n i f i c a n t amount o f l i t e r a t u r e i s a v a i l a b l e concern i ng t h e app I i c a t i o n o f paper chromatography t o t h e s e p a r a t i o n o f t h e iodoamino-acids. A few o f t h e r e l e v a n t pub1 i c a t i o n s have been c i t e d . 4 5 - 5 3 256

I&Lci
R f Values o f Thyroxine, Analogs,

and R e l a t e d Compounds (Paper Chromatography) M o b i l e Phase 6 7 Rf

ComDound

I
0.89 0.91 0.69 0.26 0.46 0.58

2
0.47 0.70 0.06

3
0.45 0.63 0.11 0.40 0.26 0.18

4 0.45 0.65 0.11 0.90 0.25 0.28

5
0.51 0.63 0.68

8 0.70 0.85 0.85 0.62

9
0.28 0.36 0.47

10
0.43 0.78

II
0.22 0.33 0.42 0.35 0.26

12

T4 T3 T2 TI T

0.43 0.58 0.62

0.86 0.86 0.82 0.87

0.07 0.25

'I
T MIT DIT

0.89 0.17 0.17 0.67 0.72 0.59 Reference #

0.39

Mobile Phase:
I. 2. 3. 4. 5.
6. 7. 8.

9.
10.

I I.
12.

n-Butanol:2N acetic a c i d ( I : I ) n-Butanol :&hano1 :0.5N NH40H ( 5 : 1 :2) n-Butano I :d I oxane :2& m 4 0 H ( 4 : I :5 1 C o l l i d i n e (conc. NHt,OH vapor) n-5utanol :ethanol :2N NH40H (5:I :2) n-Butanol :2N NH40H T5:2) n-Butanol :Zv f o r m i c a c i d n-Butanol :6F NH40H lsoamyl alcohol : t e r t - a m y l a l c o h o l :6N NH40H ( 1 : I :2-upper t e r t - a m y l a l c o h o l :2N NH40H:hexane ( 5 : 6 : I ) I, I-dimethy I propanol- I s a t u r a t e d w i t h 6N NH40H 3% sodium c h l o r i d e

46
47 48

46
49 49

50
phase)
50 45 53 52 53

ALEX POST AND RICHARD J. WARREN

Table 6 D e t e c t 1on Met hods Used i n Paper Chromatography o f Thyroxine

A comprehensive d e s c r i p t i o n o f t h e f o l l o w i n g d e t e c t i o n methods i s l i s t e d i n Reference 46 - they a r e o n l y b r i e f l y descr i bed here :

'I.

C e r i c - a r s e n i t e reagent: T h i s method o f d e t e c t i o n depends upon t h e l i b e r a t i o n o f i o d i n e from t h e compounds by o x i d a t i o n w i t h c e r l c s u l f a t e . The i o d i n e - c o n t a i n i n g compounds appear as w h i t e spots on a y e l l o w background. As l i t t l e as 0.1 pg o f L-T4 and L-T3 and 0.01 pg o f I- c o u l d be detected. A d e t a i l e d account of t h e a p p l i c a t i o n o f t h i s method can be found i n Reference 139.

2.

F e r r i c h l o r 1 d e - f e r r i c y a n ide-arsenious a c i d reagent: &el I n and Virtanen"' demonstrated t h a t t h e c a t a l y t i c a c c e l e r a t i o n by ' o f t h e simultaneous r e d u c t i o n o f I f e r r l c h l o r l d e and f e r r i c y a n t d e t o y i e l d a m i x t u r e of T u r n b u l l ' s b l u e and Prusslan b l u e was a s e n s i t i v e d e t e c t i o n method f o r iodothyronines and i o d o t y r o s i n e s (0.002 p g f o r each o f t h e amino a c i d s and 0.001 pg f o r I-.)

3.

Starch: Datta, e t a I .49 showed t h e use o f a s t a r c h spray, an overspray w i t h KI03, and exposure t o UV l i g h t t o be a s e n s l t i v e d e t e c t i o n reagent. A t r a n s i e n t b l u e c o l o r from as l i t t l e as 0.05 pg o f an iodoaminoa c i d can be detected.

Less s e n s i t i v e d e t e c t i o n reagents a r e :

4.

D l a t o t i z e d s u l f a n i l l c acid:

10-20 pg o f t h y r o x i n e can be detected. A range o f c o l o r s from r e d d i s h p u r p l e (L-T4) t o orange ( L - T I ) i s obtained.

258

SOD1 UM LEVOTHY ROX I N E

Table 6 (continued)

5.

D i a z o t i zed N,N-di e t h y I su I f a n i lami de: T h i s r e a g e n t i s o n l y s l i g h t l y more s e n s i t i v e t h a n t h e above, and t h e c o l o r s a r e i n t h e p u r p l e range.

6.

N i n h y d r in : A l e s s s p e c i f i c reagent, s i n c e it r e a c t s w i t h amino a c i d s . L-T4 appears as a p u r p l i s h brown c o l o r r a t h e r t h a n t h e t y p i c a l p u r p l e . The l i m i t o f d e t e c t i o n o f L-T4, L-T3, and L-T2 i s I ~ 9 . ~

7.

Ultraviolet light: These compounds absorb s t r o n g l y i n UV l i g h t and can be d e t e c t e d i f 20-30 pg a r e p r e s e n t .

A u n i ue r e a g e n t (Emerson's)'41 was used by E i s d o r f e r and P o s t 4 t o qua1 i t a t i v e l y and q u a n t i t a t i v e l y d e t e r m i n e t h e presence and amount o f L-T2 and L-T3 i n L-T4. The r e a g e n t i s p r e p a r e d by d i s s o l v i n g 4 - a m i n o a n t i p y r i n e h y d r o c h l o r i d e i n aqueous sodium c a r b o n a t e s o l u t i o n . The r e a g e n t i s s e n s i t i v e i n d e t e c t i n g I Pg o f each o f t h e iodoamino a c i d s f o r m l n g a range o f c o l o r s : L-T2 (orange), L-T3 ( r e d ) , and L-T4 ( p u r p l e ) .

D e t e c t i o n o f l a b e l e d iodoarnino a c i d s can b e accomplished by:

I.
2. 3.

Neutron a c t i v a t i o n a n a l y s i s Radioautography (X-ray f i Im) Automatic chromatographfc scanners f i t t e d w i t h a GeigerM I Ie r detector u

259

Table 7 R f Values o f T h y r o x i n e , Analogs, and R e l a t e d Compounds ( T h i n Layer Chromatography) (see notes)

6

M o b i l e Phase 4 5
Rf

8
0.18 0.50 0.58
0.62 0.07 0.04

10

0.55 0.73

0.25 0.36 0.75

0.38 0.46 0.50

0.70

0.48 0.59 0.66 0.53 0.23 0.19

1.28 1.76

0.89 1.13

0.14 0.34 0.48 0.90

0.24 0.30 0.27 0.31 0.14 0.09

0.58
0.49 0.38 0.27 0.12 0.18 0.30

1MIT DIT

0.87

0.18 0.73

0.29 0.24

0.67 0.15
NOTES

0.39 0.17

0.83 0.72

Mob; I e Phase
I. n-Butanol : c h l o r o f o r m (4:7) s a t u r a t e d
w i t h NH40H atmosphere

Adsorbent Kieselguhr G

Detect ion Rad ioscan

Ref #

55
56

2.

30% NHhOH (w/v):methanol : c h l o r o f o r m (0. I :2:2, v / v )

S i l i c a Gel H

Ceric sulfate: sodium a r s e n i t e : methy I ene b I ue

57

Table 7 ( c o n t i n u e d )
V

.-

a , + a,

.-

Mob i l e Phase

Adsorbent
L

Detect ion
V
C

Ref #

.a ,
L LL

3.
4.

Tert-amy alcohol:acetone:NH40H ( 2 5 : 8 : 7 , v/v)

Tert-but-nol:tert-amyl alcohol: NH40H :met hy I e t hy I ketone :H20

0 0 L >

5.

(Rf &ven as r e Z a t i v e Rf of I-, Rf of I- not r e p o r t e d ) Tert-amyl alcoho1:dioxane:iN NH40H 20% S i I i c a Gel G+ (2:2: I ) 80% C e l l u l o s e MN 300
(Rf given as reZative Rf of I-, Rf of I- not r e p o r t e d ) Same as 5 Ethano I :methy I e t h y I ketone:ZN NH40H ( I :4: I )
G O !I -

m r n

ul D

+I

0 t

ul

a ,

Kieselgel G
ul

N i n h y d r i n , PdCI2
c .z

58
59

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N -

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-0

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Eastman Sheets #6060 ( S i l i c a Gel)

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Q)

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ul 0

a ,

ul

*
Lo

c3

a ,

a ,

( -0 .-0 -ulna 3 D n

Diazotized s u l f a n i l i c acid, PdC I 2

N + -

D V a, .._.N -

t c

60

o m w

D .-

uz

0 -0

a,

ul

a ,

%W 'F V

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9
7.

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Formic acid:H20

( I:5)
u3 -

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rub

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Same as 5
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a ,

Ln

60
u .. u .- O .L U

ul

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Cel I u l o s e Powder
a,
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3

I : a ,

Ferric chloride: f e r r i cyan i de: arsenious a c i d

a ,
(0

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61

8.

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Z

a,

9.
10.

Ethy1acetate:methanoI:ZN NH40H (100:40:60, V / V )

U

Ch1oroform:methanol : f o r m i c a c i d ( 7 0 : 15: 15, v / v )

v
M

..

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2 I

E 0 1 ' 8 P I

Same as 7
a,

Same as 7
c3

61

ul

..

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-

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m

0 .-

L3

a,

.-. I .

Ceric sulfate: sod i um a r s e n i t e

.. 2

62

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S i l i c a Gel G
(0

(not r e p o r t e d )

0 +

V .-

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..

ALEX POST AND RICHARD J. WARREN

T a b l e 5 l i s t s t h e R f o f s e v e r a l o f t h e iodoaminoacids i n d i f f e r e n t systems. D e t e c t i o n methods a r e l i s t e d i n T a b l e 6. Several Whatman papers have been used: Whatman I , 3, 4, 3MM b e i n g t h e most p o p u l a r . I n addition, t h e procedure has been c a r r i e d o u t i n t h e ascending, descending and c i r c u l a r modes. I n each case, i t has been t h e e x p e r i e n c e o f t h e a u t h o r s t h a t , under t h e p r o p e r c o n d i t i o n s o f temperature, equi I i b r a t i o n time, and l e n g t h o f run, t h e Rfs o b t a i n e d w i t h any o f t h e s e modes gave e s s e n t i a l l y e q u i v a l e n t r e s o l u t i o n from day t o day and l a b o r a t o r y t o Iaboratory Dei od I n a t I on e f f e c t s d u r i ng paper chromatog r a p hy have been e v a l u a t e d and described I n Section 4.

Paper chromatography, u s i n g f o u r d i f f e r e n t s o l v e n t systems on Whatman # I paper, was used t o e s t a b l i s h t h e chromatographic p u r i t y o f t h e L - t h y r o x i n e sodium r e f e r e n c e substance p r i o r t o i t s a d d i t i o n t o t h e B r i t i s h .Pharmacopoeia.54 e f h y r o x i n e v a l u e s a r e r e p o r t e d f o r t h e compounds I i s t e d i n T a b l e 7. 6.32 T h i n Layer Chromatography (TLC)

T h i n l a y e r chromatography o f f e r s some advantages t o paper chromatography i n t h a t b e t t e r s e p a r a t i o n s a r e general l y o b t a i n e d w i t h h i g h e r l o a d i n g s . However, r e p r o d u c i b i l i t y o f R f values i s oftentimes d i f f i c u l t t o o b t a i n because o f t h e b a t c h t o b a t c h d i f f e r e n c e s i n adsorbents, t e m p e r a t u r e v a r i a t i o n s w i t h i n a l a b o r a t o r y , and e q u i l i b r a t i o n t i m e s used by d i f f e r e n t i n v e s t i g a t o r s . Thus, t h e d a t a p r e s e n t e d i n T a b l e 7 s h o u l d be c o n s i d e r e d i n l i g h t o f t h e s e v a r i a b l e s ; and t h e a n a l y s t should be expected t o a l t e r t h e m o b i l e phase, a l t h o u g h s l i g h t l y , t o e f f e c t a s u i t a b l e separation. The r e f e r e n c e s c i t e d 5 5 - 6 2 i n d i c a t e t h e v a r i e t y o f systems a v a i l a b l e f o r t h e s e p a r a t i o n o f t h e s e compounds. Chapters from s e v e r a l t e x t ~ 4 6 , ~ 3 > @ o n t a i n a d d i t i o n a l c i nformation. A c r i t i c a l t h i n l a y e r chromatographic a n a l y s i s o f L - t h y r o x i n e sodium was made by a j o i n t committee o f t h e Pharmaceutical S o c i e t y o f G r e a t B r i t a i n and t h e B r i t i s h Pharmacopoeia p r i o r t o e s t a b l i s h i n g t h e sample as a r e f e r e n c e s ~ b s t a n c e . 5 ~F i v e d i f f e r e n t m o b i l e phases on f i v e d i f f e r e n t adsorbants were used t o e s t a b l i s h i t s p u r i t y .

262

SODIUM LEVOTHYAOXINE

Schorn and W i n k l e r S 5 s y s t e m a t i c a l g a t e d more t h a n two dozen s o l v e n t systems on S i I p l a t e s i n t h e s e p a r a t i o n o f L-T4, L-T3, L-T2, LThe r e s u l t s c e a r l y showed t h e v i a b i l i t y o f t h i s t o t h e separa i o n o f t h e iodoaminoacids. 6.33 Col umn Chromatography

y investica g e l G I and I-. technicrue

As column chromatography i s a r a t h e r nons p e c i f i c t e r m t o d e s c r i b e a s e D a r a t i o n orocedure. w have e d e l i neated t h i s t e c h n i q u e i n t o ' f o u r s p e c i f i c t y p e s : Ion exchange, g e l f i l t r a t i o n , gas l i q u i d , and h i g h performance I i q u i d chromatography. T h e i r i n d i v i d u a l a p p l i c a t i o n s a r e d e s c r i b e d i n f o l lowing s e c t i o n s . 6.331 I o n Exchange Chromatography ( I E C )

Resin column chromatography has been e v a l u a t e d and employed by many i n v e s t i g a t o r s i n t h e separat i o n and q u a n t i t a i t o n o f iodoamino a c i d s and i o d o t h y r o n i n e s i s o l a t e d from b i o l o g i c a I mater i a I ~ . 6 ~ - 7 8 I though t h e s e A procedures a r e amenable t o a s s a y i n g t h y r o x i n e and t h y r o i d powders, t h e new s e p a r a t i o n t e c h n i q u e s d e s c r i b e d i n S e c t i o n s 6.332, 6.333, and 6.334, have, i n g e n e r a l , s u p p l a n t e d i o n exchange chromatographic s e p a r a t i o n s . A n i o n i c r e s i n s , Dowex I-X2 and 50-X4 (Dow Chemical Co., Midland, Mich.), u s i n g ammonium a c e t a t e , sodium a c e t a t e or ammonium formate b u f f e r s a t pH rnages from 3.2-5.6, w i t h o u t o r c o n t a i n i n g up t o 30% e t h a n o l , have been used t o s e p a r a t e s e v e r a l o f t h e i o d o t h y r o n i n e s . Automated procedures f o r d e t e c t i n g t h e components i n e f f l u e n t s have a l s o been reported.68J73J74J77J7&? The c o n v e n t i o n a l c e r i c a r s e n i t e r e a c t i o n d e t e r m i n a t i o n f o r i o d i n e i s t h e most f r e q u e n t l y used d e t e c t i o n method. The a p p l i c a t i o n o f c a t i o n exchange r e s i n s t o s e p a r a t e t h e i o d o t h y r o n i n e s has been r e p o r t e d . 71J 7 7 J 7 8 The c i t e d r e f e r e n c e s deal a l m o s t e x c l u s i v e l y w i t h two iodoaminoacids and two i o d o t h y r o n i n e s , MIT, DIT, T3 and T4, r e s p e c t i v e l y . R e c e n t l y , Sorimachi and U i 7 9 r e p o r t e d t h e s e p a r a t i o n o f e i g h t d i f f e r e n t i o d o t h y r o n i n e s on (1.0 x 15 cm) c a t i o n exchange r e s i n , AG 50W-X4 (30-35 pm), e q u i l i b r a t e d w i t h 0.04 M ammonium a c e t a t e b u f f e r , pH 4.7, c o n t a i n i n g 30% ( v / v ) e t h a n o l a t 5OoC and a g r a d i e n t o f I n c r e a s i n g pH. The

263

ALEX POST AND RICHARD J. WARREN

g r a d i e n t c o n s i s t e d o f s t a r t i n g b u f f e r and 0.65 N NaOH. D e t e c t i o n was by t h e i o d i n e c a t a l y z e d c e r i c - a r s s n l t e r e a c t i o n . T a b l e 8 l i s t s t h e e l u t i o n volumes o f a s e r i e s o f lodoaminoa c i d s and i o d i d e o b t a i n e d by t h i s procedure. Table 8 E l u t i o n Volumes o f lodoam noac ds Com pou nd Iodide MIT DIT TI T2 T3 T4 3'-T I 3,3'-T2 3 , 5 -T2 3,3 ,5' -T3 Approximate E l u t on Volumes ( m l 1 6 37 50 80 80 I06 91 96 I13 88 96 Gel F i l t r a t i o n Chromatography (GFC)

' ' '

6.332

The appl i c a t i o n o f g e l f i l t r a t i o n s e p a r a t i o n o f t h e i o d o t h y r o n i n e s has been p r i m a r i l y used t o d e t e r m i n e t h e i r i n d i v i d u a l c o n t e n t s I n b i o l o g i c a l samples. The i n f o r m a t i o n p r o v i d e d i n T a b l e 9 i n d i c a t e s t h e chromatog r a p h i c systems used t o s e p a r a t e t h e iodoaminoacids i s o l a t e d from t h e s e samples. Each o f t h e c i t e d r e f e r e n c e s d e s c r i b e s t h e i m p o r t a n t s t e p s r e q u i r e d t o p r e p a r e t h e s e columns (e.g., t h e i r dimensions, mesh s i z e o f t h e g e l p a r t i c l e s , e t c . ) , t h e d e t e c t i o n systems used ( g e n e r a l l y t h e c e r i c - a r s e n i t e r e a c t i o n , u l t r a v i o l e t absorption, l i q u i d s c i n t i l l a t i o n counting o f tagged i s o l a t e s , e t c . ) , and t h e p r e c i s i o n and accuracy o f t h e p a r t i c u I a r v a r i a t i o n employed by t h e r e s p e c t i v e i n v e s i t g a t o r . B l a s i and DeMasiB0 have l i s t e d t h e p a r t i t i o n c o e f f i c i e n t s , Kd, o f s e v e r a l t y r o s i n e s and t h y r o n i n e s as o b t a i n e d from t h e Sephadex G-25 column and t h e i r e l u t i o n conditions. From t h e s e data, r e l a t i o n s h i p s between t h e s t r u c t u r e o f t h e compound and t h e e l u t i o n volume can be e s t a b l i s h e d . The Kd v a l u e s a r e l i s t e d I n T a b l e 10.

264

S O D I UM LEVOTHY ROX INE

Table 9 Gel F i l t r a t i o n Chromatographic S e p a r a t i o n Systems

Co I umn Mater i a I

E1uent (a )
0.01 ,N NaOH
tert-amyl alcohol saturated w i t h N 2 - NH40H

Compounds Separated DIT, T3, T4

# 81

Ref

Sephadex G-25 Sephadex G-25

T3, T4

82

Sephadex G-25 Sephadex LH-20(a)

0.02 - NaOH N

Ty, MIT, DIT, T, T I , ( b ) T2, T3, T4

80
83

MIT, DIT, T3, T4 ethy I acetate: methanol : 2 N NH40H ( l00:25: 10, T/v)

Sephadex G-25 Sephadex G- I 5

0.1 N NaOH 0.007 - NaCl N

I-, T3, T4
T3, T4

84

fa)

0.02 - NaOH N
I nc.

85
NJ

fa)Pharmac i a F i ne Chemi ca I s,

, P i scataway ,

I n a d d i t i o n 3 ' ,5'-d i i o d o t h y r o n i ne and fb)3- i o d o t h y r o n i ne. 3,3' ,5'- t r i io d o t h y r o n i ne were a I so separated.

T a b l e 10 Kd Values of l o d o t h y r o n i n e s and R e l a t e d Compounds Comoound (a ) Kd 0.32 0.36 0.52 0.52 0.93 1.13 I .95 2.35 4.40 5.20

TY MIT DIT T 3- l o d o t h y r o n i ne T2 3 , 5 '-d i io d o t h y r o n i ne T3 3,3',5'-tri iodothyron ine T4 (a)l.5 m i n 0.5 ml 0.02 - NaOH g N

265

Table I I Gas L i q u i d Chromatographic S e p a r a t i o n Systems Compound Separated T4, T3 MIT, DIT, T T4, T3, T2 MIT, D I T Derivative
N,O-d p i v a l y l met hy e s t e r

Co I umn 0.5% SE-30 on Gas Chrom Q 3.8% SE-30 on D i a t o p o r t S 1 % polysulfone on Gas Chrom Q

Co I umn Temperature Program I30-305OC IOo/mi n 25OoC

Detector
FID(~)

Amount I nj e c t e d

Ref

# 66

ug

N,O-b s t r i f I u o r o a c e t y methy I ester N,O-d p i v a l y I met hy e s t e r N, 0-d p i v a l y l methy e s t e r TMS (CJ TMS

FID

0.01 umol

86

T4, T3, MIT, DIT T4, T3, MIT, DIT T4, T3, MIT, DIT T4, T3, T2, MIT, DIT, T T4, T3, T2 T4, T3, T2, MIT, DIT, T

232OC 282OC 285OC 75-250C 8 4.6'/rnin 225-235OC @ 5O/min 285OC

ECD f b )

ng P9 50-150 ng 20 ng

87
87 88 89

3% OV-17 on Gas Chrom P

3% O V - l on Gas Chrom Q 0.5% SE-30 o n DMSC-treated f d ) Chromosorb G 5% OV-17 on Gas Chrom Q 5% OV-17 on Gas Chrom Q

ECD
ECD

FIC

N,O-dipivalyl methyl e s t e r N,O-dipivalyl methyl e s t e r

FID
ECD

< I ug 3 ng

90 90

Table I I (continued) Compound Separated T4, T3, T2 DIT, Ty T4, T3, T DIT, MIT, Ty T4, T3, T2, T , M l T DIT, Ty
ru

Der i v a t iv e TMS TMS

Co I umn

Co I umn TemDerature

Detector FID

Amount Injected 50 ng 3-15 u g

Ref

# -91 92

2% SE-33 on Gas Chrom Q 3% OV-17 o n Gas Chrom Q

I 50-280'C

@ 1o0/min
165'C @ 3 m i n FID t o 265' @ 10 m i n 135-255'C @ 5'/min 180-225OC FID
ECD

TMS

1 % OV-1 on C h romosor b W HP

'L4 llg

93

T4, T3, T2, T, TMS MIT, DIT, Ty T4, T3, T2 T4, (f)T3, T2, DIT T4, T3, T2 T4, T3, T2 T4, T3, TMS N,O-dimethyl methyl e s t e r TFAA(9) methyl e s t e r s N,O-d i p i v a l y I methy I e s t e r s TMS

I % OV- I on Chromosorb WHP 3% O V - 1 7 o n D i a t o m i t e CQ 3% OV-1 on Gas C h r m Q

2.3% O V - l on Gas Chrom Q 2.3% OV-1 on Gas Chrom Q

0.3-1.5 5-20 ng

ng

93 94

8 25'/min

(el
25OoC

FID FID ECD

ECD

fe)
29OoC 165-285'C

1.4-2.5 6-8 ng
4-16

ng

96 96 97

1 % OV-l on Gas Chrom Q

FID

8 1o0/min

Tab I e I I (continued 1 Compou nd Separated Der i v a t i ve N,O-dipivalyl methyl e s t e r

Col umn 3% DEXS 1 L 300 GC on Chromosorb WHP

Co I umn Temper a t ure

Detector

Amount Injected

#
98

Ref

T4, T3

305C

ECD

0.25-3

ng

faFID

= Flame I o n i z a t i o n D e t e c t o r

ECD = E l e c t r o n Capture Detector

OD

fCTrimethylsl l y l d e r i v a t i v e dDimethylchlorosi feVariable,

ffAs

lane

depending on which compounds a r e t o be separated

t h e sod i u sa I t , h y d r a t e m

SODIUM LEVOTHYROXINE

6.333

Gas L i q u i d Chromatography (GLC)

S i n c e t h e iodoaminoacids a r e n o t v o l a t i l e and t h u s n o t amenable t o a gas c h r o m a t o g r a p h i c analysis, the preparation o f suitable stable v o l a t i l e derivatives p r i o r t o analysis i s a prerequisite. The f i r s t s u c c e s s f u l gas chromatog r a p h i c s e p a r a t i o n of T4, T3, DIT, MIT, and Ty, a s t h e i r N , O - d i p i v a l y l m e t h y l e s t e r d e r i v a t i v e s , was r e p o r t e d by Stouffer, e t S i n c e t h e n t h i s t e c h n i q u e s has been c r i t i c a l l y e v a l u a t e d because o f i t s i n h e r e n t s e n s i t i v i t y , speed o f a n a l y s i s , and a p p l i c a b i l i t y t o t h e q u a n t i t a t i o n o f t h e s e compounds i n b i o l o g i c a l p r e p a r a t i o n s . As i t i s beyond t h e scope o f t h i s monograph t o p r o v i d e p r e c i s e d e t a i l s o f t h e gas chromatographic methods, a t a b u l a t i o n o f the various r e p o r t s i s l i s t e d i n Table 1 1 . I t i s recommended t h a t t h e c i t e d r e f e r e n c e s be r e f e r r e d t o f o r t h e p r e p a r a t i o n o f t h e v o l a t i l e d e r i v a t i v e s , t h e use o f i n t e r n a l s t a n d a r d s f o r q u a n t i t a t i v e a n a l y s i s , and f o r t h e p r e p a r a t i o n o f t h e column s u b s t r a t e s . From t h e i n f o r m a t i o n I n T a b l e I 1 i t i s h a t t h e two f a v o r e d d e r i v a t i v e s a r e t h e N,Omethyl e s t e r and t h e TMS. The advantage o f t h e t h a t t h e e s t e r s have g r e a t e r s t a b i l i t y . However, r e a two-step s y n t h e s i s , whereas t h e l a t t e r can be n a s i n g l e step but a r e s e n s i t i v e t o moisture. 6.334 H i g h Performance L i q u i d Chromatography

apparent d i p i va I y I former i s they requ prepared

High performance I i q u i d chromatography (HPLC) has been used by Karger, e t a l . 9 9 t o s e p a r a t e T4, T3 and T2 i n about two m i n u t e s i n a m o b i l e phase o f b u t a n o l and m e t h y l e n e c h l o r i d e on a s i l i c a g e l column c o a t e d w i t h a m i x t u r e o f p e r c h l o r l c a c i d and sodium p e r c h l o r a t e . On a s i m i l a r system T4, MIT and DIT s e p a r a t e d i n l e s s t h a n e i g h t m i n u t e s . DuPont I n s t r u m e n t s l o o r e p o r t e d t h e s e p a r a t i o n o f T4 and T3 on a s t r o n g c a t i o n exchange (SCX) column. Both of t h e s e methods showed e x c e l l e n t s e n s i t i v i t y , i n t h a t nanogram q u a n t i t i e s c o u l d be r e a d i l y d e t e c t e d u s i n g h i g h l y s e n s i t i v e UV d e t e c t o r s a t 254 nm. T h y r o x i n e and t r i i o d o t h y r o n i n e have a l s o been s e p a r a t e d i n l e s s t h a n 12 m i n u t e s on a M i c r o p a k
269

ALEX POST AND RICHARD J. WARREN

C-18 ( r e v e r s e phase) column u s l n g a methanol-ammonium c l t r a t e mobi l e phase.101 Waters Associates102 r e p o r t e d a s l m i l a r s e p a r a t i o n on a C I & o r a s i I column u s i n g a mob i l e phase cons i s t i n g o f a c e t o n i t r i le-n-butanol :0.005 M sodium p e r c h l o r a t e . A c l e a r s e p a r a t i o n was e f f e c t e d i n l e s s t h a n 20 minutes.

6.4

Neutron Act i v a t i on Ana I ys i s

Neutron a c t Iv a t i o n ana I ys 1 s was used by A I soszo3 t o d e t e r m i n e t h e L - t h y r o x i n e sodium c o n t e n t i n t a b l e t s . In this procedure, t h e t a b l e t was i r r a d i a t e d f o r 5 m i n u t e s I n a n e u t r o n f l u x of = 2.5 x 1012n/cm2/sec and q u i c k l y t r a n s f e r r e d t o a c o u n t i n g t u b e f o r measurement o f 1281. Comparison w i t h standards o f potassium i o d i d e I r r a d i a t e d f o r t h e same t i m e y i e l d e d t h e c o n t e n t o f L - t h y r o x i n e sodium. Interfering n u c l e a r r e a c t i o n s were n e g l l g l b l e , and t h e e r r o r was l e s s t h a n 2%. Neutron a c t i v a t i on ana I ys Is was a I so used by G I obe I, e t a I . 104 t o determl ne t h e r e 1 a t i v e amounts o f L - t h y r o x i ne ( T 4 ) and 3 , 5 , 3 ' - t r I i o d o t h y r o n i n e (T3) i n serum. These hormones were removed from t h e p r o t e l ns by pass i ng 20 m I o f serum ( a t pH I I ) t h r o u g h a Dowex IX-2 i o n exchange column i n t h e a c e t a t e form. The e l u a t e was c o n c e n t r a t e d and chromatographed on a c e l l u l o s e t h i n l a y e r p l a t e t o s e p a r a t e t h e T4 and T3 from t h e i n o r g a n i c i o d i d e . The i s o l a t e d T4 and T3 f r a c t i o n s were i r r a d i a t e d I n a t h e r m a l n e u t r o n f l u x o f 5 x 1O2n/cm2/sec u s i n g a T r i g a Mark I r e a c t o r , f o l l o w e d by i d e n t i f i c a t i o n and measurement of induced 1281 a c t i v i t y w i t h a germanium ( L i ) sol i d s t a t e d e t e c t o r . The I i m i t s o f d e t e c t i o n were 5 ng. Schmoelzer and Muel Ier1O5 used a s l m l l a r approach b u t separated t h e T4 and T3 o n a QAE Sephadex A-25 column p r i o r t o activation analysis.

6.5 Po I a r o g r a p h i c Ana I y s i s
P o l a r o raphy was employed by Wacholz and P f e i f e r t o assay I h y r o x i n e ~ O 6 and t o d e t e r m i n e t h e t h y r o x i n e c o n t e n t o f t h y r o l d powders and t a b I e t s . I n t h e assay o f t h y r o x i n e , 5-50 pg of sample i n I m i o f 2 N n i t r i c a c i d i s heated a t 6OoC f o r I hour. A f t e r c o o l i n g aKd t h e a d d i t i o n o f 5 m l o f 0.06 - sodium h y d r o x i d e , t h e N
270

SOD1 U M LEVOTHY R OX INE

s o l u t i o n I s deoxygenated w i t h n i t r o g e n . The t h y r o x i n e c o n t e n t I s determined by comparison o f wave h e i g h t of t h e sample a t E& -0.6 t o -0.7V w i t h t h a t o f standards. The method i s s u f f l c l e n t l y s e n s i t i v e t o determine t h e t h y r o x i n e c o n t e n t o f spots i s o l a t e d by t h i n l a y e r chromatography. I n t h e assay f o r t h y r o x i n e I n t a b l e t s and powders, p r i o r e x t r a c t i o n procedures a r e r e q u i r e d t o remove t h e t h y r o x i n e and o t h e r i o d l n a t e d amino acids. A f t e r s e p a r a t i o n by t h i n l a y e r chromatography, e l u t i o n o f t h e t h r y o x i n e spot, c o n c e n t r a t i o n and n l t r a t i o n , 1 0 6 t h e wave h e i g h t a t t h e p r e v i o u s l y s p e c i f i e d E+ i s obtained. 6.6 K i n e t i c Methods o f A n a l y s i s

The a p p l i c a t i o n of k i n e t i c measurements of t h e i o d i n e c a t a l y z e d c e r i c arseni t e reaction108,109 has been u t i I lzed f o r t h e d e t e r m i n a t i o n o f t h y r o x i n e i o d i n e i n chromatograph I c e l u a t e s . 76~1*0,111 The r e p o r t e d methods a r e rapid, have h i g h p r e c i s i o n and a r e s e n s i t i v e , g e n e r a l l y d e t e c t i n g less than I ng o f T4. 6.7 Double-losotope D i l u t i o n A n a l y s i s

A double-isotope d e r i v a t i v e assay f o r serum iodot h y r o n In e s l l 2 (L-T4 and L-T3) has been mod i f l e d and improved upon by Hagen, e t a1.8 I n t h i s procedure, t h e unknown t h y r o x i n e i s labeled by formation o f an a c e t y l d e r i v a t l v e l l 3 u s i n g t r i t i u m - l a b e l e d a c e t i c anhydride. As t h e s p e c i f i c a c t i v i t y o f t h e t r i t i a t e d d e r i v a t i v e i s known, t h e t h y r o x i n e c o n t e n t of t h e sample can be c a l c u l a t e d . Losses I n t h e complex p u r i f i c a t i o n steps a r e accounted f o r by t h e a d d i t i o n of a h i g h s p e c i f i c a c t i v i t y 1311-labeled t h y r o x i n e .
6.8 Determination o f Stereoisomeric P u r i t y

I n f o r m a t i o n presented by t h e J o i n t Committee of t h e Pharmaceutical S o c i e t y of Great B r i t a i n and t h e B r i t i s h Pharmaceutical Committee54 i n d i c a t e s t h a t a l I D-thyroxine c o n t a i n s t r a c e amounts o f t h e L-isomer. A method f o r d e t e r mining t h e amount o f L-T3, an i n t e r m e d i a t e i n t h e s y n t h e s i s o f D-T4, has been reported.45 An a d a p t a t i o n o f t h i s method has been appl l e d t o D-T4 c o n t a i n i n g less than I % o f t h e Li somer. 114

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ALEX POST AND RICHARD J. WARREN

6.9

Equi I i b r i u m D i a l y s i s

Several i n v e s t r g a t o r s 115-117 have used equ i I ib r i urn d i a l y s i s t o d e t e r m i n e t h e f r e e t h r y o x i n e i n serum. A c r i t i c a l s t u d y o t h i s p r o c e d u r e was made b y Lee and P i leggT.115 The e f f e c t s of pH, i n c u b a t i o n t i m e and temperature, b u f f e r c o m p o s i t i o n and c o n c e n t r a t i o n , p r o t e i n c o n c e n t r a t i o n , and specimen d l u t i o n were s t u d i e d . U s i n g 1 3 1 1 - L - t h y r o x i n e and a r e u s a b l e p l a s t l c d i a l y s i s c e l I , r e c o v e r i e s of 92-96% were o b t a i n e d when t h e d i a l y s i s was r u n a g a i n s t 0.05 M phosphate pH 7.6 b u f f e r a t 37OC f o r 18 hours. W i t h i n - r u n and betweenr u n p r e c i s i o n s were 10.6% and 14.2%, r e s p e c t i v e l y . Fang and Selenkow,'" using t h e conventional d i a l y s i s bag and 1251-L-thyroxine, determined t h e f r e e t h y r o x i n e cont e n t a f t e r d i a l y s i s a t 4 O C , a g a l n s t pH 7.4 phosphate b u f f e r f o r 18-24 hours. B i r d and A b i o d u n l Z 7 employed e q u i l i b r i u m d i a l y s i s and i o n exchange chromatography t o d e t e r m i n e free t h y r o x i n e . I o n exchange chromatography was used t o s e p a r a t e t h e 1251 i o d i d e from t h e 1251-L-thyroxine p r i o r t o d e t e r m i n i n g t h e f r e e thyroxine content. The l i t e r a t u r e i s r e p l e t e w i t h a s i g n i f i c a n t number of p u b l i c a t i o n s d e s c r i b i n g m o d i f i c a t i o n s o f e q u i l i b r i u m d y a l y s i s o r a c o m b i n a t i o n of t h i s p r o c e d u r e w i t h o t h e r s e p a r a t i o n t e c h n l q u e s . l 1 8 - Z 2 2 The c i t e d papers o f f e r a good b a s i c background t o t h e a p p l i c a t i o n of t h i s method t o t h e s t u d y o f t h e t h r y o x i n e - p r o t e i n b i n d i n g phenomenon.

7.

Methods of A n a l y s i s

A Compilation

The f o l l o w i n g t a b l e s (12, 13, 14 and 15) i n c l u d e t h e r e f e r e n c e s t o a n a l y t i c a l procedures f o r t h e a n a l y s i s of chemicals, t a b l e t s , powders, and serum and t i s s u e . S e v e r a l a d d i t i o n a l r e f e r e n c e s which were n o t c i t e d i n t h e s p e c i f i c sections a r e included i n t h e fol lowing compi'lations Two r e v i e w a r t i c l e s , by CahnmannlZ3 and by Ral I , e t a l .i24, a r e a l s o noted.

272

SODIUM LEVOTHYROXINE

Table 12 Analysis o f Thyroxine Chemicals Ana l y s i s : See Reference # :

I dent I f i c a t ion
T I t r I metry : I odornet r ic N-Bromosuccinimide Coulometric S p e c i f i c Ion E l e c t r o d e Cer i rnetry Chromatography:

3 ,

3, 4, 54, 125, 1 2 6

1, 3, 37, 107 38 39, 4 0 43

129 45, 54, 130, 131, 132, 1 3 3 54, 55, 58, 59, 61, 62, 107, 130, 131, 134 68, 69, 77, 79, 1 3 6 120, 127, 137 86, 87, 89, 90, 91, 92, 93, 94, 97, 98, 138 101, 142, 143 103 97, 106, 107 76, 78, 111 5, 6, 7, 107, 144 68, 77, 78, 110, 136, 145, 146, 147 54 54
Table 13 A n a l y s l s o f Thyroxine T a b l e t s

PC
TL C

I EC G FC GLC
HP L C

Neutron A c t i v a t i o n Po Ia rog r a p hy Kinetic Spect rophotornet r y Automation Electrophoresis Phase S o l u b i l i t y

Ana I ys i s Titrimetry: I odometr i c

See Reference #

1, 3, 4

273

ALEX POST AND RICHARD J. WARREN

Table 13 (continued Chromatog rap hy : PC TLC Pol arography Spectrop hotometry

148 107, 144, 149 107

107, 144, 148, 149, 150, 151, 152, 153

Table 14

A n a l y s i s o f Thyroxine Powders Ana I ys i s Cer i metry Chromat o g rap hy P C T LC I EC GLC See Reference # 61 154 59, 61, 149, 155, 156 154 94, 97, 157 149, 150, 156

S pectrophotometry

Titrlmetry

1
Table 15

Analysis o f Sera and/or Tissues f o r Thyroxine and Analogs Anal y s 1 s Cer i met r y See Reference # 68, 69, 71, 73, 74, 75, 76, 77, 78, 79, 120, 133, 145, 146, 158, 159, 160, 161 67, 69, 70, 72, 72, 73, 74, 76, 77, 120, 117, 136, 145, 146, 147, 159, 163, 164 85, 110, 137, 165, 166, 167 87, 90, 98, 138 104, 105
274

Chromatography : I EC
GFC GF C GLC

Neutron Act i v a t ion

SODIUM LEVOTHYROXINE

T a b l e 15 ( c o n t i n u e d ) Kinetic D i a iy s i s Spectrometry Au toma t ion Radloimmunoassay Competitive P r o t e i n Binding

211

215, 116, 117, 118, 719, 168, 169, 170, 171, 172 163, 169, 173 73, 74, 78, 136, 145, 146, 147, 159, 170 174, 175, 176

105, 128, 170, 171, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186; 187188 70, 135, 172 162

Nephelometry E I e c t r o p hores i s F I uoromet r y

8.

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G. Schmoelzer and K. M u e l l e r , I n t . J . A p p l . R a d i a t . Isotop., 22, 509 (1971). E. WachhoE and S. P f e i f e r , Pharmazie, 27, 43 (1972). E. Wachholz and S P f e i f e r , Pharmazie, 27, 97 (1972) E. B. S a n d e l l and I . M. K o l t h o f f , M i k r o z i r n . Acta, 9 (1937). V. S t o l c , M i k r o c h m. Acta, - 710. 1961, G. Knapp, e t a l . , Anal. Chem., 46, 724 (1974). H. P. d e v r i e s , Z. K I i n . Chem K n n . Biochem., 13, 97 Biochem.. 13. (1975). J . K. Whitehead and D. B e a l e C I i n . Chim. Acta, - 710 4, ( 1959). G. A. Hagen, e t a l . , A n a l y t . B i o c h m . , 38, 517 (1970)H. Neudecker and E. S c h l e f f e e, Arzneim.-Forsch., 432 (1971). N . D. Lee and V . J . P i l e g g i , C I i n . Chem., - 166 (1971 ) 17, V. S. Fang and H. A. Selenkow, C I i n . Chem., - 185 16, (1970). R. B i r d and M. 0. Abioudun, C l i n . Chim. Acta, - 323 48, (1973). F. W i Ison, e t a l Am. J . C I i n . P a t h o l . , 62, 383 (1974). J . Hermann and H. L. Krueskemper, K I i n . Wochenschr., 46, 1285 (1968). K S t e r l i n g and M. A. Brenner, J . CI i n . I n v e s t . , 45, 153 (1966). J . H. Oppenheimer, e t a l . , J . C l i n . I n v e s t . , - 1769 42, (1963). S. H. Ingbar, e t a t . , J . C I i n . I n v e s t . , 44, 1976 (1965). H. J . Cahnmann, Methods I n v e s t . Diagn. E n d o c r i n o l . , 27 (1972). J . E . Ral I , e t a l . , Methods I n v e s t . Diagn. E n d o c r i n o l . , -, 204 (1972). I S. E. Saheb and M. A. Sanassian, J . Pharm. S c i . , 60, 1259 (1971). P. Z. Anthony and J . E. Geever, J . Pharm. Sci., 394 ( 1 9 5 8 ) . G. Hccman, E n d o k r i n o l o g i e , 57, 176 (1971). A. P. H i I l i e r , A c t a E n d o c r i z l . , - 32 (1975). 78, w - HeersPink and G . J . OP deWeegh, C I i n . Chim. Acta, 39, - 327 (1972) * S. J . P a t t e r s o n and R. L. Clements, A n a l y s t , 89, 328 (1964).

. 1,

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47,

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ALEX POST AND RICHARD J. WARREN

131. 132. 133. 134.

135.

136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150.

252.
152. 153. 154.

155.
156.

157.

158.

J . M. Ensor and P. Kendal I - T a y l o r , E n d o c r i n o l 46, 527 (1970). P. R. Larsen, Metabolism, 20, 609 (1971). A . J . M a t t y and C. C. Thornburn, J . E n d o c r i n o l . , 46, 417 (1970). T. S o f i a n i d e s , e t a l . , Proc. SOC. Exp. B i o l . Med., 123, 646 (1966). S. Hamada and S . H. Ingbar, J . Chromatogr., - 352 61, (1971 1. R. J . B l o c k and R. H. Mandl, Ann. N.Y. Acad. Sci., 87 (1962). R. H. Osborn and T. H. Simpson, J . Chromatogr., 34, 110 (1968). N. N. N i h e i , e t a l . , A n a l y t . Biochem., 43, 433 (1971). C. H. Bowden, e t a l . , Biochem. J . , 2, 93 (1955). R. Gmelin and A . V i r t a n e n . Acta Chem. Scand.. 13. 1469 (1959). E. Emerson, J . Org. Chem., g, 417 1943). A p p l i c a t i o n High1 i g h t s #19, Waters A s s o c i a t e s , I n c . , Framingham, MA. L i q u i d Chromatography R e p o r t # I 16, DuPont I n s t r u m e n t s , W i lmington, DE. E. Wachholz and S . P f e i e r , Pharma i e , - 459 (1969). 24, G. K e s s l e r and V . J . P i eggi, C I i n . Chem., - 339 14, (1968). G. K e s s l e r and V. J . P i eggi, C I i n . Chem., - 382 16, (1970). H. Y . Yee, e t a l . , C I i n Chem., 622 ( 1971 1. R. Lemieux and J. M. Ta mage, J . Pharm. Pharmacol 94 (1966). J . E. Moody, J r . , e t a l , J . Pharm. Sci., 57, 634 ( I968 D. L. Sondack, J. Pharm S c i . , 62, 1342 (1973). I. C o v e l l i , e t a t . , Ana y t . B i o s e m . , 42, 82 (197 1. J . H. Graham, e t a l , J Pharm. S c i . , 63, 763 ( 1 9 4 ) . J. H. Graham. J . Pharm. Sci , 64, 1 3 9 3 7 1 975). S . k b l o g l u , e t a l . , E n d o c r i n o l . , 78, 231 (1966). H. Frey, Scand. J. C I i n . Lab. I n v e s t . , 470 (1964). J o i n t Committee o f t h e Pharmaceutical S o c i e t y and t h e S o c i e t y f o r A n a l y t i c a l Chemistry on Recommended Methods f o r t h e E v a l u a t l o n o f Drugs, A n a l y s t , Lond., - 328 (1967). 92, R. B l l o u s , e t a l . , J . Pharm. S c i . , 60, 1270 (1971). E. Makowetz, e t a l . , Mlcrochem. J., 194 (19661.

.,

102,

17,

16,

10,

280

SODIUM LEVOTHYROXINE

159. 160. 161. 162. 163. 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182. 183.
184. 185. 186. 287. 188.

K. Horn, e t a t . , Z . K I i n . Chem. u k i n . B ochem., 99 (1972). N. E. K o n t a x i s and D. E. P i c k e r i n c l . J. CI n. E n d o c r i n o l .,I Metab., 18, 774 (1958). J . D. A c E n d , Biochem., g , 177 (1957). E. E. G u s s a k o v s k i i , e t a l . , Mol. B i o l . , 7, 598 (1974 J . A. Hathaway, e t a t . , Am. J . CI i n . Path., - 635 53, (1970). V. V. Row, e t a l . , C I i n . Chim. Acta, 31, 473 (1971). C. H. G. I r v i n e , Proc. A s i a Oceania C z g r . E n d o c r i n o I , 401 (1974). R; Liewendahl, e t a l . , Acta E n d o c r i n o l . , 793 ( 1 9 7 1 ) . P. J . D a v i s and R. I . Gregerman, J. CI i n . E n d o c r i n o l . Metab., 30, 236 (1970). R. R. C a E l i e r i and S. H. Ingbar, Methods Enzyrnol 36, 126 (1975). R. A. Pages, e t a l . , Biochem., 2773 (1973). W. C. G r i f f i t h s , e t a l . , C I i n . Biochem., 5, 13 (1972 N . M. Alexander and J . F. Jenni ngs, CI in.-Chem., 20, 553 ( 1974). S . Harnada, e t a l . , J. C I i n . E n d o c r i n o l . Metab., 2 , 66 ( I 970). I . Posner, J . Lab. C I i n . Med., 57, 314 (1961). A . Bercy, J . BeIge R a d i o l . , 5 7 , 1 8 5 ( 1 9 7 4 ) . M. I . Surks, e t a l . , J. C l i n T l n v e s t . , 52, 805 ( 1 9 7 3 ) . I . J . Chopra, e t a l . , J . C l i n . E n d o c r i n o l . Metab., 36, 31 I ( 1 9 7 3 ) . F. W. S p i e r t o , e t a t . , C l i n . Chim. Acta, 5 , 281 ( 1 9 7 4 ) . B. Murphy and C. Patee, J . C I i n . E n d o c r i n o * 26, 247 (1966) * 24, 3. Murphy and C. Patee, J . C I i n . E n d o c r i n o - 187 ( I 964 1 * R. Ekins, C I i n . Chim. Acta, 5, 453 ( 1 9 6 0 ) R. Ekins. e t a l . , C I l n . BiocFem., 2, 253 ( 969) * 46, F. J . L. Crombag, e t I., C l i n . Ch%. Acta - 345 ( 1 973). D. E. D a l r y m p l e and R D. U t i g e r , J . Lab. C l i n . Ped., 75, 325 ( 1 9 7 0 ) . S . Barbadoro, Can. J . Med. Technol., 35, 5 ( 1 9 7 3 ) . 38, H. Sel igson and D. Se igson, C I i n . C h x . Acta, - 199 (1972) * S. C. Thorson, e t a l . , Acta E n d o c r i n o l . , 64, 630 ( 1 9 7 0 ) . S. Nobel and F. B a r n h a r t , C I i n . Chem., 509 ( 1 9 6 9 ) . G. Hocman and L. Hegedus, E n d o k r i n o l o g i e , - 194 55, (1969).

2,

.,

67,

., -

12,

15,

261

METHOTREXATE

Arthur R. Chamberlin, Andrew P.K. Cheung, and Peter Lim

ARTHUR R . CHAMBERLIN etal.

Contents 1.

Description 1.1 Name, Formulae, Molecular Weight 1.2 Isomeric Forms 1.3 Appearance, Color, Odor Physical P r o p e r t i e s 2.1 Infrared Spectrum 2.2 Proton Magnetic SpeCtrUm 2.3 Carbon-13 Magnetic Spectrum 2.4 U l t r a v i o l e t spectrum 2 . 5 Mass Spectrum 2.6 Optical Rotation 2.7 D i s s o c i a t i o n Constants 2.8 S o l u b i l i t y Synthesis Stability 4.1 Bulk 4.2 S o l u t i o n

2.

3.

4.

5. M e t a bol i s m

6. Methods of A n a l y s i s 6.1 Elemental Analysis

6.2 Equivalent Weight Determination 6.21 Nonaqueous t i t r a t ion 6.22 Complex-formation t i t r a t i o n Biological Assay 6.31 Microbiological Assay 6.32 Enzymic Assay Polarographic Assay Spectrophotometric Analysis 6.51 Fluoromet r i c 6.52 U l t r a v i o l e t / v i s i b l e Chromatography 6.61 Paper 6.62 Thin-Layer 6.63 column 6.64 High Speed Liquid Proton Magnetic Resonance

6.3

6.4 6.5
6.6

6.7

284

METHOTREXATE

7.
8.

Acknowledgment References

285

ARTHUR R . CHAMBERLIN eta/.

Description
1.1 Name, S t r u c t u r a l and Empirical Formulae, Molecular

Weight Methotrexate is N-[4-{[(2,4-diamino-6-pteridinyl)met hy 1 met hy 1 ] amine } benz oy1 g l u t ami c a c i d ] Frequent 1 y the name i s abbreviated t o MTX. Methotrexate a l s o i s known as ~-amino-l0-methylfolic a c i d and amethopterin and i s i d e n t i f i e d by t h e National Cancer I n s t i t u t e code number NSC-740.

'

6'

FooH

COOH CZ0Hz2N8O5

Mol. w t .

454.46

Isomeric Forms The presence of an asymmetric carbon i n t h e glutamic a c i d moiety provides f o r o p t i c a l isomerism. Unless sp2cif ied, commercially a v a i l a b l e methotrexate i s prepared from L-glutamic a c i d , Recently, L e e and co-workers' prepared methotrexate s t a r t i n g with D-glutamic a c i d . The D-enantioxer of methotrexate was a c t i v e a g a i n s t L-1210 i n t h e mouse and had less t o x i c e f f e c t s t h a n methotrexate i t s e l f , t h e L-enant iomer

1.2

Appearance, Color, Odor Methotrexate i s a bright yellow-orange, o d o r l e s s powder. It g e n e r a l l y is hydrated t o t h e e x t e n t of 8 t o lo$ w a t e r . I t a l s o has been prepared a s t h e hydrochloride ( 1:0.3) and hydrate (1:2.5)*.
li.

1.3

P r i v a t e communication from D r . H.B. Wood, J r . , of t h e National Cancer I n s t i t u t e and M r . D.F. Worth of Parke, Davis and Co.

286

METHOTREXATE

2.

P h y s i c a l Properties 2.1 Infr --a r e d Spectrum --

Recorded as a s u s p e n s i o n i n m i n e r a l o i l , t h e spectrum i n F i g u r e 1 shows r e l a t i v e l y broad s t r u c t u r e s , i n d i c a t i n g t h e complexity of t h e molecule and s u g g e s t i n g t h e l a c k of c r y s t a l l i n i t y of t h e sample. T a b l e I g i v e s t h e assignments t o t h e major bands. TABLE 1 I n f r a r e d Assignments f o r Methotrexate
I -

I R Absorption B a n d b )

Int ---e r p r e t a t i o n
H20,

2.90-3.10

-W2)

3 .O0-4.03

-COOH
-C-(

5.90-6.10
6.20, 6.50-6.60

COOH, -C-N-)

R B

Aryl systems Amide I1

H

6.50-6.60
11.9

H
2.2

ElH
H

P r o t o n Magnetic Resonance Spzctrum (pmr) The pmr spectrum i n F i g u r e 2 w a s r e c o r d e d on a Varian A&-A s p e c t r o m e t e r w i t h t h e sample a s a s o l u t i o n i n DMSO-de. The chemical s h i f t s are i n ppm r e l a t i v e t o TMS d e s i g n a t e d a s 0.00.
Table I1 g i v e s t h e s t r u c t u r a l assignments t o t h e resonances i n F i g u r e 2 .

287

-I
0.10 O
w V
N

2 K
Q

z 0.20
0

a , a ,

03 .0 0.40

7 8 9 10 WAVELENGTH - microns

11

12

13

14

15

FIGURE 1

INFRARED SPECTRUM OF METHOTREXATE

w
L-

m
X

a
L

d
U

N
L u

289

TABLE I1
pnr Assignments for Methotrexate

Assignments
_ _ _ I _

s i c a l Shifts

(ppm)

Multiplicity
s( broad)
S

J(Hz)

H2N-2

H-7 H-9
HSC-11

~ - 2 ~ , 6 ~ ,5'3 ' ,
H-8'
H u H-B9y H(COOH, HOH)

7.42 8.64 4.81 3.21 6.81,7.78

8.21

s( broad)
S

----8.2

4.43 1.70-2.60 6.21

d q
s( broad)

8 .O
7 .o

---

The values agree reasonable w e l l w i t h those reported by Pastore2 who studied the p m spectra of methotrexate i n solutions a t pH 7.5.

METHOTREXATE

2.3

Carbon-13

magnetic spectrum (cmr)

The "cmr spectrum i n Figure 3 w a s recorded on a Varian XL-100 spectrometer with t h e sample as a s o l u t i o n i n IMSO. The assignments given i n Table 111 f o r Figure 3 are i n general agreement with those reported by Ewers and co-workers,' but minor d i f f e r e n c e s e x i s t . TABLE I11
13C Assignment For Methotrexate (TMS 0 .OO ppm)

Chemical S h i f t ( ppm) 162.67

Assignment c-2

160.01 148.31 148.96 150.94

rv

3
1

c-4
C-6 c-7 c-8a

121.66 40 (amid DMSO)

51.96
121.24 128.94 111.12 150.85

~ - 2 1 , C-6' c-31, c-5'

C-4a c-9 c-11 c-1

166.41
54.82 26.17 39.55

c-7' ca

c-4 '

c-B

173.96 174 .ll

2.1; Ultraviole ---=thet Figure

CY U-CoOH Y -COOH

spectru~ UV spectrum of methotrexate i n 0.1 N NaOH. The longest wavelength maximum appears a t 372 n and m i s a s c r i b a b l e t o t h e diaminopteridine moiety. The i n t e r mediate wavelength maximum occurs a t 303 nm and i s due primarily t o t h e aminobenzoyl group. The s h o r t wavelength maximum is a t 258 nm and is a t t r i b u t e d t o both chromophores. In 0.1 N HC1 methotrexate expsriences a hypsochromic s h i f t r e s u l t i n g i n a UV spzctrum (Figure 5 ) t h a t e x h i b i t s

UV s p e c t r a were recorded on a Cary Model. 14 and t h e molar absorpt i v i t i e s a r e based on anhydrous methotrexate

291

i
W

I -

a
X
W

I-

L
z
LL

H 9 a
I 0
W

n
v)

(? F

E'
u
m
W

(3
U

292

I
1
d

1
I

I
1

I
I

0
I n

P
0 0 d

I-

2 2
w

IX w
& I

m a
W

a
I -

t;
0

0 I Iw

z
LL

a
Z
0 0 0

3
I -

u w

v)

t;
0 > a
E
0
I n
hl
J

3
3 t

12

LL

293

ARTHUR R. CHAMBERLIN

eta:.

250

300

350 NANOMETERS

400

FIGURE

294

METHOTREXATE

maxima a t 307 and 243 nm. The W d a t a are summarized i n T a b l e I V and i n g e n e r a l are i n agreement w i t h t h o s e
r e p o r t e d by S e e g e r and co-workers4, TABLE I V A b s o r p t i o n S p a c t r a of M e t h o t r e x a t e
I

Solvent buffer

0.1 N HC1 0.1 M

~ ~ 6 T. r 7s i
0.1 N NaOH

2.5

Mass Spectrum M e t h o t r e x a t e it self d o e s not y i e l d a s a t i s f a c t o r y

HoNever, t r e a t m e n t mass spectrum because of non-volat i l i t y of m e t h o t r e x a t e w i t h a TMS r e a g e n t a f f o r d s a m i x t u r e of tri-, tetra-, and psnta-TMS d e r i v a t i v e s t h a t d o e s g i v e u s e f u l mass spzctral d a t a . That s e v e r a l TMS-containing d e r i v a t i v e s are formed i s n o t s u r p r i s i n g , c o n s i d e r i n g t h e number and v a r i e t y of f u n c t i o n a l g r o u p s i n v o l v e d . The mass s p s c t r a l f r a g m e n t a t i o n p a t t e r n o b t a i n e d f o r t h e tri-, tetra-, and psnta-TMS d e r i v a t i v e s is summarized as follows:
RZ-NH

I
R1=R,=TMS: Rl,R2=1TMS,

m/e 319 I 1H; m/e 247

'1
Rl,R,=lTMS,

C OM OT S

I Rl=R2=TMS

m / e 452 1 H m / e 380

M1bR1=R2=R3=TMS m / e 814 M-, R1=R2=TMS, R,=H m/e 742 Mf-R1,R2=1TMS, l H , R3=H m/e
M-CH, m / e 727 MH T S m/e 652 - OM M-HOTMS-CH, m/e

670

637

295

ARTHUR R . CHAMBERLIN et al.

2.6

Optical Rotation
46

21
589
546

= 20.4 =

2 0.6'
0.8'

(c 1, N/10

NaOH)

26.9

2.7

Neither t h e a c i d i c nor t h e b a s i c pKa f o r methotrexate has been r e p o r t e d . However, Albert and co-workers5 reported t h a t t h e basic pKa values f o r 2 , b d i a m i n o p t e r i d i n e w e r e < 0.5 and 5.32. Kallen and Jencks' r e p o r t e d t h e a c i d i c pKa values f o r p-aminobenzoylglutamic a c i d a s 4.83 and 3.76. By spectrometry, w e found a pKa of 5.60 2 0.03, which i s a s s i g n a b l e t o t h e diaminopteridinyl moiety. Methotrexate is p r a c t i c a l l y i n s o l u b l e i n water, alcohol, chloroform, and e t h e r . I t i s f r e e l y s o l u b l e i n d i l u t e s o l u t i o n s of a l k a l i n e and carbonates; it i s s l i g h t l y s o l u b l e i n d i l u t e hydrochloric acid (1 i n 2 ) .

D i s s o c a t i n Constants ~ i-o- - -

2.8 ___-- i l i t y 7 Solub

3. Synthesis
The f i r s t reported s y n t h e s i s of methotrexate, t h a t by Seeger and co-workers' is a s follows:

CH2Br

CHBr
CHO

+H-N

C-NH-

COOH

-------+C

---

These s p e c i f i c r o t a t i o n values a r e based on anhydrous methotrexate.

296

METHOTREXATE

T h i s r e a c t i o n o f t e n i s r e f e r r e d t o as t h e Waller r e a c t i o n , because Waller i n i t i a l l y p r e p a r e d p t e r o y l g l u t a m i c a c i d by an analogous method. T h e o r e t i c a l l y , the s u b s t i t u t i o n on t h e p y r a z i n e r i n g c a n be at t h e C-6 o r t h e C-7 p o s i t i o n . Proof of s t r u c t u r e is based on t h e p t e r i n e c a r b o x y l i c a c i d o b t a i n e d from a n a l k a l i n e p3rmanganat.e o x i d a t i o n of m e t h o t r e x a t e . Of t h e two a c i d s p o s s i b l e , o n l y t h e C-6 h a s been found. D i s t i n c t i o n between t h e two p o s s i b l e a c i d s h a s been based on comparative paper chromatography, a l t h o u g h pmr or c m r a l s o c o u l d be u s e d . Because t h e Waller r e a c t i o n g e n e r a l l y y i e l d s impure p r o d u c t s t h a t are v e r y d i f f i c u l t t o p i r i f y, many r e s e a r c h e r s have a t t e m p t e d t o improve t h e s y n t h e s i s of m e t h o t r e x a t e . Among t h e more s u c c e s s f u l a t t e m p t s are t h o s e by Taylor The r e s p e c t i v e methods and by P i p 2 r and Montgomery. are o n t l i n e d as f o l l o w s : Taylor

NC

CHZC1

I DMF

R=N-( g l u t a m y l )

297

ARTHUR R . CHAMEERLIN eta/.

Piper and Montgomery

NH2 NH2

PMABG = N-( p-methylaminobenzoy1)glutamic acid DMAC = N,N-dimethylacetamide

These improved procedures not only y i e l d products t h a t are easier t o purify, but t h e s u b s t i t u t i o n on t h e pyrazine is unambiguous.

4. ----a b i l i t y St
Bulk When stored i n a cappsd, brown b o t t l e a t room temperature, samplings removed over a twelve-month period yielded i n d i s t i n g u i s h a b l e UV and papsr chromatographic d a t a , These r e s u l t s i n d i c a t e t h a t methotrexate is stable f o r a t least one y e a r under these conditions. Solution As d i l u t e s o l u t i o n s ( 0 . 5 m and 0.05 mg/ml) i n p g H 7 .O aqueous sodium bicarbonate maintained a t room temperat u r e i n darkness and under laboratory illumination, a l i q u o t s removed over a 24-hour period y i e l d e d i d e n t i c a l papergrams, within experimental e r r o r s . O t h i s basis, these s o l u t i o n s n were considered stable under t h e s e conditions. 4.2

4.1

298

METHOTREXATE

Under s t r o n g l y a c i d i c aqueous conditions, t h e amide i s s u b j e c t t o hydrolysis, y i e l d i n g N10-methyl-4-amino-4deoxypteroic a c i d and glutarnic a c i d . Under highly a l k a l i n e aqueous conditions, e s p x i a l l y a t e l e v a t e d temperatures, t h e p r i n c i p a l decomposition products a r e N1'-methylfolic acid, N1'-methylpteroic acid, and glutamic a c i d - - a l l as t h e carboxylate i o n s . Photodecompwition of c e r t a i n p t e r i n e s is well documented .I1 However, no s p e c i f i c r e p o r t on t h e photochemistry of methotrexate has been found i n t h e literature.

5. Metabolism
I n man, a very l a r g e p o r t i o n of t h e methotrexate administered is excreted unchanged, 'la i n d i c a t i n g t h a t l i t t l e metabolism of t h i s drug occurs. Johns and Loo" reported t h a t methotrexate is metabolized r a p i d l y by r a b b i t l i v e r aldehyde oxidase, and t h e metabolite w a s i d e n t i f i e d as 4-amino-k-deoxy-7hydroxy-N1O-methylpteroylglut amic acid (7-hydroxy-MTX)

Johns and Valerino13 have observed t h a t , when methotrexate i s incubated with cecal c o n t e n t s from t h e a mouse i n v i t r o , ~-amino-~-deoxy-N1O-methylpteroic c i d i s produced. Levy and Goldman'* have demonstrated t h a t t h i s metabolite can be produced from methotrexate by a c t i o n of carboxypiptidases from s t r a i n s of Psudoxona_t.

6. Methods of Analysis -Table I V p r e s e n t s t h e r e s u l t s from a r e p r e s e n t a t i v e elemental a n a l y s i s of methotrexate ( r e f e r e n c e standard)

6.1 ---- Analysis Elemental

TABLE V

Elemental Analysis of Methotrexate Element

C H N

- h oy -Ter 52.85

Found 52.72

4.88

4.85
24.51

24.66 Adjusted f o r t h e found water

299

ARTHUR R . CHAMEERLIN e t a / .

6.2

Equivalent Weight Determinations

_ _ l _ l I _ _

6.21

Nonaqueous T i t r a t i o n

D e Carnevale e t a1.l d e s c r i b e d a n e q u i v a l e n t weight d e t e r m i n a t i o n based on a sodium methoxide t i t r a t i o n i n p y r i d i n e t o an a z o - v i o l e t end p o i n t . Because t h e b a s i s of t h e t i t r a t i o n i s a n a c i d l b a s e n e u t r a l i z a t i o n , it l a c k s s p e c i f i c i t y . Consequently, t h e method has not been employed widely as a n a s s a y f o r m e t h o t r e x a t e .

6.22 Complex-Formation -----r a t i o n Tit

G u e r e l l o h a s described a second t i t r a t i o n by which e q u i v a l e n t w e i g h t s of m e t h o t r e x a t e samples can be obtain??. The aethod i s basad on t h e complexation between the Ca and t h e glutamyl moiety and t h e r e b y a f f o r d s higher s p e c i f i c i t y than an acidlbase neutralization. Because g l u t a m i c a c i d c o n t a i n i n g i m p u r i t i e s a r e commonly found i n m e t h o t r e x a t e samples, r e s u l t s from t h i s t i t r a t i o n must be i n t e r p r e t e d c a r e f u l l y .

6.3 ---------c a l Assay Biologi 6.31 M i c r o b i o l o g i c a l Assay

S e v e r a l m i c r o b i o l o g i c a l a s s a y s are d e s c r i b e d i n t h e 1 i t e r a t ~ r e . l ~ l l are r e l a t i v e l y n o n s p e c i f i c and A very time consuming and t h u s have l i t t l e u s e f u l n e s s f o r routine analyses.

Werkheiser and co-workers have developed an enzymatic a s s a y f o r m e t h o t r e x a t e u s i n g f o l i c acid reductase M e t h o t r e x a t e b i n d s v e r y t e n a c i o u s l y and s t o i c h i o n e t r i c a l l y t o t h i s enzyme and i s determined c o l o r o m e t r i c a l l y by t i t r a t i o n of t h e drug w i t h t h e enzyme.

6.32 - - - - Assay Enzymic ~ -

6.4

P o l a r o g r a p h i c Assay

Asahi has r e p o r t e d p o l a r o g r a p h i c r e d u c t i o n of m e t h o t r e x a t e . H e a t t r i b J t e s t h e f i r s t wave as being t h e r e d u c t i o n t o dihydromethotrexate, t h e second wave a s being t h e r e d u c t i v e c l e a v a g e of t h e CH2-N bond, and t h e t h i r d wave a s being t h e r e d u c t i o n t o 2,4,-diamino-6-methyl-5,6,7,

8-tetrahydropteridine , 300

METHOTREXATE

6.5

S p e c t r o p h------- t r i c A n a l y s i s otome

F l u o r o m e t r i c methods2' have been used w i d e l y i n t h e a n a l y s i s of m e t h o t r e x a t e . The methods a r e based on a n o x i d a t i v e t r a n s f o r m a t i o n of m e t h o t r e x a t e t o the presumed p t e r i n e c a r b o x y l i c a c i d which f l u o r e s c e s i n t e n s e l y . Because many of t h e i m p u r i t i e s commonly found i n m e t h o t r e x a t e a l s o undergo t h e same r e a c t i o n , t h e s e methods l a c k s p c i f i c i t y u n l e s s t h e o x i d a t i o n i s preceded by a s e p a r a t i o n scheme i n which m e t h o t r e x a t e i s i s o l a t e d . 6.52

6.51 --- o r o m e t r i c A n a l y s i s Flu

--l t r a v i o l e t / V i s i b l e A n a l y s i s U

Because commercial m e t h o t r e x a t e s a n p l e s a r e impure, and because t h e o r g a n i c i m p u - i t i e s have W absorpt i o n c h a r a c t e r i s t i c s t h a t a r e similar t o t h o s e of methotrexate, d i r e c t u v / v i s s p s c t r o p h o t o n e t r i c a n a l y s i s i s e n t i r e l y t o o n o n s p e c i f i c t o be of any v a l u e i n t h e q u a n t i t a t i v e a n a l y s i s of m e t h o t r e x a t e . P r a c t i c a l l y a l l t h e contaminants l i k e l y t o be p r e s e n t i n a sample of met h o t r e x a t e- -N1' -me t h y 1p t e r o i c a c i d , and so f o r t h - w m l d i n t e r f e r e w i t h a direct W o r v i s i b l e method.

Nichol and co-workers21 have r e p o r t e d t h e s e p a r a t i o n s of m e t h o t r e x a t e from related compounds on H Whatman No. 1 w i t h p 7.0, 0.1 M sodium phosphate and w i t h pH 5.0, 0.1 M sodium acetate. I n our l a b o r a t o r y , w e have used 0.5% NaHC03 and 0.5% Na2C03 w i t h t h e sane papzr. Balazs and co-workers22 r e p o r t e d a r a p i d a s s a y f o r methotrexate based on ps.per chromatographic s e p a r a t i o n followed by W measurement of t h e i s o l a t e d m e t h o t r e x a t e component. The l i m i t a t i o n s of t h i s a s s a y a r e t h e accuracy of t h e r e f e r e n c e UV v a l u e and t h e r e c o v e r y of m e t h o t r e x a t e from t h e papergram. The molar a b s o r p t i v i t y a t 303 nm f o r m e t h o t r e x a t e i n N / 1 0 NaOH r e p o r t e d by B a l a z s and co-workers i s i n e r r o r ; it should be 24,830. The a s s a y procedure f o r m e t h o t r e x a t e c i t e d i n USP XVIII is s i m i l a r t o t h e a s s a y r e p o r t e d by Balazs et a 1 e x c e p t t h a t t h e r e f e r e n c e s t a n d a r d i s USP m e t h o t r e x a t e

6.61 -Paper

301

ARTHUR R . CHAMEERLIN etal.

which i t s e l f i s impure.

6.62 Thin-layer
Copenhaver and O'Brienz3 have used ionexchange thin-layer chromatography t o s e p a r a t e a number of f o l i c a c i d analogs including methotrexate The cationexchange r e s i n was AG50W-X4, and the developing solvent was 154 Na2HF04'12 H20, p H 8.5 b u f f e r containing 0.1 M mercaptoethanol

6.63 Column
Heinrich and c o - ~ o r k e r sreported t h e ~~ use of Dowex 1-chloride w i t h very d i l u t e HC1 or N@ to separate f o l i c acid and r e l a t e d compounds. Noble described a p u r i f i c a t i o n procedure based on the use of a Dowex 1-acetate w i t h p 3.2, M a c e t a t e b u f f e r . Oliverio2' H cited t h e use of DEAE c e l l u l o s e and pH 8 phosphate b u f f e r gradient t o s e p a r a t e f o l i c acid analogs. G a l l e l l i and YokoyamaZ7 developed a methotrexate assay procedure e n t a i l i n g a DEAE c e l l u l o s e s e p a r a t i o n followed by a spectrophotometric measurement of t h e i s o l a t e d component.
The use of DEAE cellulose t o separate f o l i c a c i d analogs is w e l l e s t a b l i s h e d . The p r i n c i p a l l i m i t a t i o n s of t h i s method are t h e t i m e required t o pack t h e column and t h e t i m e needed t o develop t h e chromatogram.

Speed Liquid In our work w i t h other f o l i c a c i d antagonists, cat ion exchange high speed l i q u i d chromatography (HSLC) has been very e f f e c t i v e i n separating c l o s e l y r e l a t e d p t e r i d i n e s . Taking advantage of t h i s s e l e c t i v i t y , we have developed a HSLC method of a n a l y s i s f o r methotrexate that is specific, f a s t , and s e n s i t i v e . W use it t o assay e b u l k and formulated methotrexate. The procedure r e q u i r e s a reference methotrexate sample of known purity, which i s used a s an e x t e r n a l reference o r i n conjunction w i t h a n i n t e r n a l reference that, i n our laboratory, has been 2-amino-bmethylpyridine . The column, l m x 3mm 0 .D. Vydac Cation Exchange Packing, i s held a t 55' during t h e developnent w i t h p 4.30, 0 . 1 M KH2P04 a t a f l o w rate of H 1.0 ml/min. The e f f l u e n t is monitored by a UV-detector set a t 254 nm. With t h e d e t e c t o r s e t a t i t s highest s e n s i t i v i t y , s o l u t i o n s a s d i l u t e d a s 1 pg methotrexate/ml can be i n j e c t e d

6.64 High

302

METHOTREXATE

d i r e c t l y and d e t e c t e d . A second procedure employing a l m x 3mm O.D. column packed with reverse-phase phenyl and a mobile phase H of 5% MeOH i n 0.05 M KH2P04, p 7.0, b u f f e r a l s o has been developed and found u s e f u l , The reverse-phase system is less s e n s i t i v e t o minor p v a r i a t i o n s r e s u l t i n g from sample H i m p u r i t i e s . For t h i s reason, t h i s column produces high p r e c i s i o n more r e a d i l y .

6.7 Proton Magnetic Resonance Assay

K t h o t r e x a t e has been assayed by q u a n t i t a t i v e p r on a Varian A-&A spectrometer. Accurately weighed m portions of methotrexate and of an i n t e r n a l standard (2,4-dimethoxy-5-methylpyrimidine) were dissolved i n DMSO, and t h e spectrum was recorded. The psrcentage of anhydrous methotrexate i n t h e sample then was c a l c u l a t e d according t o t h e formula

MTX = Wr x A s x Ws Ar

454.4 - - 142 x 5.

where W r = weight of i n t e r n a l standard used W s = weight of methotrexate sample

A r = i n t e g r a t e d a r e a of i n t e r n a l standard He s i n g l e t (8.02 6 )

As

= i n t e g r a t e d a r e a of methotrexate

H7 proton

(8.64

6)

154.2 = molecular weight of i n t e r n a l standard

454.4 =

molecular weight of anhydrous M X T

P = p u r i t y of t h e i n t e r n a l standard

Although t h e pmr method may lack s e n s i t i v i t y and possibly s p e c i f i c i t y i n complex mixtures, it i s one method of assay t h a t does not r e q u i r e a methotrexate reference sample of known p u r i t y . Thus, it may be u s e f u l i n cases i n which no reference material is a v a i l a b l e .

303

ARTHUR R. CHAMBERLIN etal.

7 , Acknowledgments ---Supported by C o n t r a c t N01-CM-33723 from t h e d i v i s i o n of Cancer Treatment, N a t i o n a l Cancer I n s t i t u t e , N a t i o n a l I n s t i t u t e s of Health, Department of Health, Education, and Welfare. The o p i n i o n s e x p r e s s e d are t h o s e of t h e a u t h o r s and not n e c e s s a r i l y t h o s e of t h e N a t i o n a l Cancer I n s t i t u t e .

The a u t h o r s wish t o acknowledge t h e t e c h n i c a l a s s i s t a n c e r e n d e r e d by Ms. Leslie Pont, M s . Barbara Senuta, Ms. F l o r e n c e Yoshikawa, M r . Martin S t r u b l e and I r John Jee of S t a n f o r d Research I n s t i t u t e .

304

METHOTREXATE

8.

Reference?
1.

2.

3.

Lee, A.P. Martinez, and L . Goodman, J . Med. J 326 (1974) , Chem. l E . J . Pastore, Ann. N.Y. Acad. S c i . -185 43 (1971). 9 U . Ewers, H. Gunther, and L . Jaenicke, Chem. B e r .
W.W.

4.
5.
6.
7.

- 3351 (1973) * 106,

D.R. Seeger, D.B. Coslllich, J . M . Smith, and M.E. Hultquist, J . Am. Chein. SOC. 2, 1753 (1943). A . A l b e r t , D.J. Brawn, and G . Chessman, J . Chem. SOC., 4219 (1952). R.G. Kallen and W.P. Jencks, J . B i o l . Chem. 24,

531.~5(1956)

The United S t a t e s Pharlnacopzia, The XVIII Revision (1970), p. 418. 8. D . R . Seeger, J.M. Smith, and M.E. Hulquist, J . Am. Chem. S O C . 69, 2567 (1947) and Biology of P t e r i d i n e s , 9 . E .C. Taylor,'Chernistry Proceedings of the Fourth I n t e r n a t i o n a l SympDsium on P t e r i d i n e s , Toba, 19s9, I n t e r n a t i o n a l Academic P r i n t i n g Co., Ltd., Tokyo, 1970, p. 79. 10* J . R . Pipzr and J . A . Montgoaery, J . H e t . Chem. 273 (1974) * 11. R.L. B l a k e l y , The Biochemistry of F o l i c Acid and Related P t e r i d i n e s , Nort h-Holland Pub1i s h i n g Compnny, London, 1959, p. 77. l l a . I b i d p.495. 56, 12. D.G. Johns and T.L. Loo, J . Pharm. S c i . - 356

1, 1

13. D.G. Johns and D.M. Valerino, Ann. N.Y. Acad. S c i .

(1957) *

- 384 (1971). 186,

14.
15.
16.
17.

C.C.

Levy and P. Goldman, J . Biol. Chem. -242,

2933

(1967) *
R.C.D. D e Carnevale, J . Dobrecky, and L.O. Guerello, 113, Rev. F a m . (Buenos Aires) - 15 (1971). L.O. Guerello, Rev. Asoc. Bioquim. Argent. - 33 34,

(1969) *
J.H. H.D. Burchenal, G.B. Waring, R.R. E l l i s o n , and R e i l l y , Proc. SOC. Exp. B i o l . N.Y. i8 603 ', ( 1951) ; A . Z Smolyanskaya and N.N. Agadzhanova, vop. Onkol 13, 58 (1957) Chem. Abst 46, 26036; D.E. Hunt a n r R . F . P i t t i l l o , Cancer R e s . - 1095 28,

.,

(1958)

305

ARTHUR R . CHAMBERLIN etal.

18.

19.
20.

W.C. Werkheiser, S . F . Zakrzewski, and C.A. Nichol, J . Plannacol Exp. Therap. 162 (19.52) Y, Asahi, Yakugaku Zasshi 1570 (1959), Chea. Abst 54, 10593d. S.G. CcGkrabarti and I . Bernstein, C l i n . Chem. 1157 (1959); S . F . Zakrzewski and C.A. Nichol, J . Biol Chem. --- 364 (1953); M.V. Freeman, J . 205, Pharmacol. E X ~ .Therap. --- 1, (1957) and 121, 120,

E,

m,

9,

21.

22. 23. 24. 25.

s,454 (1969) 'V.C. M.K. Heinrich,

357 (1957)

S . F . Zakrewski, and A.D. Welch, Proc. 272 (1953); S.F. Zakrrewski and C . A . Nichol, J . Biol. Chea. 205, 352 (1953). M.K. Balazs, C.A. Anderson, and K-Lim, J . Pharm. sci. 2002 (1968). J.H. Copmhaver and K.L. O'Brien, Anal. Biochem. C.A. Nichol,

154 (1958) *

So. Exp. Biol. Med.

5,

z,

26. 27.

Dewey, and G.W. Kidder, J . Chromatog. - 296 (1959). 2, E.P. Noble, Biochem. Prep. 8, 20 (1961). V.T. Oliverio, Anal. Chem. 263, (1951). J . F . G a l l e l l i and G. Yokoyama, J . Pharm. S c i .

3,

55,

306

METHYCLOTHIAZLDE

James A . Raihle

JAMES A. RAIHLE

Contents
1.

Description 1.1 Nomenclature 1.11 Chemical Names 1.12 Generic Name 1.13 Trade Names 1.2 Formulae 1.21 Empirical 1.22 Structural 1.3 Molecular Weight 1.4 Elemental Composition 1.5 General Physical Properties 2.1 Infrared Spectrum 2.2 Raman Spectrum 2.3 Nuclear Magnetic Resonance Spectrum 2.4 Ultraviolet Spectrum 2.5 Mass Spectrum 2.6 Melting Range 2.7 Differential Thermal Analysis 2.8 Dissociation Constant 2.9 Solubility 2.10 Crystal Properties Synthesis Stability-Degradation Drug Metabolic Products

2.

3.
4. 5.

6. Methods of Analysis 6.1 Titrimetric Methods 6.11 Argentimetric Titration 6.12 Potentiometric Titration 6.2 Chromatographic Methods 6.21 Column Chromatography 6.22 Paper and Thin-Layer Chromatography 6.3 Spectrophotometric Methods 6.4 Polarographic Method

7. References

308

METHYCLOTH lAZl DE

Methyclothiazide

1 .

Description

1.1 Nomenclature
1.11 Chemical Name

amide 1,l-dioxide.

Methyclothiazide is 6-chloro-3-(chloromethyl) -3,4-dihydro-2-methy1-2H-1,2,4-benzothiadiazine-7-su1fon(1) It is also known as 6-chloro-3-

ch1oromethy1-3,4-dihydro-2-methy1-7-su1famoy1-1,2,4-benzothiadiazine lyl-dioxide; 6-chloro-3-chloromethyl-2-methyl7-sulfamyl-3,4-dihydro-1,2,4-benzothiadiazine 1,l-dioxide (2) and by many slight variations of the particular nomenclature. The GAS Registry No. is [135-07-91.

1.12 Generic Name Methyclothiazide 1.13 Trade Names Enduro@ 1.2 Formulae 1.21 Empirical gHl 1C12N304S 2 1.22 Structural and Aquatensen@

309

JAMES A. RAIHLE

1.3 Molecular Weight 360.23 1.4

0

Elemental Composition

C 30.00; H 17.77; S 17.80.

3.08; C1

19.68; N

11.66;

1.5 General Methyclothiazide occurs as a white to practically white crystalline powder which is principally odorless. 2. Physi'cal Properties 2.1 Infrared Spectrum (IR)

The infrared spectrum of methyclothiazide (NF Reference Standard, Lot No. 69196) is presented in Figure 1. The spectrum of a KBr pellet is taken on a Perkin-Elmer Model 521 Spectrophotometer. The assignments for the characteristic bands in the IR spectrum are listed in Table I . (3) Table I Characteristic Bands in the IR Spectrum of Methyclothiazide Wavelength (em-l) 3270 and 3360 1595 and 1502 1155 and 1330 (doublet) Characteristic of

NH stretching vibration of sulfonamide group

C =

C stretch of aromatics

S = 0 stretching vibrations of sulfonamide groups

This spectrum is consistent with that published by Fazzari 4 and co-workers. ( )

30 1

2.5

WAVELENGTH 4

(MICRONS) 5

9 10

12

15

FIGURE 1

- INFRARED SPECTRUM
0F METHY CLOTHIAZIDE

JAMES A. RAIHLE

2.2

Raman Spectrum

The raman spectrum of the methyclothiazide reference standard was determined in the solid phase on a Cary Model 83 Spectrophotometer. The assignments of the characteristic bands as shown in Figure 2 are listed in Table 11. (3) Table I1 Characteristic Bands in the Raman Spectrum of Methyclothiazide Wavelength (cm-l)
3270 and 3363 1600 1160

Characteristic of NH stretching vibration of sulfonamide group C = C stretch of aromatics

s = 0 stretching vibrations of sulfonamide groups

2.3

Nuclear Magnetic Resonance Spectrum ("El)

The 60 MHz NMR spectrum of methyclothiazide is presented in Figure 3. The spectrum was determined in deuterated acetone (d6) on a Varian T-60 Spectrometer. Spectral assignments are given in Table 111. (5)

312

AlISN31NI
0
0

0
Qo

0 9

0 el

313

FIGURE 3

- NUCLEAR MAGNETIC RESONANCE

SPECTRUM OF METHYCLOTHIAZIDE

d
8.0

7.0

6.0

5.0

4.0 PPM (6)

3.0

2.0

1.o

METHYCLOTH IAZIDE

Table 111
NMR Assignments for Methyclothiazide

Assignment Aromatic proton at carbon 8 Aromatic proton at carbon 5 Exchangeable protons: NH, NH2 Methyne proton at carbon 3 Methylene protons of chloromethyl group at carbon 3 Methyl protons of 2-methyl group

Number of Protons 1 1 3 1 2

Chemical Shift (ppm) 8.23 7.22 6.87 5.53

Multiplicity
S

Mb ()

T
D

4.07

2.77

2.4 Ultraviolet Spectrum (W)

The UV spectrum of methyclothiazide prepared as a 1 in 100,000 solution in methanol is shown in Figure 4 . The spectrum exhibits three maxima and two minima characteristic of substituted benzothiadiazines. The maxima are at 226 nm (Em = 39,300), 267 nm (Em = 21,250) and ca 311 nm (Em = 3,300). Minima were observed at 240 nm and 290 nm. The spectrum is consistent with previously published re4 ports by Furman ( 6 ) and Fazzari, et. al. ( ) 2.5 Mass Spectrum

The mass spectrum shown in Figure 5 was obtained using an Associated Electrical Industries Model 902 Mass Spectrometer with an ionizing energy of 50 eV and a temperature of 150C. Methyclothiazide yields a spectrum with a base peak at m/e 359. Subsequent fragments, Table IV,
315

JAMES A. RAIHLE

FIGURE 4

- ULTRAVIOLET SPECTRUM
OF METHYCLOTHIAZIDE

Ly

a m
0
VI

=
m

200

250

300

350

WAVE LENGTH (nm)

316

FIGURE 5, MASS 5PEClRUM OF MElHYCLOlHIAZIDE

I
250

'

'

'

'
300

I,,

.I,

.#I ,II

;
350

I,,

317

JAMES A. RAIHLE

reflect the loss of the chloromethyl and sulfonamide groups as well as the fragmentation of the ring system. (7) Table IV High Resolution Mass Spectrum of Methyclothiazide Mass Found 358.9565 309.9711 291.9629 229.9914 166.0280 131.0615 123.9952 96.9847 90.0113 42.0345 2.6 Relative Intensity 3.06
100.00

Error (mu) -0.32 -1.21 1.18 -0.26 -1.78 0.57 -0.22 0.16 0.25 0.13

Composition

- L c !
9 1 1 8 8 8 8 8 6 5 3 2 9 7 7 7 7 3 2 5 4

! 0
3 3 3 2 2 2 1 0 1 1

s
2 2 2 1 0 0 0 0 0 0

c135 2
1

4
4 3 2 0 0 0 0 0 0

0.56
6.02

1
1
1

3.52 5.42 7.89 5.95 6.77 63.94 Melting Range

1 1 1
0

Methyclothiazide melts with rapid decomposition between 225C and 227C. Slight discoloration of the solid may be observed at about 215C.

2.7

Differential Thermal Analysis

The thermogram depicted in Figure 6 shows a large endothermic melt between 224C and 231C. The thermogram shows a subsequent exothermic response confirming the visual observation of rapid decomposition.

318

FIGURE 6

DIFFERENTIAL THERMAL ANALYSIS CURVE OF METHYCLOTHIAZIDE

20

40

140 160 in0 200 1 OC (CORRECTED FOR CHROME1 ALVMEL THERMOCOUPLES) ,

40
80

100

no

220

240

JAMES A. RAIHLE

2.8

Dissociation Constant

The pKa of methyclothiazide was determined by the titrimetric method by extrapolation of data from acetonewater mixed solvents to 100% water. The pKa is 9.4 (proton lost). 2.9 Solubility

The following solubilities have been determined for methyclothiazide at room temperature. Solvent Water Chloroform Benzene n-Butanol PEG-600 Ethano1 Acetonitrile Methanol Acetone Pyridine Solubility 0.06 0.03 < 1 1 1 11 5 > 10 - 200 - 200
N

National Formulary Descriptive Term( 1 ) Very Slightly Soluble Very Slightly Soluble Very Slightly Soluble

mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml

------Slightly Soluble ----

Sparingly Soluble Freely Soluble Freely Soluble

2.10 Crystal Properties The X-ray powder diffraction pattern of methyclothiazide was determined by visual observation of a film obtained with a 143.2 mm Debye-Scherrer Powder Camera. An Enraf-Nonius Diffractis 601 Generator; 38 KV and 18 MA with nicker filtered copper radiation; ) = 1.5418 was , employed. A listing of d-spacings and intensities is presented in Table V. ( ) 8

320

METHYCLOTH IAZIDE

Table V X-Ray Powder Diffraction Pattern d-Spacings and Intensities

dA
9.8 7.75 7.5 7.2 6.25 5.75 5.3 5.1 4.85 4.56 4.42 4.3 4.11 4.00 3.90 3.75B 3.6 3.52 3.44 3.32 3.30 3.12 3.07 3.00
3. Synthesis

111 1 5 50 30 5 20 50 5 10 100 80 20 15 20 2 2 20 3 20 5 5 8 5 8 5

dA 2.95 2.90 2.81 2.72 2.68 2.62 2.51 2.42 2.27 2.24 2.15 2.11 1.99 1.88 1.85 1.81 1.77 1.75 1.73 1.69 1.64 1.5 B 1.35B 1.3 B

I/I1
5 LO 60 3 5 15 10 3 15 10 3 5 8 3 5 8 2 3 3 2 4
4 3 3

Methyclothiazide is synthesized by the reaction sequence shown in Figure 7. Sprague (9) described the reaction of 4-amino-6-chloro-1,3-benzenedisulfonamide with urea to form the 3-keto derivative. Close, et. al. (10) have preferentially alkylated the more acidic cyclic sulfonamide with methyl iodide to form 6-chloro-2-methyl-3oxo-7-sulfamyl-3,4-dihydro-l,2,4-benzothiadiazine 1,ldioxide. This intermediate is readily ring opened by alkaline hydrolysis and then re-cyclized with chloroacetaldehyde to form the desired product.
321

Figure 7 Synthesis of Methyclothiazide

+HpNCNHz
N

z 0=0 z
0

II

+
z
I
N

CH31

322

6
I
N

I 0 I

METHYCLOTH IAZIDE

4.

Stability-Degradation

Methyclothiazide is stable in the solid state and under ordinary ambient conditions. It is rapidly decomposed in boiling acidic solutions to 2-methylsulfamyl-4-sulfarnyl-5chloroaniline. In alkaline solution it rapidly loses one chlorine atom to form the 3-hydroxymethyl analog. This product has been shown to degrade further under severe conditions, however none of the alkaline degradation products contain primary aromatic amine centers. Solutions buffered at pH 4.0 show 22% hydrolysis after 7 days at 60"C, 10% after 28 days at 40"C, but only 2% after 28 days at 25C. Solutions buffered at pH 6.0 gave 1.6% hydrolysis after 7 days at 6OoC, and less than 1% after 28 days at 40C or 25C.
5.

Drug Metabolic Products

No report of metabolic products related to methyclothiazide has been published.

6. Methods of Analysis
6.1 Titrimetric Methods 6.11 Argentimetric Titration The compendia1 procedure for the purity determination of the drug substance is based upon the argentimetric titration of the chloride liberated after reflux in methanolic potassium hydroxide. (1) The equivalent weight is 360.23 since only the chlorine from the 3-chloromethyl group is liberated during reflux. The method is specific since this group is added during the final step of the synthesis and a limit of 0.02% free chloride is imposed on the drug substance. 6.12 Potentiometric Titration Methyclothiazide can be titrated as an acid using either tetra-butylamonium hydroxide in chlorobenzene (11) or potassium hydroxide in isopropanol (12) as the titrant. The solvents are pyridine and acetone, respectively. Methyclothiazide consumes two equivalents of base per
323

JAMES A. RAIHLE

mole of drug substance. The first equivalent is from the neutralization of the free sulfamyl group. The exact location for the reaction of the second equivalent has not been determined, however, it may result from rapid hydrolysis of the 3-chloromethyl function. 6.2 Chromatographic Methods 6.21 Column Chromatography Fazzari (13) has published a collaborative study on a column chromatographic method for the analysis of methyclothiazide from tablets. The drug is eluted from a 0.1 NaHC03-Celite column with chloroform and measured directly at 267 nm by W spectrophotometry. The precision of the method was 99.8 4 1.64% on a commercial preparation of 2.5 mg tablets. 6.22 Paper and Thin-Layer Chromatography Paper chromatography has been applied by Pilsbury and Jackson (14) for the rapid detection and identification of thiazide diuretics in tablets, gastric fluid, and urine. Identification is accomplished by ascending reverse phase chromatography using tributyn treated paper and developing for 20 minutes at 90C with a phosphate buffer (pH 7.4). The thiazides are located by ultraviolet light (254 nm) and confirmed by the red color given by alkaline sodium 1,2-naphthaquinone-4-sulfonate spray reagent. Paper chromatography can also monitor the extent of manufacturing by-products. Ascending chromatography using butanol saturated with 3% ammonium hydroxide and descending chromatography using butano1:acetic acid:water (50:15:60) have been employed. Visualization is by ultraviolet light. Thin-layer chromatography using the system ch1oroform:methanol:ammonium hydroxide (170:30:2 on 0.25 ) mm silica gel GF254 plates and ultraviolet detection rapidly isolates and identifies the common manufacturing intermediate s ,

324

METHYCLOTH IAZIDE

6.3

Spectrophotometric Assays

Methyclothiazide can be determined after acid hydrolysis to 2-methylsulfamyl-4-sulfa~l-5-chloroaniline by a modified Bratton-Marshall procedure. This procedure without prior acid hydrolysis also monitors diazotizable substances in the drug substance. (1) Ultraviolet absorption at 267 nm is seldom directly employed since the intermediates and degradation products have similar absorption spectra. The ultraviolet procedure has been utilized after prior separation by chromatography (13) or for non-specific content uniformity measurements. (1)

6.4 Polarographic Method

The current compendia1 assay for methyclothiazide tablets is a polarographic assay in an aqueous system containing 6% v/v dimethylformamide and 0 . 1 _M tetra-n-butylammonium chloride as the supporting electrolyte. A mercury anode is used in conjunction with the DME since the halfwave potential of methyclothiazide occurs in the region where potassium ions from classical agar electrodes would interfere.
7.

References 1. The National Formulary, 14th Ed., Mack Publishing G o . , Easton, PA (1975). 2. The Merck Index, 8th Ed. , Merck and Go. Rahway, NJ (1968).

, Inc.,

3. Washburn, W., Abbott Laboratories, Personal Comunication.

4 Fazzari, F. R., Sharkey, M. F. , Yaciw, C. A. and . Brannon, W. L., J. Ass. Offic. Anal. Chem., 2,
1154, (1968). 5. Egan, R. S., Abbott Laboratories, Personal Communication.

325

JAMES A. RAIHLE

6. Furman, W. B., J . Ass. Offic. Anal. Chem., 1111, (1968).

51,

7. Mueller, S . , Abbott Laboratories, Personal Communication.

8 Quick, J. E. , Abbott Laboratories, Personal . Communication.

9. Sprague, J. M., Ann. N. Y. Acad. Sci. , 71, 328, (1958).

10.
J. H. , and Versten, M. , J. Am. Chem. SOC. , 8 , 2

Close, W. J., Swett, L. R., Brady, L. E., Short, 1132 (1960).

. 11. Luebke, D 'R., Abbott Laboratories, Personal Communication.

12. Chang, S . L., Acta. Phann Sinica, 7, (1959). (Anal. Abstr., 1, 1909, (1960)).

13. Fazzari, R. , J. Ass. Offic. Anal. Chem., (1973). 14.

56, 677,

Pilsbury, V B., and Jackson, J. V., J. Pharm. . - , 713, (1966). Pharmacol., J 8

326

M ETRONIDAZOLE

Lorraine L. Wearley and Gaylord D. Anthony

LORRAINE L. WEARLEY AND GAYLORD D. ANTHONY

Cantents 1. Description
1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor
2. Physical Properties

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9

3.
4.

Infrared Spectrum Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Mass Spectrum Optical Rotation Melting Range Differential Scanning Calorimetry Thermogravimetric Analysis Solubility

Metabolism Pharmacokinetics 5.1 5.2 5.3 5.4 Titrimetric Analysis Spectrophotometric Analysis Colorimetric Analysis Chromatographic Analysis 5.41 Thin Layer Chromatography 5.42 Gas Chromatography 5.5 Polarographic Analysis

5. Methods of Analysis

6. Synthesis

7. References
8.

Acknowledgments

328

METRON IDAZOLE

1. Description 1.1 Name, Formula, Molecular Weight

Metronidazole is 1-(2-hydroxyethyl) -2-methyl- 5n i troimidazole

N02a
'SHSN303 l4olecular Weight: 171.16

CH2 CHZOH

1.2

ADDearance. Color. Odor Metronidazole is a white t o pale yellow, odorless c r y s t a l l i n e powder.

2.

Physical Properties

2.1

Infrared S p e c t m The infrared absorption spectrum of a metronidazole reference standard compressed i n a KBr p e l l e t is shown i n Figure 1. The following assignmenfs have been made f o r absorption bands i n Figure 1.
Band (an-1)

Assignment
OH s t r e t c h

32 30 3105 1538 6 1375 1078 830

C=CH; C-H s t r e t c h

NO2; N-0 s t r e t c h
C-OH; C-0 s t r e t c h

C-N02; C-N s t r e t c h

329

Figure 1
Infrared Spectrum of Metronidazole

METRON I DAZO LE

2.2 Nuclear Magnetic Resonance Spectrum The M I spectrum of metronidazole in deuterated acetic acid is shown in Figure 2. Below are the assignments of the major signals. Positions of absorption bands are reported as shifts downfield from the signal of the protons in te ramethylsilane which was used as internal standard.

Assignment
, C
\\

155 singlet 241 (triplet) 274 (triplet) 481 (singlet)

- CH3 CH2

--ez OH -

a -, C

$ 2

-cFr2-oH H

2 . 3 Ultraviolet Spectrum

Metronidazole exhibits an absorption maxima at n about 274 run. using 0.1 N sulfuric acid i methanol as solvent. The molar absorptivity in this solvent is 6333. The ult2aviolet absorption spectrum is shown in Figure 3. 2.4 Mass Spectrum The low resolution mass spectrum of metronidazole is shown h3Figure 4. Structure assignments are as follows: !YE 171 154 125 125 41 Assignment
% Relative Intensity

M**(molecular ion)
M-OH

12.5
40 .

M - NO2
M-NOZ-H

22.1 25.7 100

M - CH3CN

331

. . . . 5.0 .
l
00

. . m ). .60 . . . (T . ,
'

. . . . . 7.0 . . . . . . .8.0 . . . . . . . . . . . , . . . . ? . . . . . 91)

tm

'

"

'

'

'

PD

tm

o m

*, *

0 1u

I . . .
ko

I
1

1
74

. . r....l....,

. . . II . . .. . , . . . . l

..

I
I

Figure 2

4.a

..I

. . . . ,

I . . . . ' .

..

....LA

..

Nuclear Magnetic Resonance Spectrum of Metronidazole

Figure 3

u1 traviolet Spectrum of Metronidazole i 0.1 N Sulfuric Acid i Methanol n n

E
1
J
NBMINFIL- MRSSi-

Figure 4
Mass Spectnnn of Metronidazole

METRON IDAZOLE

2.5

Optical Rotation Metronidszole exhibits no optical activity.

2.6 Melting Range Theomelting range given in the USP XIX is 19 5' 163 C. 2.7 Differential Scanning Calorimetry The DSC thermogramoof metronidazole obtained at a heating rate of 20 C/minute is shown in Figuse 5, The endothermic change observed at about 163 C corresponds to the melting of the compound. 2.8 Thermogravimetric Analysis
The TGAof metronidazole obtained a t a heatinq r a t e o f 10C/minute i s shown i n f i g u r e 6. 4

to

2.9

Solubility Solubilities in various solveps at 2S0C are given in the following table. Solvent Water Methanol Ethanol Chlorofo m Heptane Solubility, mg/ml 10.5 32.5 15.4
3.8

co.01

3. Metabolism

Stambaugh et a1.6 found that the major urinary excretioA products of metronidazole in man and 0 - 1 mice were unchanged metronidazole, 1-(2-hyroxyethyl)-2-hydroxymethyl-5-nitroimidazole and their ether glucuronides

335

OaN3 OX3
336

Figure 5
DSC Thennogram of Metronidazole

2 -

.- m c+

E W E R A T W E OC
Figure 6

------

TGA of Metronidazole

LORRAINE L. WEARLEY AND GAYLORD D. ANTHONY

These products represented 70-90% of the total urinary nitro fraction. Minor metabolites were reported to be 1-(2 -hydroxyethyl) 2 -carboxylic acid-5-nitraimidazole and 1-acetic acid-2-methyl-5-nitroimidazole(see Fig. 7). Oxidation of the methyl group of metronidazole appears to occur more facilely than the hydroxyethyl group. Nitro reduction products have not been found in animal or human urine. The major vaginal products in females given oral doses of drug were found by Manthei et a1 to be unchanged drug, and 1-(2 -hydroxyethyl) - 2 -hydroxymethyl- 5-ni troimidazole. These products were also present in the urine. In addition a fluorescent lipophilic product thought to be a cyclized lactone was found.
4.

Phannacokinetics In a study on healthy females receiving single and multiple doses of 200 mg metronidazole tablets, Welling and Monro reported the biological half-life to be 6.2 hr. Serum concentration data fit a one compartment open model. Steady state serum concentrations of metronidazole on a regimen of 200 mg twice daily averaged 7.07 ug/ml maximum and 2.47 ug/ml m i n h . Ings et al, studied the distribution of p4C]jmetronidazole in rats. They found that oral doses were rapidly absorbed from the gastrointestinal tract; and rapidly equilibrated between blood and most tissues. Radioactivity was found to concentrate in the liver, kidney, gastro-intestinal tract and vaginal secretions. The half life of clearance was longest for the gastro-intestinal tract. I.V. doses showed similar distribution.

5.

Methods of Analysis 5.1 Titrimetric Analysis The titration with perchloric acid is the method of choice to assay metronidazole. The sample is dissolved in acetic anhydride, and warmed slightly to effect solution. After cooling, one drop of mlachite green T.S. is added, and the titration with 0.1 N perchloric acid to a yellow-green endpoint is carried out. A blank determination is
338

METRON IDAZOLE

Figure 7: Pathways proposed by Stambaugh et al. for the metabolism of metronidazole in man. (1) metronidazole (2) the corresponding ether glucuronide (R = glucuronide) (3) 1 - (2-hydroxyethy1)2-hydroxymethyl-5-nitroimidazole (4) its corresponding glucuronide (5) 1-acetic acid-2-methyl- S-nitroimidazole (6) 1-(2- hydroxyethyl) - 2-carboxylic acid-5-nitroimidazole

339

L O R R A I N E L. WEARLEY A N D G A Y L O R D D. ANTHONY

performed and any necessary correction is One equivalent of the compound is t i t r a t e d .

5.2 Spectrophotometric Analysis

?ae

Spectrophotometric analysis of metronidazole m y be carried out using 0 . 1 N sulfuric acid in methanol a s the solvent. The ultraviolet absorption maxima is a t about 274 nm. 5.3 Colorimetric Analysis
5.31 Metronidazole can be analyzed colorimetrically by reducing the n i t r o group t o t h e corresponding m i n e , which is subsequently determined by diazotization and coupling w i t h N- (l-naphthyl) - ethylenediamine dihydrochlor ide (Brat tonMarshall Reagent) .11

5.32 A variation of the above method involves the alkaline hydrolysis of the n i t r o group of metronidazole. The nitrous acid produced diazotizes sulfanilamide i n acidic m e d i u m t o form a diazonium s a l t . After coupling with Bratton-Marshall reagent the concentrat i o n is d e t e n i n e d spectrophotmetrically by canparison t o n i t r i t e standards which have been arried through the colorimetric procedure

15

5.4 Chromatographic Analysis 5.41 Thin Layer Chromatography - Several TLC systems and corre onding Rf values are smnarized below. Pi

340

METRONIDAZOLE

Solvent System Acetone

Absorbent Detection Silica Gel

1

E
0.65 0.76

Ch1orofonn:Methanol: Silica Gel Water:Acetic Acid

74:20:4:2

Benzene:methanol: ammonium hydroxide

79:20:1

Silica Gel

0.36

Ch1orofonn:methanol: Silica Gel water:acetic acid 70:24 :4: 2

1.

1, 2

06 .6

Spray Vith 1% aqueous titanium trichloride; heat at 130 C for 3 minutes. Spray with 1% Dimethyln aminobenzaldehyde i 2 N HC1. Saturate plate with t-butyl hypochlorite vapors. Spray with aqueous 1% starch/l% potassium iodide solution. (This spray has been found to be much more sensitive than #1.)
5.42

2.

Gas-Liquid Chromatography - Metronidazole can be chranatographed as the trimethyl silyl derivative. The silyl derivative is prepared by dissolving metronidazole in a 1:l mixture of dimethylfomide and bis (trimethylsilyl)trifluoroacetamide. The silylation reaction is compete in 30 minutes at room temperature.1 Instrumental Conditions Column: 6 ft. glass column packed with 3% OV-1 on Gas Chrom Q Column Temp.: 160' C Carrier: Nitrogen at 70 ml/min Detector: Hydrogen Flame Ionization Retention Time: 4 . 1 minutes

341

LORRAINE L. WEARLEY AND GAYLORD D. ANTHONY

-0.6 V

Figure 8 Polarograph of Metronidazole, 2% Solution in pH 3.8f 0.2 Buffer

342

METRONIDAZOLE

5.5 Polarographic Analysis A suitable polarographic analysis of metronidazole may be carried out at a concentration of approximately 5 ug/ml in a 2% solution of pH 3 . 8 + 0.2 buffer. The scan shown in figure 8 was ohained on such a solution, using differential pulse mode, 3 electrode system. For less concentrated solutions addition of a maxima suppressant may be necessary. Peak maximuni value is approximately -0.23 volts vs saturated calomel ele~trode.~
6.0 Synthesis

2-methyl-imidazole (I) is nitrated by reacting with nitric acid in the presence of sulfuric acid catalyst. The resulting 5-nitro product (11) can then be reacted with either chloroethanol or ethylene oxide to produce 1-(2-hydroxyethy1)-2methyl-5-nitroimidazole (111). See figure 9.

CICH2CH20H or H 2 C ~ F H 2 0

NwH3
CH2CH20H

Figure 9 Synthesis o f Metronidazole

343

LORRAINE L. WEARLEY AND GAYLORD D. ANTHONY

7.

References
1. Damascus, J., Searle Laboratories, personal conmumication. 2. Aranda, E., Searle Laboratories, personal cammicat ion. 3. Hribar, J., Searle Laboratories, personal conmumication. 4. Marshall, S., Searle Laboratories, personal cammication. 5. Aranda, E., Searle Laboratories, personal camnrnicat ion. 6. Stambaugh, J., Feo, L., Manthei, R., J, Phaxm. and Fxp. Therapeutics, Vol. 161, No. 2, 1968. 7. Manthei, R., Feo, L., Stambaugh, J., Wiadmosci Parazytologiczne T. XV, Nr. 3-4, 1969. 8. Welling, P., Monro, A., Arzneim - Forsch (drug Research) 22, Nr. 1 2 (1972). 9. Ings, R., McFadzian J., Onnerod, W., Xenobiotica, 1975, Vol. 5, No. 4, 223-235. 10. USP XIX, p. 327. 11. Bratton, A., Marshall, E., Babbitt, D., Herdrickson, A., J. Biol. Chm., 128, 537, (1939). 12. Lau, E., Yao, C., Lewis, M., Senkwski, B., J. Phaxm. Sci., 58, No. 1, 55, (1969). 13. Quirk, P., Chow,T., Searle Laboratories, personal cammication. 14. Wood, N., Chow, R., Searle Laboratories, personal commmication.

8.

Acknowledgments The authors wish t o express special thanks t o Dr. J. W i t t for the information on synthesis; t o Dr. J. Opperman for his assistance i n preparing the sections on metabolism and pharmacokinetics; to Mr. S. Marshall and Mrs. M. Gerry for generating and interpreting the data on TGA, DSC and Polaror graphy; to M, J. Damascus and Dr. J. Hribar for the information on IR, W, and Mass Spectra; and t o Ms. J. Janik for her secretarial assistance i n preparing t h i s manuscript.

344

NITROFURANTOIN

Donald E. Cadwallader and Hung Won Jun

DONALD E. CADWALLADER AND HUNG WON JUN

T a b l e of Contents 1. Description N a m e , F o r m u l a , and Molecular Weight 1. 1 Appearance, C o l o r , Odor and T a s t e 1. 2

2. P h y s i c a l P r o p e r t i e s 2.1 Ultraviolet S p e c t r a 2.2 Infrared Spectrum 2 . 3 N u c l e a r Magnetic Resonance S p e c t r u m 2.4 Dissociation Constant 2.5 Melting Range 2.6 Crystal Properties 2.61 C r y s t a l Shape 2 . 6 2 C r y s t a l Size 2.63 Hydrates 2.7 Salts 2.8 Solubility 2.81 Solubility in Aqueous Media 2.82 Solubility in Organic Solvents
3 . Synthesis

4. Stability
4.1 4.2 Stability to Light and M e t a l Shelf-Life and S t o r a g e Conditions

5 . Metabolism

6 . Methods of Analysis 6.1 Identification

6.2 6. 3 6.4 Color Reaction T e s t E l e m e n t a l Analysis Chromatographic Systems 6.41 Thin L a y e r Chromatography 6.42 P a p e r Chromatography 6 . 4 3 Column Chromatography

346

NITROFURANTOIN

T a b l e of Contents, continued.

6.5

Quantitative Analysis 6.51 A s s a y of Dosage F o r m s 6 . 5 2 Quantitative D e t e r m i n a t i o n in Biological S a m p l e s

7. B i o p h a r m a c e u t i c s and P h a r m a c o k i n e t i c s 7.1 Absorption

7.2 7. 3

7.4
7.5

Distribution Elimination Bioavailability Pharmacokinetics

8. R e f e r e n c e s

347

DONALD E. CADWALLADER AND HUNG WON JUN

I. Description
1. 1 N a m e , F o r m u l a , and M o l e c u l a r Weight Nitrofurantoin is N - ( 5 -Nitro-2-furfurylidene) -1aminohydantoin; I - ( 5- N i ro-2-furfuryliden a r e the hydantoin. F u r a d a n t i n and Macrodantin m o s t commonly u s e d t r a d e m a r k s ; 11 additional a r e listed in the M e r c k Index (1).

smino)

CH2gHgN4O5

,C

/
0
238.16

Mol. w t . :

1.2 A p p e a r a n c e , C o l o r , Odor and T a s t e Nitrofurantoin i s d e s c r i b e d as lemon-yellow, o d o r l e s s c r y s t a l s o r fine powder, having a b i t t e r t a s t e (1,2).

2. P h y s i c a l P r o p e r t i e s
2.1 U l t r a v i o l e t S p e c t r a Values of E (170, l c m ) in w a t e r a t 367.5 and 265 n m a r e found to be 760 and 540, r e s p e c t i v e l y (3). T h e m o l a r extinction coefficients, at 367 and 265 n m a r e 13,100 and 17, 300, r e s p e c t i v e l y ( 4 ) .

340

NITROFURANTOIN

When t h e U V s p e c t r u m of nitrofurantoin i n 270 d i m e t h y l f o r m a m i d e ( D M F ) w a s s c a n n e d f r o m 260 to 400 n m , t w o maxima o c c u r r e d a t 265 and 367 n m . T h e s p e c t r u m shown in F i g u r e 1 w a s obtained f r o m a solution of 10.00 m g of n i t r o f u r a n t o i n / l i t e r of 270 D M F in w a t e r ( 3 ) . F i g u r e 2 shows the effect of pH on m a x i m a l w a v e I length and E (170, c m ) v a l u e s of n i t r o f u r a n t o i n ( 3 ) . K H 2 P 0 4 - NaOH and b o r i c acid - NaOH buffer s y s t e m s w e r e employed. T h e s p e c t r a l shift w a s n o t due to hydantoin r i n g - opening in the alkaline solution s i n c e the shift w a s r e v e r s i b l e upon reacidification ( 3 ) . 2.2 Infrared Spectrum T h e i n f r a r e d s p e c t r u m of nitrofurantoin (Norwich P h a r m a c a l Reference Standard Purity) a s a m i n e r a l oil m u l l is shown in F i g u r e 3. I n t e r p r e t a t i o n of the s p e c t r u m w a s m a d e by M i c h e l s ( 3 ) using C r o s s , Stevens and Watts ( 5 ) a s a r e f e r e n c e . Table I IR Band A s s i g n m e n t s f o r Nitrofurantoin Wavelength ( m i c r o n s ) 3.05 5.6-5.75 6.6-7.45 10.4 8.05 Assignment NH hydantoin C = 0 d, - n i t r o f u r a n 2,5-disubstituted furan asymmetrical C-0-C symmetrical C-0-C

9. a

349

DONALD E. CADWALLADER AND HUNG WON JUN

1.c

.e

z
Q
K

m
4

NANOMETERS
F i g u r e 1. U l t r a v i o l e t S p e c t r u m of Nitrofurantoin (Coleman 124)
350

NITROFURANTOIN

390

r
-

380

a z

370

360

1 0

F i g u r e 2. U l t r a v i o l e t A b s o r p t i o n of N i t r o f u r a n t o i n as a F u n c t i o n of pH

351

4000 3000
100

2000

1500

1000

900

800

700

F R

80 60

Lu

2 k
v)

2
fi

40

20
0

rcc 5 0

.rl

Figure 3 . Infrared Spectrum of Nitrofurantoin (Perkin Elmer 467)

M
U

a c n

(d

E 5

a,

a,

NITROFURANTOIN

2. 3 N u c l e a r Magnetic R e s o n a n c e S p e c t r u m T h e n u c l e a r magnetic r e s o n a n c e s p e c t r u m f o r nit r ofurantoin ( N o rwic h P h a r m a c a l R e f e r e n c e S t a n d a r d P u r i t y ) in DMSO - d6 containing a t e t r a m e t h y l s i l a n e a s the i n t e r n a l r e f e r e n c e i s shown in F i g u r e 4 ( 3 ) . T h e s p e c t r a l a s s i g n m e n t s a r e presented in T a b l e I1 ( 3 ) . T a b l e I1 NMR S p e c t r a l A s s i g n m e n t s f r Nitr furantoin Band ( p p m , d ) Singlet 4.40 Doublet 7. 15 ( J = 4 ) Doublet 7 . 6 2 ( J = 4 ) Singlet 7 . 8 3 B r o a d Singlet 1 1 . 4 (exchangeable) 2.5 2 . 4 Dissociation Constant M i c h e l s ( 3 ) d e t e r m i n e d the pKa of the f r e e a c i d to b e 7 . 0 using t h e method d e s c r i b e d by Stockton and Johnson ( 6 ) . A pKa of 7 . 2 i s a l s o r e p o r t e d f o r n i t r o furantoin ( 1). 2 . 5 Melting Range Nitrofurantoin m e l t s a t 270-272' C.
(1,7).

No. of P r o t o n s 2
1

Assignment hydantoin CH2 f u r a n 3H f u r a n 4H -CH=Nhydantoin NH

1 1 1

solvent

353

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Figure 4. NMR Spectrum of Nitrofurantoin (Varian A60A)

NITROFURANTOIN

2.6 Crystal Properties 2.61 C r y s t a l Shape C r y s t a l l i z a t i o n f r o m diluted a c e t i c a c i d yields needle-like c r y s t a l s (1). 2.62 C r y s t a l S i z e T h e c r y s t a l s i z e of n i t r o f u r a n t o i n h a s been found to affect the d e g r e e of emesis a n d the r a t e s of g a s t r o i n t e s t i n a l a b s o r p t i o n and u r i n a r y excretion following o r a l a d m i n i s t r a t i o n (8). T h e d i f f e r e n t c r y s t a l s i z e s of n i t r o f u r a n t o i n w e r e p r e p a r e d by recrystallization f r o m nitromethane. 2.63 Hydrates It h a s b e e n found that n i t r o f u r a n t o i n c a n e x i s t i n anhydrous and monohydrate f o r m ( 9 ) . Anhydrous n i t r o f u r a n t o i n and p r e v i o u s l y dried n i t r ofurantoin monohydrate b e c o m e h y d r a t e d only a t v e r y high humidity (>92% R . H. ). N i t r o f u r a n toin monohydrate d o e s not l o s e o r gain m o i s t u r e upon s t o r a g e a t v a r i o u s r e l a t i v e humidities i n the r a n g e of 31-9270 R.H. Monohydrate f o r m i s v e r y s t a b l e i n terms of retaining w a t e r a t 5OoC. 2. 7 S a l t s T h e s o d i u m s a l t of nitrofurantoin i s available and is u s e d t o p r e p a r e p a r e n t e r a l f o r m u l a t i o n s ( 1 0 ) . Aqueous solutions of the s a l t a r e v e r y u n s t a b l e .

355

DONALD E. CADWALLADER AND HUNG WON JUN

2 . 8 Solubility 2 . 8 1 Solubility in Aqueous Media

Solubilities of nitrofurantoin in aqueous m e d i a have been r e p o r t e d f o r v a r i o u s t e m p e r a t u r e and pH conditions. T h e s e solubility values a r e p r e s e n t e d in T a b l e 111. Table I11 Solubilities of Nitrofurantoin i n Aqueous Media T e m p e r a t u r e , OC
@

Solubility ( m g / l ) References.

24 24 24 25 30 37 37 37 37 37 45

5 7 d. H 2 0 7 d. H 2 0 1.12 4.8 5 d. H 2 0 7 7.2 d. H 2 0

76. 3 131.1 79.5 190 113.4 154 125 167.8 174. 1 312. 1 374 251.2

11 11 11 1,12 11 13 14 11 11 11 13 11

2 . 8 2 Solubility in Organic Solvents

The solubilities of nitrofurantoin in v a r i o u s organic solvents a r e p r e s e n t e d in T a b l e IV.

356

NlTR 0 FUR A N T 0 I N

T a b l e IV Solubilities of Nitrofurantoin in Organic Solvents:% Solvent Acetone Dime thy lfo r t n a m i d e Ethanol 47.570 70 % Solubility ( m g / l ) 5,100 80 ,000 189 712 5 10

9 570
Glycerin Peanut Oil Polyethylene glycol 300 Propylene glycol 2070

600
20.7 15,100 1,560

;::The solubility values w e r e r e p o r t e d i n R e f e r e n c e s ( 1 ) and (15). T h e t e m p e r a t u r e w a s not indicated.

357

DONALD E. CADWALLADER AND HUNG WON JUN

3.

Synthesis

Nitrofurantoin c a n b e p r e p a r e d by the r e a c t i o n of 1aminohydantoin a s the sulfate (16) and as the hydrochlor i d e (17) with 5 - n i t r o f u r f u r a l diacetate in isopropylalcohol-water media. Details of the production of n i t r o furantoin w e r e d e s c r i b e d by S a n d e r s -- ( 1 8 ) . T h e e t at. r e a c t i o n s c h e m e is shown in F i g u r e 5. Nitrofurantoin w a s a l s o p r e p a r e d by t h e condensation of 1-aminohydantoin with 5-nitro-2-furaldehyde (19). Another method f o r the s y n t h e s i s of nitrofurantoin w a s d e s c r i b e d by J a c k (20). 4. Stability 4. 1 Stability to Light and M e t a l Nitrofurantoin c r y s t a l s and its solutions a r e d i s colored by alkali and by e x p o s u r e to light, and a r e decomposed upon contact with m e t a l s o t h e r than s t a i n l e s s s t e e l and aluminum ( 2 ) . Since n i t r o f u r a n toin solutions a r e photosensitive, all analytical o p e r a tions m u s t b e conducted under subdued light. Nitrofurantoin solutions a r e a l s o e x t r e m e l y s e n s i t i v e to alkali; t h e r e f o r e , a l l g l a s s w a r e m u s t b e analytically clean and d r y f o r a s s a y p r o c e d u r e s . 4 . 2 Shelf-Life and S t o r a g e Conditions S t o r a g e conditions of nitrofurantoin and o r a l suspensions a r e r e c o m m e n d e d t o be in tight, lightr e s i s t a n t c o n t a i n e r s ( 2 ) . Recommended shelf-life of tablets and suspensions i s f i v e y e a r s when s t o r e d a t r o o m t e m p e r a t u r e and in r e g u l a r g l a s s c o n t a i n e r s (21).

358

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359

DONALD E. CADWALLADER AND HUNG WON JUN

When the products a r e packaged in l i g h t - r e s i s t a n t c o n t a i n e r s and s t o r e d a t r o o m t e m p e r a t u r e , the d r u g showed negligible l o s s of potency o v e r a f i v e - y e a r period of t i m e . T h e nitrofurantoin products should not be s t o r e d w h e r e t e m p e r a t u r e s a r e expected to exceed 86'F.

5. Metabolism
Reckendorf -- ( 2 2 ) r e p o r t e d that l a r g e amounts of e t al. nitrofurantoin (30 -50%) of a n o r a l l y and intravenously a d m i n i s t e r e d d o s e w e r e r e c o v e r e d intact in the u r i n e of r a t , dog and m a n . Beutner _ - ( 2 3 ) a l s o found a s i m i l a r r e et at. covery in urine after o r a l administration. T h e s e studies suggest that nitrofurantoin undergoes metabolic t r a n s f o r mation in t h e body to a significant extent. T h e possible metabolic pathways of nitrofurantoin a r e not completely elucidated i n t h e l i t e r a t u r e . However, nitrofurantoin would follow somewhat s i m i l a r pathways in m e t a b o l i s m to that f o r n i t r o f u r a z o n e which undergoes reduction in the n i t r o g r o u p and hydrolysis in the aaomethane linkage.

6. Methods of Analysis
6. 1 Identification
Nitrofurantoin may b e identified by i t s melting point (270-272OC) and by m e a n s of i t s c h a r a c t e r i s t i c infrared spectra (see Figure 3 ) .

6. 2 Color Reaction T e s t
A n u m b e r of c o l o r r e a c t i o n t e s t s f o r the identification of nitrofurantoin has b e e n p r e s e n t e d in a publication ( 2 4 ) .

360

NITROFURANTOIN

6. 3 Elemental A n a l y s i s
T h e r e s u l t s of a n e l e m e n t a l a n a l y s i s of nitrofurantoin (Norwich P h a r m a c a l C o . , Lot. No. E3769) a r e presented in Table V (25). Table V E l e m e n t a l Analysis of Nitrofurantoin Element
C

y Theory o
40. 34 2.54 23.53 33.59

y Found o
40.22 2.57 23.53

H
N
0

6.4

Chromatographic S y s t e m s 6.41 Thin Layer Chromatography T h e following T L C s y s t e m s have b e e n found suitable f o r detection of p o s s i b l e postulated impurities (26). Solvent S y s t e m I Acetone: 90 Glacial Acetic Acid: Methanol 5 5 (v/v)

361

DONALD E. CADWALLADER AND HUNG WON JUN

Rf = 0 . 8 1 on B r i n k m a n Silica Gel plate without f l u o r e s c e n t indicator as visualized by s h o r t wave u l t r a v i o l e t light. It w a s found that the l i n e a r dynamic range w a s 0.25-1.10 p g / s p o t . Solvent S y s t e m I1 Acetone: 80 Benzene: Glacial a c e t i c a c i d 20 1 (v/v)

Rf = 0 . 9 5 on Brinkman Silica Gel plate without f l u o r e s c e n t indicator as visualized by s h o r t - w a v e ultraviolet light and H2SO4 c h a r r i n g . Other T L C p r o c e d u r e s f o r the identification and s e p a r a t i o n of nitrofurantoin have been d e s cribed (27).

6.42 P a p e r Chromatography
T h e u s e of paper chromatography i n the a n a l y s i s of nitrofurantoin and o t h e r n i t r o f u r a n compounds h a s been r e p o r t e d by B r e i n l i c h ( 2 8 ) , who examined s e v e r a l solvent s y s t e m s and methods f o r identification of the s p o t s .

6.43 Column Chromatography

e t al. Bender -- (29) u s e d column c h r o m a t o g r a p h i c p r o c e d u r e s to s e p a r a t e nitrofurantoin f r o m o t h e r components of u r i n e s a m p l e s and s u b sequently d e t e r m i n e d the d r u g concentrations by s p e c t r o p h o t o m e t r i c a s say.

362

NITROFURANTOIN

6 . 5 Quantitative Analysis 6.51 Assay of Dosage F o r m s The methods of analysis that a r e used o r the determination of nitrofurantoin depend on i t s ultraviolet absorption c h a r a c t e r i s t i c s . The U. S. P. XIX ( 3 0 ) d e s c r i b e s a spectrophotometric determination of nitrofurantoin in an acidic 27'0 dimethylformamide -wate r solution. This g e n e r a l method i s applicable to nitrofurantoin in tablet, capsule and suspension dosage f o r m s w h e r e the excipients a r e U V non-absorbing. Under conditions w h e r e separations a r e not made, any drug degradation products a r e reasonably expected to be reflected in the value of the U V ratio; conv e r s e l y , constancy of the U V r a t i o a s compared to the initial formulation value can be taken a s evidence f o r stability (31). A polarographic ( 32) and gravimetric ( 3 3 ) determination of nitrofurantoin in tablets have been described.

6 . 5 2 Quantitative Determination in Biological

Samples Paul -- (34) utilized the ultraviolet abet al. sorption of nitrofurantoin to determine drug concentrations in r a t urine followed by extraction with e t h e r , but the absorption maximum was pH dependent a s indicated by Stoll and c o - w o r k e r s (35).

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DONALD E. CADWALLADER AND HUNG WON JUN

In 1965 Conklin and Hollifield (36) i n t r o duced the nitromethane-Hyamine p r o c e d u r e f o r the d e t e r m i n a t i o n of nitrofurantoin i n u r i n e , and this method h a s been established a s the p r e f e r r e d a s s a y f o r nitrofurantoin in biological s a m p l e s . T h e original p r o c e d u r e h a s been modified to a s s a y the d r u g in whole blood o r p l a s m a (37). T h i s p r o c e d u r e h a s a sensitivity of 2 r g / m l and i s specific f o r nitrofurantoin. However, this method w a s inadequate f o r quantitating nitrofurantoin blood levels in s u b j e c t s who r e ceived n o r m a l therapeutic d o s e s . Mattock, McGilveray and C h a r e t t e (38) improved the n i t r o methane-Hyamine method f o r the determination of nitrofurantoin in blood s a m p l e s s o that s m a l l e r volumes a r e r e q u i r e d ( 0 . 8 ml) and the sensitivity i s g r e a t e r ( 0 . 2 u g / m l ) . A modified nitromethaneHyamine method w a s d e s c r i b e d by Hollifield and Conklin (39) f o r the a s s a y of nitrofurantoin in u r i n e i n the p r e s e n c e of phenazopyridine and i t s metabolites

e t at. Buzard -- ( 4 0 ) developed a c o l o r i m e t r i c method f o r the a n a l y s i s of nitrofurantoin in plasma o r s e r u m of the r a t a f t e r v a r i o u s o r a l d o s e s . J o n e s e t at. (41) d e s c r i b e d a polarographic method f o r the d e t e r m i n a t i o n of nitrofurantoin i n u r i n e . T h e sensitivity l i m i t of a s s a y i s l p g / m l . S e v e r a l a u t h o r s (42, 43) have r e p o r t e d polarog r a p h i c p r o c e d u r e s f o r the a s s a y of the drug.

364

NITROFURANTOIN

A microbiological p r o c e d u r e f o r the a s s a y of nitrofurantoin in u r i n e w a s a l s o d e s c r i b e d by J o n e s e t a l . ( 4 1 ) . Gang and Shaikh (44) u s e d a n indicator o r g a n i s m i n a t u r b i d i m e t r i c method f o r the a s s a y of nitrofurantoin and o t h e r n i t r o f u r a n d e r i v a t i v e s in s e r u m and u r i n e s a m p l e s .

--

A column c h r o m a t o g r a p h i c method f o r the d e t e r m i n a t i o n of nitrofurantoin i n u r i n e w a s r e ported by Bender e t a l . ( 2 9 ) .

Stone (45) and P u g l i s i (46) have m e a s u r e d a t r a c e of nitrofurantoin i n m i l k by the c o l o r i m e t r i c and s p e c t r o pho t o m e t r i c methods , r e s p e c tive 1y Both p r o c e d u r e s a r e b a s e d on t h e conversion of nitrofurantoin to 5 -nitrofurfuraldehyde phenylhyd r a z o n e and a r e followed by the e x t r a c t i o n and concentration on a c h r o m a t o g r a p h i c column. F i n a l d e t e r m i n a t i o n s depend on the development of a blue c o l o r by the addition of Hyamine base.

7. Biopharmaceutics and P h a r m a c o k i n e t i c s
7 . 1 Absorption
Nitrofurantoin i s efficiently and rapidly a b s o r b e d a f t e r o r a l a d m i n i s t r a t i o n ( 4 7 ) . Limited d r u g a b s o r p tion o c c u r s when nitrofurantoin is a d m i n i s t e r e d r e c t a l l y (48). T h e f i r s t evidence that a b s o r p t i o n and e x c r e t i o n w e r e affected by d i f f e r e n c e s in p a r t i c l e s i z e of the d r u g w a s published in 1967 ( 8 ) . T h i s paper r e p o r t e d on the r e l a t i o n s h i p of p a r t i c l e s i z e of nitrofurantoin to emesis in dogs and to absorption and u r i n a r y

365

DONALD E. CADWALLADER AND HUNG WON JUN

excretion in man and r a t s . It was found that l a r g e r c r y s t a l s of the drug caused l e s s e m e s i s in dogs and slower absorption in man and r a t s . Thus, i t was concluded that the u s e of l a r g e c r y s t a l s of nitrofurantoin ( M a c r o d a n t i n e could minimize a d v e r s e effects of this drug such a s nausea and vomiting by slowing the r a t e of absorption in the gastrointestinal tract. Nitrofurantoin occasionally causes nausea, vomiting, drowsiness, headache and skin r a s h e s (49). Incidence of these reactions may be reduced to some degree by administration of the drug with food (50,51). Bates and co-workers (52) studied the effect of food on the absorption of nitrofurantoin f r o m c o m m e r cial dosage f o r m s . They found considerably i n c r e a s e d absorption in nonfasting a s compared to fasting subjects.

7 . 2 Distribution
After absorption into the blood circulation, n i t r o furantoin is rapidly distributed into most body fluids (53 ) . During n o r m a l o r a l therapeutic regimen, blood o r plasma levels of the drug a r e usually v e r y low, in the neighborhood of 1 p g / m l . However, nitrofurantoin levels about 3-5 t i m e s g r e a t e r than this a r e usually found in these fluids when drug i s administ e r ed intravenous Iy o r intramuscularly. Detailed studies of the absorption and distribution of nitrofurantoin have been c a r r i e d out by Buzard e t al. (54) in small animals.

366

NITROFURANTOIN

7. 3 Elimination
The biological half-life of nitrofurantoin in man a p p e a r s to b e 30 min. o r l e s s (55, 56, 57, 22). In clinical studies in human subjects, u r i n a r y drug concentration of 200 to 400 m g / l i t e r have been reported (50, 58). Renal excretion involves glomerular f i l t r a tion and active tubular secretion. S c h i r m e i s t e r --. et a t (59)found that the clearance of nitrofurantoin in human subjects was lower in acid urine than in alkaline urine. During renal impairment, nitrofurantoin u r i n a r y excretion was significantly reduced (60). Conklin and Wagner (61)and Conklin -- (62) e t al. reported that a s much a s 20% of the intravenous dose of nitrofurantoin sodium was excreted in hepatic bile in the dog.

7.4 Bioavailability
Bioavailability of nitrofurantoin has received a considerable attention in recent y e a r s . Cadwallader (63)presented a monograph on the bioavailability of nitrofurantoin in the special APhA Bioavailability pilot project. The general c h a r a c t e r i s t i c s and experimental c r i t e r i a f o r bioavailability testing of nitrofurantoin w e r e discussed. More recently, Meyer and co-workers (64)evaluated the bioavailabilities of 14 commercially available nitrofurantoin p r e p a r a tions by in vivo and -in vitro procedures. They found that some products that m e t the official Compendia1 requirement, w e r e l e s s bioavailable than other p r o ducts tested. E a r l i e r studies by various authors

367

DONALD E. CADWALLADER AND HUNG WON JUN

(65, 66, 67) a l s o r e p o r t e d bioavailability p r o b l e m s a s s o c i a t e d with the u s e of c o m m e r c i a l n i t r o f u r a n t o i n tablets. An updated monograph on the bioavailability of nitrofurantoin w a s r e c e n t l y p r e s e n t e d by Cadwallader (68). 7. 5 P h a r m a c o k i n e t i c s In m a n , p l a s m a levels of nitrofurantoin a p p e a r t o decline exponentially with a half-life v a l u e of about 20 to 30 m i n u t e s (56, 57, 22). U r i n a r y e x c r e t i o n and b i o t r a n s f o r m a t i o n a p p e a r to b e mainly a n d equally r e s p o n s i b l e f o r the elimination of nitrofurantoin. The o n e - c o m p a r t m e n t m o d e l a p p e a r s to be adequate f o r d e s c r i b i n g the k i n e t i c s involved in nitrofurantoin absorption and elimination.

8. R e f e r e n c e s
T h e M e r c k Index, 8 t h e d . , M e r c k and C o . , I n c . , Rahway, N . J . , 1968, p. 738. 2. T h e United S t a t e s P h a r m a c o p e i a , 19th e d . , M a c k Publishing C o . , E a s t o n , Pennsylvania 1975, p. 341. 3. J. G. M i c h e l s , Norwich P h a r m a c a l C o . , P e r s o n a l Communication. 4. V. E g e r t s , J. S t r a d i n s a n d M. S h i m a n s k a , "Analysis of 5 - n i t r o f u r a n D e r i v a t i v e s , t r a n s l a t e d by J. S c h m a r a k , Ann A r b o r S c i e n c e P u b l i s h e r s , Ann A r b o r , Michigan, 1970, p. 83. 5. A. H. J. C r o s s , S. G. E. S t e v e n s , a n d T . H. E. W a t t s , J . Appl. C h e m . , London, 1, 562 (1957). 6. J. Stockton and C. R. Johnson, J. A m , P h a r m . A s s o c . , Sci. E d . , 33, 383 (1944). 7. T h e N i t r o f u r a n s , Vol. 1, "Introduction to the N i t r o f u r a n s , Eaton L a b o r a t o r i e s , Norwich, N. Y. 1958, p. 16. 8 . H. E. P a u l , K . J . H a y e s , M . F. P a u l and A . R. Borgmann, J. P h a r m . S c i . , 882 (1967).
1.

56,

368

NITROFURANTOIN

9.
10.
1 1.

12.

13.

14. 15.

16.

17. 18. 19. 20. 21. 22. 23.

B. G. P a t e l , Norwich P h a r m a c a l C o . , P e r s o n a l Communication. F u r a d a n t i n a Sodium P a r e n t e r a l , Eaton L a b o r a t o r i e s , Norwich, N. Y. L. K. Chen, M.S. T h e s i s , U n i v e r s i t y of G e o r g i a , 1975. H. E. P a u l and M. F. P a u l i n E x p e r i m e n t a l C h e m o t h e r a p y , vol. 11. R. J. S c h n i t z e r and F. Hawking, e d s . , A c a d e m i c P r e s s , New Y o r k , N . Y . , 1964, p. 334. T . R. B a t e s , J . M. Young, C . M. Wu a n d H. A. R o s e n b e r g , J. P h a r m . S c i . , 63, 643 (1974); T . R. B a t e s , SUNY a t Buffalo, P e r s o n a l Communication. M. F. P a u l , R . C. B e n d e r , and E. G. Nohle, A m e r . J . P h y s i o l . , 197,580 (1959). T h e N i t r o f u r a n s , vol. 1, "Introduction to the N i t r o f u r a n s , I ' Eaton L a b o r a t o r i e s , Norwich, N. Y . , 1958, pp. 14-15. A . S w i r s k a , J . L a n g e , and Z . Buczkowski, P r z e m y s l . C h e m . , 11 ( 3 4 ) , 306-308 (1965). Through C. A . , 52, 14079 b (1958). C. J. O'Keefe, Norwich P h a r m a c a l G o . , P e r s o n a 1 C ommunication , H. J. S a n d e r s , R. T . E d m u n d s , and W. B. Stillman, Ind. Enp. C h e m . , 47, 358 (1955). K. H a y e s , U. S. P a t e n t , 2,610, 181 (1952). D. J a c k , J. P h a r m . P h a r m a c o l . , 1 , 1 Suppl., 108 T (1959). J. F. S t a r k , Norwich P h a r m a c a l C o . , P e r s o n a l C ommuni ca ti on. H. K. Reckendorf, R . C a s t r i n g i u s , a n d H . Spingler, Med. Welt, 816 (1963). E. H. B e u t n e r , J. J . P e t r o n i o , H. E. Lind, H. M. T r a f t o n , and M. C o r r e i a - B r a n c o , Antibiotics Ann., 1954-1955, 988 (1955).

15,

369

DONALD E. CADWALLADER AND HUNG WON JUN

24.

25. 26. 27.

28. 29. 30.

31. 32.

33. 34.

35. 36.

V. E g e r t s , J. S t r a d i n s , and M. Shimanska, I'Analysis of 5-Nitrofuran D e r i v a t i v e s , I ' t r a n s lated by J. S c h m a r a k , Ann A r b o r Science P u b l i s h e r s , Ann A r b o r , Michigan, 1970, p. 18. Atlantic M i c r o l a b , Inc. , Atlanta, Georgia. M. J. Cardone, Norwich P h a r m a c a l Co., P e r s o n a l Communication. V. E g e r t s , J. S t r a d i n s , and M. Shimanska, "Analysis of 5-Nitrofuran D e r i v a t i v e s , t r a n s lated by J . S c h m a r a k , Ann A r b o r Science P u b l i s h e r s , Ann A r b o r , Michigan, 1970, pp. 126127. J. Breinlich, Dtsch. Apotheker. Z t g . , 104, 535(1964). R . C . B e n d e r , E . G. Nohle, and M. F . P a u l , Clin. C h e m . , 2, 420 (1956). T h e United S t a t e s P h a r m a c o p e i a , 19th e d . , Mack Publishing Co. , Easton, Pennsylvania, 1975, p. 342. R. A. Boice, Norwich P h a r m a c a l C o . , P e r s o n a l Communication. V. E g e r t s , J. S t r a d i n s , and M. Shimanska, "Analysis of 5-Nitrofuran D e r i v a t i v e s , ' I t r a n s lated by J. S c h m a r a k , Ann A r b o r Science P u b l i s h e r s , Ann A r b o r , Michigan, 1970, pp. 7374. Ibid. , pp. 39-40. H. E. P a u l , F. L. Austin, M. F. P a u l and V. R . E l l s , J. Biol. C h e m . , 180,345 (1949). R . G. Stoll, T. R . B a t e s and J. S w a r b r i c k , J. P h a r m . S c i . , 62, 65 (1973). J . D. Conklin and R . D. Hollifield, Clin. C h e m . , 11, - 925 (1965).

370

NITROFURANTOIN

37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

J . D. Conklin and R. D. Hollifield,

690 (1966).

w.,3,
16,

G. L. Mattock, I. J . McGilveray, and C. Charette, ibid., 820 (1970). R. D. Hollifield and J. D. Conklin, ibid.,

16,

335 (1970).

61,

J. A . B u z a r d , D. M. V r a b l i c , and M. F. P a u l , Antibiotics and Chemotherapy, 6, 702 (1958). B. M. J o n e s , R. J . M. Ratcliffe and S. G. E. S t e v e n s , J. P h a r m . P h a r m a c o l . , 17,525 (1965). T . S a s a k i , P h a r m . Bull. (Tokyo), 2, 104 (1954). H. P. M o o r e and C . R. G u e r t a l , J. A s s o c . Offic. Agr. C h e m i s t s . , 43, 308 (1960). D. M. Gang and K. Z. Shaikh, J. P h a r m . S c i . ,

L. R. Stone, J. A s s o c . Offic. A g r . C h e m i s t s , 44,

462 (1972).

2 (1961).

E. P u g l i s i , J. A s s o c . Offic. A g r . C h e m i s t s ,
30 (1961).

44,

J. 1. J. G.

D. Conklin, R. J. S o b e r s , and D. L. Wagner, P h a r m . S c i . , 58, 1365 (1969). D. Conklin, P h a r m a c o l o g y , S, 178 (1972). C a r r o l l and R . V. Brennan, J. U r o l . , 71, 650

( 1954).

51. 52.

P. F. MacLeod, G. S. R o g e r s , and B. R . Anylowar, Intern. R e c o r d Med. and Gen. P r a c t . C l i n . , 169, -- 5 6 1 (1956). A. G. S m i t h , J . A m . Med. A S S O C . , 195,1061 (1966). T . R. B a t e s , J. A. S e q u e i r a , and A. V. T e m b o , Clin. P h a r m a c o l . T h e r a p . , 63 (1974).

16,

371

DONALD E. CADWALLADER AND HUNG WON JUN

5 3.

M. F. P a u l , H. E . P a u l , R . C. B e n d e r , F. Kopko, C. M. H a r r i n g t o n , V. R . E l l s , and J. A. B u z a r d , Antibiotics and C h e m o t h e r a p y , 10,287

(1960).

54.

J . A. B u z a r d , J . D . Conklin, E. O'Keefe and M. F. P a u l , J . P h a r m a c o l . Exptl. T h e r a p . 131,

38 (1961).

55. 56. 57. 58.

W. M. Bennett, I. S i n g e r , and C. H. Coggins, J. Am. Med. A s s o c . , 214, 1468 (1970). V . D. Kobvletzki. Med. W e l t . .. 19. 2010 (19681. . . , C. M. Kunin, Ann. I n t e r n a l Med. , 1 5 1 (1967). W. A . R i c h a r d , E. R i s s , E. H. K a s s a n d M . Finland, A.M.A. Arch. Internal Med.,
- I

67, 96,

4 3 7 (1955). 59.

60.

61.
62. 63.

J . S c h i r m e i s t e r , F. Stefani, H. Willmann, and W. H a l l o u e r , Antimicrobiol. Agents and C h e m o t h e r a p y , 1965, p. 223. J. S a c h s , T . G e e r , P. Noell, and C. M. Kunin, New Engl. J. M e d . , 91032 (1968). , J . D. Conklin and D. L. Wagner, J. P h a r m a c o l . ,

- 140 (1971). 43,

J . D. Conklin and R . J. S o b e r s and D . L. W a g n e r , B r . J. P h a r m a c o l o g y , 48, 2 7 3 (1973). D. E. C a d w a l l a d e r , "Nitrofurantoin, I f T h e APhA Bioavailability P i l o t P r o j e c t , A m e r i c a n P h a r m a c e u t i c a l Association, Washington, D. C. J u l y 1973 M. C. M e y e r , G. W . A. Slywka, R. E. Dann and P. L . Whyatt, J . P h a r m . S c i . , 63, 1693 (1974). I. J. M c G i l v e r a y , G. L. Mattok, a n d R . D. H o s s i e , J. P h a r m . P h a r m a c o l . , 23, 246 S ( 1 9 7 1 ) .

64. 65.

372

NITROFUR ANT0 IN

66. G. L. Mattok, R . D. H o s s i e , and I. J . McGilveray

67. 68. Can. J. P h a r m . S c i . , 1,8 4 (1972). I. J. McGilveray, G. L. Mattok, and R . D. Hossie Rev. Can. Biol. 32, 99 (1973). D. E. Cadwallader, J. Am. P h a r m . A S S O C . , NS15, 413 (1975).

T h e a u t h o r s w i s h to thank D r . John S t a r k and o t h e r p e r s o n n e l at the Norwich P h a r m a c a l Company f o r t h e i r valuable a s s i s t a n c e and support. T h e excellent secretarial support of M r s . Kay W. Oliver i s acknowledged.

373

PIPERAZINE ESTRONE SULFATE

Zui L. Chang

ZUI

L. CHANG

Contents Analytical Profile 1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, color, Odor 1.3 Elemental Composition 2. Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Raman Spectrum 2.6 Optical Rotation 2.7 Melting Range 2.8 Differential Thermal Analysis 2.9 Solubility 2.10 Crystal Properties 2.11 Dissociation Constant 2.12 Fluorescence 2.13 Hygroscopic Behavior 2.14 Sublimation
3.

Piperazine Estrone Sulfate

Synthesis

4. Stability
5.

Degradation

Drug Metabolic Products and Pharmacokinetics

6. Method of Analysis

6.1 Identification 6.2 Chromatographic Analysis 6.21 Thin-Layer Chromatography 6.22 Gas-Liquid Chromatography 6.3 Spectrophotometric Analysis 6.4 Colorimetric Analysis 6.5 Nitrogen Analysis 6.6 Titration

376

PlPERAZlNE ESTRONE SULFATE

7.
8.

Acknowledgements References

377

ZUI L. CHANG

1. Description 1.1 Name, Formula, Molecular Weight Piperazine estrone sulfate is estra-1,3,5 (10)trien-l7-one, 3-(su1fooxy)-, compound with piperazine (1:1. y 2 )

HO-SO'

II

Piperazine Estrone Sulfate c18H2205 s' C4H10N2 Molecular Weight 436.56

1.2 Appearance, Color, Odor Piperazine estrone sulfate is a white to yellowish white, fine crystalline powder. It is odorless. 1.3 Elemental Composition C-60.53; H-7.39; N-6.42; 0-18.32; S-7.34. 2. Physical Properties 2.1 Infrared Spectrum The infrared spectrum of piperazine estrone sulfate is presented in Figure 1. The spectrum was measured in the solid state as a potassium bromide dispersion. The following bands (cm-1) have been assigned for Figure 1.3 a. 3400-2300 cm-l broad complex of bands due mainly to N-H stretching vibrations of the amine salt.
378

FIGURE 1
2.5 3

- INFRARED

S P E C T R U M O F PIPERAZINE E S T R O N E SULFATE
WAVELENGTH (MICRONS)
4

1 0

12

15

0 4
( D

4000

3500

3000

2 500 FREQUENCY (CM-1)

2000

1500

1000

700

ZUI L. CHANG

b. c. d.

1730 cm-l

characteristic C=O stretching vibration of the 17-keto group.

1600 and 1490 cm-I characteristic skeletal stretching vibrations of the aromatic ring. 1040 cm-l due to S-0 linkage

2.2 Nuclear Magnetic Resonance Spectrum (NMR) The nuclear magnetic resonance spectrum of piperazine estrone sulfate as shown in Figure 2 was obtained on a Varian HA-100 NMR Spectrometer in deuterated dimethylsulfoxide (d6) containing tetramethylsilane as the internal standard. The spectral peak assignments4 are presented in Table I. 2.3 Ultraviolet Spectrum (W) When the W spectrum of 0.1% solution of piperazine estrone sulfate in 0.04% sodium hydroxide solution was scanned from 400 to 210 nm, two maxima and two minima were observed (Figure 3).l The maxima are at 275 nm ( c = 838) and 268 nm ( c = 851). The minima occur at 272 nm and 239 nm. The spectrum was obtained with a Cary Model 14 Recording Spectrophotometer. 2.4 Mass Spectrum The mass spectrum shown in Figure 4 was obtained using an Associated Electrical Industries Model MS-902 Mass Spectrometer with an ionizing energy of 70 eV. The mass spectrum of piperazine estrone sulfate indicates the presence of estrone, piperazine, and a sulfur-oxygen constituent, but it does not yield a molecular ion f o r the complete chemical entity. This is attributed to the following behavior: (a) Dissociation of the amine salt into the free amine (piperazine) and the free acid (estrone hydrogen sulfate).
(b) Possible thermal decomposition of estrone hydrogen sulfate to estrone, S02, OH, etc.

380

FIGURE 2 - NUCLEAR MAGNETIC RESONANCE SPECTRUM OF PIPERAZINE ESTRONE SULFATE

ZUI L. CHANG

Table I
NMR Spectral Assignments for Piperazine

Estrone Sulfate Proton Assignment Chemical Shift (ppm)

Mu 1 tip 1i c i ty

f l
0

7.17

doublet J=9. 5Hz

QIH
0
H

6.92

Mu1 t ipl e t

5.5

Broad

2.89

Singlet

382

PIPERAZINE ESTRONE SULFATE

Table I Cont.
Proton Assignment Chemi c a 1 Shift (ppm)

Mu1 tip 1ic i ty

3.0

Comp 1ex

C -CH,

0.84

Singlet

383

ZUI L. CHANG

0.8

0.7

0.6 =

I
\

FIGURE 3 - ULTRAVIOLET SPECT RUM SPECTRUM OF PIPERAZINE ESTRONE SULFATE SULFATE

0.5

0.4

0.3

0.2

0.1

2 00

250

300

350

WAVELENGTH (nm)

384

FIGURE 4 - MASS SPECTRUM OF PIPERAZINE ESTRONE SULFATE

I '

ZUI L. CHANG

The mass spectrum assignments of the prominent ions and subsequent fragments are shown in Table I1 and Figure 5.5 2.5 Raman Spectrum The Raman spectrum of piperazine estrone sulfate a s shown in Figure 6; was obtained in the solid state on a Cary Model 83 Spectrometer. The following bands (cm-l) have been assigned for Figure 6.3
a.

1733 ane1

due to the C=O stretching of the 17-keto group.

b.
c.

1608 cm-l due to skeletal stretching mode of the aromatic ring. 1050 cm-l due to s-0 linkage

2.6 Optical Rotation A 1% solution of piperazine estrone sulfate in 0.4% sodium hydroxide solution exhibited a rotation of [cu]~~ determined on a Perkin-Elmer Model 141 + 87.8" when Po1ar imeter . 6 2.7 Melting Range Piperazine estrone sulfate melts at about 190C to a light brown, viscous liquid which re-solidifies, on further heatin and finally melts at about 245C with decomposition.

f3

2.8 Differential Thermal Analysis (DTA) The DTA curve obtained on a Dupont Model 900 Analyzer as shown in Figure 7 confirms the observed melting characteristics described in section 2.7. 2.9 Solubility Approximate solubility data obtained at room temperature are given in the following table:'s8

386

PIPERAZINE ESTRONE SULFATE

Table I1 High Resolution Mass Spectrum of Piperazine Estrone Sulfate Found Mass 270.1620 213.1287 185.0960 172.0887 146.0725 86.0843 85.0771 63.9618 47.9669 Calculated Mass 270.1620 213.1279 185.0966 172.0888 146.0732 86.0844 85.0766 63.9619 47.9670
5 - - --C H N O

18 15 13 12
10

22 17 13 12

0 0

2 1

0
0 0

0
0

1
1

LO
10 9
0

0
2 2 0 0

1
0 0 2 1

0 0

4 4
0

0
1

387

ZUI L. CHANG

FIGURE 5

- FRAGMENTATION PATHWAYS OF PIPERAZINE ESTRONE SULFATE

bN>1+*

y ' &
H

HO-S-0 O II

II

II

HO-S-0

m / a 350 not observed

...I-

--

m/a 64

m/a 270

[ so]+'

[..m]+'
m/a 172

[ H o r n ] "
388

m/a 146

FIGURE 6
2000

- RAMAN SPECTRUM OF PIPERAZINE ESTRONE SULFATE

10 40
I

10 80
I

10 60
I

10 20
I

loo0
I

800
I

600
I

400
I

200
I

80 -

80

60 -

- 60 - 40 - 20

0 2000

I 1 I 1 I
I
I

10 80

10 60

10 40

10 20

l0 o0

800

600

400

200

RAMAN SHIFT ACM-

FIGURE 7

DIFFERENTIAL THERMAL ANALYSIS CURVE OF PIPERAZINE ESTRONE SULFATE

Ly

A -

s
(D

I -

d '

0 n

v -

1
0

I
450

50

100

150

200

250

300

3 50

400

T " C (CORRECTED FOR CHROME1 ALUMEL THERMOCOUPLES)

PlPERAZlNE ESTRONE SULFATE

Solvent Water
95% Ethanol

Solubility (mg/ml) 8
7.4

Chloroform Ether Acetone Benzene Methylene dichloride Isopropanol

1
Practically insoluble 0.2 Practically insoluble 1.6 Practically insoluble
57
35

0.1

Sodium hydroxide

Propylene glycol Mineral oil Sesame oil

2.10 Crystal Properties The X-ray powder diffraction pattern of piperazine estrone sulfate was determined by visual observation of a film obtained with a 1 4 3 . 2 mm Debye-Scherrer Powder Camera (Table 111). An Enraf-Nonius Diffractis 601 Generator; 38 KV and 18 MA with nickel filtered copper radiation; ) = 1 . 5 4 1 8 , were employed.9 , 2.11 Dissociation Constant The apparent pKa value of the unprotonated piperazine nitrogen (proton gained) was found to be 3 . 6 , by titration in acetonitrile-water ( 8 0 1 2 0 , v/v) with aqueous sodium hydroxide. Attempts to find systems to extrapolate the pKa to 100% water were unsuccessful. The pKa value of the protonated piperazine nitrogen (proton lost) was found to be 9 . 7 by titration in pyridine-water mixtures with methanolic KOH, and extrapolation to 100% water. 8

391

ZUI L. CHANG

Table 111
X-Ray Powder Diffraction Pattern d-Spacings and Intensities

dA

I/I

dA -

__.

if1
2 2 5

16.5 7.7 7.4 6.6 6.0 5.67 5.23 4.55B 4.38 4.22 4.05 3.86 3.77 3.61 3.54 3.4013 3.22 3.05

10
10

2.98 2.92 2.86 2.73 2.53 2.46 2.37 2.32 2. 28 2.21 2.16B 2.11 2.07B 1.95 1.89 1.85B 1.75B 1.70
392

40 40
20 30

10 5

8 1
5 5 1 5 1

40
20 80 60 5 30
100

8
2 2 3 5
3

5 2 20 5 1

PlPERAZlNE ESTRONE SULFATE

2.12 Fluorescence Piperazine estrone sulfate does not exhibit fluorescent properties in either methanol or in an alkaline aqueous solution. However, it does exhibit fluorescence at 488 nmwhen excited at 465 nm in 65% sulfuric acid solution.6 The strong sulfuric acid converts the piperazine estrone sulfate to estrone which reacts with sulfuric acid to yield the fluorescent species. 2.13 Hygroscopic Behavior Piperazine estrone sulfate was not hygroscopic when e posed to a relative humidity of 40-50% for three weeks.3 Piperazine estrone sulfate does not absorb moisture at 79% relative humidity, and is only very slight1y hygroscopic (0.89%) at 100% relative humidity. 7 2.14 Sublimation Piperazine estrone sulfate did not sublime when it was stored at 1 5 C for one month.8 No evidence of 0' sublimation was noted when it was heated with a hot stage to 204c.7
3.

Synthesis

Piperazipg estrone sulfate was first prepared by Hasbrouck in 1951. The compound can also be prepared from estrone by a fast, complete conversion reaction using a dimethylformamide/ sulfur troxide complex as the sulfating reactant. Excess dimethylformamide is the solvent. Th reaction is completed by the addition of piperazine.'11 Stability-Degradation Piperazine estrone sulfate was found to be stable when refluxed in water for 3 hours. But it degrades completely after 3 hours in refluxing 1 3 hydrochloric acid to yield estrone and piperazine sulfate. It degrades slightly in refluxing 1 sodium hydroxide after 3 hours to yield less than 10% free estrone.12 4.

393

ZUI L. CHANG

Piperazine estrone sulfate yields about 10% free estrone when heated at 105OC for one month. The rate of hydrolysis of piperazine estrone sulfate to estrone at 90C was studied over the pH range of 2.5-9.1. The extent of degradation was determined by a spectrophotometric measurement in 0.1 1 sodium hydroxide solution.13 The hydrolysis of piperazine estrone sulfate to estrone follows first order kinetics with respect to the piperazine estrone sulfate concentration remaining. Futh rmore, the degradation is first order with respect to t' 2 hydrogen ion concentration, resulting in a 10-fold rate increase with each pH unit decrease.13 Some rate cons ants at different pH and temperature values are shown in I? oles IV and V. The activation energy of the degradatit n reaction, Ea (obtained from the slope of the plot of 11 K, as a function of 1/T where T is the absolute temp rature), for three pH levels is shown in Table V. Drug Metabolic Products and Pharmacokinetics The drug substance is hydrolyzed to estrone in acidic media. The known metabolic intercon ersions of the estrone are summarized in Figure 8.1Z Purdy's15 work suggests that estrone, as the sulfate conjugate, is an important transport form of estrone in human plasma. Urinary excretion studies of sodium estrone sulfate have been performed by Twombly and Levitz16, and Brown. 17 Biliary excretion was also studied by Twombly and Levitz.16
A very exhaustive study of urinary and biliary metabolites was described by Jirku and Levitz. 18

5.

Quantitative determination of plasma estrogens by radioimmmunoassay has been developed by Vega. l9

394

PlPERAZlNE ESTRONE S U L F A T E

Table IV First Order Rate Constants of Piperazine Estrone Sulfate as a Function of pH at 90C Initial Concentration mg. /ml. 0.3 0.3
1.5

pH 2.50 3.03

No. of Samp1e s 8
8

kl -

95% Conf. Lim. X lo4, hour 1,830 + 250 -1

480 + 25 396 + 16 -

3.07
3.40

12

1.5 0.3 1.5 1.5 1.5 1.5 1.5

13 7 15 15 15 15 13

3.57 3.88 4.26

+ 130 + 201 59.l

14 16
2.9

22.4 + 1.5 -

4.86
5.16 5.98 6.11 6.48

1.5
1.5 1.5 1.5 1.5 1.5 1.5

14
15

+ 3.63 + 1.85 + 1.12 +

7.51

1.03 .68 .77

.44

.68 + .30 .72 + .17 .42 + .12 -

6.88
7.50 7.90 8.43 9.10

14
13 15 13 12

+ .14 - .11
22 - .08 +

.18+ .11 -

395

ZUI L. CHANG

Table V F i r s t Order Rate C o n s t a n t s and A c t i v a t i o n E n e r g i e s of P i p e r a z i n e E s t r o n e S u l f a t e a t Three pH l e v e l s

pH

2.50

pH

3.03

pH

3.57

kl x

lo4,
90C.
8OOC.
7OOC.

hr.- 1,830 695 262

z +

250 31 15

480 +_ 25 150 + 4.6 52.2 7 2.2 -

130 44.8 14.6

+
f

16 4.0 2.3

Arrhenius R e l a t i o n s h i p Linear C o r r e l a t i o n Coeff. A c t i v a t i o n Energy, Ea 1.000 24,100 .999 27,500 1.000 27,100

396

FIGURE 8

z 0 2! c

Ly

v)

c 4

METABOLIC PATHWAYS OF PIPERAZINE ESTERONE SULFATE

&

c 4

3 -

Ly

Ly

v)

Ly

HO

HO

2 -h yd rox yertrone

HO

\
178 -errradio1

OH

I 0

397
W

(0

HO
2-methoxyestrone estriol

F (

Q E

Ef

X 0

P)

P
HO

M a -h y drox y estrone (also 168-)

HO
15a-hydroxyestradiol 16-epiestriol

ZUI L. CHANG

6. Methods of Analysis
6.1 Identification The presence of piperazine may be identified with or thin-layer chromatography (Section quinone T. S. 6.21). The presence of estrone hydrogen sulfate may be identified by thin-layer chromatography (Section 6.21). The presence of free estrone may be ident with a 2.5% solution of @-naphthol in sulfuric acid thin-layer chromatography (Section 6.21). 6.2 Chromatographic Analysis 6.21 Thin-Layer Chromatography A number of thin-layer chromatographic systems on silica gel have been described for the separation of hydrolysis products of i erazine estrone sulfate from the parent substance. 1 9 6 2f 9 $2 piper azine estrone sulfate chromatographs as the piperazine and estrone sulfate moieties. Various systems,methods of detection and Rf valves are summarized in Table VI.
9

Idied or

6.22 Gas-Liquid Chromatography Piperazine estrone sulfate can not be directly chromatographed. However, the principal degradation product, estrone, may be silylated with BSA and chromatographed using 3% QF-1 on Gas Chrom Qf2, or it may be silylated with a silylating mixture containing N-trimethylsilylimidazole, BSA and trimethylchorosilane in the ratio 1:30:bf, and chromatographed using 10% SE-30 on Chrom0 sorb W AW .

6.3 Spectrophotometric Analysis Direct spectrophotometric analysis of piperazine estrone sulfate is applicable provided significant quantities of interferring contaminents are not present. The drug substance may be examined directly in a methanol solution at 269 nm ( 6 = 860) or in 0.1 - sodium hydroxide N solution.6

398

Table VI
TLC Systems f o r P i p e r a z i n e E s t r o n e S u l f a t e

Solvent System Chloroform: Methanol (5:4) n-Propanol: Ethanol : Conc. Ammonia (2:1:2) Chlorof o m : Methanol (3:1 ) n-Butanol: Water: Conc. Ammonia (1: 1: 1) Chloroform: Methanol: S t r o n g e r Ammonia TS (85:15: 1 )

Rf

Detection
A r senomolybda t e spray

Piperazine Origin 0.42

Rf Estrone Sulfate

Rf Estrone 0.82 0.92

RefePence

0.64
0.78

6
21

S h o r t wave W o r 10% S u l f u r i c Acid i n Ethanol S h o r t wave W o r 10% S u l f u r i c Acid i n Ethanol s h o r t wave W o r 10% S u l f u r i c Acid i n Ethanol I o d i n e Vapor

( 0 D (0

0.03

0.35

0.72

21

ZUI L. CHANG

The estrone content of piperazine estrone sulfate can be determined with a suitable spectrophotometer at 238 nm after the conversion of piperazine estrone sulfate to estrone with the use of hydrochloric acid.l The degradation product, estrone,may be quantitated in the drug substance by a liquid-liquid extraction into chloroform and comparison to an estrone reference standard at 280 nm. Alternately, the chloroform extract may be evaporated and the residue dissolved in 65% suifuric acid solution. The estrone is dehydrated to a species which fluorescence at 488 nm with excitation at 465 nm.6 Colorimetric Analysis Piperazine estrone sulfate may be determined as dehydrated estrone using a phenol-sulfuric acid mixture as the col r development reagent, The chromophore absorbs at 522 nm.40 Piperazine estrone sulfate may also be determined using 65% sulfuric acid as the reagent. The reaction pro-6 duct with sulfuric acid has a yellow chromophore at 4 5 0 nm. These colorimetric methods may be used for the analysis of piperazine estrone sulfate in tablets or creams. The primary degradation product, estrone, can be removed by prior chloroform extraction. Nitrogen Analysis The amount of nitrogen present in piperazine estrone sulfate may be determined by converting the sulfuric nitroqgn to ammonia and titrating with 0.05 acid. Titration Piperazine estrone sulfate may be potentiometrically titrated in pyridine-water mixtures using methanolic potassium hydroxide and glass-calomel electrodes.8
7.

6.4

6.5

6.6

Acknowledgements

The author wishes to thank Mr. V. E. Papendick and Mr. J. A. Raihle for their review of the manuscript, and Miss Sandra Hudson for typing and drawing of the manuscript.
400

ZUI

L. CHANG

8. References 1.

The National Formulary, 14th Revision, Mack Publishing Co., Easton, PA (1974). Chemical Abstracts Service Registry No. 7280-37-7.

2.

3. Washburn, W., Abbott Laboratories, Personal Communication.

4. Cirovic, M., Abbott Laboratories, Personal

Comunicat ion.
5.

Mueller, S., Abbott Laboratories, Personal Communication.

6. Chang, 2. L., Abbott Laboratories, unpublished work.

7. Yunker, M., Abbott Laboratories, Personal Communication.

8. Wimer, D. C., Abbott Laboratories, Personal Communicat ion.

9. Quick, J., Abbott Laboratories, Personal Communication.

10. Hasbrouck, R. B., Assignor to Abbott Laboratories, U. S. Patent 2,642,427. 11. Lex, C. G., Assignor to Abbott Laboratories, U. S. Patent 3,525,738. 12. Williamson, D. E., Abbott Laboratories, Personal communication. 13. Borodkin, S., Abbott Laboratories, Personal Communication. 14. Kohn, F. E . , Abbott Laboratories, Personal Communication.
15.

Purdy, R. H., Engel, L. L., and Oncley, J. L., J. Biol. Chem., 236, 1043, (1961).
401

ZUI L. CHANG

16.

Twombly, G H., and Levitz, M., Am. J. Obstet. and Gynec., 80, 889, (1960).

17. Brown, J. B., J. Obstet, and Gynec. Brit. Emp., - 795, (1969). 66, 18. Jirku, H. and Levitz, M., J. Clin. Endocr., 615, (1969). 19. Vega., S. M., Abbott Laboratories, Personal Communication. 20. Abbott Laboratories, J. Am. Med. Assoc., 149 (5), 443, (1952). 21. Birch, R. A., Dale, B. J. and Earl, J. M., Abbott Laboratories, Personal Communication. 22. Luebke, D. R., Abbott Laboratories, Personal Communication.

2,

402

PROCARBAZINE HYDROCHLORIDE

Richard J. Rucki

RICHARD J. RUCK1

INDEX
Analytical Prof il e

Procarbazine H y d r o c h l o r i d e

1.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor Phys i ca 1 Proper t ies 2.1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 U l t r a v i o l e t Spectrum 2.4 F1uorescence Spectrum 2.5 Mass Spectrum 2.6 O p t i c a l R o t a t i o n 2.7 M e l t i n g Range 2.8 D i f f e r e n t i a1 Scanning C a l o r i m e t r y 2.9 Thermogravi met r ic Anal ys i s 2.10 Solubi 1 i t y 2.11 C r y s t a l P r o p e r t i e s 2.12 D i s s o c i a t i o n Constant Synthesis
S t a b i 1 i t y and Degradation

2.

3.
4. 5.

Drug Metabolic Products Toxi c i t y Methods o f A n a l y s i s 7.1 Elemental A n a l y s i s 7.2 Thin-Layer Chromatographic A n a l y s i s 7.3 D i r e c t Spectrophotometric A n a l y s i s 7.4 Coulometric A n a l y s i s 7.5 Pol arog raph ic Ana 1ys i s 7.6 T i t r i r n e t r i c A n a l y s i s Acknowledgements References

6.
7.

a.
9.

404

PROCARBAZINE HYDROCHLORIDE

1.

Description

1.1

Name, Formula, Molecular Weight P roca r baz i ne hyd roch 1o r i de i s N- i sop ropy 1-a(2-methylhydrazino)-~-toluamid~ y d r o c h l o r i d e . h

Procarbazi ne H y d r o c h l o r i d e C12H19N30-HCl 1.2 Molecular Weight: 257.76

Appearance, Color, Odor White t o p a l e ye1 low c r y s t a l 1 i n e powder w i t h a s l i g h t c h a r a c t e r i s t i c odor.

2.

Phys ic a l P r o p e r t i e s

2.1

I n f r a r e d Spectrum ( 1 R ) The i n f r a r e d spectrum o f procarbazine hydroc h l o r i d e i s presented i n F i g u r e 1 ( 1 ) . The instrument used was a Perkin-Elmer Model 621 G r a t i n g Spectrophotometer. The sample was dispersed i n FluorolubeR t o r e c o r d t h e spectrum i n t h e r e g i o n o f 4000-1340 cml and i n m i n e r a l o i l f o r the r e g i o n o f 1340-400 cm-l. The f o l l o w i n g assignments have been made f o r the bands i n F i g u r e 1 ( 1 ) . Band (cm-) 3277 and 3200 3035 2961 and 2853 2760-2300, main band a t 2725 Assignment
NH s t r e t c h Aromatic CH s t r e t c h A 1 i p h a t i c CH s t r e t c h
NH :

405

FIGURE 1 I n f r a r e d Spectrum of Procarbazine Hydrochloride

25 .

WAVELENGTH (MICRONS) 6 7 8

1 0

1 1 2 4

1 8

22

3550

33NWlllWSNVW %

406

4000

3500

3000

2500

2000

10 70

10 40

I loo

800

500

200

FREQUENCY (CM-1

PROCARBAZINE HYDROCHLORIDE

1556

1660 and 1636 1299

1361 and 1351

C=O stretch (Amide I ) Amide I t Amide I l l

857
2.2

Aromatic CH out-ofp 1 ane bend i ng

Nuclear Magnetic Resonance Spectrum (NMR) The N M R spectrum shown in Figure 2 was obtained by dissolving 50.4 mg o f procarbazine hydrochloride in 0.5 ml of DMSO-d6 containing tetramethylsilane as internal reference. The spectral assignments are shown in Table 1 (2).

Table 1 Chemi ca 1 Shift 6 (ppm) 1.20 2.72 4.23 Coup 1 i ng Constant , J (in Hz)

Protons

Mu1 tip1 ici ty

Doub 1 et Singlet Mu1 ti p let Sing 1 et Doub 1 e t Doublet Doublet Singlet (broad)
407

6.0

--

4.18

6.0

--

10.33

7.55 7.99 8.36 6.33-

8.5

--

8.5 7.0

FIGURE 2

N M R Spectrum o f Procarbazine Hydrochloride

0)

408
1

3 PPM (8)

PROCARBAZINE HYDROCHLORIDE

2.3

U I t r a v i o l e t Spectrum (UV)
The u l t r a v i o l e t spectrum o f procarbazine hydroc h l o r i d e i n the r e g i o n o f 350 t o 200 nm i s shown i n F i g u r e 3. ( 3 ) . The s p e c t r u q e x h i b i t s one maximum a t 232 nm ( E = 1.3 x 10 ) and a m i n i m m a t 213 nm. The s o l u t i o n c o n c e n t r a t i o n was u 0.01 mg/ml i n 0 . 1 N h y d r o c h l o r i c a c i d and t h e q u a r t z c e l l width-was 1 cm.

2.4

FI uorescence Spectrum
E x c i t a t i o n and emission scans were c a r r i e d o u t f o r procarbazine h y d r o c h l o r i d e i n s o l u t i o n s o f 0 . 1 N h y d r o c h l o r i c a c i d , 0.1N sodium hydroxi d e a n 7 water. No fluorescence, however, was observed under these c o n d i t i o n s (2).

2.5

Mass Spectrum The low r e s o l u t i o n mass spectrum shown i n F i g u r e 4 was o b t a i n e d u s i n g a Varian MAT CH5 mass spectrometer, i n t e r f a c e d w i t h a V a r i a n d a t a system, w i t h an i o n i z i n g energy o f 70 eV ( 4 ) . The d a t a system accepted t h e o u t p u t o f t h e spectrometer, c a l c u l a t e d t h e masses, compared t h e i r i n t e n s i t i e s t o the base peak and p l o t t e d t h i s i n f o r m a t i o n as a s e r i e s o f l i n e s whose h e i g h t s were p r o p o r t i o n a l t o t h e intensities. The molecular i o n o f t h e f r e e base was observed a t m/e 221. The H C l moiety was observed a t m/e 36. The ions a t m/e 191, 177 and 163 c o r respond t o a loss from t h e f r e e base o f NHCH 3 NHNHCH , and NHCH(CH ) r e s p e c t i v e l y . The ions a ? m/e 149 and 3j$correspond t o t h e loss o f C H by M c L a f f e r t y rearrangement from m/e I91 an9 177, r e s p e c t i v e l y . The base peak a t m/e 118 r e s u l t s from t h e l o s s o f NHNHCH f r o m
m/e

163.

A h i g h r e s o l u t i o n scan confirmed t h e r e s u l t s of t h e low r e s o l u t i o n spectrum. Table I I l i s t s t h e elemental compositions f o r t h e i o n s as

409

RICHARD J. RUCK1

FIGURE

U l t r a v i o l e t Spectrum o f Procarbazi ne Hydrochlor i de

0.5

0.4 -

z a

0.3-

$ m a

200

250 300 NANOMETERS

35c

410

FIGURE

Mass Spectrum o f Procarbazine Hydrochloride

10 0

90
80
A l l S N 31N I 3A I1 V 138

70
60

411

50
40

30
1 0

20

0 1 1

50

10 0

Iio
m/e

200

2 0

RICHARD J. RUCK1

determined by h i g h r e s o l u t i o n mass spectroscopy (4). Table I I High R e s o l u t i o n Mass Spectrum o f Procarbazine H y d r o c h l o r i d e Found Mass 118.0428 135.0698 149.0753 163.0870 177.1170 191.1288 221.1533 2.6 Calcd. Mass 118.0419 135.0684 149.07 1 5 163.0872

C
8

H
6

N
0

9
9 11

1
2 2

9
11
12

177.11 54 191. I 3 1 1
221.1529 Optical Rotation

15
17

1 1

12

19

Procarbazine h y d r o c h l o r i d e e x h i b i t s no o p t i c a l a c t i v i t y (3). 2.7 Me1 t i ng Range According t o USP X I X , procarbazine hydroc h l o r i d e m e l t s a t about 223"C, w i t h decornp o s i t i o n (5). 2.8 D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC)

DSC s p e c t r a f o r procarbazine h y d r o c h l o r i d e a t a scan r a t e o f 10C/min. e x h i b i t e d an endotherm a t about 23OoC, where m e l t i n g was accompanied by sample decomposition. The endotherm was f o l l o w e d immediately by a slow exotherm (con t inu i n g decompos it ion). The observed temperature o f t h e melting/decomposit i o n endotherm i s dependent upon i n s t r u m e n t a l c o n d i t i o n s and, t h e r e f o r e , i s n o t c h a r a c t e r i s t i c o f t h e compound.
2.9 Thermogravimetric A n a l y s i s (TGA)

A thermogravimetric a n a l y s i s performed on
412

PROCARBAZINE HYDROCHLORIDE

procarbazine h y d r o c h l o r i d e e x h i b i t e d n o l o s s o f w e i g h t from 30-150"C. A t about 150"C, decomposition weight losses began and cont i n u e d t o 500C (upper l i m i t o f i n s t r u m e n t ) . 2.10 Solubi 1 i t y Approximate s o l u b i l i t y data o b t a i n e d a f t e r 3 hours a t 25C a r e g i v e n i n Table I l l (6,7). Table I l l S o l u b i l i t y o f Procarbazine H y d r o c h l o r i d e Solvent Water 95% Ethanol Absolute Ethanol Methanol Ace tone Diethyl Ether Petroleum E t h e r (30-60") C h 1o r o f o r m Benzene 3A A l c o h o l l s o p r o p y l Alcohol Cyclohexane Ethyl Acetate D ioxane Acetonitri l e Carbon Tet rach l o r i d e Methylene C h l o r i d e 2.11 Crystal Properties The x-ray powder d i f f r a c t i o n p a t t e r n of p r o carbazine h y d r o c h l o r i d e i s presented i n Table I V (8). Instrument C o n d i t i o n s Instrument Genera t o r
GE Mode XRD-6 Spect rogon i o meter 50 K V , 2.5 m~ 413

Solubi 1 i t y (mg/ml)
200 27

8
59 4.5
1 <0.1 2 <0.5 12 1 <0.5

<o.

<O. 5
0.7
<O. 5

<0.5
<O. 5

RICHARD J. RUCK1
0

Tube T a r g e t Optics

Goniometer Detector

Recorder Samp 1 es

Copper (Cu Ka = 1.5418 A) 0.1" D e t e c t o r s l i t M.R. S o l l e r s l i t 3" Beam s l i t 0.0007'' N i F i l t e r 4" Take o f f a n g l e Scan a t 0.2" 2 W m i n u t e A m p l i f i e r gain-16 coarse 8.7 f i n e Sealed p r o p o r t i o n a l c o u n t e r t u b e and DC v o l t a g e a t . plateau. P u l s e h e i g h t s e l e c t i o n El 5 v o l t s , Eu o u t . Rate meter T.C. 4, 2000 c / s f u l l scale. Chart speed 1"/5 minutes Prepared by g r i nd i ng a t room temperature. Table I V Procarbazine Hydrochloride
0

20

d (A)+:

- Oft /

''

7.8719 0.24 11.240 7.1380 0.46 12.400 4.9225 0.48 18.020 0.23 18.640 4.7601 0.17 19.800 4.4838 4.3596 0.40 20.370 0.61 21.550 4.1235 22.550 3.9428 0.35 3.7185 0.30 23.930 3.5352 1 .oo 25.190 0.10 3.4530 25.800 0.14 28.800 3.2090 0.04 28.410 3.1415 0.22 29.550 3.0228 0.23 29.650 3.0128 2.6564 0.07 33.740 ;fd ( i n t e r p l a n a r d i s t a n c e ) = nX/2 s i n 0 1 **l/lo = r e l a t i v e i n t e n s i t y ( h i g h e s t i n t e n s i t y = 1.00)

414

PROCARBAZINE HYDROCHLORIDE

2.12

D i s s o c i a t i o n Constant The d i s s o c i a t i o n c o n s t a n t f o r p r o c a r b a z i n e h y d r o c h l o r i d e was determined t i t r i m e t r i c a l l y u s i n g 0.1N sodium hydroxide and a s o l u t i o n i o n i c s t r e n g t h o f 0.1. The v a l u e f o r t h e pKa determined by t h i s method was 6.8 ( 9 ) .

3.

Synthesis Procarbazine h y d r o c h l o r i d e may be prepared by t h e r e a c t i o n scheme shown i n F i g u r e 5. 4-Formylbenz o i c a c i d isopropylamide ( I ) i s combined w i t h m e t h y l h y d r a z i n e i n dimethylformamide t o prepare t h e c o r r e s ponding methylhydrazone ( I I ) , which i s then reduced, u s i n g p a l l a d i u m charcoal c a t a l y s t , t o N - i s o p r o p y l a-(Z-methylhydrazino) -p-toluamide ( I IT). Hydrogen c h l o r i d e i s added t o t h e r e a c t i o n m i x t u r e o f I l l conv e r t i n g i t t o the h y d r o c h l o r i d e o f N-isopropyl-a(2-methyl h y d r a z i no) -e-toluami de ( I VT.

4.

S t a b i 1 i t y and Degradation Procarbazine h y d r o c h l o r i d e , i n t h e presence o f m o i s t u r e o r i n aqueous s o l u t i o n , undergoes o x i d a t i o n by atmospheric oxygen. T h i s a u t o x i d a t i o n has beeg 2 r e p o r t $ $ t o be c a t a l y z e d by metal ions such as M n and Cu (10,ll). The major products o f t h i s o x i d a t i o n a r e N- i sopropyl -a- (2-methylazo) -p- to1 uami de ( I I ) and N-Tsopropyl-a-(2-methyl hydrazof;o)-p-toluamide ( I 1 IT. To a much l e s s e r e x t e n t , t o l u i c a c i d isopropylamide ( I V ) and 4-formylbenzoic a c i d i s o propylamide (V) can a l s o be formed (12,13). The o x i d a t i v e degradation scheme i s shown i n F i g u r e 6. I n a d d i t i o n , procarbazine h y d r o c h l o r i d e i s v e r y s e n s i t i v e t o u l t r a v i o l e t l i g h t (14), n e c e s s i t a t i n g t h e use o f l o w - a c t i n i c glassware d u r i n g analyses. I n closed, amber b o t t l e s a t room temperature, degrad a t i o n o f procarbazine h y d r o c h l o r i d e i n t h e s o l i d s t a t e i s slow, and t h e compound remains s u i t a b l y s t a b l e f o r a t l e a s t t h r e e years (15). A n i t r o g e n atmosphere has been found t o r e t a r d degradation. U t i l i z i n g spectrophotometric (16) and p o l a r o g r a p h i c time s t u d i e s , i t was determined t h a t p r o c a r b a z i n e
415

FIGURE 5
Synthesis o f Procarbazine Hydrochloride

CH3 H I HC-N-CI

IIo

+ C H ~HN - N H Z ---+ I

CH3 H 0 H H;-i-!!OC=N-NCH3 I
I

H I

I CH3

(I)

CH3

CH3 H 0 H H H h - l ! l - ! o C H 2 - N - N - - C H 3I I

+ HCI -HCI

CH3 H 0 H H HC-l!l-: I O C H z - N - N IC H 3 I

I
CH3

(Im

CH3

(nI)

FIGURE 6
Deg rada t ion o f Procarbaz ine Hydroch l o r i de
0-0-0

y 3

0=0

c)l
I"

6F

HC-NH-leCH2-NH-NH-CH3
I
I 2

CH3

(-2H)
I

7
I

HCI
0

(I)
Y LI

I r I

I
Y

\2HI

IN

0-0-0

CH3

I m

0=0 I

0-0-0

CH3

HC-NH-!o
f

CH2-N=N-CH3
I 2
2

0
0

41 7

4 I"

66
(If)
Y

r
0-0-0

HC

CH3

-NH-

II

:oCH=N-NH-CH3

z II r

I"
r z
I

CH3

tm,

m I
I"

0-0-0

o=o

r--0

ni I"

QR

r 0 I z

II

cc

RICHARD J. RUCK1

h y d r o c h l o r i d e degrades r a p i d l y i n a l c o h o l i c media ( i n c l u d i n g a c i d i f i e d and deaerated a l c o h o l ) and more s l o w l y i n aqueous media. S t a b i l i t y was b e s t i n aqueous a c i d and decreased w i t h i n c r e a s i n g pH (13,16).

5.

Drug M e t a b o l i c Products The metabolism o f the t u m o r - i n h i b i t i n g p r o c a r b a z i n e h y d r o c h l o r i d e proceeds according t o a s i m i l a r p a t t e r n i n man, dog and r a t (17). The major m e t a b o l i t e s o f procarbazine h y d r o c h l o r i d e a r e N - i sopropyl -a- (2methyl azo) - to1 uami de (AZO) , NTi sopropy 1 terephtha 1 amic a c i d $AC), methylhydrazin: and carbon d i o x i d e (17-24). A l a r g e p o r t i o n o f t h e drug i s e x c r e t e d i n u r i n e as t h e i n a c t i v e TAC, w h i l e b o t h TAC and t h e a c t i v e m e t a b o l i t e A20 have been detected i n b l o o d (17, 20, 21).

A p h o t o m e t r i c method f o r t h e q u a n t i t a t i v e determinat i o n o f procarbazine h y d r o c h l o r i d e i n blood plasma, u t i l i z i n g e x t r a c t i o n w i t h e t h a n o l / c h l o r o f o r m and o x i d a t i o n o f t h e hydrazine group w i t h f e r r i c y a n i d e , i s described by Raaflaub (17, 25).
The major m e t a b o l i c pathways c o n s i s t o f r a p i d o x i d a t i o n (microsomal and non-microsomal) of p r o c a r bazine t o A20 f o l l o w e d by N2-C cleavage o f A20 t o 4- formy 1ben zo ic ac id isop ropy 1 am ide and me t h y 1 hydrazine. While several authors (17, 20, 26) suggest t h a t cleavage occurs a f t e r AZO i s isomerized t o 1- sop ropy 1 -a- (2-methy 1 hyd razono-2- t o 1 uami de i (HYDRAZONE), B a g g i o l i n i , e t a l . (18) present d a t a which suggests t h a t the major pathway i s d i r e c t cleavage o f AZO. The m e t a b o l i c scheme proposed by the l a t t e r i s presented i n F i g u r e 7. The chemical names o f t h e compounds i n F i g u r e 7 a r e l i s t e d i n Table V.

Although t h e mode o f c y t o t o x i c a c t i o n o f procarbaz i n e h y d r o c h l o r i d e has n o t y e t been c l e a r l y d e f i n e d , t h e r e i s evidence t h a t the drug a c t s by i n h i b i t i o n of p r o t e i n , RNA and DNA s y n t h e s i s (27-29) The N-methyl group o f procarbazine h y d r o c h l o r de and i t s azo m e t a b o l i t e , p o s s i b l y i n t h e form o f a methyl f r e e r a d i c a l (30), has been found t o be essent a1 f o r
418

FIGURE 7
Metabolic Products o f Procarbazine Hydrochloride

CH3-NH-NH2

cm1
I

HC-NH-C I CH3

%3-y (Ip)
C=O

HN=N-CH3

1
(Y)
Oxidation
b y rnr,rosomol
hydroxylase

I N M I = O x i d a t i o n 0th.r
IDH)

than by ( M I

* O l i d o t i o n by N A D - linked d.hydroprnora
Yost

+=

important

atmpa

RICHARD J. RUCK1

b i o l o g i c a l a c t i v i t y as a tumor i n h i b i t o r (30-32). It has a l s o been suggested t h a t hydrogen peroxide, produced d u r i n g procarbazine o x i d a t i o n t o t h e azo compound, may be r e s p o n s i b l e f o r t h e c y t o t o x i c e f f e c t

(33, 3 4 ) .
Table V M e t a b o l i t e s o f Procarbazine H y d r o c h l o r i d e Referred t o i n Figure 7

I.
I I.

1- sopropyl -ai

I II.
IV. V.

(2-methy 1h y d r a z i no) -e-toluamide hydroch l o r i de (procarbazine h y d r o c h l o r i d e ) N- i s o p r o p y l - a - (2-methy lazo) - p - to1 uamide methylhydrazine 4-formylbenzoic a c i d isopropylamide - isopropy 1 tereph tha lami c a c i d N-

6.

Toxicity The major drug t o x i c i t i e s i n acute and c h r o n i c animal s t u d i e s were hematologic w i t h g r a n u l o c y t e depression, thrombocyte depression and anemia. Reticuloendothel i a l system lymphocytic d e p l e t i o n , marrow c e l l depress i o n , t e s t i c u l a r atrophy and mucous membrane u l c e r a t i o n were f u r t h e r evidence of i n v i v o c y t o t o x i t y (35). Leukemia and pulmonary tumors i n mice, and mammary adenocarcinomas i n r a t s , have been observed subsequent to procarbazine h y d r o c h l o r i d e a d m i n i s t r a t i o n i n h i g h doses. The o r a l LD i n mice and r a t s was d e t e r mined t o be 1320 + 66 mg%g and 785 2 3 4 mg/kg, respectively. I n r a b b i t s the o r a l LD was 147 2 11.5 50 mg/kg (35) *

7.

Methods o f A n a l y s i s

7.1

Elemental A n a l y s i s

A t y p i c a l elemental a n a l y s i s o f a sample o f p r o carbazine h y d r o c h l o r i d e i s presented i n Table V I

(36).

420

PROCARBAZINE HYDROCHLORIDE

Table V I

E lementa 1
Element

Ana l y s i s o f Procarbazi ne H y d r o c h l o r i d e

% Theory

2 Found
56.17 7.90
16.29 14.00

H
N

55.91 7.82
16.30

c1
7.2

13.75
Thin-Layer Chromatographic A n a l y s i s

The f o l l o w i n g TLC procedure i s u s e f u l f o r sepa r a t ing N- isop ropy 1 -a- (2-methy 1azo) -p- to1uam i de and E-Tsop ropy 1-a- (2- met hy 1 hyd razono) - p toluamide from procarbazine h y d r o c h l o r i d e (77). The procedure i s used t o e v a l u a t e t h e s t a b i l i t y o f the dosage form. Using s i l i c a g e l G F p l a t e s (about 300 p t h i c k ) and e t h y l a c e t a t e as t h e developing s o l v e n t , the e q u i v a l e n t o f 1 mg of p r o c a r b a z i n e h y d r o c h l o r i d e i n 2.5% (w/v) c y s t e i n e h y d r o c h l o r i d e i n methanol i s s p o t t e d on t h e p l a t e and s u b j e c t e d t o ascending chromatography. After the s o l v e n t f r o n t has ascended about 15 cm, t h e p l a t e i s a i r d r i e d and observed under shortwave u l t r a v i o l e t r a d i a t i o n . The p l a t e i s then sprayed e i t h e r w i t h m o d i f i e d E h r l i c h ' s reagent o r w i t h g l a c i a l a c e t i c a c i d f o l l o w e d by 10% aqueous f e r r ic c h l o r i de: 5% aqueous potass ium f e r r i c y a n i d e (1:l). The approximate R values a r e as f o l l o w s :

- i sopropyl-aN7.3

Procarbazine h y d r o c h l o r i d e (2-methyl hydrazono) 2-toluamide N- isopropy 1-a- (2-methyl azo) -Eto1 uami de

0.0

0.6
0.7

D i r e c t Spectrophotometric A n a l y s i s The procarbazine h y d r o c h l o r i d e c o n t e n t (as base e q u i v a l e n t ) i n capsules may be determined spect r o p h o t o m e t r i c a l l y by u s i n g the f o l l o w i n g p r o cedure. An amount o f capsule f i l l i s mixed and a p o r t i o n o f t h e powder e q u i v a l e n t t o a p p r o x i -

421

RICHARD J. RUCK1

m a t e l y 25 m o f procarbazine base i s weighed. g The powder i s mixed w i t h 100 m l o f 0.1N hydroc h l o r i c a c i d and the m i x t u r e i s f i l t e r F d t h r o u g h d r y f i l t e r paper, d i s c a r d i n g t h e f i r s t 15 m l o f f i l t r a t e . A s u i t a b l e a l i q u o t o f t h e remaining f i l t r a t e i s d i l u t e d w i t h 0.1N h y d r o c h l o r i c a c i d t o g i v e a s o l u t i o n c o n t a i n i L g about 12.5 ug/ml. The absorbance o f the s o l u t i o n i s measured a t 232 2 2 nm u s i n g 0.1N h y d r o c h l o r i c a c i d as r e f erence. The amount o f procarbazine base i s c a l c u l a t e d by comparison t o the absorbance o f .a s o l u t i o n o f p r o c a r b a z i ne h y d r o c h l o r i d e r e f e r e n c e standard s i m i l a r l y prepared and measured ( 3 8 ) .

7.4

Cou 1 ome t r ic Ana 1y s is Procarbazine h y d r o c h l o r i d e can be assayed b o t h as the drug substance and i n the dosage form by c o u l o m e t r i c t i t r a t i o n . Using a p l a t i n u m genera t i n g system and a p o l a r i z e d p l a t i n u m i n d i c a t i n g system, a constant c u r r e n t of 96.5 mA i s a p p l i e d t o the sample i n s o l u t i o n w i t h O.5M potassium i o d i d e s o l u t i o n , b u f f e r e d a t pH 8.x 2 0 . 1 . The t i t r a t i o n r e s u l t s i n the q u a n t i t a t i v e o x i d a t i o n o f t h e hydrazo moiety o f procarbazine hydroc h l o r i d e t o t h e azo group, making t h e method quite specific. Each coulomb o f e l e c t r i c i t y i s e q u i v a l e n t t o 1335 pg of procarbazine hydroc h l o r i d e (39).

A coulometric t i t r a t i o n w i t h e l e c t r o l y t i c a l l y generated bromine has been used t o determine procarbazine h y d r o c h l o r i d e i n capsules (40). T h i s method i s n o t a s s p e c i f i c as t h e above method o f O l i v e r i - V i g h , e t a l . , s i n c e e l e c t r o c h e m i c a l l y generated bromine n o t o n l y o x i d i z e s t h e hydrazine moiety b u t also s u b s t i t u t e s i n t o t h e phenyl r i n g as f o l l o w s (39):

422

PROCARBAZINE HYDROCHLORIDE

(CH3)2-CH-NH-CD

CH2NH-NH-CH 3+4 Br2+2H20

(CH ) - C H - N H - C U 3 2

7.5

Pol arograph ic Ana 1ys is Polarographic a n a l y s i s o f procarbazine hydroc h l o r i d e i s u t i l i z e d as b o t h an i d e n t i t y and an assay method f o r t h e dosage form (5), an i d e n t i t y f o r the drug substance (41), and a s t a b i l i t y t e s t i n g procedure f o r the dosage form (42, 43) and drug substance. I n pH 12 B r i t t o n - R o b i n s o n b u f f e r , the halfwave p o t e n t i a l f o r the o x i d a t i o n o f procarbazine h y d r o c h l o r i d e occurs a t about -0.16V versus a s a t u r a t e d calomel r e f e r e n c e e l e c t r o d e and the d i f f u s i o n c u r r e n t i s proport i o n a l t o c o n c e n t r a t i o n . The p o l a r o g r a p h i c wave i s a t t r i b u t e d t o the o x i d a t i o n o f the h y d r a z i n e t o the azo moiety (-N=N-) and moiety (-NH-NH-) i s , t h e r e f o r e , s p e c i f i c f o r t h e i n t a c t drug substance. A p o l a r o g r a p h i c procedure u t i l i z i n g an e l e c t r o l y t e of aqueous sodium acetate:absolute ethanol ( 2 : l v/v) has a l s o been r e p o r t e d

(13, 44).
7.6
T i t r i m e t r i c Analysis Procarbazine h y d r o c h l o r i d e i s assayed by d i s s o l v i n g t h e sample i n water and t i t r a t i n g w i t h 0.1N sodium hydroxide. The endpoint i s d e t e r minzd p o t e n t i o m e t r i c a l l y , u s i n g a glass-calomel

423

RICHARD J. RUCK1

e l e c t r o d e system. Each m l o f 0.1N sodium hydrox i d e i s e q u i v a l e n t t o 25.78 mg of-C H N OsHCI (5). A l t e r n a t i v e l y , the endpoint m& aetermined v i s u a l l y u s i n g p h e n o l p h t h a l e i n T.S. as T i t r i m e t r i c assay o f proi n d i c a t o r (41, 45). carbazine h y d r o c h l o r i d e has a l s o been r e p o r t e d u s i n g t i t r a n t s o f aqueous s i l v e r n i t r a t e s o l u t i o n and p e r c h l o r i c a c i d i n g l a c i a l a c e t i c a c i d (13, 46). Bromide-bromate, potassium f e r r i c y anide, and n i t r i t e t i t r a t i o n s have been attempted w i t h l i m i t e d success (13). T i t r a t i o n w i t h 0.1N i o d i n e l e d t o unfavorable s t o i c h i o m e t r y , causez by l o c a l i z a t i o n o f excess t i t r a n t d u r i n g the a n a l y s i s (13, 39).

8.

Acknowledgements The a u t h o r wishes t o acknowledge the assistance of t h e Research Records O f f i c e and t h e S c i e n t i f i c L i t e r a t u r e Department o f Hoffmann-La Roche Inc.

424

PROCARBAZINE HY DROCH LORlDE

9.

References

1.
2.

3.
4. 5. 6.

7.
8.
9.
10.

Waysek, E., Hoffmann-La Roche Inc., Personal Communication. Johnson, J.H., Hoffmann-La Roche I n c . , Persona 1 Communication. Corrado, C . , Hoffmann-La Roche Inc., Personal Communication. Benz, W., Hoffmann-La Roche I n c . , Personal Commun ic a t ion. United States Pharmacopeia XIX, p. 410 (1974). Bass, E . , Hoffmann-La Roche Inc., Personal Commun ic a t ion. Uy B a r r e t a , E., Hoffmann-La Roche Inc., Personal Communication. Munroe, M., Hoffmann-La Roche Inc., Personal Commun ic a t i o n . P h i l i p , C . , Hoffmann-La Roche Inc., Personal Commun ic a t i o n . Aebi, H., Dewald, B. and Suter, H., Helv. Chim.

Acta,
11.
12.

48,

656 (1965).
e t al.,

Erlenmeyer, H.,

876 (1964).
13.
14.

HeZv. Chim. Acta, 47,

15.

16.

17.
18.

Carstensen, J.T., Hoffmann-La Roche Inc. Unpubl i s h e d Data. Weber, S. and R i g a s s i , L., Hoffmann-La Roche I n c . , Unpublished Data. G a l l e l l i , J.F., U n i t e d S t a t e s Department of Health, Education and Welfare, Unpublished Data. B o e h l e r t , J., Hoffmann-La Roche Inc. , Unpublished Data. B i l l m e y e r , A., Hoffmann-La Roche Inc., Personal Cornmun i c a t ion. Raaflaub, J. and Schwartz, D.E., Experientia,

21,

19.
20. 21.

M., Dewald, B. and Aebi, H., Biochem. (1969). Reed, D . , Wittkop, J. and Prough, R. , Fed. Proc., - 346 (1970). 29, 0 1 i v e r i o , V.T. , e t a 1. , Cancer Cltemother. Rep.,
Baggiolini,

44 (1965).

PharmacoZ.,

18,2187

42,

1 (1964). Schwartz, D.E.

, ExpeAentia, 3, (1966). 212

425

RICHARD J. RUCK1

22.

23. 24. 25. 26. 27. 28. 29. 30.

C a r t e r , S.K. (ed.), Proc. Chemother. Conf. on Procarbazi ne (Ma t u l ane:NSC-772 13) : Development and A p p l i c a t i o n , Bethesda, Md., 1970, p . 24. B a g g i o l i n i , M. and B i c k e l , M.H., L i f e S c i . , 5, 795 (1966). 432 Adamson, R.H., Ann. N.Y. Acad. Sci., (1971). Raaflaub, J., Hoffmann-La Roche I n c . , Unpublished Data. Berneis, K., e t a l . , HeZv. Chim. Acta, 2157 (1963). , Ezperient&, 23, Rutishauser, A. and Bol l a g , \-I. 222 (1967). W e i t z e l , G., e t a l . , 2. PhysioZ. Chem., 443

179,

46,

S a r t o r e l l i, A.C. and Tsunamura, S., MoZec. Phar2, 275 (1966). Dost, F.N. and Reed, D.J., Biochem. PharmacoZ., - 1741 (1967). 16, Schwartz, D.E., B o l l a g , W. and Obrecht, P.,

( 1967) .

348,

macoz.,

33. 34. 35. 36.

Arzneim.-Forsch., 17, 1389 (1967). K r e i s , W., Proc. Am. Assoc. Cancer Res., 2, 39 ( 1966) . Berneis, K., e t a l . , Expeuientia, 19, 132 (1963).
J e l l i f f e , A.M. and Marks, J. ( e d s . r N a t u l a n (Ibenzmethyzin), B r i s t o l : John W r i g h t and Sons Ltd., 1965, p. 4. Data on F i l e , Hoffmann-La Roche Inc., N u t l e y , New Jersey. S c h e i d l , F., Hoffmann-La Roche Inc., Personal Communication. B o e h l e r t , J . , Hoffmann-La Roche I n c . , Unpubl i s h e d Data. Houghton, R.E., Hoffmann-La Roche Inc., UnDub1 ished Data. O l i v e r i - V i g h , S., e t a l . , J . Pharm. Sci., 1851 (1971). B e r a l , H. and Stoicescu, V., Pharm. ZentraZh., - 469 (1969). 108, Van Dyk, M.A., Hoffmann-La Roche I n c . , Unpubl i s h e d Data. Colarusso, R.J. and Scerbo, A., Hoffmann-La Roche Inc., Unpublished Data.

37.
38.

39.
40.

60,

41.
42.

426

PROCARBAZINE HYDROCHLORIDE

43.

Johnson, J. B. and Venture1 l a , V.S., BUZZ. Parentera2 Drug ASSOC., 2 5 , 239 (1971).

44. 45.

46.

Levin, M., Hoffmann-La R z h e I n c . , Unpubl ished Data. Levin, M. and Mahn, F., Hoffmann-La Roche I n c . , Unpub 1 i shed Data. Kragh, Hoffmann-La Roche I n c . , Unpublished Data.

427

PROMETHAZINE HYDROCHLORIDE

Charles M. Shearer and Susan M. Miller

CHARLES M. SHEARER AND SUSAN M. MILLER

CONTENTS
1.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor 2. Physical Properties 2.1 I n f r a r e d Spectrum 2.2 U l t r a v i o l e t Spectra 2.3 Nuclear Magnetic Resonance Spectrum 2.4 Mass Spectra 2.5 Melting Range 2.6 D i f f e r e n t i a l Scanning Calorimetry 2.7 S o l u b i l i t y 2.8 Crystal P r o p e r t i e s 2.81 X-Ray D i f f r a c t i o n 2.82 O p t i c a l 2.9 Micelle Formation 2.10 I o n i z a t i o n Constant 2.11 Metal Complex and Charge Transfer Complex Formation 2.12 Adsorption and Protein Binding Properties 2.13 Optical A c t i v i t y 2.14 Dipole Moment 2.15 P a r t i t i o n Coefficients 3. Synthesis 4. S t a b i l i t y and Degradation 5. Metabolism and Pharmokinetics 6. I d e n t i f i c a t i o n 7. Methods of Analysis 7.1 Elemental Analysis 7.2 Phase S o l u b i l i t y Analysis 7.3 Direct Spectrophotometric Analysis 7.4 Colorimetric Analysis 7.5 Electrochemical Analysis 7.6 T i t r i m e t r i c Analysis 7.7 Gravimetric Analysis 7.8 Fluorometric Analysis 7.9 Microbiological Analysis 7-10 Separation Methods of Analysis 7.101 Paper Chromatography 7.102 Thin Layer Chromatography 7.103 Gas Chromatography 7.104 Column Chromatography 7.105 Electrophoresis 7.106 Counter Current P a r t i t i o n 8. References

430

PROMETHAZINE HYDROCHLORIDE

1 .

Description 11 .

Name, Formula, Molecular Weight Promethazine hydrochloride is 10H-phenothiazine-102 ethanamine, N,N,d-trimethyl-, monohydrochloride, or 10- t (dimethy1mino)propya phenothiazine, monohydrochloride () 1. Additional names are 10-(2-dimethylamino-2-methylethyl) phenothiazine hydrochloride and N-(2'-dimethylamino-2'methy1)ethylphenothiazine hydrochloride. The most commonly used trade name is Phenergan. The empirical formula is C1,H20N2S-HC1 with a molecular weight of 320.88.

I CH, CH(C5 )N(Ch

*HC1

The CAS Registry number is 60-87-7 for 10H-phenothiazine-10ethanamine, N,N,o(-trimethyl and 58-33-3 for thehydrochloride ealt of this compound. 12 . Appearance, Color, Odor White to faint yellow, practically odorless, cryetalline powder. Slowly oxidizes, and acquires a blue color, on prolonged exposure to air () 1. 2 . Physical Properties 21 . Infrared Spectrum An infrared spectrum of a mineral oil dispersion of promethazine hydrochloride (USP Reference Standard Material) was obtained on a Perkin Elmer Model 467 grating infrared . spectrophotometer ( 3 ) . This spectrum is shown in Figure 1 The Spectral band assignments are listed in Table I This . spectrum compares favorably with that presented by Sunshine and Gerber ( ) in which the promethazine (presumably the 4 hydrochloride salt) is dispersed in a potassium bromide pellet. A mineral oil dispersion is the preferable technique for this material because the possibility exists for salt interchange between the bromide and chloride if potassium bromide is used. Ultraviolet Spectra The ultraviolet spectra of USP Reference Standard promethazine hydrochloride in water is presented (7) as Fig. ure 2 Table I1 gives ultraviolet spectral data obtained on the USP Reference Standard in various solvents. The Merck Index (2) describes the ratio of absorbance by .-.. lo(A249/A298> = 8 0 8 8
431

22 .

WAVELENGTH MICRONS

h)

FREQUENCY (CM-' )

Figure 1

Infrared Spectrum of Promethazine Hydrochloride (USP Reference Standard), Mineral O i l Dispersion

433

WAVE LENGTH (nm)

Figure 2

U l t r a v i o l e t Spectrum of Promethazine Hydrochloride (USP Reference Standard) water Solvent

0

Fc V h

CHARLES M. SHEARER AND SUSAN M. MILLER

Table I I n f r a r e d Spectral A s si gnment s f o r Promethazine Hydrochloride (5, 6) Vibration Mode Wave Number (ern-') CH s t r e t c h i n g (Nujol) 28QO -3000 H+ s t r e t c h i n g 2 200-2480 aromaticCrC S t r e t c h i n g 1591 CH, and % bending 1430-1470 C , bending H 1378 C-N s t r e t c h i n g of 1334 tertiary mine o u t of plane CH bending 850-860, 757 and 731 of d i s u b s t i t u t e d aromatic Table I1 U l t r a v i o l e t S p e c t r a l C h a r a c t e r i s t i c s f o r Promethazine Hydrochloride (7) Solvent water 0.1 N HC1 USP alcohol 2.3 249 297 249 297 253 302
a -

E -

89.9 10.6 89.4 10.6 95.5 11.4

28,770 3,400 28,690 3,400 30,640 3,660

Nuclear Magnetic Resonance Spectrum The nuclear magnetic resonance spectrum of promethazine hydrochloride dissolved i n deuterochloroform containing tetramethylsi lane as the i n t e r n a l standard is presented (8) a s Figure 3. The s p e c t r a l peak assignments a r e l i s t e d i n Table 111.

NMR Spectral Assignments of Promethazine Hydrochloride

Chemical S h i f t 1.50 2.85 3.5-4.3 4.6-5 0 6.8-7.5 2.4

Table I11 Protons

(d)

E 2 I*
N-C&-CH N-%

- -

-2

Sp l i t t i n g doublet sing 1 t e mu1t i p l e t mu1ti p l e t

broad

---

Mass Spectra

The mass spectrum of promethazine hydrochloride (USP Reference Standard Material) was obtained with a MS-902 double focusing, high r e s o l u t i o n mass spectrometer (9). The
434

01

9
Figure 3

- Nuclear Magnetic
Standard)

Resonance Spectrum of Promethazine Hydrochloride (USP Reference Solvent deuterochloroform

S3U /SN3f N/ 3 A / N 7 3 &

436

Figure 4

Mass Spectrum of Promethazine Hydrochloride (USP Reference Standard)

P

a, P

v1

m m

L)

PROMETHAZINE HYDROCHLORIDE

n~/z

probe temperature w a s 15OoC and t h e i o n i z a t i o n e l e c t r o n beam energy was a t 70 eV. High r e s o l u t i o n d a t a were compiled and t a b u l a t e d w i t h t h e a i d of t h e PDP-8 D i g i t a l computer. These d a t a showed t h e molecular i o n (formula C l,H20N2S) t o have a measured mass of 284.1359 compared t o a c a l c u l a t e d mass of 284.1347. Figure 4 i s a b a r graph of t h e mass spectrum, with t h e molecular i o n a t 284. The base peak a t 72 (C H N) a r i s e s from t h e s i d e chain by cleavage of t h e C-C 4 10 bond which i s p t o both t h e s i d e chain n i t r o g e n and t h e Loss of t h i s fragment c r e a t e s t h e h e t e r o c y c l i c nitrogen. peak a t nJ"2 212, and a proton t r a n s f e r before cleavage gen213. Loss of t h e complete s i d e chain r e s u l t s i n erates 198, and e l i m i n a t i o n of s u l f u r from t h e 212 fragment gives t h e o t h e r predominant peak a t d z 180. This spectrum and r o u t e of fragmentation i s i n agreement w i t h t h a t presented by G i l b e r t (10) and Audier (11) and Fales (12). A chemi c a l i o n i z a t i o n spectrum showed t h e parent peak a t M + 1, t h a t i s , dz 285 ( 9 ) .

dz

d~

mJ"

Melting RanPe The observed melting range (13) f o r t h e USP Reference Standard promethazine hydrochloride i s 219.5C-220.50C with decomposition using t h e USP Class I a procedure. The melting range i s increased with increased h e a t i n g r a t e s . The Merck Index (2) r e p o r t s a value of 23OOC with decomposition. D i f f e r e n t i a l Scanning Calorimetry (DSC) The D C thermogram (14) of promethazine hydroS c h l o r i d e (USP Reference Standard) i s shown a s Figure 5. The thermogram w a s obtained a t a h e a t i n g r a t e of 10C/minute i n a n i t r o g e n atmosphere u t i l i z i n g a Perkin-Elmer DSC-2. The thermogram e x h i b i t s no endotherms o r exotherms o t h e r than t h a t a s s o c i a t e d with t h e decomposition melt. Solubility The following s o l u b i l i t y d a t a were obtained a t unc o n t r o l l e d room temperature (7). Solvent ethyl acetate u ab s o 1 t e e t h ano 1 i sopropanol USP ethanol me thano 1 chloroform water

2.5

2.6

2.7

85 9 150 320

335
500

437

I0
Figure 5

220

200

10 8
OC

10 6

Differential Scanning Calorimetry Curve of Promethazine Hydrochloride (USP Reference Standard)

PROMETHAZINE HYDROCHLORIDE

2.8

Crystal Properties

2.81

of promethazine hydrochloride (USP Reference Standard), obtained with a P h i l l i p s diffractometer using C u K a r a d i a t i o n i s presented (14) i n Figure 6. The calculated Itd" spacings (Table I V ) a r e i n good agreement with those obtained by Rajeswaran and Kirk (15).
X-Ray D i f f r a c t i o n P a t t e r n of Promethazine Hydrochloride

X-Ray D i r a c t i o - X-rayf f powdern d i f f r a c t i o n p a t t e r n The

d(Ao 7.85 7.11 6.93 6.61 5.72 5.60 5.23 4.89 4.39 4.23 3.99 3.86 3.80 3.65 3.53 3.45 2.82

*
0.28 0.59 0.32 0.63 0.47 0.54 0.61 0.94 1.00 0.25 0.17 0.52 0.16 0.47
0.46

Table I V

d(Ao
3.40 3.36 3.25 3.18 3.10 3.06 3.03 2.96 2.85 2.80 2.73 2.57 2.51 2.35 2.27

I/Io
0.12 0.10 0.62 0.30 0.38 0.20 0.08 0.06 0.12 0.12 0.12 0.15 0.16 0.07 0.09

0.10

Optical C r y s t a l Properties Promethazine hydrochloride has been described as c o l o r l e s s rods and i r r e g u l a r fragments. I n p a r a l l e l polarized l i g h t the e x t i n c t i o n i s p a r a l l e l and t h e s i g n of t h e elongation i s positive. The r e f r a c t i v e i n d i c e s are: o ( = 1.617, p = 1.691, f = 1.733 a l l ? 0.002 (16). Escobar (17) describes the c r y s t a l l i n e s t r u c t u r e of promethazine hydrochloride. The c r y s t a l l i n e parameters a r e given. The space group i s P2(1)-C with four molecules per u n i t c e l l . Projections of t h e s t r u c t u r e along t h e OZ and OY a x i s a r e illustrated

Micelle Formation Florence and P a r f i t t (18, 19) have determined t h e c r i t i c a l micelle concentration of promethazine hydrochloride by t h e use of nuclear magnetic resonance and by p measurements. Ionic s t r e n g t h H e f f e c t s are a l s o discussed. Stevens (20) discusses t h e
439

2.9

80-

70-

$ 50k !
% 4030

1 1

20

"6

I0

I2

1 ' 4

1 6

I8

20

22 24 213

26

28

30

32 34

k k

Figure 6

- X-Ray

D i f f r a c t i o n P a t t e r n f o r F'romethazine Hydrochloride (USP Reference S t a n d a r d )

PROMETHAZINE HYDROCHLORIDE

e f f e c t s of p and temperature upon t h e c r i t i c a l m i c e l l e conH c e n t r a t i o n . Zografi (21) and V i l a l l o n g a (22) d i s c u s s t h e s u r f a c e a c t i v i t y of promethazine hydrochloride a t t h e a i r solution interface. I o n i z a t i o n Constant The i o n i z a t i o n constant f o r promethazine hydroc h l o r i d e has been reported a s 9.1 using t h e solubi1i;y method i n water (23) and as 9.1 using a potentiometric t i t r a t i o n with varying amounts of methanol i n water as t h e s o l v e n t and e x t r a p o l a t i n g t h e values obtained t o zero percent methanol (24). Metal Complex and Charge Transfer Complex Formation Promethazine hydrochloride has been shownto complex with F e ( I I 1 ) , Co(II1) and Mn(II1) t o produce rose-red colored s o l u t i o n s (25). The u l t r a v i o l e t and v i s i b l e s p e c t r a of t h e c o b a l t complex has been published (26). The complex with Pd(I1) has been used as a method of a n a l y s i s (27). The cobaltous thiocyanate complex with promethazine has been i s o l a t e d and c h a r a c t e r i z e d (28). I n t h e presence of promethazine t h e polarographic wave corresponding t o t h e r e d u c t i o n of oxygen disappears, and a new wave appears a t a more negative p o t e n t i a l . The l a t t e r apparently r e p r e s e n t s t h e reduction of t h e phenothiazine oxygen complex (29). Charge t r a n s f e r complexes of bromine and i o d i n e with promethazine which form i n a c e t o n i t r i l e have been detected by t h e use of wnductometric t i t r a t i o n s (30). I n f r a r e d s p e c t a of t h e i o d i n e complex showed a new band a t 1700-1650 cm-' (30). Association c o n s t a n t s of promethazine with 1 , 4 dinitrobenzene i n carbon t e t r a c h l o r i d e o r chloroform were measured by nuclear magnetic resonance (31). Charge t r a n s f e r s p e c t r a l s t u d i e s of promethazine i n chloroform, a c e t o n i t r i l e and ethanol-acetone, with bromanil, chlora n i l , p-benzoquinone, m-dinitrobenzene and tetracyanoethylene have been reported (32,331. These e l e c t r o n acceptors have a l s o been used a s spray reagents f o r d e t e c t i n g promethazine on t h i n - l a y e r chromatography p l a t e s (34). Adsorption and P r o t e i n Binding P r o p e r t i e s Promethazine hydrochloride i s adsorbed from aqueous s o l u t i o n onto t a l c , k a o l i n , and a c t i v a t e d charcoal (35,361. I t i s bound by carrageenan, f u r c e l l a r e n , carboxymethyl c e l l u l o s e , sodium a l g i n a t e , p e c t i n , gum tragacanth, gum a r a b i c , l o c u s t bean gum, gum agar and quince seed mucilage (37). I t can a l s o bond t o s u r f a c e a c t i v e agents such as polysorbate 80 (38) and sodium polyethylenesulfonate (39). The bonding of 441 2.12 2.10

2.11

CHARLES M. SHEARER AND SUSAN M. MILLER

promethazine hydrochloride t o bovine serum albumin has been studied (40, 41). Optical A c t i v i t y Promethazine as normally synthesized i s a r a c m i c - mixture. The o p t i c a l isomers have been separated by use of dibenzoyl-D-tartaric acid (42). Both t h e (+> and (-1 forme The o p t i c a l obtained i n D t h i e manner decompose a t 220-221OC. activityp] of (+) promethazine i s + 7 . 6 O : t h a t of the (-1 form is -7.6! The t o x i c i t y , antihistaminic a c t i v i t y and c e n t r a l nervous a c t i o n of t h e separated o p t i c a l isomers and t h e racemic promethazine a r e t h e same (42).
D i o l e Moment - ot p rs t h e d i p o l e moment f o r promethaz i n e base a t 25OC as p(D) = 2.05 f 0;Ol. A second paper by

2.13

2.14

t h e same author (44) discusses the conformation of the s i d e chain with r e s p e c t t o t h e r i n g portion of t h e molecule. P a r t i t i o n Coefficients Burger (45) has studied t h e d i s t r i b u t i o n of promethazine hydrochloride between 0.5 N HC1 and various organic phases. The r e s u l t s he obtained a r e given i n Table V. Doyle ( 4 6 ) discusses the e f f e c t of solvent composition on the part i t i o n of amines i n general. Table V P a r t i t i o n Coefficients of Promethazine Hydrochloride between 0.5 N HCL and Vartous Organic Phases (45)

2.15

K = conc. i n 0.5 N HC1

organic phase bu tan0 1 chloroform amyl a c e t a t e ethyl acetate benzene ethyl ether hexane

conc. i n organic phase

K 0.37
0.15 1.14 2.70 5.25 15.20 18.20

Synthesis The most d i r e c t synthesis of promethazine involves the r e a c t i n g of phenothiazine with 2-chloro-l-dimethylaminopropane i n t h e presence of various bases such as sodium hydroxide (47, 48, 491, eodamide (47, 50, 511, potassium hydroxide (48) phenyl l i t h i u m (52) and calcium carbonate (53). This s y n t h e t i c r o u t e i s i l l u s t r a t e d i n Figure 7. The s a l t can then be prepared by t r e a t i n g t h e promethazine base with hydrogen chloride.

3.

442

PROMETHAZINE HYDROCHLORIDE

Figure 7 Synthesis of Promethazine

443

CHARLES M. SHEARER AND SUSAN M. MILLER

A second synthesis is via the Grignard reaction ( 5 4 , 55). Phenothiazine is refluxed with methyl iodide and magnesium. To this is then added 2-chloro-1-dimethylaminopropane to give promethazine.
1-Phenothiazine-2-hydroxypropane p-toluenesulfonate when heated with dimethylamine gave promethazine (52).

Promethazine can also be synthesized by first reacting 2-bromo-2'-amino-diphenylsulfide with l-dimethylamino-2chloropropane in xylene solution with sodamide present, to give 2-bromo-2I - ( 2"-dimethylaminopropyl)-amino-diphenylsulfide. To bring about cyclization, this product is heated with potassium carbonate and copper powder in dimethylformamide (56). Stability and Degradation Promethazine is decomposed primarily by oxidation and/or photolysis. Oxygen has been shown by polarography to form a 9. complex with promethazine ( 5 7 , 2)
4.

Promethazine hydrochloride when refluxed in an aqueous solution produced 10-methyl phenothiazine, acetaldehyde and dimethylamine ( 5 8 ) . It was concluded that the cleavage of the promethazine was probably due to a free radical mechanism and oxygen in some way caused an activation of the molecule. The same products were formed when a promethazine hydrochloride (pH 5 ) solution was placed in an ampule, sealed 5) under nitrogen and irradiated with a fluorescent light ( 9 . However, Yamamoto found no degradation in a similar solution of promethazine under nitrogen when exposed to a light intensity of 5000 luxes at 3 5 O (60). Aqueous solutions of promethazine hydrochloride stored at room temperature in diffused daylight for 2 days to six months decomposed to promethazine sulfoxide, 9, 9 dioxopromethazine, N-demethylpromethazine and several more unidentified compounds (61, 62) Phenothiazine was identified as a major degradation product when a solution of promethazine hydrochloride was exposed to sunlight (63).

A kinetic study was reported by Stevens ( 6 4 ) . The pH was controlled with Sorenson citrate buffer containing 0.1% EDTA and adjusted to constant ionic strength. The sample flask was held at constant temperature in the dark and the solution was bubbled with 4 at a flow rate of 10 ml/min. The first order rate constants are given for the pH range 1 2 to 5.0. . Within this range promethazine is most unstable
444

PROMETHAZINE HYDROCHLORIDE

a t a pH around 4.3. A t pH's between 5.5 and 7 t h e degradat i o n no longer followed f i r s t o r d e r k i n e t i c s . The s t a b i l i t y of promethazine hydrochloride i n s o l u t i o n i s improved by t h e a d d i t i o n of ascorbic acid (65, 66, 67, 681, c y e t e i n e (651, sodium m e t a b i s u l f i t e (69) o r r o n g o l i t e (68, 69). I n s e v e r a l of t h e s e p u b l i c a t i o n s t h e e f f e c t of pH upon t h e s t a b i l i t y of t h e m a t e r i a l was discussed. Metabolism and Pharmokinetics Hansson and Schmiterlow (70) i n v e s t i g a t e d t h e e x c r e t i o n and metabolism of S35 labeled promethazine i n rats. Maximum e x c r e t i o n t a k e s place during t h e f i r s t 24 hours. The drug w a s r e a d i l y metabolized, p r i m a r i l y t o t h e sulfoxide. Two o t h e r u n i d e n t i f i e d metabolites were a l s o found. The sulfoxi d e w a s found as t h e only m e t a b o l i t e i n t h e b r a i n and l i v e r . Rueiecki and Wysocka-Paruszewska (71) i n another s t u d y on rats found similar e x c r e t i o n p a t t e r n s most i n t e n s i v e d u r i n g t h e f i r s t 72 hours a f t e r a d m i n i s t r a t i o n and l a s t i n g no more t h a n 5 days. S i x o r seven, depending upon t h e dose, metab o l i t e s were found i n t h e urine. These m e t a b o l i t e s were not i d e n t i f i e d chemically, but c h a r a c t e r i z e d by Rf on t h i n l a y e r chromatography. I n t h e organs (kidney, spleen, lungs and stomach) 24 hours a f t e r t h e drug w a s administered, only t r a c e s of promethazine and i t s metabolites were found. These metabolites were t h e same as found i n t h e urine. I n the blood 24 hours a f t e r a d m i n i s t r a t i o n , no unchanged promethaz i n e and only t r a c e s of two metabolites were found. Metabol i t e s of promethazine i n r a t s were c h a r a c t e r i z e d as beingproduced by s u l f o x y l a t i o n , aromatic hydroxylation and N-demethyk a t i o n . I n human v o l u n t e e r s promethazine w a s found t o be excreted primarily as t h e glucuronide conjugate (72). Robinson (73, 74) has shown t h a t hydroxylation (two d i f f e r e n t products), d e a l k y l a t i o n and demethylation of promethazine occur i n r a t l i v e r homogenate.

5.

Identification Several c o l o r r e a c t i o n s f o r t h e i d e n t i f i c a t i o n of proKirk (75) d e s c r i b e s t h e methazine a r e given i n Table V I . preparation of many of t h e l e s s e r known reagents. Bradford (76) p r e s e n t s a systematic procedure f o r t h e i s o l a t i o n of promethazine from b i o l o g i c a l m a t e r i a l and dosage forms. The i d e n t i f i c a t i o n of t h e i s o l a t e d m a t e r i a l i s by u l t r a v i o l e t spectrophotometry. DeLeenheer (77) d e s c r i b e s a system f o r combining p r e p a r a t i v e gas-liquid chromatography with microi n f r a r e d spectroscopy f o r t h e i d e n t i f i c a t i o n of drugs. I n f r a red spectroscopy can be used d i r e c t l y on t h e raw m a t e r i a l f o r

6.

445

CHARLES M. SHEARER AND SUSAN M. MILLER

Table V I I d e n t i f i c a t i o n Color Tests f o r Promethazine Hydrochloride Reagent Palladium c h l o r i d e Ferric chloride Conc. $ SO,, Conc. H N 4 Mandelin Reagent Marquis Reagent Buckingham's Reagent F r a h d e ' s Reagent Chloroplatinic acid Bromine w a t e r 1% F'hos phomolybda t e a c i d 10%K,,Fe(CNl6 10%QFe(CN)6 SO, -HN$ (1: 1 ) Mecke I s Reagent R e i c k a r d ' s Reagent F l u e k k i g e r ' s Reagent V i t a l i s Reagent S c h n e i d e r ' s Reagent 0.3 M P e r i o d i c a c i d i n 1 N %SO,, Ammonium c e r r i c n i t r a t e i n 5% n i t r i c a c i d Conc. $ SO,, , t h e n 0.1% E h r l i c h ' s Reagent 10% Chloramine p l u s CHC 1 , Fuming H N 4 V a n i l l i n w i t h $SO,, Response red-purple p r e c i p i t a t e red color fuschia color magenta t o yellow-orange pink c o l o r magenta c o l o r b r i l l i a n t red t o magenta ptnk c o l o r f l a t purple c r y s t a l s red fading t o c o l o r l e s s violet precipitate yellow p r e c i p i t a t e yellow p r e c i p i t a t e pink t u r n i n g yellow purple color pink c o l o r red c o l o r yellow-pink c o l o r brownish-green c o l o r red c o l o r brownish-red c o l o r red color orange c o l o r v i o l e t color red color pink, red c o l o r Reference 142 142 14, 143 14, 143 14, 143 14, 143 14 14, 75 14 14, 143 143 143 143 143 75 75 75 75 75 144 145 146 147 147 143, 147 147

NaN4

446

PROMETHAZINE HYDROCHLORIDE

its identification () 1. Edge and Wragg (78) discuss the identification of promethazine and isopromethazine. Several identification tests are described by Clarke ( 9 . He also 7) gives a microchemical test to distinguish promethazine hydrochloride from promethazine 8-chlorotheophyllinate. Several authors describe microcrystalline identification tests for prornethazine. Andres (80) gives a description of the crystals and also photomicrographs of the products of promethazine reacted with platinum bromide, ammonium reineckate and gold chloride. Promethazine from tabletswas identified by these tests. An amorphous precipitate was obtained with picric acid. Bogs (81) obtained a crystalline product 0 (mp 159-16 C) by treating promethazine hydrochloride with sodium 4,4'-dichlorodiphenyldisulfimide.

7 Methods of Analysis .
Elemental Analysis The elemental analysis of promethazine hydrochloride USP Reference Standard is presented below. 71 .

E 1ement
H N
C

% Calculated 63.59

% Reported (8)

65 .9

63.61 10.76

c1 S

8.73 11.05

6.59 86 .8
99 .0

99 .9

Phase Solubility Analysis Phase solubility analysis was carried out using acetone as the solvent ( 3 . 1) Experience in this laboratory has shown that 24 hours with vibration at 31.2OC is sufficient for equilibration to be attained. A purity of 99.7 ? 0 5 w s obiained. .% 7 9 Direct Spectrophotometric Analysis . Promethazine may be assayed by ultraviolet spectrophotometry at 249 nm in water or at 256 nm in ether (82). Ultraviolet spectrophotometric methods are used (1) for the analysis of promethazine hydrochloride syrups, injections and tablets after separation from the inert ingredients by a biphase extraction or a partition column. Pellerin (83) describes a method which involves first, forming ion pairs of promethazine with lauryl sulfate or dioctylsulfosuccinate, second, extracting these from an aqueous into a non-miscible organic solvent and third, determining the concentration of promethazine by ultraviolet spectrophotometry. Separation prior to spectrophotom etric determination by partition column chromatography (63, 841, ion exchange chromatography (85, 86) and reverse phase partition column

72 .

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CHARLES M. SHEARER AND SUSAN M. MILLER

Salvesen (88) chromatography (87) have been reported. d e s c r i b e s a q u a n t i t a t i v e i n f r a r e d spectrophotometric determination of pr ome thaz ine

Colorimetric Analysis Cavatora (89) d e s c r i b e s methods f o r determining promethazine i n t h e presence of promazine and chlorpromazine by r e a c t i n g i t w i t h mercuric s u l f a t e t o form a colored species. Promethazine hydrochloride can be determined by forming a colored product with sodium 1 , 2 naphthoquinone-4e u l f o n a t e i n 50% s u l f u r i c acid (90). Many common i n e r t i n g r e d i e n t s i n pharmaceutical preparations do not i n t e r f e r e w i t h t h e assay. Promethazine hydrochloride can be oxidized by FeN&,(S0,,)2 and n i t r i c acid t o form a product which can be estimated spectrophotometrically (91). Ryan (27) d e s c r i b e s a c o l o r i m e t r i c assay f o r promethazine by forming a colored complex of it with palladium chloride. The method i s s t a b i l i t y i n d i c a t i n g w i t h r e s p e c t t o t h e o x i d a t i o n of promethazine t o t h e sulfoxide. A s i m i l a r method i s discussed by Cavorta (89). Mercaldo and Gallo (92) have automated t h i s method of a n a l y s i s using Technicon AutoAnalyzer equipment. Hetzel (93) d e s c r i b e s a c o l o r i m e t r i c a s s a y f o r compounds having a phenothiazine moiety by forming a yellow colored n i t r a t i o n product. The method w a s used f o r assaysof phenothiazine drugs i n b i o l o g i c a l samples. Oxidation of promethazine hydrochloride by h e a t i n g i t with ammonium p e r s u l f a t e gave a colored product with absorption maxima a t 520 n (94). The method w a s applied t o m physiological s a l i n e and t o syrups by e x t r a c t i n g t h e promethazine base from a b a s i c s o l u t i o n i n t o e t h e r and t8gh e x t r a c t i n g t h e hydrochloride s a l t i n t o 0.1 N HC1. Sodium c h l o r i t e r e a c t s with promethazine hydroc h l o r i d e t o produce a r o s e - v i o l e t c o l o r which can bemeasured a t 533 run (95). The r e a c t i o n between promethazine hydrochloride and p-benzoquinone can be applied t o t h e determination of promethazine. The method was used f o r b i o l o g i c a l samples (96).

7.4

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PROMETHAZINE HYDROCHLORIDE

Beer's law is followed in the range 2.0-3.2 mg/50 ml for promethazine hydrochloride reacted with 2-nitro-1,3indandione in acetic acid, when measured at 540 run ( 7 . 9) The reaction between antimony pentachloride and promethazine in dichloroethane can be used for the quantitative determination of the drug in solutions, or in biological media ( 8 . 9) Eriochrome Black T with promethazine forms salts which can be extracted into chloroform and measured at 510520 nm ( 9 . 9) Promethazine hydrochloride can be determined photometrically after extraction as ion-pairs with methyl orange (0) 10. An automated application of the method is presented. Promethazine hydrochloride has been analyzed by the formation of the photooxidation products which are free radicals characterized by their strong absorption in the visible region. An automated system for the generation and determination of the free radicals is described ( 0 ) 11. Promethazine can be quantitatively precipitated as the reineckate salt and redissolved in acetone and determined spectrophotometrically ( 0 ) 12. Promethazine can be determined by precipitating it with molybdophosphoric acid (103) or tungstophosphoric acid (104) and then redissolving it in dimethylformamide-methanol solution and measuring it- spectrophotometrically. Electrochemical Analysis Kabaskalian and McGlotten (105) studied the polarographic oxidation of promethazine and other phenothiazine tranquilizers. A gold micro-electrode was used. The effects of pH, concentrations and temperature on the position and mag nitude of the anodic wave were investigated. Meckle and Discher (106) found that controlled poterr tial electrolysis is suitable for the coulometric determination of promethazine. In 14 N $SO, promethazine is quantitatively oxidized to the free radical at + 0 . N vs SCE and to the sulfoxide at + 0.98V. Controlled potential electrolysis at + 0.70V was suitable for the analysis of the raw material. Hynie (107) and Dusinsky (108) investigated the use of oscillographic polarography for analysis of promethazine.

75 .

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CHARLES M. SHEARER AND SUSAN M. MILLER

Promethazine can be n i t r a t e d with conc. n i t r i c a c i d and t h e r e s u l t i n g product determined by polarography (109). T i t r h n e t r i c Analysis Promethazine h y d r o c h l o r i d e can be t i t r a t e d by t h e Pifer-Wollish method u s i n g c r y s t a l v i o l e t a s t h e endpoint i n d i c a t o r (1). M a i n v i l l e and Chatten (110) d e s c r i b e a s i m i l a r t i t r a t i o n u s i n g a c e t o n i t r i l e a s t h e s o l v e n t , 0.1 N perc h l o r i c a c i d i n dioxane a s t h e t i t r a n t and endpoint determina t i o n by e i t h e r t h e c o l o r change of c r y s t a l v i o l e t o r by potentiometry u s i n g a glass-calomel e l e c t r o d e combination. This procedure was s a t i s f a c t o r y f o r t h e drug s u b s t a n c e b u t n o t f o r promethazine h y d r o c h l o r i d e t a b l e t s . Kerney (111) uaed acetone a s a s o l v e n t i n a Pifer-Wollish t y p e t i t r a t i o n . Promethazine h y d r o c h l o r i d e can be determined by a two phase (chloroform and a c i d ) t i t r a t i o n u s i n g sodium l a u r y l s u l f a t e a s t h e t i t r a n t and methyl yellow a s t h e i n d i c a t o r (112, 113, 114). This method h a s been a p p l i e d t o promethaz i n e hydrochloride t a b l e t s (115). Sodium d i o c t y l s u l f o s u c c i n a t e has a l s o been used as t h e t i t r a n t (116). Oxidimetric ti t r a t i o n s of promethazine h y d r o c h l o r i d e w i t h c e r r i c s u l f a t e and w i t h potassium bromate (potassium bromide added) have been s t u d i e d (117, 118, 119, 120). The endpoint can b e determined by potentiometry, v i s u a l l y o r by t h e dead s t o p method. S i m i l a r methods u s i n g e l e c t r o g e n e r a t e d c e r r i c i o n were d e s c r i b e d by P a t r i a r c h e (121, 122). Lead t e t r a a c e t a t e h a s been used a s t h e o x i d a t i v e t i t r a n t f o r promethazine h y d r o c h l o r i d e w i t h endpoint d e t e r m i n a t i o n employing platinum f o i l as i n d i c a t o r and a calomel e l e c t r o d e a s t h e r e f e r e n c e (123). Promethazine h y d r o c h l o r i d e can be t i t r a t e d w i t h e i l i c o t u n g s t i c a c i d determining t h e endpoint e i t h e r conduct o m e t r i c a l l y (124) o r by t h e c o l o r change of congo r e d (125). The method has been a p p l i e d t o drug dosage forms. Promethazine b a s e a f t e r e x t r a c t i o n from a sodium b i c a r b o n a t e s o l u t i o n i n t o chloroform h a s been t i t r a t e d w i t h an a r y l s u l f o n i c a c i d i n dioxane u s i n g dimethyl yellow a s t h e endpoint i n d i c a t o r (126). The c h l o r i d e p o r t i o n of promethazine h y d r o c h l o r i d e h a s been determined by t h e use of a c h l o r i d e i o n s e l e c t i v e e l e c t r o d e (127). Promethazine h y d r o c h l o r i d e has been t i t r a t e d w i t h 0.1 N sodium hydroxide f o r t h e proton o f t h e a c i d i c p o r t i o n of t h e s a l t by d i s s o l v i n g it i n dimethylformamide and 7.6

450

PROMETHAZINE HYDROCHLORIDE

using thymol blue for endpoint determination ( 2 ) 18. Danek (129) precipitated promethazine reineckate, redissolved it in acetone-water and determined the amount of the reineckate present by titrating it with silver nitrate. Promethazine can be quantitatively precipitated with mercuric chloride and the excess mercury determined by a complexometric titration ( 3 ) 10. Dilute solutions of promethazine hydrochloride in water, when treated with a known equal volume of 0 1 % reineckate salt solution gives a precipitate which is .5 removed by filtering and the excess reineckate determined by potassium bromate titrations (131). Promethazine hydrochloride in dosage forms was determined by using an anion exchange resin and titration of the eluted promethazine free base (132, 133). Gravimetric Analysis Gravimetric analysis of promethazine hydrochloride can be carried out by precipitating it as the styphnate (1341 reineckate (135) , molybdophosphone complex (103) , or silicotungstate ( 3 ) 16. A different type of gravimetric determination of promethazine involves the oxidation and subsequent bromination of it. The resulting compound was extracted from base with chloroform, the organic layer evaporated and the residue weighed (137). Fluorometric Analysis Promethazine hydrochloride can be oxidized in 50% glacial acetic acid with hydrogen peroxide and heat to give a stable product which can be quantitatively determined by This procedure can be used to deterits fluorescence ( 3 ) 18. mine promethazine in blood samples ( 3 ) 19. Another fluorometric procedure utilizes the fluorescent product produced by diluting into dimethylsulfoxide a solution of promethazinc in sulfuric acid. The procedure has also been used for analysis of promethazine hydrochloride in biological samples. Microbiological Analysis . Solution of greater than 0 5 mg/ml promethazine hydrochloride can be assayed by a microbiological method using J. anthracin ( 4 ) 11. 7.10 Separation Methods of Analysis 7.101 Paper Chromatography Promethazine has been chromatographed on
451

77 .

78 .

79 .

Table V I I Paper Chromatography of Promethazine Eluent Butyl alcohol: amyl alcohol: acetic acid: water, 25: 75: 12:1% Butano1:water:citric acid, 50:50: 1 Acetone:l M sodium acetate:l M acetic acid, 1 :20: 5 0 : Butanol: acetic acid:water, 4 1: 2 Butano1:acetic acid:water, 4 1:5 : n-Butanol:acetic acid:water, 60:15: 25 Phosphate buffer (pH 6 5 .) 01 M NaCl:methanol, 5 l . : 01 M NaCl:formamide, 5:l . Dichloroethane:acetic acidswater, 20: 8:2 5% aqueous (NH,, I2 :isobutanol, 1 1 SOc : Paper Reference 2045b

& S

Whatman No. 1 Whatman No. 1

S & S 2043

06 .6 07 .
0.83 0.85

148 149 150 151 70 152

Whatman No. 1 Munktell OB CM2 paper CM-82, ion exchange CM-82 CM-82
S & S 2043 or 2045

03 .1 04 .0 05 .1 06 .9 06 .2

153 153 153 154 155

Whatman No. 1

Table IX TLC Systems Using Silica Gel as Adsorbent Eluent Ethano1:water: acetic acid; 20:20: 1 0 3 4 in chloroform .% Cyclohexane:acetone; 50: 50 : Cyclohexane: diethylamine; 9 1 Methano1:ammonia; 100: 15 . Ch1oroform:methanol; 50: 50 Benzene: ethanol: m o n i a ; 95: 15: 1 Methanol: acetone; 12: 88 Water: ethanol; 4 96 : 1sopropanol:isopropyl ether; 16:84 Methano1:methyl acetate:cyclohexane; 18: 49: 33 Ethyl acetate: isopropanol:m o n i a ; 70: 25: 5 Ethyl acetate:dichloroethane: ammonia; 80: 20:5 Benzene:dioxane: annnonia; 10:80: 10 Acetonermethanol: ammonia; 50: 50: 1 95% Ethanol Methanol 2 Cyc1ohexane:benzene:methanol; 75: 15: 10 Methanol2 Ac etone2 . Support prepared with 0 1 M NaXSOC or 01 M KHSq . 2Support prepared with 01 N KOH or 0 1 N NaOH . .

,Rf04 .1 0.29 0.15 06 .2 0.61 04 .4 04 .1 0.26 02 .1 01 .6 04 .7 0.73 0.56 0.77 0.53 0.23 0.45 04 .6 04 .7 0.37

Reference 159 164 165 166 166 167 168 169 169 169 169 170 170 171 172 173, 174 173, 174 173, 174 173, 174 173, 174

m
0

CHARLES M. SHEARER AND SUSAN M MILLER

paper under v a r i o u s c o n d i t i o n s . S o l v e n t systems and o t h e r p e r t i n e n t d a t a are given i n Table V I I . Methods o f d e t e c t i o n a r e given i n Table V I I I . S e v e r a l o f t h e s e systems (148, 152, 154) have been used i n a n a l y z i n g u r i n e and o t h e r b i o l o g i c a l materi a l s f o r metabolic products of promethazine. Table V I I I Spray Reagents f o r D e t e c t i o n of Promethazine on Paper Chromatography Reagent Dragendorff's Reagent s u l f u r i c acid-ethanol s u l f u r i c acid n i t r i c acid palladium c h l o r i d e f e r r i c chloride cerric sulfate E h r l i c h ' s reagent 7.102 Color orange red red rose orange rose rose rose Reference 148 148 156 156 156 156 156 156

Thin Layer Chromatography The v a r i o u s e l u e n t systems f o r t h i n l a y e r chromatography of promethazine u s i n g S i l i c a Gel a s adsorbent a r e given i n Table IX. Systems u s i n g o t h e r a d s o r b a n t s a r e included i n Table X. Table X I l i s t s s p r a y r e a g e n t s and o t h e r d e t e c t i o n methods s u c c e s s f u l l y used i n analyzing promethazine. D i r e c t conversion of promethazine t o t h e s u l f o x i d e on t h e p l a t e , and subsequent a n a l y s i s by u l t r a v i o l e t spectroscopy h a s a l s o been d e s c r i b e d (157, 158). S e p a r a t i o n o f phenot h i a z i n e was achieved u s i n g a p g r a d i e n t from 7.0 t o 8.5 on H S i l i c a Gel l a y e r s (1591, and on alumina (160).

Table X TLC Systems Using Adsorbents Other Eluent Adsorbent alumina benzene: e t h a n o l ; 98: 2 benzene: e t h a n o l ; 95: 5 benzene: e t h a n o l ; 90$10 ch1oroform:ethanol; 98: 2 chloroform: e t h a n o l ; 95: 5 chloroform: n-propanol: 25% anunonia; 98: 1: 1 acetone:chloroform; 15: 85 ether:petroleum e t h e r ; 1: 1 cyclohexane: e t h a n o l ; 85: 15

Than S i l i c a Reference

EL

.34 .66 * 73 .74 .80 .88 .70 .55 67

161 161 161 161

161
161 161 161 161

454

PROMETHAZINE HYDROCHLORIDE

isobutyl alcohol saturated SO, w i t h 5% (NH, I2 chloroform: acetone: 25% ammonia; 50: 50: 1 chloroform: e t h y l a c e t a t e : 25% ammonia; 50: 50: 1 7.1013

.54 .96 .90

162 163 163

Gas Chromatopraphy Gas chromatography has been used t o a n a l y z e promethazine and t o s e p a r a t e i t from o t h e r p h e n o t h i a z i n e s . The column c o n d i t i o n s and n e c e s s a r y d a t a f o r t h e v a r i o u s methods a r e given i n Table X I I . The v a r i o u s p h e n o t h i a z i n e s have been c h a r a c t e r i z e d by gas chromatography of t h e i r p y r o l y s i s p r o d u c t s (180). Gas chromatography of promethazine h a s been used i n combination w i t h m i c r o - i n f r a r e d s p e c t r o s c o p y (181). Table D e t e c t i o n Methods Used i n Reagent A n i l i n e vapors, t h e n bromine vapors I o d o p l a t in a t e s p r a y I o d i n e vapors 1.0% w/v s e l e n i o u s a c i d i n conc. & SO, Concentrated Q SO, Folin's reagent Ceric s u l f a t e i n s u l f u r i c a c i d Marquis r e a g e n t Palladium c h l o r i d e Dragendorff ' s r e a g e n t Dime t h y 1aminob enz a l d ehyd e Furfural i n s u l f u r i c acid KMnO,, S u l f u r i c acid: e t h a n o l ; 1: 9 Perchloric acid Phos phomol ybdic a c i d 7.104

XI TLC of Promethazine Reference Color 175 brown changing t o green 176 violet 176, 162 brown

red-purple fuschia red-violet red yellow-red red orange rose rose yellow rose red rose

168 170 170 170 177 172, 178 176, 178 176 176 176 179 179 179

Column Chromatography Promethazine can be s e p a r a t e d from o t h e r dosage form components and drugs by p a r t i t i o n chromatrography (63, 841, i o n exchange chromatography (85, 86, 132, 133) and r e v e r s e phase chromatography (87).
455

Table X I 1
G a s Chromatography of Promethazine Hydrochloride

Column

Temperature 175' 175O 175O 175O 240" 2200 205 "

Retention Time 7.2 min. 21.7 min. 27.9 min. 17.1 min.

Reference 182 182

6 f t x 3 m i.d.;

0.07% SE-30 on 120-170 mesh g l a s s beads

6 f t x 3 mm i.d., 0.08 PDEAS on 120-170 mesh g l a s s beads

6
Q)

6 f t x 3 mm i.d., 1.1%XF-1150 on 100-120 mesh Chrmosorb W-HMDS

W2 M 6 f t x 3 mm i.d.; 1.08% C - O on 100-120 mesh G a s Chrom P 5% Apiezon L on 4 f t x 4 mm i.d.; 100-110 mesh Anachrom ABS

182
182 183 184 185

1.8 m e t e r x 4 m i.d.; 3.8% SE-30 on 80-100 mesh D i a t o p o r t S

6 f t x 5 nun i.d.; 2% SE-30 on 80-100 mesh G a s Chrom S

PROMETHAZINE HYDROCHLORIDE

Pound and Sears (186) discuss the use of high pressure liquid chromatography for the analysis of promethazine hydrochloride. Electrophoresis Promethazine was separated from other phenothiazines by paper electrophoresis (Whatman No. 2 paper) using a glycol-HC1 electrolyte (pH 3 . 3 4 ) . Visualization was by Dragendorff, iodoplatinate, palladium chloride or cerric sulfate spray ( 8 ) 17. Counter Current Partition Promethazine was separated from promazine and chlorpromazine using counter-current partition between chloroform and hydrochloric acid ( 8 ) 18. 7.106 7.105

457

CHARLES M. SHEARER AND SUSAN M. MILLER

8.

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(1965). 121.

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z ,

127.
128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144.

Acta, 49, 25(1970). A. Berka, V. Prochazkova and J. Zyka, Cesk. Farm., 13, 121(1964). CA: 61: 12640b. S Haque, Z . B l a g o j z i c and K. Nikolic, Acta Pharm. . Jugoelav. , 107(1967). C A : s : 98695g. H. Thieme, Pharmazie, 11,460(1956). S Roleki and Z. Zakrzewski, Farm. Pol., 25, 621 . (1969). C A : Z : 103796e. E. Pungor, K. Toth and M. K. Papay, Chem. Anal. (Warsaw) , 17,947(1972). C A : z : 922933. F. Balaefalvy and Z. Toth, Acta Pharm. Hung., 33, 73(1963). C A : E : 4977e. A. Danek, Acta Polon. Pharm., 18,229(1961). C A : S : 8842d. K. Gramberg and Z. Blagojevic, Acta Pharm. Jugoslav. 14, 17(1964). CA:G:9748b. A. Olech, Acta Pol. Pharm., 29, 57c1972). C A : E : 52421~. S M. Blaug and L. C. Zopf, J. Am. Pharm. Assoc., . 45, 9(1956). A. J i n d r a and 0. M o t l , Ceekoslov. Farm., 632 (1952). C A : c : 5071e. S Uyeno and H. Oiehi, J. Pharm. SOC. Japan, 72, . 443(1952). C A : S : 7708g. P. Spacu and E. Antonescu, Acad. Rep. Papulare Romine, S t u d i i Cercetari Chim. 247(1959). C A : a : 7436d. J. Blazek and V. Mares, Cesk. Farm., 15, 349(1966). C A : s : 22266f. E. S z a b o k e , Acta Pharm. Hung., 2, 212(1964). C A : g : 15936e. J. B. Ragland and V. J. Kinroee-Wright, Anal. Chem., 36, 1346(1964). S L. Tompeett, Acta Pharmacol. Toxicol., 26, 298 . (1968). E. A. Martin, Can. J Chem., 44, 1783(1966). . Z. Bruno, Rev. Farm, Bioquim. Univ. Sao Paulo, 247(1972). CA:E:61345u. C. Foeeoul, J. Pharm. Belg., 5, 202(1950). H. Auterhoff, Arch. Pharm., 14(1952). C A : S : 10537b. I. Simonyi and G. Tokar, 2 Anal. Chem., 212, 317 . (1965). C A : g : 12975h.

17,

L,

,L,

10,

&,

463

CHARLES M. SHEARER AND SUSAN M. MILLER

145. 146. 147. 148. 149. 150. 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. 161. 162. 163. 164. 165. 166. 167. 168. 169.

L. M. Atherden, Pharm. J, . 115(1965). C A : s : 1402l h H. Vasbinder and H. R. van der S i j d e , Pharm. Weekblad. l7, 610(1962). CA:58: 6648a. E, W. Neuhoff and H. A u t e r G f f , Arch. Pharm,, 288, 400(1955). M, Frahm, E. F r e t w u r s t and K. Soehring, Klin. Wochschr. , 34, 1259(1956). C A : Z : 13981b. A. S Curry and H. Powell, Nature, . 1143(1954). R. L. Tabau and J. P. Vigne, Bull. SOC. Chim. France 1958, 458. C A : Z : 16125d. A. Wankmuller, Apoth.-Ztg. , 5 , 127(1953). C A : s : 41838. K. S j o b e r g and G. Tufvesson, Acta Vet. Scand., 4, 209(1963). CA:g:5878c. Z. I. El-Darawy and Z. M. Mobarak, Pharmazie, 28, 37 (1973). CA: 78:144057g. E. V i d i c a n d T . S c h u e t t e , Arch. Pharm., 295, 342 (1962). CA: 57: 7388f. J. Blazek, C z k . Farm. IS, 314(1966). CA: 65: 199330 J. Vecerkova, M. Sulcova and K. Kacl, P h a r m z i e , 3, 22(1962). CA:57:4761i. J. Kofoed, C. Z r c z a k - F a b i e r k i e w i c z and G, H, W. Lucas, Nature, 147(1966). J. Kofoed, C. Korczak-Fabierkiewicz and G. H W. . Lucas , J. Chromatog. , 23, 410(1966). L. Kraus and E. Dumont, J. Chromatog., 56, 159 (1971). L Kraus and E. Dumont, Z. Anal. Chem., 252, 380 . (1970). M. Sarsunova, B. Kakac, V. Schwarz, V. Maly and J. P r o t i v a , Pharmazie, 2, 752(1966). C A : E : 1083056 A. N o i r f a l i s e and M. H. Grosjean, J. Chromatog., 16, 236(1964). A. D e Leenheer, J. Chromatog., 75, 79(1973). M. R. Gasco and A. Bodrato, Farmaco, Ed. P r a t . , 26, 337(1971). C A : Z : 52876h. A. N o i r f a l i s e , J. Chromatog., 19,68(1965). I. Sunshine, Am. J. C l i n . Pathol., 40, 576(1963). C A : S : 8540b. A. N o i r f a l i s e , A c t a Clin. Belg., 20, 273(1965), CA: 64: 7971h. W. N French, F. Matsui, D. C. Robertson and . S. J. Smith, Can. J. Pharm. Sci., 1 ,27(1975). 0 E. Roeder, E. Mutschler and H. Rochelmeger, Pharmaz i e , 24, 238(1569).

195,

173,

g,

464

PROMEfHAZl NE HYDROCHLORIDE

170.

171.

172.
173. 174. 175. 176. 177.

178.
179.

180.
181.

182.
183. 184. 185. 186. 187. 188.

L a u f f e r , E. Schmid and F. W e F s t , A r z n e i m i t t e l Forsch, 2, 1965(1969). CA: 72:47413p. J. P. Comer and I. Comer, J.Pharm. S c i . , 56, 413 (1967). J. J. Thomas and L. Dryon, J. F'harm. Belg., 2, 481 (1964). CA: 63: 1660d. W. W. F i k e , Anal. Chem., 38, 1697(1966). I. Sunshine, W. W. Fike and H. Landesman, J. Forens i c S c i . , 1 , 428(1966). CA:66:16972s. 1 V. C l a r k e and E. R. Cole, J. Chromatog., 24, 259 (1966). A. N o i r f a l i s e , J. Chromatog., 61(1965). 2 Margasinski, R. Danielak, T. Pomazanska and . H. Rafalowska, Acta Polon. Pharm., 2, 5(1964). CA:G: 8941h. J. Baumler and S. R i p p s t e i n , Pharm. Acta Helv., 2, 382( 1961). H, E b e r h a r d t , 0. W. Lerbs and K. J. Freundt, Arzneimittel-Forsch, 2, 804(1963). CA: 60: 15000h. C. R. Fontan, N. C. J a i n and P. L Kirk,Mikrochim. . 326. C A : g : 37OOg. Ichnoanal. Acta, 3, A. DeLeenheer, J. Chromatog., 74,35(1972). C A : Z : 47866n. A. MacDonald, Jr. and R. T. Pflaum, J. Pharm. S c i . , 53,887(1964). C. L. Bramlett, J. Assoc. Offic. Anal. Chem., 49, 857(1966). A. Deleenheer, J. Chromatog., 74, 35(1972). M. W. Anders and G J, Mannering, J. Chromatog., 1, . 258(1962). N. J. Pound and R. W S e a r s , Can. J. Pharm. S c i . , 8, . 84(1973). A. Voicu and I. Popa, Farmacia (Bucharest), 21, 373 (1973). C A : E : 19631e. G. M. Nano, A t t i Accad. S c i . Torino, C l a s s e S c i . Fie., Mat. Nat., 95, 639(1960). CA:61:14668b.
S,

20,

465

RIFAMPIN

Gian G. Gallo and Pietro Radaelli

GlAN G. G A L L 0 AND PIETRO RADAELLI

TABLE OF CONTENTS
DESCRIPTION 1.1 Name 1.2 Formula and Molecular Weight 1.3 P o t e n t i a l Stereoisomerism 1.4 Appearance, Color and Odor

PHYSICAL PROPERTIES 2.1 Spectra 2.11 Ultraviolet Spectra 2.12 Infrared Spectra 2.13 Nuclear Magnetic Resonance Spectra 2.131 Proton Magnetic Resonance Spectra 2.132 Carbon Magnetic Resonance Spectra 2.14 Mass Spectra 2.2 Ionization Constants 2.3 Polarography 2.4 Optical Rotation 2.5 C r y s t a l Properties 2.51 X-ray Diffraction 2.52 Thermal Analysis 2.521 Melting Range 2.522 D i f f e r e n t i a l Scanning Colorimetry 2.6 Distribution Properties 2.61 Solubility 2.62 Lipid-Water P a r t i t i o n 2.621 Organic S o l v e n t d a t e r P a r t i t i o n 2.622 Silicon O i l d a t e r P a r t i t i o n 2.623 Surface Activity

STABILITY 3.1 S t a b i l i t y a s Powder 3.2 S t a b i l i t y i n Solution

4.

SYNTHESIS

RlFAMPlN

5.

METHODS OF ANALYSIS Elemental Analysis 51 . I d e n t i f i c a t i o n Tests 5.2 Spectrophotometric Methods 5.3 5.31 Spectrophotometric Assay on B u l k Product 5.32 Spectrophotometric Assay on Pharmac e u t i c a l Preparations 5.33 Spectrophotometric Assay on Biological Fluids F luorome t r i c D termina t ion e 5.4 Analysis by Complex F ormation 5.5 Volumetric Methods 5.6 5.7 Chromatographic Methods 5.71 T h i n Layer Chromatography 5.72 Column Chromatography 5.73 Paper Chromatography 5.74 High Pressure Liquid Chromatography Microbiological Methods 5.8 5.81 Diffusion P l a t e Assay Methods 5.82 S e r i a l Tube Dilution Methods 5.83 Turbidime t r i c Methods

6 .

PROTEIN BINDING
PHARMACOKINETICS AND METABOLISM I N MAN REFERENCES

7 .
8.

469

GlAN G. GALL0 AND PIETRO RADAELLI

1. 1.1
NAME

DESCRIPTION

Rifampin (1) i s designated by IUPAC r u l e s as 2,7(Epoxypentadeca[l ,11,13]trienimino)naphtho~2,1 - b l f u r a n = 1 11(2H) -dione, 5,6,9,17,19,21 -hexahydroxy-23-methoxy-

2,4,12,16,18,20,22-heptamethyl=8-CN-(4-methyl-l-pipe=
r a z i n y l ) f o r m i m i d o y l l - 2 1 -acetate. However, i n t h e l i . t e r a t u r e , r i f a m p i n has been p r e f e r e n t i a l l y known as 3- [[( 4-methyl-1 - p i p e r a z i n y l ) iminolmethyl] r i f a m y c i n SV, according t o t h e o r i g i n a l nomenclature o f r i f a m y c i n s (2-4). Rifampin (5,6) i s t h e USAN name f o r t h e compound, r i f a m p i c i n i s t h e i n t e r n a t i o n a l n o n p r o p r i e t a r y name (7) and other t r i v i a l names used a r e r i f a m y c i n A P and r i M f a l d a z i n e (8,9). 1.2 FORWIA AND MOLECULAR WEIGHT

36

35

C43H58N401 2

Mol.Wt. = 822.95

470

R I F AMP IN

T h i s numbering system, according t o t h e s p e c i a l nomenclature o f rifamycins, i s adopted i n t h e present profile. The numbering system according t o IUPAC r u l e s i s :

The molecule o f r i f a m p i n i s u s u a l l y described as c o n s i s t i n g o f a naphthohydroquinone chrornophoric p a r t spanned by an a l i p h a t i c c h a i n c a l l e d t h e ansa w i t h a p i p e r a z i n e s i d e c h a i n attached. POTENTIAL STEREOISOMERISM Rifarnpin, a semi-synthetic a n t i b i o t i c (8) even though i t c o n t a i n s 9 asymmetric carbon atoms and 3 double bonds, i s found o n l y as one isomer because a l l t h e isomerogenic centers belong t o t h e n a t u r a l p a r t o f t h e molecule and o n l y one isomer i s s p e c i f i c a l l y produced by t h e microorganism ( 4 , I O ) .

1.3

471

GlAN G. G A L L 0 AND PIETRO RADAELLI

1.4

APPEARANCE, COLOR A D ODOR N Rifampin i s a red-orange, odorless, c r y s t a l l i n e powder.

2. 2.1
SPECTRA

PHYSICAL PROPERTIES

2.1 1 U l t r a v i o l e t Spectra
UV-VIS spectrophotometry has been used f o r s t r u c t u r a l determination o f various rifamycins t o obtain s p e c i f i c i n f o r m a t i o n on t h e chromophoric p a r t o f t h e molecule. I n p a r t i c u l a r , t h e V I S maximum, which undergoes a hypochromic e f f e c t and a s m a l l hypsochromic s h i f t w i t h s t r o n g acids, i s c h a r a c t e r i s t i c o f t h e naphthohydrg quinone form c a r r y i n g t h e a c i d i c i o n i z a b l e f u n c t i o n (1 1 13), and t h e auxochromic e f f e c t on t h e same V I S maximum depends on t h e nature o f t h e s u b s t i t u e n t s i n p o s i t i o n 3

( 14-1 7).
The UV spectrum o f r i f a m p i n , recorded on a P e r k i n Elmer model 4 0 0 0 4 spectrophotometer i n aqueous phosphate b u f f e r pH 7.38 (8), e x h i b i t s t h e a b s o r p t i o n maxima given i n Table I. Table I U l t r a v i o l e t absorption o f ri f ampi n X,ax' nm
E

237 255 334 475

33 , 200 32,100 27,000 15,400

The v a r i a t i o n o f t h e UV-VIS spectrum o f r i f a m p i n w i t h pH ( f i g u r e 1) i n d i c a t e s t h e presence o f an i o n i z a b l e f u n c t i o n , a t t r i b u t e d t o t h e a c i d i c 8-hydroxyl group i n p e r i p o s i t i o n t o t h e hydroquinonic h y d r o x y l

(13,18,19).
I n f r a r e d Spectra The i n f r a r e d s p e c t r a o f v a r i o u s r i f a m y c i n s u s u a l l y have been r e p o r t e d i n t h e l i t e r a t u r e b u t o n l y
472

2.12

ELP

215

210

230

1.0

yyI

lbo

270

PBO

2vo

1rm

310

WAVUENGIH

r*ILLIMICRONS

Figure 1
cc1
C

- W spectra of

I-*

rifampin i n methanol-water (2:3) solution a t different pH's

pH 0 5 .;
W

...........

pH 2.5;

-.-.-.-.-.= 1.8; pH ---------_ 3.5-1 1 .o

P
I I 4
0

. -. .

.-.

01

-%

5.

01

GlAN G. G A L L 0 AND PIETRO RADAELLI

occasionally have been discussed (1 9/20). Exhaustive s p e c t r a l assignments have only recently been made (21). The infrared spectrum of rifampin i n chloroform solution was reported by Maggi e t al. (8). Figures 2 and 3 are the spectra of rifampin recorded as mineral o i l m u l l and i n deuterochloroform solution, respectivelg w i t h a Perkin-Elmer mod.421 spectrophotometer. Interpretation of the spectrum o f rifampin (21) i s given i n Table 11. I n CDCl solution, rifampin does not e x i s t 3 a s the zwitterion, while i n solid s t a t e it seems to. Table I1 Infrared spectra of rifampin

I absorption band, cm-1 R

lineral o i l m u l l 35003300 CDCl solution 3 3480 2970, 2930 and 2880 2820 2800 3300-2300 1715

Interpretation u0H-bonded uCH3 uCH30 uCH3-N uNH and vNH-bonded and vOH-bonded vC0 a c e t y l uCO furanone ( i n so. lution i n t r a Hbonded) amide I

*
n

3200-2300

1715
1735 1670 and 1610 1570 1540 and 1520 1255,1050 and 1020 Non-transparen 2.13

1640 1 620
1570 1540 and 520 1255,1040 and 1020

vc=c

amide I1 vC-0-C a c e t y l

region of the m neral o i l

Nuclear Maanetic Resonance Spectra 2.131 Proton Magnetic Resonance SDectra 'H-NMR spectroscopy has been widely used as a fundamental t o o l for s t r u c t u r a l determination of various rifamycins (18,19,22-24).
474

WAVELENGTH [MICRONS)

25

1 0

12

15

20

25

3500

2500

Moo 1900 1800 17$XJOJE@&~

1400 1300 1200 1100 loo0

900

800

700

600

500

400

Figure 2

I spectrum o f rifampin i n mineral o i l m u l l . R

WAVELENGTH 'MICRONS)

2.5

1 0

1 2

1 5

20

25

3500

3000

2500

zoo0 1 0 90

10 1400 1300 1200 1 0 lo00

900

8 b

700

603

5'20

AX

Figure 3

IR s p e c t r u m of r i f a m p i n i n CDCl s o l u t i o n . 3

R I FAMP IN

The H-NMR spectrum o f r i f a m p i n i n C D C l a t 60 MHz has been r e p o r t e d and some assignments 3 F i g u r e 4 i s t h e spectrum o f r i f a m p i n i n g i v e n (8). M C D C l recorded a t 100 MHz w i t h a V a r i a n )(L-l00-15 N R specqrometer. Complete i n t e r p r e t a t i o n o f t h e spectrum has been made (25) and i s g i v e n i n Table 1 1 1. 2.132 Carbon Magnetic Resonance S p e c t r a %NMR spectroscopy has been r e c e n t l y used f o r s t r u c t u r a l d e t e r m i n a t i o n i n t h e f i e l d o f r i f a m y c i n s (24,26-28). 13 F i g u r e 5 i s t h e FT C proton-noisedecoupled NMR spectrum o f r i f a m p i n i n CDCl3 recorded a t 25.2 MHz w i t h a V a r i a n XL-100-15 N R spectrometer, M equipped w i t h an FT accessory. Interpretation o f the spectrum has been made (25) and i s g i v e n i n T a b l e I V . Mass Spectra The mass s p e c t r a o f some r i f a m y c i n s have been s t u d i e d and f r a g m e n t a t i o n p a t t e r n s have been i n t e r p r e t e d (29). I n p a r t i c u l a r , t h e most c h a r a c t e r i s t i c peaks correspond t o M?, CMICH30Hlt and t o t h e chromop h o r i c ions. F i g u r e 6 i s t h e spectrum o f r i f a m p i n obt a i n e d under 70eV e l e c t r o n impact i n DIS a t 200OC w i t h a P e r k i n Elmer 270 spectrometer. Interpretation of this spectrum has been p u b l i s h e d (30). The spectrum was o n l y p a r t i a l l y c h a r a c t e r i s t i c as t h e compound decomposes i n t h e i o n source and M'f and CMLH30HIf were absent, w h i l e t h e peak a t m/e 398 corresponded t o t h e chromophoric ion. The mass spectrum o f r i f a m p i n was a l s o obt a i n e d under f i e l d d e s o r p t i o n i o n i z a t i o n c o n d i t i o n s a t w i r e c u r r e n t 12 mA w i t h a V a r i a n MAT 731 spectrometer I t e x h i b i t e d t h e m o l e c u l a r i o n a t m/e 822 and (31). t h e [M+1 I peak corresponded t o t h e i s o t o p i c c o n t r i b u t i o n . No f r a g m e n t a t i o n peaks were observed.

2.14

2.2

IONIZATION CONSTANTS The i o n i z a t i o n p r o p e r t i e s o f r i f a m y c i n s have been used i n con j u n c t i o n w i t h UV-VIS spectrophotometry (see S e c t i o n 2.1 1) t o o b t a i n i n f o r m a t i o n on t h e chromophoric

477

1%

P U P

'

'

Figure 4

- 'H4MR

spectrum a t 100 M z of rifampin i n C K l , H

solution.

RlFAMPlN

Table

I11

'H-NMR d a t a o f r i f a m p i n i n C K l n s o l u t i o n [ M u l t i p l i c i t y , c h e m i c a l s h i f t s (6, ppm) and v i c i n a l i n t e r p r o t o n coupl i n g c o n s t a n t s (J,Hz)l.

Proton
NI-i CH4l

M u 1t i p l a )
S S

6
8.22 2.9-3.3 2.4-2.8 2.34 11.4-14.0 b) 13.16 b) 1.82 2.22 6.3-6.8 5.92 2.26 3.78 3.2-4.2 b) 1.70 3.04 1.52 4.96 1.22 3.58 5.00 6.20 2.10 0.88 1.01 0.58 -0.33 2.06 3.05

I
I

I
I

CH2-2' ,6' CH2-3 ' ,5 ' N-CH3 OH-8 , OH-4 CH3-1 3 CH~-14

m
m
S

c) c)

bs
S S

c) 15 and 5 5 and 9 and 7 9 and 1

H-19 H-20 H-21 OH-23 H-22 H-23 H-24 H-25 H-26 H-27 H-28 H-29 CH3-30 CH3-31 CH3-32 CH3-33 CH3-34 CH3-36 CH3-37

dd ddq dd bs ddq dd ddq dd dds dd dd d

S

1 and 1.5 and 7 1 1.5 and 10 I 10 and 1 and 7 1 1 and 10 10 and 1.5 and 7j I 1.5 and 7 7 and 13 ' 13

d d d d
S S

7 7 7 7

a ) s z s i n g l e t ; d = d o u b l e t ; d d = d o u b l e t of d o u b l e t s ; d d q = d o u b l e t of d o u b l e t s o f q u a r t e t s ; m = m u l t i p l e t ; bs=broad s i g n a l ; b) it e x c h a n g e s w i t h D20 c ) not d e t e r m i n e d

479

480

Figure 5

13 C proton-noise-decoupled FT CDCl s o l u t i o n .

S .rl

C .rl

4-

0 4-

h l

h l

.rl

UJ

c,

NMR s p e c t r u m a t 25.2 MHz of r i f a m p i n i n

0

c,

I4

3 I4

Q,

u)

RlFAMPlN

Table

IV

16,
I

'IC-NMR data o f rifampin i n CDC13 solution. [Chemical s h i f t s ppm) and one-bond coupling constants ( I J , H a .
1

I I

I
I

I
I

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 CH=NC-2'C-3'C-5'C-6'CH,-NJ

138.6 105.9"' 110.8') 147.8 112.8a) 174.3 1 20.3a) 169.3 104.4') 117.8") 195.3 108.7 21.5 7.6 169.6 129.4 135.0 123.2 142.6 38.6 70.7 33.4 76.7 37.6 74.4 39.5 76.7 118.7 142.6 20.7 17.8 10.9 8.5 8.8 171.9 20.7 57.0 134.4 50.2 53.9 53.9 50.2 45.8

/
I

I
I

130 130

1 50 150 1 50 125 140 125 140 125 140 125 140 155 190 130 130 130 130 130

,
1

130 140 1 70 130 130 130 130 130

,
I
I

a)

These assignments may be interchanged

481

L
AlISN31NI 3AIlVl3H

o = s s " = o

I
I

482

I
I

Figure 6

- MS spectrum of
cc
0

rifampin a t 70 eV i n DIS a t 200%.

R I FAMPlN

p a r t o f t h e molecule and as a q u a n t i t a t i v e method o f a n a l y s i s ( 1 1 - 1 3 18). The pK v a l u e s f o r r i f a m p i n have been determined s p e c t r o p h o t o m e t r i c a l l y (see f i g . 1 ) and p o t e n t i o m e t r i c a l l y i n s o l u t i o n i n water and i n m e t h y l c e l l o s o l v e Rifampin water ( 4 : l ) and a r e r e p o r t e d i n T a b l e V. e x i s t s i n w a t e r s o l u t i o n as t h e z w i t t e r i o n w i t h i s o e l e c t r i c p o i n e q u a l t o 4.8 (8,13).

Table V I o n i z a t i o n constants o f r i f a m p i n

pKa
proton l o s t p r o t o n gained

*KS

Attribution h y d r o x y l a t C-8 p i p e r a z i n e N-4

IReference

1.7 3.6 17.9 6.7

I,13,18,19
113

R i f a m p i n i o n i z e s i n non-aqueous s o l v e n t s , i.e., in g l a c i a l a c e t i c a c i d t h e b a s i c p i p e r a z i n e n i t r o g e n can be t i t r a t e d w i t h p e r c h l o r i c a c i d (32).

2.3

POLAROGRAPHY The p o l a r o g r a p h i c behaviour o f r i f a m y c i n s has been d e s c r i b e d and t h e presence o f an o x i d a t i o n o r r e d u c t i o n wave a t about 0 v o l t s X E i n d i c a t e s t h e hydroquinone o r quinone ( 1 4/33-35) system, r e s p e c t i v e l y . These polar o g r a p h i c p r o p e r t i e s p e r m i t amperometric t i t r a t i o n o f r i f amyc i n s (36). R i f a m p i n i n methanol-acetate b u f f e r s o l u t i o n , pH 5.9, shows an o x i d a t i o n wave w i t h El12 = 4 . 1 0 v o l t s. SCE, a t t r i b u t e d t o t h e hydroquinone system ( 8 ) , and i n aqueous phosphate b f f e r , pH 6.88, t h e r e i s a l s o a r e d u c t i o n wave w i t h E 2 -1.66 v o l t s SCE, n o t a t t r i b u t e d (37). Sano e t a l . ( 3 8 ) r e p o r t e d an o x i d a t i o n SCE, w i t h o u t o t h e r p o t e n t i a l o f E1/2 = 4 . 0 6 v o l t details.

=.

y/

s.

E.

2.4

OPTICAL ROTATION The o p t i c a l r o t t i o n f o r r i f a m p i n was r e p o r t e d by Sano e t a l . (38) : C a l k +10.60 ( G O . 5% i n CDC13).

483

GlAN G. G A L L 0 AND PIETRO RADAELLI

CRYSTAL PROPERTIES 2.51 X-ray D i f f r a c t i o n S i n g l e - c r y s t a l X-ray d i f f r a c t i o n was used t o deduce the s t r u c t u r e of the p-iodoanilides of rifamycin B and rifamycin Y (10,39-42). T h e s i n g l e - c r y s t a l X-ray d i f f r a c t i o n method was applied t o rifampin c r y s t a l l i z e d w i t h 5 water molecules i n t h e orthorombic system (43). The X-ray powder d i f f r a c t i o n p a t t e r n of rifampin, i s reported i n Figure 7 (44) and Table V I g i v e s the values according t o the ASTM rules. The grinding causes the c r y s t a l l i n i t y of rifampin t o disappear and an amorphous form t o o r i g i n a t e , a s shown i n Figure 8.
2.5 Thermal Analysis 2.521 Melting Range Rifampin melts w i t h decomposition a t 183-1 88% (open c a p i l l a r y g l a s s tube), 2.522 D i f f e r e n t i a l Scanning Calorimetry U n t i l 1 now, D C has not been applied S t o the f i e l d of rifamycins. T h e heating curve of rifampin has been obtained on a Du Pont D i f f e r e n t i a l Thermal Analyzer mod.990 w i t h a temperature r i s e of 100C/min and i s reported i n f i g . 9 (45). I t shows an endotherm a t 193OC corresponding t o t h e melting, immediately followed by an exotherm corresponding t o t h e r e c r y s t a l l i z a t i o n of t h e melt, which t h e n decomposes exothermically a t about 240%. 2.52

DISTRIBUTION PROPERTIES 2.61 S o l u b i l i t y The approximate s o l u b i l i t i e s of rifampin i n various s o l v e n t s have been determined a t room temperat u r e (32). The r e s u l t s a r e reported i n Table VII, according t o t h e d e f i n i t i o n used by t h e US Pharmacopeia X I X , Rifampin i s s o l u b l e i n a c i d i c and a l k a l i n e water (8). Q u a n t i t a t i v e s o l u b i l i t y d a t a f o r rifampin were reported, a s shown i n Table VIII.
2.6

484

Figure 7

- X-ray

powder d i f f r a c t i o n pattern of rifampin before grinding.

T a b l e VI X-rav powder d i f f r a c t i o n p a t t e r n of r i f a m p i n before q r i n d i n q , a c c o r d i n q t o ASTM r u l e s

Rifampin ( s a m p l e before g r i n d i n g )

Rad. Cuka

= 1.5418 A Filter N i

d(i)
0

1/11

hkl

d(i)

1/11

hkl

I/I

Spectrometer

17.16 12.43 11.11 10.76 94 .0 8.80 78 .6 75 .1 69 .1 65 .1 62 .1 56 .0

2 8 3 23 35 2 14 4 2 44

1 1

51 .8 4.90 46 .3 44 .3 4.12 40 .5 39 .5 38 .2 36 .7 3.44 3.38 2.96

loo

13 17 2
5 2 3 6 8 8 5

3a
_ L ,
d

-_d

Figure 8

- X-ray

-20

10
I

3"

. a

2w

powder diffraction pattern of rifampin after grinding

Figure 9

- Heating

curve of rifampin

R I F AMP IN

Table

VII

Approximate s o l u b i l i t i e s of rifampin P a r t s of s o l v e n t required f o r 1 p a r t of rifampin from from from from from from from Descriptive term freely soluble soluble soluble soluble s l i g h t l y soluble s l i g h t l y soluble very s l i g h t l y soluble practically insoluble practically insoluble practically insoluble practically insoluble practically insoluble practically insoluble practically insoluble
I

Solvent chloroform methanol dimethylformamide dimethylsulphoxide e t h a n o l 95 per c e n t acetone benzene c o r bon t e t r ac h l o r i d e n-hexane cyclohexane n-butanal propyleneglycol glycerol carbowax 400
I

1 t o 10 10 t o 30 10 t o 30 10 t o 30 100 t o 1,000 100 t o 1,000 to00 t o 10,000

Table VIII

-- u b i l i t i e s Sol

of rifampin

chloroform dichloromethane , ethyl acetate dioxane methanol I acetone l n-hexane j petroleum e t h e r water pH 7.3 w a t e r pH 4.3 water pH 7.5 w a t e r pH 2.0 O.tN Hcl 1 phosphate b u f f e r pH 7.4

I I

1
I

'

349 216

I
I

I
38

108
39 16 14 0.43 0.33 25oc
,

i
I

2.5
1.3

2.8

99.5 !2O0.O , 9.9

'room
;37oc

46
47
'
d

489

GlAN G. G A L L 0 AND PIETRO RADAELLI

Lipid-water p a r t i t i o n 2.621 Organic solvent-water P a r t i t i o n The study of the p a r t i t i o n between water and organic s o l v e n t s a s an i n d i r e c t measure of t h e lipid-water p a r t i t i o n has not been generally applied t o t h e f i e l d of rifamycins. T h e p a r t i t i o n of rifampin i n the system n-octanol/aqueous phosphate buffer, pH 7.4 was determined by Seydel (47) t o be K=15.6, w h i l e C u r c i e t al. (48) have measured i t i n the system benzene/ aqueous b u f f e r s i n the range pH 5.5-7.0, andKequaled 9.7; a t pH 7.5, K=9.0; a t pH 8.0, K=4.6. 2.622 S i l i c o n oil-water p a r t i t i o n The lipid-water p a r t i t i o n has been obtained f o r v a r i o u s rifamycins from Rf values i n part i t i o n reverse-phase t h i n l a y e r chromatography on S i l i c a g e l p l a t e s impregnated w i t h s i l i c o n o i l as stat i o n a r y phase and water-acetone a s mobile phase (49-51 ). The f r e e energy parameter RM=log(- 1 -1) (52), was used f o r q u a n t i t a t i v e c o r r e l a t i o n s between s t r u c t u r e and a n t i b a c t e r i a l (49,50) and a n t i v i r a l (51) a c t i v i t i e s . RM values were obtained f o r rifampin and a r e reported i n Table IX. Table I X RM values f o r rifampin
R

2.62

RM
0.029

mobile phase
30% acetone i n water (v/v)

l s t a t i o nary phase silicon silicon oil oil

Ref.
49

-0.293

40% acetone i n water (v/v)

2.623

50

Surface a c t i v i t y Rifamycins have been shown t o have s u r f a c e a c t i v i t y , from the v a r i a t i o n of t h e surface tension of b u f f e r water s o l u t i o n s w i t h concentration (53 I 54).

490

RlFAMPlN

The s u r f a c e p r o p e r t i e s of rifampin depend on t h e pH of t h e medium. I n the a l k a l i n e t o n e u t r a l range, rifampin i s a weak s u r f a c t a n t and no a s s o c i a t i o n s a r e observed; i n the a c i d i c range (pH 4-0) a pronounced lowering of the s u r f a c e t e n s i o n w i t h conc e n t r a t i o n i s observed, and m i c e l l e formation i s apparent a t a c o n c e n t r a t i o n of about 10-5 m o l e / l i t e r

(55)
3.
3.1
STABILITY

STABILITY A POWDER S Rifampin i s very s t a b l e i n the s o l i d s t a t e i n s e a l e d c o n t a i n e r s a t room temperature, a s described i n Table X (56). Rifampin i n t h e s o l i d s t a t e i s s t a b l e a l s o a t temperatures u p t o 70OC, a s reported by Sano e t a l . (38). STABILITY I SOLUTION N The s t a b i l i t y of rifampin i n aqueous s o l u t i o n has been widely i n v e s t i g a t e d and t h e c o n d i t i o n s and t h e transformation products a r e reported i n Table XI. L i k e o t h e r rifamycins, rifampin undergoes d e s a c e t y l a t i o n on a l k a l i n e t r e a t m e n t , y i e l d i n g t h e corresponding 25-desa c e t y l d e r i v a t i v e without s u b s t a n t i a l l o s s of a n t i b a c t e r i a l a c t i v i t y (57). I n mildly a l k a l i n e aqueous s o l u t i o n s and i n the presence of atmospheric oxygen a t room temperature, rifampin transforms i n t o rifampin quinone; t h i s oxidation can be prevented by sodium ascorbate. Under the same c o n d i t i o n s and a t 60-70OC, rifampin y i e l d s t h r e e main transformation products, which were ident i f i e d by Maggi e t a1.(22) a s 25-desacetyl-rifampin, 25-desacetyl-23-acetyl-rifampin and 25-desacetyl21 - a c e t y l - r i f ampin, Sano e t a l . (38) have s t u d i e d t h e s t a b i l i t y o f rifampin a t 250 i n aqueous s o l u t i o n s a t d i f f e r e n t pH's: it decomposes r a p i d l y i n a c i d i c o r a l k a l i n e c o n d i t i o n s , but slowly i n n e u t r a l ones. 3-Formylrifamycin SV i s the main decomposition product of rifampin i n aqueous 3.2

491

Table St -a b i l i t y

of rifampin i n t h e s o l i d s t a t e a t room temperature 1 2 months

(56)
41 months

st o r t i n g
' UV kssay

21 months

30 months

%
c

Microbig UV Microbig UV Microbig UV Microbig UV Microbig l o g i c a l Assay l o g i c a l Assay l o g i c a l Assay l o g i c a l Assay l o g i c a l Assay % % Assay % % Assay % % Assay % % Assay %

96.4
P

99.0

100.0
TLC
I

100.2
TLC

95.4

97.3
TLC

( D

t ~forrnvlrifarifampin

I I
i

95.9

TLC

TLC

jI

I
i1

I
I
I

1
-

1
!
!

absent traces

1-1.5% 1-1.5%

1
11

1.5-2%
1-1 .!j%

2.54%
1-1.5%

1.5-2%
1-1.5%
!
1

: rifampin Noxide

I
i

25-desacetyl rifampin acetyl-rifampin

,1
1

0.5

1%

j 1.5

- 2%

1.5

- 2% 1 1 - 1.5%
I
I

1-1.5%

i
I

R I F AMP IN

Table

XI

S t a b i l i t y o f r i f a m p i n i n aqueous s o l u t i o n s

3-formyl-rifamycin SV

+
r i f ampin

r i f ampin-quinone

pH 2.3, 20-22OC (8) tic1 0,1N, 37% (47)

pH 8.2, 20-22% (8)

NaOH 5% i n e t h a n o l water ( 1 : l ) 2022% (57)

-i
t
I

pH 8 . 2 (22) 60-70%

25-desacetyl-21-acetylrifampin

bl 25-desace t y l - 2 3 - a c e t y l r i f am p i n I

493

GlAN G. GALL0 AND PIETRO RADAELLI

acidic medium (8). Seydel (47) has determined the decomposition r a t e of rifampin i n 0 1 tC1 vs. temperature .N and, from the Arrhenius plot, calculated the activation energy, &la=l9.2 Kcal/mole/degree. Rifampin does not lower i t s microbiological activi t y i n the various conditions reported i n Table XII. Table X1 I S t a b i l i t y of the a n t i b a c t e r i a l a c t i v i t v of rifamPin i n solution. Conditions
water-dimethylf ormamide ( 95 :5) a t 5%
d imet hylsulphoxide a t 150

Concentration( mg/ml)

Time

Ref

5 days %

58

10
I

8 months
I

59
I

water-ethanol (8:2) a t 4% o r 20%

i
6O,6l

8 weeks

4. SYNTHESIS Rifampin i s a semi-synthetic rifamycin, obtained by condensation o f 1-amino-4-methylpiperazine w i t h 3formyl-rifamycin SV i n peroxide-free tetrahydrofuran a t 100-150C. The 3-formyl-rifamycin SV (14) was obtained from rifamycin B, the fermentation product (62-64) , by the chemical procedure reported i n t a b l e XIII. Gianantonio e t al. (65) have patented the production of rifampin by reacting rifamycin S d i r e c t l y w i t h formaldehyde, tert-butylamine and MnO and condensing w i t h 2 1 -amino-4-methylpiperazine (see t a b l e XIII).

494

R I F AMP IN

Table X l l l Synthesis of r if a mpin

STREPTOMYCES MEDITERRANEI

1
oxidation
w

reduction

HC 3*
0

CH2-COOH RlFAMYClN B(62-64) (fermentation product)

I !
'0

RlFAMYClN 0 ( 66) Acidic hydrolyais

in aerated aqueous solution

OH OH

oxidation reduction

OH 0

RlFAMYClN SV(67) Mannich reaction and reduction tart-but lamine, dn02 in tetrahydrofuran
2) l-amino4 -methylpiperazine (65)

OH OH

Hc 3& f

CH2-N /RI

/
OH OH "c c >mty 3@H- ~ l a ;ih : OH i-NnN-CH3

OH 'R2 MANNICH DERIVATIVE OF RlFAMYClN SV (15)

Oxidation in acidjc conditions

OH 3-FORMYL RlFAMYClN SV (14,681

v
RIFAMPIN ( 8 )

495

GlAN G. G A L L 0 AND PIETRO RADAELLI

5.
5.1
Element

METHODS OF ANALYSIS

ELEMENTAL ANALYSIS Theoretical

H
N 0
5.2

62.76 7.10 6.81 23.33

IDENTIFICATION TESTS R i f a m p i n i s i d e n t i f i e d and d i f f e r e n t i a t e d from t h e o t h e r r i f a m y c i n s by i n f r a r e d and n u c l e a r magnetic resonance spectroscopy, f i e l d d e s o r p t i o n mass s p e c t r o metry, p o t e n t i o m e t r i c t i t r a t i o n , d i f f e r e n t i a l scanning c a l o r i m e t r y and e l e m e n t a l a n a l y s i s , I n o r d e r t o d e f i n e t h e homogeneity o f t h e r i f a m p i n sample, chromatographic procedures (paper and t h i n l a y e r ) and t h e r m a l a n a l y s i s a r e used. C o l o r i m e t r i c t e s t s based on o x i d a t i o n were deTo 5 m l o f aqueous r i f a m p i n s o l u t i o n ( a b o u t scribed, 1 mg/ml), I m l o f 10% (w/v) ammonium p e r s u l p h a t e i n phosphate b u f f e r , pH 7.38, i s added: t h e c o l o r t u r n s from orange-yellow t o v i o l e t - r e d (69). A s i m i l a r method, employing f e r r i c n i t r a t e as o x i d i z i n g agent, was d e s c r i b e d by Eidus e t a l . (70,71), who used i t t o i d e n t i f y t h e presence o f r i f a m p i n and d e s a c e t y l r i f a m p i n i n biological fluids.

5.3

SPECTROPHOTOMETRIC METHODS 5.31 S p e c t r o p h o t o m e t r i c assay on b u l k p r o d u c t The V I S maximum a t 475 n i n aqueous phosm phate b u f f e r s o l u t i o n pH 7.38 w i t h an a b s o r p t i v i t y (g/a) v a l u e o f 18.7 (see S e c t i o n 2.11) enables r i f a m p i n t o be q u a n t i t a t i v e l y assayed. The main i m p u r i t i e s o f r i f a m p i n a r e 3f o r m y l r i f a m y c i n SV and rifampin-quinone. The two p r o d u c t s can be s p e c t r o p h o t o m e t r i c a l l y q u a n t i t a t e d : t h e

f i r s t one by an e x t r a c t i o n method u s i n g n-hexane:ethyla c e t a t e ( 2 : l ) (13) and t h e second one by a d i f f e r e n t i a l s p e c t r o p h o t o m e t r i c method which t a k e s advantage o f t h e r e d u c t i o n by sodium ascorbate o f t h e quinone ( 1 3).
496

RlFAMPlN

Spectrophotometric assay on p h a r m a c e u t i c a l preparations R i f a m p i n can be determined i n pharmaceutic a l p r e p a r a t i o n s by u s i n g t h e d i f f e r e n t i a l spectrophot o m e t r i c method (72), m o d i f i e d by P a s q u a l u c c i e t a l .

5.32

(73)

Spectrophotometric assay i n b i o l o n i c a l fluids Numerous s p e c t r o p h o t o m e t r i c methods f o r det e r m i n i n g r i f a m p i n i n b i o l o g i c a l f l u i d s have been des c r i b e d i n t h e l i t e r a t u r e , b u t owing t o t h e i r low sens i t i v i t y t h e y were s a t i s f a c t o r i l y a p p l i e d o n l y t o t h e d e t e r m i n a t i o n o f r e l a t i v e l y h i g h l e v e l s (more t h a n 5pg/ml). The methods were based on t h e e x t r a c t i o n o f r i f a m p i n and i t s m e t a b o l i t e s w i t h o r g a n i c s o l v e n t s and on t h e d e t e r m i n a t i o n o f t h e V I S absorbance o f t h e o r ganic e x t r a c t . The e x p e r i m e n t a l c o n d i t i o n s a r e summarized i n T a b l e XIV.

5.33

5.4

FLUORQMETRIC DETERMINATION

R i f a m y c i n s do n o t e x h i b i t n a t u r a l fluorescence. A q u a n t i t a t i v e method was developed f o r r i f a m y c i n B (82), based on i t s t r a n s f o r m a t i o n i n t o t h e t r i a c e t y l d e r i v a ti v e R i f a m p i n was determined f l u o r e m e t r i c a l l y by t r a n s f o r m i n g i t w i t h hydrogen p e r o x i d e (83) i n t o a f l u o r e scent product. The maximum f l u o r e s c e n c e develops i n an aqueous carbonate-bicarbonate b u f f e r , pH 9.2 a t 4-80 nm, when t h e e x c i t a t i o n wavelength i s 370 nm. The r e l a t i v e fluorescence i n t e n s i t y i s l i n e a r w i t h concentrations o f r i f a m p i n i n t h e range 0.1 t o 10 pg/ml.

ANALYSIS BY COMPLEX FORMATION R i f a m y c i n s complex w i t h m e t a l l i c ions, as r e p o r t e d f o r r i f a m y c i n L (34) and f o r r i f a m y c i n S (84). R i f a m p i n i n methanol s o l u t i o n g i v e s a complex when t r e a t e d w i t h an aqueous s o l u t i o n o f A l C l i n t h e r a t i o 2:l (85); t h e i n t a b i l i t y c o n s t a n t i n waqer-methanol ( 1 : l ) i s 3.4.10 Complex f o r m a t i o n was demonstrated t o t a k e p l a c e a l s o w i t h Hg*, Cu", Ag' and Fe(86).

5.5

8 .

497

GlAN G. G A L L 0 AND PIETRO RADAELLI

Table X I V Spectrophotometric methods f o r t h e d e t e r m i n a t i o n o f r i f a m p i n and i t s m e t a b o l i t e s i n body f l u i d s .

Body fluid

1
I

1 Compound
Extraction solvent

I
benzene iso-amyl alcohol

Analytical Assayed ar wavelength stated i n :abrorpt i v i t y the r e f 2 9/11 rence

bile, urine urine, serum

RtDA

urine urine, serum, b i l e and t i s s u e s urine

I$;;ne-hexane

I R
I

butanol-hexane

(4:l)
1

:;;p;ol-hexane

I R
II
/
I
R

teeth

butanol-hexane

(4:l)
1;; L e t n ;o h pa e ethylalcoholethylacetate

serum

aerum
~~ ~

(1 :1)
urine cyclohexaneb u t y l acetate blue l i g h t f i

I(i :i)

498

AD+R
RtDA
R R R

*,
eference 475nm and 335nm ( c a l i b r a t i o n curve)

75

335nm ( c a l i b r a t i o n curve) 478nm ( c a l i b r a t i o n curve)

76

478nm (19.6) 478nm ( C a l i t i o n curve)

77

482nm (15.6)

79

475nm (15.5)

80

' t i o n curve)

RlFAMPlN

The f o r m a t i o n o f t h e complex w i t h A1C13 was employed f o r q u a n t i t a t i v e d e t e r m i n a t i o n o f r i f a m p i n i n bulk, i n pharmaceutical f o r m u l a t i o n s and i n u r i n e (87), by measuring t h e cherry-red c o l o r a t 507nm i n methanolwater (49:l) o r i n methanol-water-butanol-hexane (1 9:

1 :4:1),

5.6

VOLUMETRIC METHODS

Rifampin i n capsules was determined by a volum e t r i c method (88): t h e a n t i b i o t i c was o x i d i z e d w i t h an excess o f f e r r i c c h l o r i d e , which was then t i t r a t e d iodometr i c a l l y .

57 .

CHROWTOGRAPHIC METHODS

T h i n l a y e r chromatography TLC has been w i d e l y used i n t h e f i e l d o f r i f a m y c i n s (89,90) and t h e c o l o r e d spots a r e d i r e c t l y located. Many TLC procedures were developed f o r t h e a n a l y s i s o f r i f a m p i n and i t s m e t a b o l i t e s i n body f l u i d s . The Rf values were shown t o be dependent on t h e concent r a t i o n s (91). The spots were q u a n t i t a t e d by u s i n g t h e bioautographic technique (92) o r t h e d e n s i t o m e t r i c one (93) o r by v i s u a l e s t i m a t i o n (94) or, f i n a l l y , by t h e e l u t i o n method f o l l o w e d by m i c r o b i o l o g i c a l assay (95). A reverse,,phase p a r t i t i o n TLC procedure has been des c r i b e d f o r t h e d e t e r m i n a t i o n o f r i f a m p i n (96) :s i l a n i z ed S i l i c a g e l p l a t e s were used as s t a t i o n a r y phase, w i t h phosphate b u f f e r , pH 7 c o n t a i n i n g 0.1 % sodium ascorbate as mobile phase ( o t h e r mobile phases are reported), The c o l o r e d spot was e l u t e d and measured spectrophotometrically. Reverse-phase p a r t i t i o n TLC has been used f o r s t r u c t u r e - a c t i v i t y c o r r e l a t i o n s (see S e c t i o n 2.622). 5.72 Column chromatography The c o n t e n t o f r i f a m p i n and i t s m e t a b o l i t e s i n urine, b i l e and serum, was determined by e x t r a c t i o n w i t h c h l o r o f o r m and by column chromatography, f o l l o w e d by spectrophotometry (97). The l i q u i d - s o l i d chromatography was c a r r i e d o u t u s i n g a g l a s s column 7 c long, m 4 mm I.D., packed w i t h S i l i c a g e l G, b u f f e r e d a t pH 6, and chloroform and chloroform-methanol i n p r o g r e s s i v e l y
499

5.71

GlAN G. G A L L 0 AND PIETRO RADAELLI

i n c r e a s i n g amounts (5%, 10% and 16.6%) as s o l v e n t , a t a f l o w r a t e o f 0.15 ml/min. R i f a m p i n and i t s m e t a b o l i t e s were t h e n q u a n t i t a t e d by r e a d i n g t h e absorbance o f t h e e l u a t e s a t 475 nm. 5.73 Paper chromatoqraphy A paper chromatographic t e c h n i q u e f o r det e r m i n a t i o n o f r i f a m p i n and 2 5 - d e s a c e t y l r i f a m p i n was used f o r u r i n e and b i l e (74). Descending chromatography was c a r r i e d o u t on Whatman 3MM paper u s i n g methanol: no c t a n o l ( 4 : l ) as s t a t i o n a r y phase and aqueous b u f f e r , pl-l 6 , as m o b i l e phase f o r 6 hr. The i n t e n s i t y o f t h e red-orange s p o t s was measured by an A n a l y t r o l photoden s i t o m e t e r (mod.Rl3-450 n f i l t e r ) o r t h e m a t e r i a l s dem termined b i o a u t o g r a p h i c a l l y . Akimoto e t a l . (98) r e p o r t e d a paper chromatographic method f o r t h e s e p a r a t i o n o f r i f a m p i n and i t s m e t a b o l i t e s , u s i n g e t h y l acetate-water-dimethylformamide ( 1 O : l O : l ) . 5.74 H i g h pressure l i q u i d chromatography R i f a m p i n has been f r e q u e n t l y c i t e d as an Rifampin, emample o f t h e u s e f u l n e s s o f HPLC (99-102). 25desacetylrifampi11, r i f a m p i n quinone and 3 - f o r m y l r i f a m y c i n SV were separated (99) under t h e f o l l o w i n g c o n d i t i o n s : DuPont 820 Chromatograph equipped w i t h UV d e t e c t o r , ODS Permaphase column a t 50% and 1,000 p s i , w a t e r t o methanol w i t h l i n e a r g r a d i e n t 8%/min as m o b i l e phase and l m l / m i n f l o w r a t e .

5.8

METHODS M i c r o b i o l o g i c a l met hods have been w i d e l y d e s c r i b e d f o r t h e d e t e r m i n a t i o n o f r i f a m p i n potency i n b u l k produ c t s , i n p h a r m a c e u t i c a l f o r m u l a t i o n s and i n body f l u i d s , as l i s t e d i n t h e r e v i e w by B i n d a e t a l . (103). These methods can be c l a s s i f i e d as a) d i f f u s i o n p l a t e methods, b) s e r i a l t u b e d i l u t i o n methods and c) t u r b i d i m e t r i c methods, 5.81 D i f f u s i o n p l a t e assay methods These methods were used t o d e t e r m i n e r i fampin i n serum, b i l e and u r i n e as w e l l as i n o t h e r body f l u i d s and organs (fragments o f organs and t i s s u e s were

MICROBIOLOGICAL

500

RlFAMPlN

appropriately treated f o r extracting rifampin). They can be d i v i d e d i n d i f f e r e n t c l a s s e s a c c o r d i n g t o techn i c a l aspects as r e p o r t e d i n T a b l e XV, where o t h e r exp e r i m e n t a l d e t a i l s a r e summarized, The c y l i n d e r - p l a t e t e c h n i q u e d e s c r i b e d by Grove e t a l . (104) has been adopted i n L e p e t i t S.p.A. (105) 5.82 S e r i a l t u b e d i l u t i o n methods T h i s method, f i r s t a p p l i e d by C l a r k e t a l . (115) t o r i f a m i d e and r i f a m p i n , has been used f o r t h e d e t e r m i n a t i o n o f r i f a m p i n i n serum by v a r i o u s l a b s . Some d e t a i l s a r e r e p o r t e d i n T a b l e XVI. 5.83 T u r b i d i m e t r i c met hods A t u r b i d i m e t r i c m i c r o b i o l o g i c a l assay o f r i f a m p i n u s i n g an a u t o m a t i c a n a l y z e r was developed by S i m o n c i n i e t a l . (120). The method i s based on t h e measurement o f t h e o p t i c a l d e n s i t y o f t h e b a c t e r i a l suspension ( K l e b s i e l l a Pneumoniae ATCC 10031 o r E s h e r i c h i a c o l i ATCC 10536 as t e s t microorganism) i n a D i f c o c u l t u r e medium c o n t a i n i n g r i f a m p i n , a f t e r i n c u b a t i o n a t 370 f o r 3.5 hr.

6 PROTEIN BINDING The b i n d i n g o f r i f a m p i n t o blood serum and plasma p r o t e i n s i n man and o t h e r a n i m a l species was w i d e l y studied. The d a t a o b t a i n e d c o v e r a r e l a t i v e l y l a r g e range, p r o b a b l y because o f t h e d i f f e r e n t methodologies used and o f t h e many parameters t h a t can i n f l u e n c e t h e phenomenon (1 21 ) I n v i t r o experiments were c a r r i e d o u t by C u r c i e t a l . w i t h a d i a l y s i s technique, and t h e y r e p o r t e d t h a t a t t h e c o n c e n t r a t i o n s reached i n v i v o , about 7 5 4 5 % o f Inr i f a m p i n b i n d s w i t h serum p r o t e i n s (48,122-125). t e r e s t i n g l y , t h e y found t h a t PAS ( p a r a - a m i n o - s a l i c y l a t e ) i n c o n c e n t r a t i o n s from 100 t o 200 pg/ml decreases t h e By an u l t r a c e n t r i b i n d i n g o f t h e a n t i b i o t i c (122). f u g a t i o n technique, Seydel (47) o b t a i n e d a percentage o f r i f a m p i n bound t o bovine albumin i n v i t r o i n t h e range from 50 t o 70%. From t h e s e data, K e b e r l e e t a l . (126) d e r i v e d t h a t each albumin molecule has two b i n d i n g Mashimo (127) has found t h e p r o t e i n s i t e s f o r rifampin.

501

Table M i c r o b i o l o q i c a l ass= Technical aspects

XV

of r i f a m p i n by d i f f u s i o n p l a t e assay methods Medium Agar ( D i f c o 0263-02) Microorganism ISarcina , l u t e a (ATCC 9341) Reference

-h e t

metal c y l i n d e r s containing l i q u i d t o be assayed wells ( l o m m ) c o n t a i n i n g t h e l i q u i d t o be assayed

1 05

wells (9mm)

d i s k s (6mm) soaked i n the l i q u i d t o be assayed d i s k s (9mm) disks disks

d i s k s (9mm)

1
~

Salt-glucose-peptone b r o t h ( P a s t e u r Inst. Codex)

'

1 I
I

k o r c i n a l u t e a (IP 5345)

112

d i s k s (9mm)
(6mm)

C a s e i n peptone-soya peptone-agar Agar ( D i f c o 0263-02)

!Staphylococcus c o a g u l a s e lnegative ' S a r c i n a l u t e o (ATCC 9341) I

113

1 disks

'

114

I i .

RlFAMPlN

Table

XVI

M i c r o b i o l o q i c a l assays o f r i f a m p i n by s e r i a l t u b e d i l u t i o n methods. Medium non-spec i f i e d broth D i f c o sucrose broth w i t h phenol r e d mannite and phenol r e d selective broth Youmans Microorganism Sarcina l u t e a Inoculum i e f e r e nc e

0.031111o f a 1 :250 d i l u t i o n o f 18h c u l t u r e Staphylococcus 0.05ml o f a aureus (ATCC 1 :lo0 d i l u t i o n o f 18h c u l t u r e 6538P) Staphylococcus 0.05ml o f a aureus (Oxford, 1 :200 d i l u t i o n sensitive t o o f 24h c u l t u r e O.O2ug/ml) Mycobacterium 5-1 OO*1O4
tuberculosis viable units

115 116 117

118

(H37RV)
Loc kemann Mycobacterium tuberculosis (H37Rv o r F u hrmann

119

b i n d i n g o f r i f a m p i n by d i a l y s i s , u l t r a f i l t r a t i o n and u l t r a c e n t r i f u g a t i o n , a t a c o n c e n t r a t i o n o f 2Opg/ml, t o No c l e a r i n t e r p r e be lo%, 90% and 70%, r e s p e c t i v e l y . t a t i o n was g i v e n f o r t h e d i f f e r i n g data, I n v i v o experiments o f p r o t e i n b i n d i n g were c a r r i e d o u t u s i n g ' ' k - r i f a m p i n (128) and about 75% o f r i f a m p i n was found t o be bound t o human serum p r o t e i n , P i l h e u e t a l . (129) found r a t i o s o f 15 t o 40% between c e r e b r o s p i n a l f l u i d ( c o n s i d e r e d as a p r o t e i n - f r e e f l u i d ) and plasma l e v e l s . Aoyagi has r e p o r t e d (130) t h e b i n d i n g o f r i f a m p i n t o t h e serum p r o t e i n s o f v a r i o u s animals: i n t h e horse 40-46%, i n t h e r a b b i t 13-17% and i n man 17-38%. A r e c e n t s t u d y by d i a l y s i s u s i n g r i f a m p i n (131) showed t h a t 86.1% and 88.9% o f r i f a m p i n were bound t o t h e plasmas o f t u b e r c u l a r p a t i e n t s and

503

GlAN G. G A L L 0 AND PIETRO RADAELLI

healthy volunteers, r e s p e c t i v e l y . Yokosawa e t a l . (1 32) have suggested, on t h e b a s i s of e l e c t r o p h o r e s i s and microbiological assay, t h a t rifampin a s s o c i a t e s w i t h a1-t 012- and P-globulins of human serum.

7. PHARM4COKINETICS AND METABOLISM I N T h e pharmacokinetics of rifampin has been widely

s t u d i e d and t h e serum l e v e l s of rifampin a f t e r s i n g l e and repeated administration of d i f f e r e n t doses were determined by various i n v e s t i g a t o r s and have been reviewed (103). To summarize, a f t e r o r a l administration, rifampin is well-absorbed w i t h t h e maximum plasma leve l s a t 1.5-3 h r over a wide range of s i n g l e dose, i . e . , 0.1 t o 1.2 g. The l e v e l s a r e s t i l l appreciable (5-10% of the peak l e v e l ) a f t e r 12 h r only a t t h e high doses (103). Rifampin i s widely d i f f u s i b l e i n the t i s s u e s and i n t h e various body f l u i d s (105), a s indicated i n Table X V I I . T h i s i s r e l a t e d t o t h e h i g h lipotropism of r i fampin (47,413). Rifampin e l i m i n a t i o n , which m u s t be considered slow, occurs mainly through t h e b i l e b u t a l s o through the The t o t a l amount of rifampin elimu r i n e (103,128,133). inated i n the b i l e i s not proportional t o t h e dose administered (48,116,134) while t h e urinary e l i m i n a t i o n i n c r e a s e s w i t h t h e dose (77,105,109,116,133-136). I n an i n v e s t i g a t i o n c a r r i e d out over 6 days (105), about 22% was eliminated i n t h e f a e c e s and about 26% i n t h e u r i n e , I n another study, however, Riess(128) found t h a t 96 hrs a f t e r administration of 300 mg of labeled rifampin, 94% of t h e t o t a l r a d i o a c t i v i t y had been recovered i n equal percentages i n the faeces and u r i n e , The main metabolite of rifampin i n man was ident i f i e d , a f t e r i s o l a t i o n from b i l e and urine, a s 25T h i s compound i s much desacetylrifampin (74,92,94,137). less l i p o p h i l i c than rifampin and it is e a s i l y excreted i n urine (46) and not reabsorbed (138,139). Sano e t a l , (93) reported t h a t i n a d d i t i o n t o 2 5 - d e ~ a c e t y l r i f a m p i n ~ a second metabolite i s 3-formyl rifamycin SV.

504

R I F AMP IN

Table XVII Distribution of rifampin i n human tissues and f l u i d s a f t e r oral administration of a s i n g l e 450 mg dose (105)

adm i n istration 16

l2
6

\-I
13

spleen

lung

]gallbladder wall
colon wall

Dliver

36

12

s k i n muscle

12

kidney

15

mammary gland

ovarian c y s t wall

505

GlAN G. G A L L 0 AND PIETRO RADAELLI

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P.Marga1it.h

73. 74. 75. 76. 77.

November 24, 1970 P.Sensi, R . B a l l o t t a and A.M.Greco, Farmaco (Pavia) Ed.Sci., l5, 228 (1960). P.Sensi, R . B a l l o t t a , A.M.Greco and G.G.Ga!.lo, Farmaco (Pavia) Ed.Sci. , l 6 , 165 (1 961). N.Maggi, R.Pallanza and P.Sensi, A n t i m i c r o b i a l Agents Chemotherapy, 765 , (1 965). N.Maggi, L e p e t i t Q u a l i t y C o n t r o l Labs., P r i v a t e Communication (1 974). L.Eidus, G.M.Ling and A.M.T,Harnanansingh, Intern. J . C l i n . Pharmacd. , 296 (1 969). L. E i d u s and A. M.T.Harnanansingh Am.Rev.Respirat, Disease 100, 738 (1 969). G.Sferruzza and R.Rangone, Farmaco (Pavia) Ed. . rat P 19, 491 (1964). C.R.Pasqualucci, A.Vigevani, P . R a d a e l l i and G. G.Gallo, J.Pharm.Sci. 5 9 , 685 (1970). N.Maggi, S.Furesr, R.Pallanza and G.Pelizza, A r z n e i m i t t e l - F o r s c h . , l 9 , 651 (1 969). S.Sunahara and H.Nakagawa, Chest, 526 (1972). Y.Kimura, Y.Inoue, M.Takahashi and Y.Todome, Shinryo, 23, 1003 (1970). P.Abbamonte and G . S c o d i t t i , Riv.Pato1.Clin.Tuberc.

u.3,542,762, ,

( 1 961 )

and H. Pagani, Appl.Microbio1.

, 9,

325

2,

61,

42 , 319

(1969).

509

GlAN G. G A L L 0 AND PIETRO RADAELLI

78

79.
80. 81 82. 83. 84. 85. 86. 87. 88 , 89.

90.
91. 92. 93. 94.

95. 96. 97. 98. 99.

M. S i l v e s t r i n i - B i a v a t i , S Sacco, M . V i t i and N.Vespa, A t t i Simposio Naz, Rifampicina, Rome, Vol. I 1 1970, p. 53-C E d i z i o n i Rassegna Medica L e p e t i t , Milan. M, Alberg hina A t t i Tavola Rotonda R i f ampicina Taormina, 1969, p.59, E d i z i o n i Rassegna Medica, L e p e t i t , Milan, P.De Gregorio, Arch.Monaldi (Naples) 27,685( 1972) R.A.Ioffe, A n t i b i o t i k i , 18, 799 (1973). P.Sensi, C.Coronelli and A,Binaghi, Farmaco (Pavia) Ed.Prat., 1 , 292 (1960). 5 J.M.Finke1, R . F . P i t t i l l o and L.B.Mellett, Chemotherapv, 16, 380 ( 1 971). D , L e i b f r i t z , Tetrahedron L e t t e r s , 4125 (1974). P.Barza, Farmacia (Bucharest), 21, 273 (1973). P.Barza, Farmacia (Bucharest), 20, 499 (1 972). P.Barza, Farmacia (Bucharest) , 21, 435 (1 973). P.Barza, Farmacia (Bucharest), 21, 121 (1973). C . C o r o n e l l i and A.Binaghi, FarmaB.J.R.Nicolaus,c o (Pavia) Ed.Prat., l6, 349 (1961). P.Sensi, C.Coronelli and B.J.R.Nicolaus, 3. Chromatorr. 5 , 519 (1961). O.T.Kolos, L.L.Eidus, J.Chromatog., 68, 294 ( 1 972). K.Shimizu and O.Kunii, Shinryo, 23, 969 (1970). K.Sano, H.Hakusui, Japan.J.Antibiotics, 23, 416 (1 970) L.T.Tenconi and E.Beretta, E x c e r p t a Med.Intern. Congress S e r i e s , No.198, Proc.Europ.Soc.Study Drua T o x i c i t y , 11,80 (1970). E . B eretta, L e p e t i t Research Labs., P r i v a t e Communication (1 974). R.Rangone and C.Ambrosio, Farmaco (Pavia) Ed.Prat. - 237 (1971). 26, E.Beretta, L.T.Tenconi, L e p e t i t Research Labs., P r i v a t e Communication (1 969). K.Akimoto and K.Ono, Japan. J . A n t i b i o t i c s , 23, 250 ( 1 970). J.A.Schmit, R.A.Henry, R.C.Williams and J.F. Dieckman, J.Chromatoa. Sci. , 9, 645 (1971).

510

RI FAMPlN

100.

J.J.Kirkland matography",

"Modern P r a c t i c e of L i q u i d ChroW i l e y - I n t e r s c i e n c e , New York, N.Y. Bull.Soc.Chim.France,

101. 102. 103. 104.

105.

1971, p.197.
M.Martin and G.Guiochon,

1 1 (1973). 6
A.F.Michaelis, D.W,Cornish and R . V i v i l e c c h i a , J.Pharm.Sci., 62, 1399 (1973). G.Binda, E.Domenichini, A , G o t t a r d i , B.Orlandi, E . O r t e l l i , B . P a c i n i and G.Fowst, A r z n e i m i t t e l Forsch., 1907 (1971). D.C.Grove and W.A.Randal1, "Assay Method o f A n t i - ' b i o t i c s " , M e d i c a l E n c y c l o p e d i a I n c . N.Y., 1955. S.Furesz, R . S c o t t i , R.Pallanza and E.Mapelli, d e i m i t t e l - F o r s c h , I l 7 , 534 (1 967). A.Ninni, A.Di F i l i p p o , M.Del Bono and P . N a t a l i , Arch.Tisiol.(Naples) 22, 487 (1 967). H.Beeuwkes, H.J.Buytendijk and F.P.V.Maesen, Arzneimittel-Forsch. l 9 , 1283 (1 969). F . K r a d o l f e r , L.Neipp and W.Sackmann, A n t i m i c r o b i a l Agents Chemotherapy, 359 (1 966). G.Porven and G.Canetti, Rev.Tuberc.Pneumo1. [Paris) 32,707 (1 968). L . V e r b i s t and A.Gyselen, Am.Rev.Respirat.Disease,

21,

106. 107. 108. 109. 110.

111.
112.
113.
114. 115. 116. 117.

- 923 (1968). 98, J.C.Pech&e and C .Tancr\ede, Hop., 17,155 (1969).

(1 968).
H.Hussels,

Pothol.Bio1.Semaine

P.Constans, M.Saint-Paul, Y.Morin, G.Bonnaud and M.Bariety, Rev.Tuberc.Pneumo1. ( P a r i s ) , 32, 991 A c t a Tuberc.Pneumol.Bela.,

L . V e r b i s t , A c t a Tuberc.Pneumol.Beln, I 60, 288 ( 1969). j.Cla;k and A.Wallace, T u b e r c l e , 48, 144 (1967). G.Miano and G.Peruzzi, A t t i Accad.Lancisiana [Rome), 13, SuPD1. 1, 39 (1969). E.Paunescu and M.Stoinescu, F t i z i o l o a i a (Buchar e s t ) , l8, 467 (1969).

( 1969).

@, 270

511

GlAN G. G A L L 0 AND PIETRO RADAELLI

118.

G.Canetti, V.Djurovic, M.Le L i r z i n , R.Thibier and A.Lepeuple, Rev.Tuberc.Pneumo1. ( P a r i s ) 34, 93

119. 120. 121. 122.

123.

124. 125. 126.

127.

128. 129. 130. 131. 132. 133. 134.

( 1970). E.Freerksen, M.Rosenfeld and E.H.Orlowski, B e i t r . Klin.Tuberk. , 141,273 (1 970). F.Simoncini, R.Rangone and C.Calanni, Farmaco (Pavia) Ed.Prat, , 23, 559 (1 968). 84 Suppl., G.Boman, Scand.J.Respirat.Diseases, 40 (1973). G.Curci, A.Ninni and A.D'Alessio, A t t i Tavola Ro- R i f a m p i c i n a , Taormina, 1969 p.19, E d i z i o n i tonda Rassegna Medica, L e p e t i t , Milano. G.Curci, A.Ninni and U.Bellissimo, A t t i Tavola Rotonda R i f a m p i c i n a , C a t a n i a , 1969, p . 5 , E d i z i o n i Rassegna Medica, L e p e t i t , M i l a n o . G.Curci, A.Ninni and V.Fabbrocini, G i o r n . I t a 1 . Chemioterap. , l 7 , 265 (1 970). G.Curci and A.Ninni, F t i z i o l o g i a ( B u c h a r e s t l , & I Suppl., 1 9 (1971). H.Keberle, E.KrUger-Thiemer, W.Sackmann, K.Schmid and J.Seyde1, Proc. V t h I n t e r n . Congress Chemotherapy, Wien 1967, V o l . I V , p.157 K.Mashimo, "Progress i n a n t i m i c r o b i a l and a n t i cancer c h e m n e r a p y . Proc. V I t h I n t e r n , Congress Chemotherapq: vol.11, p. 1198, U n i v e r s i t y Tokyo Press , Tokyo (1 970). W.Riess, Symp.Rimactane, Ciba, Base1 1968, p.36 J.A.Pilheu, F.Maglio, A.D.Pleuz, N.M,De H o r d i e and J.A.Iannello, Torax, l 9 , 261 (1970). T.Aoyagi, Scand.J.Respirat.Disease, 84 Suppl., 44 (1973). G,Boman and V.A.Ringherger, European J.Clin. Pharmacol. , 369 (1974). A.Yokosawa, H.Arai, H.Sato, M.Motomiya and S.Oka, Sci.Rept.Tohoku Univ., Ser.C. , 19, 177 (1972). M.Serembe, Giorn.Ita1.Chemioterap. , 15,2 (1968). G.Acocella , F. B . N i c o l i s and A.Lamarina, P r o c .Vth 1ntern.Congress Chemotherapy, Wien, 1967, Vol.V, a7.

z,

512

RlFAMPlN

135.
136.

R.Marconi, C . A r t a z and D.Do, Minerva Med.,

60,

4845 (1969).
137.
P.E.Dans, R.F. j r . Mc Gehee, C.Wilcox and M. F i n l a n d , Am.J.Med.Sci. 259, 120 (1970). L.T.Tenconi, R . P a l l a n z a , E . B e r e t t a and S.Furesz, "Progress i n a n t i m i c r o b i a l and a n t i c a n c e r chemot h e r a p y . P r o c . V I 1ntern.Congress Chemotherapy'', V o l . 1 , p.346, U n i v e r s i t y Tokyo P r e s s , Tokyo

( 1 970).
138.
S.Furesz, A c t a Tuberc. Pneumol B e l a . ,

60, 266
Symp.

( 1 969).
139.
H.Keberle, K.Schmid and H.G.Meyer-Brunot, Rimactane, C i b a , Base1 1968, p.20.

513

SULFASALAZINE

J. Patrick McDonnell

J. PATRICK McDONNELL

CONTENTS Analytical Profile

Sulfasalazine

1.

Description 1.1 Name Formula, Molecular Weight 1.2 Appearance, Color Odor Physical Properties 2 . 1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2 . 3 U l t r a v i o l e t - v i s i b l e Spectrum 2.4 Mass Spectrum 2.5 M e l t i n g Range 2.6 D i s s o c i a t i o n C o n s t a n t s 2.7 Solubility 2.8 D i s t r i b u t i o n C o e f f i c i e n t Synthesis Impurities

2.

3.

4.
5.

Stability

Drug Metabolic P r o d u c t s Methods of A n a l y s i s 6 . 1 U l t r a v i o l e t Spectrophotometry 6.2 Q u a n t i t a t i v e Thin-Layer Chromatography 6 . 3 Column Chromatography 6.4 Polarography 6.5 T i t r i m e t r i c A n a l y s i s 6 . 6 High Speed Liquid Chromatography D i s t r i b u t i o n i n F l u i d s and T i s s u e s
P ha rma c okine t i c s

6.

7.
8.

9.

Determination i n Body F l u i d s and T i s s u e s 9 . 1 Spectrophotome t r y 9 . 2 Polarography 9 . 3 Paper Chromatography

1 . Acknowledgments 0

11.

References

516

SULF ASALAZIN E

SULFASALAZINE

1.

Description

1.1 Name, Formula , Molecular Weight S u l f a s a l a z i n e i s known c h e m i c a l l y a s benzolc a c i d , 14- / (2 -pyr i d inylamino) sul f o n y i l p h e n y i l a ; 2 -hyd;oxy-5 5-1 b-(2-pyr i ~ y l ~famoyl) phenyi/azg/ s a l i c y l i c a c i d (1) o r u l 4-Tpyr idyl-2-amidosul f o n y l ) -3 -carboxy-& -hydroxya zobenzene ( 2 ) . Common names f o r t h i s drug s u b s t a n c e a r e s a l a z o s u l f a p y r i d i n e and s a l i c y l a z o s u l f a p y r i d i n e (2).

-L

227-

. .

COOH

Molecular Weight 398.39 Appearance, C o l o r , Odor The compound i s a b r i g h t yellow t o brownishyellow, o d o r l e s s , f i n e powder. 2. Physical Properties 2.1

1.2

I n f r a r e d Spectrum The i n f r a r e d a b s o r p t i o n spectrum of s u l f a s a l a z i n e ( S a l s b u r y Reference Standard XP-2) i s p r e s e n t e d i n F i g u r e 1. The spectrum was t a k e n i n a potassium bromide p e l l e t w i t h a Perkin-Elmer, Model 735B I n f r a r e d Spectrophotometer. The assignments a r e a s f o l l o w s : 3400 OH s t r e t c h (bonded) 2500 1670 1680 C = 0 vibration 1630 C = C or N = N vibrations 1580 1360 C02NH v i b r a t i o n 1290 SO2 asymmetrical 1280 1278 C 0 s t r e t c h i n g v i b r a t i o n o r OH d e f o r m a t i o n 1260 1170 1195 S02NH 1135 SO2 group symmetrical 770, 790, 800 CH d e f o r m a t i o n (aromatic r i n g )

517

a
N
W rl

tn
m

8
c n
I

8
9

s
0

8 w

?
N

8
N

0
0

f
N

0 0

NOlSSlWSNVMl lN33M3d

518

SULFASALAZINE

Nuclear Magnetic Resonance Spectrum The n u c l e a r magnetic resonance spectrum o f s u l f a s a l a z i n e (U.S.P. LN231547, Lot F) i s shown i n F i g u r e 2 . The spectrum was r u n on a V a r i a n , Model HA-100 a s 100 MHZ p r o t o n spectrum u s i n g t e t r a m e t h y l s i l a n e a s t h e r e f e r e n c e l o c k s i g n a l . Sweepwidth was 1000 c y c l e s i n t h e f i e l d sweep DMF) was t h e mode. D e u t e r a t e d N , N-dimethylformamide (d7 s o l v e n t . The s p e c t r a l p a t t e r n a r i s e s from a r o m a t i c p r o t o n s on d i f f e r e n t r i n g s . These a r e found i n t h e r e g i o n of 6 . 8 ppm t o 8 . 8 ppm. Each of the s m a l l s e p a r a t e d groups c o r r e sponds t o one proton. Three groups l i e u p f i e l d of t h e r e g i o n of mixed a b s o r p t i o n s and one l i e s downfield ( 3 ) .

2.2

U l t r a v i o l e t - v i s i b l e Spectrum The u l t r a v i o l e t - v i s i b l e sDectrum o f s u l f a s a l a z i n e (U.S.P. LN231547, Lot F) i s shown i n F i g u r e 3. It was made u s i n g a Cary, Model 15 Spectrophotometer on a 1.9 x 10-%I s o l u t i o n of t h e compound. The maximum and minimum a b s o r p t i o n s a r e s i m i l a r t o t h o s e r e p o r t e d i n t h e l i t e r a t u r e (4, 5). I n t h e pH range of 1 t o 10 a ueous s o l u t i o n s o f s u l f a s a l a z i i l e ( c o n c e n t r a t i o n 1.9 x 10-$4) e x h i b i t e d s p e c t r a l a b s o r p t i o n maxima a t 235-240 nm and 350-360 nm. A b s o r p t i o n minimum was shown a t 285-290 nm. A t pH above 11.6 a b s o r p t i o n maxima were e x h i b i t e d a t 450 nm, 235-240 nm and a b o u t 290 nm (4). Mass Spectrum The low r e s o l u t i o n mass spectrum of s u l f a s a l a z i n e (U.S.P. LN231547, Lot F) was o b t a i n e d u s i n g a Varian/MAT CHq Mass Spectrometer. The sample temperature was 3600 C (See F i g u r e 4 ) . The primary f r a g m e n t a t i o n p a t t e r n i n d i c a t e s e l i m i n a t i o n of S02H (m/e = 65) from t h e molecule (M 65 = 333) followed by e l i m i n a t i o n of C 0 2 ( m / e = 45) from t h e molecule (m 109 = 289). T h i s may i n d i c a t e a n o r i g i n a l m o l e c u l a r s t r u c t u r e i n v o l v i n g c y c l i z a t i o n of t h e sulfonamide , p y r i d i n e n i t r o g e n and t h e 2 p o s i t i o n of t h e n e i g h b o r i n g phenyl group ( 6 ) . 2.4

2.3

'

2.5

M e l t i n g Range I t m e l t s a t about 255O C w i t h decomposition (1).

519

A
-3

I " "

"

'

' IW

'

'

'

'

'

'

'

'

' 600

400

2c4

LO

7
1

5 PPM
t

~ . : . : , : . ~ . ~ . : . ~ , ~ . . . ~ . . . ~ , . . .. .. .. I . . . . I . . . .
Figure 2

....

I....

. . . .

.. . . I

....

, j ....

,
I

..1

Nuclear Magnetic Resonance Spectrum

Sulfasalazine

SU LFASALAZI NE

Figure 3

U l t r a v i o l e t - v i s i b l e Spectrum

1(

1
SULFASALAZINE

O!
0.t

0.;

0.f

z 2

QJ

pH 12.3

0.E

0.4

03 .
0.2

0.1

o.a
Wavelength, MI

521

100
90

80
70

60

522

50
YO

30
26 10

50

1110

150

Figure 4
al
M
&

Mass Spectrum
rn rn

- Sulfasalazine
m rl m rn m w
rl

*rl N

v)

a l c

SULF ASALAZI NE

2.6

pK Values ( D i s s o c i a t i o n C o n s t a n t s )

The f o u r d i s s o c i a t i o n c o n s t a n t s of s u l f a s a l a z i n e determined s p e c t r o p h o t o m e t r i c a l l y a r e a s follows: pK1 = 0 . 6 ; pK2 = 2.4; pK3 = 9.7 and pQ = 11.8 ( 4 ) .

2.7

Solubility The s o l u b i l i t y of s u l f a s a l a z i n e i s a s f o l l o w s ( 1 ) : P r a c t i c a l l y insoluble i n water P r a c t i c a l l y insoluble i n ether P r a c t i c a l l y i n s o l u b l e i n chloroform P r a c t i c a l l y i n s o l u b l e i n benzene Very s l i g h l y s o l u b l e i n a l c o h o l S o l u b l e i n aqueous s o l u t i o n s of a l k a l i hydroxides

Synthesis S u l f a s a l a z i n e i s p r e p a r e d by d i a z o t i z i n g s u l f a p y r i d i n e and r e a c t i n g t h i s w i t h an a l k a l i n e s o l u t i o n of s a l i c y l i c a c i d ( 7 , 8). Other r e p o r t e d methods of p r e p a r a t i o n a r e : a ) r e a c t i n g 5 - n i t r o s o s a l i c y l i c a c i d w i t h s u l f a p y r i d i n e , and b) r e a c t i n g n i t r o s o - s u l f a p y r i d i n e w i t h 5 - a m i n o s a l i c y l i c a c i d (8) (See F i g u r e 5 ) .

3.

4.

Impurities Stability P a r t i a l c h a r a c t e r i z a t i o n of i m p u r i t i e s i n commercial s u l f a s a l a z i n e was r e p o r t e d by Stone e t a 1 (9). Of t h e t h r e e i m p u r i t i e s s t u d i e d , one was proposed t o be a p o s i t i o n a l isomer of s u l f a s a l a z i n e ; t h e second t o be polymeric r e s u l t i n g from t h e f o r m a t i o n of a benzyne i n t e r m e d i a t e ; and t h e t h i r d an u n d i a z o t i z e d s u l f a m i d e (See F i g u r e 6 ) . The compound d i d n o t degrade when d i s s o l v e d i n dimethylformamide and was s u b j e c t e d t o thermal stress a t 80 C f o r 196 h o u r s ( 1 0 ) .

5.

Drug M e t a b o l i c P r o d u c t s I n man, s u l f a s a l a z i n e i s metabolized by r e d u c t i v e c l e a v a g e , presumably by t h e g u t f l o r a , t o s u l f a p y r i d i n e and 5-aminosalicy i c a c i d . The s u l f a p y r i d i n e p o r t i o n is t h e n s u b j e c t t o & - a c e t y l a t i o n o r p y r i d i n e r i n g hydroxyla t i o n , followed by c o n j u g a t i o n t o g l u c u r o n i c a c i d , o r both. The 5 - a m i n o s a l i c y l i c a c i d moiety i s N - a c e t y l a t e d (11) (See Figure 7). In r a t s , s u l f a s a l a z i n e i s metabolized s i m i l a r t o t h a t i n man (12, 13).

523

J. PATRICK McDONNELL

Figure 5

Synthesis o f S u l f a s a l a z i n e

Example 1:

Base

Example 2:

kOOH

+Q
=O

CH 3coon 9

I NH

9
/

Pcoo
OH

Example 3:

524

SULFASALAZINE

Figure 6

S u l f a s a l a z i n e Impurities

Benzyne Intermediate

Undiazot ized Sul famide

525

J. PATRICK McDONNELL

Figure 7

S u l f a s a l a z i n e Metabolic Pathway

5 - A m i n o s a l i c ~ l i c Acid

Sul fapvridfae

COOH

&-Acetyl su1f.D-

N-Acetyl-5-aminosa l i c y l i c Acid

5 ' -HydKoxysulfaD.rr id i

&-Ace t y 1- 5 ' -hydroxmul f a m + r i d k

5 ' -Hydroxysul fapyridine -o-Rlucuranide

N4-Acetvl-5 ' -hvdroxvsul fapyridine-a-glucuronide

526

SULFASALAZINE

6.

Methods of A n a l y s i s 6.1

U l t r a v i o l e t Spectrophotometry Berggren e t a 1 (5) developed a s p e c t r o p h o t o m e t r i c procedure f o r s u l f a s a l a z i n e which c o n s i s t e d of d i s s o l v i n g t h e sample i n 0.1 E sodium h y d r o x i d e , a d j u s t i n g t h e pH t o between 4 and 5 w i t h 0 . 1 E a c e t i c a c i d and d e t e r m i n i n g t h e absorbance i n a 1 cm q u a r t z c e l l a t 359 mu. T h i s method was a l s o r e p o r t e d i n t h e 1953 E d i t i o n of T e s t s and S t a n d a r d s f o r New and N o n o f f i c i a l Remedies (14). Q u a n t i t a t i v e Thin-Layer Chromatography (TLC) Two methods of q u a n t i t a t i v e TLC a n a l y s i s of s u l f a s a l a z i n e have been p u b l i s h e d ( 1 , 15). Both u s e t h e same developing s o l v e n t r e p o r t e d by Kiger e t a 1 (16) and a r e s i m i l a r i n t h e method of p r e p a r i n g and c o n d i t i o n i n g t h e d e v e l o p i n g tank. The Powell e t a 1 method (15) c o n s i s t s of d i s s o l v i n g t h e sample and r e f e r e n c e s t a n d a r d s i n dimethylformamide/methanol s o l u t i o n , s p o t t i n g t h e s o l u t i o n s on a TLC p l a t e , d e v e l o p i n g t h e p l a t e , c u t t i n g t h e s u l f a s a l a z i n e s p o t (Rf. 0.6-0.7) from t h e p l a t e and e l u t i n g i t i n a 0.016 N sodium hydroxide s o l u t i o n . The s o l u t i o n i s f i l acetic acid t e r e d and a n a l i q u o t combined w i t h a 0 . 1 s o l u t i o n . The absorbance of t h e r e f e r e n c e s t a n d a r d and sample s o l u t i o n s a r e compared a t 360 nm u s i n g w a t e r a s a blank. The method provided i n t h e F o u r t e e n t h E d i t i o n o f t h e N a t i o n a l Formulary ( 1 ) i n v o l v e s d i s s o l v i n g t h e sample and r e f e r e n c e s t a n d a r d i n dimethylformamide, s p o t t i n g t h e s o l u t i o n on a TLC p l a t e , d e v e l o p i n g t h e p l a t e , s c r a p i n g t h e s u l f a s a l a z i n e s p o t ( R f . a b o u t 0 . 6 ) and e l u t i n g i t w i t h dimethylformamide. The r e s u l t a n t s t a n d a r d and sample s o l u t i o n a b s o r b a n c i e s a r e compared a t 406 nm. Column Chromatography Stone e t a 1 (9) r e p o r t e d a n a s s a y procedure f o r s u l f a s a l a z i n e u s i n g column chromatography. The s u l f a s a l a z i n e sample i s d i s s o l v e d i n p y r i d i n e t h e n s e p a r a t e d on a s i l i c i c a c i d column. The d e v e l o p i n g s o l v e n t system i s methyl e t h y l k e t o n e / a c e t o n e / w a t e r (16: 16: 1) and t h e flow r a t e i s 1.2 m l p e r minute. The second of t h e t h r e e bands t o e l u t e i s s u l f a s a l a z i n e which i s c o l l e c t e d and t h e s o l v e n t removed by e v a p o r a t i o n . The r e s u l t a n t sample i s t a k e n up w i t h 0.1 E sodium hydroxide and t o t h i s i s added
527

6.2

6.3

J. PATRICK McDONNELL

0.1 a c e t i c a c i d and d i l u t e d t o volume w i t h w a t e r . The absorbance o f t h i s s o l u t i o n i s determined a t 359 tun u s i n g w a t e r a s a blank. Polarography A p o l a r o g r a p h i c method of a n a l y s i s f o r s u l f a s a l a z i n e h a s been p r e l i m i n a r i l y o u t l i n e d by Nygard (17) and i n d e p e n d e n t l y a l s o by Lastovkove e t a 1 (18). Based on t h e work of t h e s e i n v e s t i g a t o r s , Nygard et (19) developed a p o l a r o g r a p h i c a s s a y method which t h e y used t o determine sulfasalazine purity. T i t r i m e t r i c Analysis Berggren e t a 1 (5) a l s o developed a t i t r i m e t r i c method of a n a l y s i s which was adopted i n t h e 1953 New and N o n o f f i c i a l Remedies (14). The sample i s d i s s o l v e d i n 2 ammonium hydroxide and d i l u t e d t o volume w i t h w a t e r . The s o l u t i o n i s heated t o 7 0 C and carbon d i o x i d e i s passed ' t i t a n i u m trithrough i t . H y d r o c h l o r i c a c i d and 0.1 c h l o r i d e a r e added and t h e mixture i s maintained a t 70 C u n t i l a l l t h e p r e c i p i t a t e has gone i n t o s o l u t i o n . It i s cooled t o 1 5 0 C and t h e e x c e s s t i t a n i u m t r i c h l o r i d e i s t i t r a t e d w i t h 0.1 f e r r i s c h l o r i d e u s i n g 2 m l of 10% ammonium t h i o c y a n a t e a s t h e i n d i c a t o r . High Speed L i q u i d Chromatography High speed l i q u i d chromatography methods o f a n a l y s i s have been used t o determine t h e p u r i t y of s u l f a s a l a z i n e b u l k powder and t o q u a n t i f y t h e l e v e l of s u l f a s a l a z i n e i n t a b l e t e d dosage forms (10, 2 0 ) . B i g h l e y e t a 1 (10)used a r e v e r s e phase C o r a s i l c18 column and 10% 2-propanol i n pH 7 . 7 phosphate b u f f e r a s t h e mobile phase t o accomplish a n a l y s i s . The s u l f a s a l a z i n e b u l k drug o r t a b l e t was d i s s o l v e d i n dimethylformamide and p r o p y l p a r a ben added a s t h e i n t e r n a l s t a n d a r d . Q u a n t i t a t i o n was e f f e c t e d by peak h e i g h t o r peak a r e a u s i n g a UV photom e t r i c d e t e c t o r ( 2 5 4 nm r a d i a t i o n u s i n g a low p r e s s u r e mercury s o u r c e ) a s t h e d e t e c t i o n sys t e m . A C o r a s i l C 1 8 column packing was used by Frahm et a 1 (20). T h e i r mobile phase was 13%a c e t o n i t r i l e i n pH 7 . 3 phosphate b u f f e r and t h e i n t e r n a l s t a n d a r d was methyl meta-nitrobenzoate. The s u l f a s a l a z i n e was d i s s o l v e d i n dimethylformamide. Q u a n t i t a t i o n was e f f e c t e d by peak h e i g h t measurement and t h e d e t e c t i o n made a t 254 nm u s i n g
528

6.4

6.5

6.6

--

SULFASALAZINE

a low p r e s s u r e mercury s o u r c e . D i s t r i b u t i o n i n F l u i d s and T i s s u e s Following i n t r a v e n o u s i n j e c t i o n i n mice, s u l f a s a l a z i n e a t t a c h e d t o c o n n e c t i v e t i s s u e and was p r e s e n t a l s o i n h i g h c o n c e n t r a t i o n s i n p e r i t o n e a l , p l e u r a l and s y n o v i a l f l u i d s , i n t h e l i v e r , and i n t h e i n t e s t i n a l lumen (21, 2 2 ) .

7.

8, Pharmacokine t i c s
The s t e a d y - s t a t e serum c o n c e n t r a t i o n s i n humans f o r s u l f a s a l a z i n e and i t s m e t a b o l i t e s o b t a i n e d by Day 5 (dose 4 grams d a i l y ) were a s f o l l o w s : Range Median S u l f a sa l a z i n e 12 u d m l 4 . 7 t o 45 u d m l T o t a l S u l f a p y r i d i n e M e t a b o l i t e s 37 t o 9 2 ug'jml 50 ug/ml T o t a l 5-Aminosalicylic Acid Less t h a n 2 ug/ml

The u r i n a r y e x c r e t i o n of unmetabolized s u l f a s a l a z i n e ranged from 1.7% t o 10%of t h e dose. Approximately 80% of t h e dose was e x c r e t e d i n t h e u r i n e a s m e t a b o l i t e s of s u l f a p y r i d i n e and o n e - t h i r d of t h e dose was e x c r e t e d a s t h e m e t a b o l i t e of 5 - a m i n o s a l i c y l i c a c i d . Feces d i d n o t c o n t a i n any s u l f a s a l a z i n e , b u t about 5% of t h e dose was p r e s e n t a s m e t a b o l i t e s of s u l f a p y r i d i n e , and a n unknown amount a s m e t a b o l i t e s of 5 - a m i n o s a l i c y l i c a c i d (11).
9.

D e t e r m i n a t i o n i n Body F l u i d s and T i s s u e s

Spectrophotometry von P o r a t (23) p u b l i s h e d two c o l o r i m e t r i c methods . . f o r t h e d e t e r m i n a t i o n of s u l f a s a l a z i n e i n serum. One was a d i r e c t measurement of t h e drug c o l o r i n a l k a l i n e serum. The serum b l a n k , however, was h i g h , c o r r e s p o n d i n g t o 1 4 ug/ml of s u l f a s a l a z i n e . I n a d d i t i o n , hemolyzed and i c t e r i c s e r a i n t e r f e r r e d c o n s i d e r a b l y w i t h t h e measurement. The o t h e r method involved a combined p r e c i p i t a t i o n and e x t r a c t i o n method. The serum b l a n k d e c r e a s e d t o 5 ug/ml and t h e i n t e r f e r e n c e from i c t e r i c s e r a diminished. B o t t i g e r (24) r e p o r t e d a d i r e c t c o l o r i m e t r i c method s i m i l a r t o von P o r a t ' s . He used an a c i d i f i e d p o r t i o n of t h e serum sample a s a r e f e r e n c e . The e f f e c t of s l i g h t hemolysis and i c t e r i c s e r a were t h u s , t o a l a r g e e x t e n t , eliminated.

9.1

529

J. PATRICK McDONNELL

Sandberg (25) p r e s e n t e d a s p e c t r o p h o t o m e t r i c method f o r d e t e r m i n i n g s u l f a s a l a z i n e i n serum and u r i n e by e x t r a c t i n g t h e compound i n t a c t and measuring s p e c t r o p h o t o m e t r i c a l l y a t 455 run. I n b i l e and f e c e s t h e drug was reduced t o s u l f a p y r i d i n e which was e x t r a c t e d and t h e n determined w i t h a s l i g h t l y modified B r a t t o n - M a r s h a l l procedure. A f t e r t h e e x t r a c t i o n s t e p , serum, u r i n e , b i l e and f e c e s gave low b l a n k v a l u e s c o r r e s p o n d i n g t o 0.3 and 1 ug/ml s u l f a s a l a z i n e i n serum and u r i n e and less t h a n 1 ug/ml i n b i l e and f e c e s . Polarography Nygard (17) d e s c r i b e d a p o l a r o g r a p h i c method f o r a n a l y z i n g s u l f a s a l a z i n e i n serum. Due t o t h e formation of a p o l a r o g r a p h i c , n o n - r e d u c i b l e a d d u c t between serum p r o t e i n s and s u l f a s a l a z i n e , 1,2-diphenyl-3,5-dioxo-4-nb u t y l p y r a z o l i d i n e ( B u t a z o l i d i n R ) was added t o d i s p l a c e s u l f a s a l a z i n e from i t s molecular a d d u c t w i t h albumin. Paper Chromatography Hanngren e t a 1 (21, 22) used paper chromatography i n c o n j u n c t i o n w i t h autoradiograms of t h e chromatograms t o s e m i q u a n t i t a t i v e l y determine s u l f a s a l a z i n e and i t s metab o l i c by-products i n u r i n e , l i v e r e x t r a c t s and i n t e s t i n a l e x t r a c t s . The developing system was a m i x t u r e of p y r i d i n d i s o - a n y 1 a l c o h o l / w a t e r (35:35:30). Whatman No. 1 f i l t e r p a p e r was used and t h e chromatograms were developed by descending chroma t o g r a p hy 9.3 9.2

Acknowledgments The a u t h o r wishes t o acknowledge t h e a s s i s t a n c e of D r . Lyle D. B i g h l e y i n reviewing t h i s a n a l y t i c a l p r o f i l e , Mary Lou B u l l a r d f o r t y p i n g t h e manuscript and Harold E . Haus f o r p r e p a r i n g t h e a r t work.

10.

11.

References

(1) N a t i o n a l Formulary, F o u r t e e n t h E d i t i o n , Mack P u b l i s h i n g Co. , E a s t o n , Pa. 667 (1975)

(2)

The Merck I n d e x , E i g h t h E d i t i o n , Merck & Co. Rahway, N . J . 930 (1968)

, Inc.

530

SULFASALAZINE

(3)

James J. Downs, P e r s o n a l Communication, Midwest Research I n s t i t u t e , Kansas C i t y , Mo. 64110 Nygard, B. , O l o f s s o n , J . & Sandberg, Acta Pharmac e u t i c a Suecica 3, 313 (1966) Berggren, A. & Hansen, S . (1952)

(4)

(5)

, Farm

Rev. 3 4 , 537

(6)

James J . Downs, P e r s o n a l Communication, Midwest Research I n s t i t u t e , Kansas C i t y , Mo. 64110 Doroswamy, K. R. & Guha, P . C. , J o u r n a l of I n d i a n Chemical S o c i e t y 23, 278 (1946)
U. S. P a t e n t 2,396,145 ( P a t e n t e d March 5 , 1946)

(7)

(8) (9)

S t o n e , J . C . & Gorby, R. , J o u r n a l of Pharmac e u t i c a l S c i e n c e s 63, 1296 (1974) B i g h l e y , L. D. & McDonnell, J. P. , J o u r n a l of P h a r m a c e u t i c a l S c i e n c e s 64, 1549 (1975) S c h r o d e r , H. & Campbell, D. E. S . , C l i n i c a l Pharmacolopy & T h e r a p e u t i c s 539 (1972)

(10)

(11)

12,

(12)

64,

Peppercorn, M. A. & Goldman, P. 240 (1973)

Gastroenterology

(13)

Peppercorn, M. A. & Goldman, P. , J o u r n a l of Pharmacology & Experimental T h e r a p e u t i c s (1972)

181,555

(14)

T e s t s & S t a n d a r d s o r New & N o n o f f i c i a l Remedies, L i p p i n c o t t , P h i l a d e l p h i a , Pa. , 256-258 (1953) Powell, D. R. & Burton, B. A . , J o u r n a l of Pharmac e u t i c a l S c i e n c e s 63, 1290 (1974) K i g e r , J. L. & K i g e r , J. G. , Annales Pharmac e u t i q u e s F r a n c a i s e s 2 4 , 593 (1966) Nygard, B . , Svenska Kem. T i d s k r . , 333 (1958)

(15)
(16)

(17)

531

J. PATRICK McDONNELL

(18) (19) (20)

Lastovkova, N. & Vackova, A . (1958)

, Cesk

Farm

1, 570
, Acts ,

Nygard, B . , O l o f s s o n , J . & Sandberg, M. Pha rma c e u t i c a Sue c i c a 3 , 343 (1966)

Frahm, L. J . , S c h e r b , Y. & McDonnell, J. P. Salsbury Laboratories (unpublished d a t a )

(21)
(22)

Hanngren, A . , Hansson, E. , S v a r t y , N. & U l l b e r g , S. , Acta Medica S c a n d i n a v i c a 6 1 (1963)

173,

Hanngren, A. , Hansson, E . , S v a r t z , N. & U l l b e r g , S . , Acta Medica S c a n d i n a v i c a 391 (1963)

173,

(23)
(24)

von P o r a t , B.

Nord Med. 24, 2070 (1944)

B o t t i g e r , L. E . , Scand. J . Chem. Lab I n v . 108 (1958)

lo,

(25)

Sandberg, M. & Hanson, K. A . , Acta Pharmaceutica S u e c i c a l , 107 (1973) 0

I n t h e p r e p a r a t i o n of t h i s A n a l y t i c a l P r o f i l e , t h e l i t e r a t u r e was reviewed t h r o u g h September, 1975.

532

TESTOLACTONE

Klaus Florey

KLAUS FLOREY

CONTENTS Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, odor 2. Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Optical Rotation 2.6 Melting Range 2.7 Differential Thermal Analysis 2.8 Solubility 2.9 Crystal Properties 3. Synthesis 4. Stability, Degradation 5. Drug Metabolism 6. Methods of Analysis 6.1 Elemental Analysis 6.2 Phase solubility Analysis 6.3 Colorimetric Analysis 6.4 Fluorometric Analysis 6.5 Non-aqueous Titration 6.6 Chromatographic Analysis 6.61 Paper 6.62 Thin Layer 6.63 column 6.64 Vapor Phase 7. Determination in Pharmaceutical Preparations 8. References 1.

534

TESTOLACTONE

Description 1.1 Name, Formula, Molecular Weiqht Testolactone is also known as A t -testololactone, l-dehydro-testololactone, 13,17 secoandrosta-1,4-dien-l7-oic acid, 13hydroxy-3-oxo-~-lactoneand D-homo-17a-oxaandrosta-1,4-diene-3,17-dione. SQ 9538.
1.

1' gH2403

M.W. 300.38

Appearance, Color, Odor Testolactone is a white to off-white, odorless crystalline powder.

1.2

Physical Properties 2.1 Infrared Spectrum The infrared spectrum (KBr pellet) of testolactone (batch 36B) is presented in Figure 1. It supports the presence of a lactone with a carbonyl stretching frequency of 1703 c m ' l and an A ring dienone with a carbonyl stretching frequency at 1650 cm'l and C=C sf.r$iching This frequencies of 1620 and 1595 cmagrees with data presented by Fried et.al. 1,12 and the spectrum given by Gual, Corfman and Rosenkrantz25. The same authors also reported frequencies in the fingerprint region26
2.

535

Figure 1.

Infra R e d Spectrum of Testolactone (batch 36B). Instrument: Perkin-Elmer 621.

KBr Pellet.

TESTOLACTONE

N u c l e a r M a g n e t i c Resonance S p e c t r u m The 60 MHz p r o t o n s p e c t r u m i s shown i n T h e r e a r e 24 p r o t o n s p r e s e n t . The F i g u r e 2. C - 1 8 and C-19 p r o t o n s o c c u r a s 3 - p r o t o n s i n g l e t s , t h e C - 1 proton i s a d o u b l e t , t h e C-2 p r o t o n i s a q u a r t e t a n d t h e C-4 p r o t o n a p p e a r s a s a b r o a d m u l t i p l e t (see T a b l e 1)24. U l t r a v i o l e t Spectrum Fried et. a l . reported max 242 n m ( 6 = 1 5 , 8 0 0 ) . An E l c r n o f 545 a t t h e same wave1 % l e n g t h was r e p o r t e d f o r S q u i b b House S t a n d a r d ( L o t # 41040-102) 27. Table I * 60 MHz NMR D a t a i n CDC13 Group
SQ 9 , 5 3 8 B a t c h #36B = 10 7.05 d; J 192 6.28 q; J = 1.5 294 J 1 , 2 = 10 6.13 m 1.25 s 1.40 s

2.2

2.3

c- 1 c- 2
c-4 C- 18 c-19

The chemical s h i f t s are i n d e l t a , w i t h i n t e r n a l r e f e r e n c e TMS. s = s i n g l e t ; d = doublet; m = multiplet;q=quartet J = c o u p l i n g c o n s t a n t i n Hz.

537

..

..

. -.

Figure 2.

NMR Spectrum of Testolactone (batch 36B) in CDC13. Instrument: Perkin-Elmer R12B (60 MHz)

TESTOLACTONE

Mass Spectrum The l o w - r e s o l u t i o n mass s p e c t r u m of t e s t o l a c t o n e ( F i g u r e 3 ) was o b t a i n e d by d i r e c t i n s e r t i o n o f t h e sample i n t o a 18OoC s o u r c e of an AEIMS-902 spectrometer. The M+ o f m/e 300 c o r r e s p o n d s t o t h e f o r m u l a o f ClgH2403. The t w o f r a g m e n t i o n s shown below a r e d i a g n o s t i c f o r t h i s structure28.
2.4

539

Figure 3.

Low-resolution mass spectrum of Testolactone (Batch 36B). Instrument: AEI-MS-902.

TESTOLACTONE

Optical R o t a t i o n 23 0 Fried e t . a l . 1 reported [ a y -44 ( c = 1 . 2 9 i n CHC13).[alD - 49.2 w a s f e c o r d e d f o r S q u i b b House S t a n d a r d L o t 41040-10227.

2.5

2.6

M e l t i n g Range
A m e l t i n g r a n g e o f 218-219O w a s

r e p o r t e d by F r i e d e t . a l . The m e l t i n g b e h a v i o r on t h e K o f l e r h o t s t a g e h a s been d e s c r i b e d 3 5 . D i f f e r e n t i a l Thermal A n a l y s i s of h o u s e s t a n d a r d L o t 4 showed a l a r g e s i n g l e endotherm a t 219OC

D.T.A.

2.7

$940-102 ,

Solubility Testolactone is s l i g h t l y soluble i n w a t e r and i n b e n z y l a l c o h o l , i s s o l u b l e i n a l c o h o l and i n c h l o r o f o r m and i s i n s o l u b l e i n e t h e r and i n s o l v e n t h e ~ a n e ~ ~ .

2.8

2.9

Crystal Properties
N o polymorph of t e s t o l a c t o n e h a v e been

d e s c r i b e d so f a r e x c e p t t h a t , t w o m o d i f i c a t i o n s were o b s e r v e d on t h e K o f l e r h o t s t a g e 3 5 . The powder x - r a y d i f f r a c t i o n p a t t e r n of t e s t o l a c t o n e i s presented i n Table 2, c o r r e s p o n d i n g t o t h e p a t t e r n i n F i g u r e 4.

541

KLAUS FLOREY

Table 2
1

dl

Relative Intensity

**

0.74 7.75 0.40 6.52 0.11 6.20 0.11 5.60 1.00 5 . 30 0.72 4.85 0.09 4.62 0.06 4.34 0.15 4.05 0.14 3.95 0.32 3.80 0.17 3.85 0.57 3.71 0.43 3.45 0.15 3.29 0.14 3.22 0.41 3.16 0.16 3.10 0.17 2.94 0.06 2.86 0.04 2.67 2.57 0.09 0.05 2.44 2.32 0.04 *d i n A = i n t e r p l a n a r d i s t a n c e 0 **b a s e d on h i g h e s t i n t e n s i t y of 1.00 R a d i a t i o n : K c l l and Ka2 Copper I n s t r u m e n t : P h i 11i p s

542

543
d

Figure 4.

a,

Powder X-ray diffraction pattern of testolactone (batch 41040-102) Instrument: Phillips

'0

JJ

KLAUS FLOREY

Synthesis Testolactone") was first isolated and identified as a bio-oxidative product of the fermentation of progesterone (I1), with Cylindrocarpon radicola by Fried, Thoma and Klingsbergl, (see Figure 5). Peterson, Thoma, Perlman and Fried2 were able to show that l-dehydro-testosterone(111) and A1,4-androstadiene-3,17-dione(Iv) are intermediates in this conversion. Conversion of testololactone(V) to testolactone('1 by the same microorganism has been observed5. other microorganisms have also been used for the production of testo19. Patents also have been lactone6'11j issued12-17. A chemical synthesis of testolactone starting with epiandrosterone acetate via epiandrololactone and dihydrotestololactone has been reportedi8

3.

544

fH3 c=o
0

&-lz2??:#
I1 I11

F i g u r e 5 . S y n t h e s i s and D e g r a d a t i o n .

KLAUS FLOREY

4 Stability, Deqradation . Testolactone is very stable as a solid. In strongly alkaline solution the lactone ring will This can also open to A' -testolic a i 2 ( I . cd'V) be accomplished by microbiological degradation. Other microbiological transformation products are 15-keto-testo-lactone(VII) l-methyl-cyclohexan-l-ol-4-keto 2,3-diproprionic acid lactone (VIII) and surprisingly 7a-methylhexahydrindan1,5-dione-4a- (3-prapionic acid) (IX)22 (see Table 4). It has been reported23 that hydrocortisone and prednisolone when exposed to ultraviolet radiation or ordinary fluorescent laboratory lighting in alcoholic solutions, undergo photolytic degradation of the A-ring. Since testolactone has the same A-ring as prednisolone it probably also is labile under these conditions. Drug Metabolism In human subjects the urinary metabolites found were unchanged testolactone and 3a, 13adihydroxy-13,17-seco-5~-and~O~ta-l-ene-l7-oic acid lactone the latter both in the free form and as the glucuronide. 5.

Bovine blood albumin contains a A4-5f3hydrogenase system which also reduces testolactone4.

546

TESTOLACTONE

6.

Methods o f A n a l y s i s 6.1 Elemental Analysis

C a l c f o r C1gH2403
C H

Found 1 27 Fried, et. a 1 House S t d . 76.29 7.81 75.89 8.23

75.97 8.05

Phase S o l u b i l i t y A n a l y s i s The p u r i t y o f S q u i b b House S t a n d a r d l o t 41040-102 was found t o be 99.6% p u r e by p h a s e s o l u b i l i t y analysis27. The s o l v e n t system used was n-propanol-methanol (2:l). Equilibration was c a r r i e d o u t o v e r 24 h o u r s a t 25OC. The e x t r a p o l a t e d s o l u b i l i t y was d e t e r m i n e d a t 25.5 mg/g of s o l v e n t . Colorimetric Analysis The r e a c t i o n o f t h e 3-ket0-A~-diene system of t e s t o l a c t o n e w i t h i s o n i c o t i n i c a c i d h y d r a z i d e t o form hydrazone w i t h an a b s o r p t i o n maximum a t 415 nm3 h a s been made t h e b a s i s f o r t h e cornpendial a s s a y of t e s t o l a c t o n e i t s e l f and i t s d o s a g e forms29. A s o t h e r s t e r o i d l a c t o n e s , tes t o l a c t o n e forms a p i n k chromogen when s u b j e c t e d t o t h e . a c t i o n o f p o t a s s i u m h y d r o x i d e , hydroxylamine and f e r r i c ~ h l o r i d e ~ ~ j i~ ~ a s b e e n used f o r a Th s h . cornpendial i d e n t i t y t e s t 2 9 . Fluorometric Analysis When t e s t o l a c t o n e i s h e a t e d i n 85% p h o s p h o r i c a c i d a t 100C f o r 30 m i n u t e s a f l u o r o g e n i s formed which on d i l u t i o n w i t h methanol e x h i b i t s e x c i t a t i o n and f l u o r e s c e n t s p e c t r a w i t h maxima a t 275 nm and 375 nm, respectively. T h i s can b e used f o r t h e d e t e r mination of t e s t o l a c t o n e i n fermentation broth. 6.4 6.3

6.2

547

KLAUS FLOREY

P r o g e s t e r o n e and 1-dehydro-progesterone interfere31. 6.5

do n o t

Nonaqueous T i t r a t i o n I t h a s been r e p o r t e d 3 2 t h a t t e s t o l a c t o n e a f t e r p r e c i p i t a t i o n w i t h sodium p h e n y l b o r a t e can be t i t r a t e d w i t h c e t y l pyridinium c h l o r i d e and thymol b l u e i n d i c a t o r w i t h a c c e p t a b l e accuracy. Chromatographic A n a l y s i s 6 . 6 1 Paper Chromatoqraphic A n a l y s i s The f o l l o w i n g paper chromatog r a p h i c s o l v e n t systems have b e n r e p o r t e d : 1. ) Methycyclohexane-carbitol 2 . ) Toluene-propylene g l y c o l 2 . 3 . ) Propylene glycol-cyclohexane-chloroform 7 6.6

s.

4.) Toluene s a t u r a t e d w i t h propylene glyco133. 5. ) Impregnation w i t h formamide-rnethanol(20:80) t h e n methyl i s o b u t y l ketone-formamide (20 :1)33, -6. ) Methylcyclohexane-chloroform (4: 1) A l l systems s e p a r a t e t e s t o l a c t o n e from p r o g e s t e r o n e , A1, $-andros ta-diene-dione and I n system 4 p r o g e s t e r o n e and testolactone. t e s t o l o l a c t o n e have g r e a t e r m o b i l i t i e s t h a n I n system 5 A I 4 - and A I 5 testolactone. t e s t o l a c t o n e ( b o t h w i t h an R f of 0.68) can be s e p a r a t e d from t e s t o l a c t o n e (Rf 0 . 6 0 ) . I n system 6 t h e o r d e r of s e p a r a t i o n ( w i t h i n c r e a s i r q Rf) i s a s f o l l o w s : t e s t o l a c t o n e ( R f 0.23) t e s t o l o l a c t o n e , t e s t o s t e r o n e , and p r o g e s t e r o n e . System 4, 5 and 6 have a l s o been used f o r q u a n t i t a t i o n f o l l o w i n g t h e g e n e r a l procedure of R o b e r t s a n d F 1 0 r e y ~ ~ .I n systems 4, 5 and 6 e l u t i o n w i t h an a c i d i f i e d m e t h a n o l i c s o l u t i o n of i s o n i c o t i n i c a c i d h y d r a z i d e was u s e d , f o l l o w e d by q u a n t i t a t i o n (see s e c t i o n 6 . 3 ) . A l t e r n a t e l y i n system 6 e l u t i o n w i t h 95% e t h a n o l and measure-

ment of absorption at 242 nm can also be used33. 6.62 Thin Layer Chromatographic Analysis The following thin-layer chromatographic solvent systems have been reported: 1.) The method of Belic and Socic19 is summarized in Table 3: Table 3. Rf-values and color reaction of steroids with 50% sulphuric acid on silica gel developed with cyclohexane/ethylacetate (1:2). Steroid
Rf

P ogesterone A -Androstene-3,17-dione Testosterone A , 4-Androstadiene-3,17-dione l-Dehydrotestosterone Tes tololactone l-Dehydrotestololactone l-Dehydroprogesterone A, 4-Andros tadien-3,17-dionea Testosteronea

0.63 0.52 0.44 0.45 0.36 0.24 0.18 0.53 0.45 0.40

Daylight Re lative Color Intensity ye1low weak blue-green strong blue-green strong bright-red intense ochre-red strong blue-green medium ochre medium pink weak bright-red intense blue-green strong

Fluorescence greenish pale-blue blue orange orange green orange ochre orange blue

adeveloped with benzene/ethylacetate(l:l).

KLAUS FLOREY

2.) Ethyl-acetate-hexane(5:S) o r chloroformmethanol(97:3) on s i l i c a g e l H F F o a t e d g l a s s p l a t e s . D e t e c t i o n by U . V . l i g h t

3 . ) Chloroform-acetone (94:6) on s i l i c a g e l SF. D e t e c t i o n by U.V. l i g h t o r s u l f u r i c a c i d s p r a y . The f o l l o w i n g Rf v a l u e s were found: t e s t o l a c t o n e 0.22; A 1 - t e s t o s t e r o n e , 0 . 2 7 ; a n d r o s t a d i e n e d i o n e 0 . 4 9 ; A1-progesterone, 0 . 5 8 ; p r o g e s t e r o n e , 0 . 6 1 li 4 . ) Butylacetate-acetone(4:l) D e t e c t i o n b y U.V. l i g h t 2 9 . 6.63 on s i l i c a g e l .

Column Chromatographic A n a l y s i s Column hromatography on 1 h a s been used t o alumina o r s i l i c a g e l '?" s e p a r a t e t e s t o l a c t o n e from o t h e r s t e r o i d s and i m p u r i t i e s i n f e r m e n t a t i o n e x t r a c t s . Petroleum e t h e r - c h l o r o f o r m (1:1) and e t h y l a c e t a t e 5 have b e e n used a s e l u t i o n s o l v e n t s . Vapor Phase Chromatoqraphic Analysis T e s t o l a c t o n e h a s been q u a n t i t a t e d together with i t s bioconversion precursors on a 6 - f t g l a s s column c o n t a i n i n g 3% SE-30 on 80-100 mesh D i a t o p o r t . Column and flamei o n i z a t i o n d e t e c t o r t e m p e r a t u r e s were 25OoC and t h e c a r r i e r g a s was h e l i u m a t 50 rnl/minll. Determination i n Pharmaceutical P r e p a r a t i o n s I n a d d i t i o n t o t h e compendia1 i s o n i c o t i n i c a c i d h y d r a z i d e assay2' (see s e c t i o n 6 . 3 ) , t h e p a p e r c h r o m a t o g r a p h i c s y s t e m s (see s e c t i o n 6 . 6 1 ) 2 and 6 h a v e b e e n u s e d a s a s t a b i l i t y i n d i c a t i n g a s s a y f o r t e s t o l a c t o n e i n s u s p e n s i o n and t a b l e t s 3 3 f o l l o w i n t h e g e n e r a l procedure of R o b e r t s and F l o r e y34 7. 6.64

550

TESTOLACTONE

8.

References F r i e d , R. W. Thoma and A . K l i n g s b e r g , J. Am. Chem. SOC. 75,5764 ( 1 9 5 3 ) . G. E. P e t e r s o n , R. W. Thoma, D. Perlman and J. F r i e d , J. B a c t e r i o l . 7 4 , 6 8 4 ( 1 9 5 7 ) . E. L. Rongone, A . S e g a l o f f , J. F r i e d and E. Sabo, J. B i o l . Chem. *,2624(1961). E . L. Rongone, Arch.Biochm.Biophys. 9 8 , 2 9 2 (1962) E. J. K u s n e r and R. D. G a r r e t t , S t e r o i d s l7, 521 ( 1 9 7 1 ) . M. Nishikawa, S. Noguchi, T. Hasegawa and I . Banno, J . Pharm. SOC. J a p a n 7 6 , 3 8 3 (1956) ; Pharm. B u l l . ( J a p a n ) 3 , 3 2 2 ( 1 9 5 5 ) , C. A. 5 0 , 13165d(1 9 5 6 ) . S. A . S z p i f o g e l , M. S . de W i n t e r and W. J. A l s c h e , R e c . t r a v . chim 7 5 , 4 0 2 ( 1 9 5 6 ) . A . Capek and 0. Hanc, F o l i a m i c r o b i o l . 5 251 (1960) C . A . 55, 3729a ( 1 9 6 1 ) . E. Kondo, S h i o n o g i Kenkyusho Numpo l O , 9 1 (1960) C.A. 54, 25026(1960) K. S i n g h and S. R a h k i t , Biochim. Biophys. Acta 1 4 4 , 1 3 9 ( 1 9 6 7 ) . T. L. M i l l e r , Biochim. Biophys. A c t a , 270,167 ( 1 9 7 2 ) . U.S. P a t e n t 2,744,120, May 1,1956;C.A. 50 14812 (1956) U.S. P a t e n t 2,823,171, F e b r u a r y 11,1958; C . A . 52, 9 2 3 5 d ( 1 9 5 8 ) . U . S . P a t e n t 2 , 8 6 8 , 6 9 4 , J a n u a r y 13,1959;C.A.53, 9295e (1959) B r i t . P a t e n t 7 9 2 , 8 0 3 , A p r i l 2,1958;C.A. 53, 2293a (1959) Gen. P a t e n t 1,021,845, J a n u a r y 2 , 1958; C . A . 54, 4 6 8 6 ( 1 9 6 0 ) . J a p a n P a t e n t 3 1 (158) J a n u a r y 1 0 ; C . A . 5 2 , 17629 ( 1 9 5 8 ) .

1.
2. 3. 4. 5. 6.

J.

7.

8.
9.

10. 11.
12. 13. 14. 15. 16.

. .

17.

551

KLAUS FLOREY

18. L. N. V o l o v e l s k i i , G. V. Knorozova and

M. Y. Yakovleva, Zh. Obshch. Khim. 37,1252 (1967)C.A. 68, 3078w ( 1 9 6 8 ) . J. B e l i c , E. P e r t o t , H. S o c i c , J . S t e r o i d Biochem 105 (1970)C. A . 73,425859 (1970) ; a l s o H. S o c i c and J. B e l i c , F r e s e n i u s J. Anal. Chem. 243,291 ( 1 9 6 8 ) . C. H. Holmlund, R. H. Blank, K. J. Sax and R. H. Evans, J r . , Arch. Biochem. Biophys. 103, 105 (1963) S. L. Neidleman, P. A . D i a s s i , B. J u n t a , R. M. Palmere and S. C. Pan, T e t r a h e d r o n L e t t e r s 44,5337 (1966). K. S c h u b e r t , K. H. Bahme, F. R i t t e r and C. Hbrhold, J. S t e r o i d . Biochem. 2, 245 (1971). W. E. Hamlin, T. C h u l s k i , R. H. Johnson and J. G. Wagner, J. Am. Pharm. Assoc. S c i . Ed. 49, 253(1963) and D. R. B a r t o n and W. C. T a y l o r , J. Am. Chem. SOC. 8 0 , 2 4 4 ( 1 9 5 8 ) , J. Chem. SOC. 1958, 2500. M. S. P u a r , The S q u i b b I n s t i t u t e , p e r s o n a l comm un i c a t i o n C. Gual, R. I. Dorfman and H. R o s e n k r a n t z , Spectrochim. Acta 1 3 , 2 4 8 ( 1 9 5 8 ) . H. R o s e n k r a n t z and C. Gual, S p e c t r o c h i m . Acta 1 3 , 2 9 1 ( 1 9 5 9 ) . B. N. Kabadi, E. R. S q u i b b and Sons, p e r s o n a l communication, P. T. Funke, The S q u i b b I n s t i t u t e , p e r s o n a l communication. N.F. X I V , 1975 p . 6 8 3 . C. A. Lendzian, The S q u i b b I n s t i t u t e , p e r s o n a l communication. E. I v a s h k i v , The S q u i b b I n s t i t u t e , p e r s o n a l communication. R . C . D . D e C a r n e v a l e Bonino, J. Dobrecky, L. 0. G u e r e l l o , Rev. Farm. 113, 1 5 ( 1 9 7 1 ) C.A. 75,67518s ( 1 9 7 1 ) .

19.

I,

20.

21.

22.

23.

24. 25. 26. 27.

28.
29. 30.

31.
32.

552

TESTOLACTON E

33. H. R. R o b e r t s , The S q u i b b I n s t i t u t e , p e r s o n a l communication. 34. H. R. Roberts a n d K. F l o r e y , J. Pharm. S c i . , 51,794 (1962). 35. K u h n e r t - B r a n d s t a t t e r , P. G a s s e r , P. D. L a r k , P. L i n d e r a n d G. K r a m e r , Microchem J. 2 , 7 1 9 (1972). 3 6 . H. J a c o b s o n , The S q u i b b I n s t i t u t e , p e r s o n a l communication. L i t e r a t u r e s u r v e y e d t h r o u g h 1973.

553

ADDENDA AND ERRATA

ADDENDA AND ERRATA

D i a t r i z o i c Acid Volume 4 , p. 151 Under 5.2, Free I o d i n e and F r e e H a l i d e , r e p l a c e sentence s t a r t i n g w i t h , "An a l t e r n a t e procedure . . . . . * I w i t h t h e f o l l o w i n g :

For d e t e c t i o n of f r e e i o d i n e and i o d i d e , t h e f i l t r a t e i s a c i d i f i e d , chloroform and sodium n i t r i t e a r e added, and t h e r e d d i s h c o l o r i n t h e chloroform l a y e r i s compared t o a s t a n d a r d s o l u t i o n ( 4 , 1 2 1 .

1sosorbi.de D i n i t r a t e V o l u m e 4 , p. 225 The correct name of t h e f i r s t a u t h o r i s Sivieri, not S i l v i e r i . Phenformin Hydrochloride V o l u m e 4 , p. 319 To t h e embarrassment and r e g r e t of t h e e d i t o r , t h e f u l l l i s t of a u t h o r s was i n a d v e r t e n t l y n o t p r e s e n t e d by him f o r t h i s P r o f i l e . The correct

t i t l e is:
Phenformin Hydrochloride Michael J. O'Hare, Hridaya Bhargava, Ruth Wasserman and Joseph E. Moody

5.

Chemical i o n i z a t i o n m a s s s p e c t r o m e t r y was used f o r t h e d e t e r m i n a t i o n of Phenformin Hydrochloride i n b i o l o g i c a l f l u i d s by S. B. Matin, J. K. Karam, P. H. Forsham and J. B. Knight, Biomedical Mass Spectrometry 1, 320 ( 1 9 7 4 ) .

6.5

An HPLC method f o r t h e d e t e r m i n a t i o n of Phenformin Hydrochloride has been developed by R. K. G i l p i n , J. A . Korpi and C . A. J a n i c k i , J. of Chromat. 1 0 7 , 115 (1975).

556

ADDENDA AND ERRATA

- *

4, p. 386

It was pointed out by Prof. H. Vanderhaeghe of Leuven, Belgium that in the structural formula on p. 386 the substituent on C15 should be H (see formula on p. 400). Also, since the absolute configuration is known (see Klyne and Buckingham, "Atlas of Stereochemistry,I' Chapman and Hall (1974)1, the structure given is that of the inactive enantiomer. The correct structural formula is:

bCH3 OCH3

Tolbutamide Volume 3, p. 513

5.

Solid probe c..emical ion-zation mass spectrometry and gas chromatography has been used for the determination of Tolbutamide and its metabolites in human plasma by S . B. Matin and J. B. Knight, Biomedical Mass Spectrometry L, 323 (1974).

Triflupromazine Hydrochloride Volume 2, p. 523

2.5

pKa The pKa of 6.5 as listed in Volume 2, p. 533 is in error. Reexamination by Dr. H. Jacobson (The Squibb Institute) gave a pKa value of 9.45, in reasonable agreement with values determined by
557

ADDENDA AND ERRATA

references 8 and 9 .
2.10 The c r y s t a l s t r u c t u r e of Triflumpromazine Hydrochloride has been determined by x-ray d i f f r a c t i o n by D . W . Phelps and A. W Cordes, Acta Cryst. g, ; 2812 ( 1 9 7 4 ) .

558

CUMULATIVE INDEX
Italic numerals refer to Volume numbers. Acetaminophen, 3 , l Acetohexamide, 1 . 1 ; 2,573 Alpha-Tocopheryl Acetate, 3.11 1 Amitriptyline Hydrochloride, 3,127 Ampicillin.2, 1;4,517 Bendroflumethiazide5, 1 Cefazolin, 4 , l Cephalexin, 4 , 2 1 Cephalothin Sodium, 1 , 3 19 Cephradine,5, 21 Chloral Hydrate, 2,85 Chloramphenicol,4 , 4 7 , 5 17 Chlordiazepoxide,1, 15 Chlordiazepoxide Hydrochloride, 1. 39; 4, 517 Chloroquine Phosphate, 5,61 Chlorprothixene, 2.63 Clidinium Bromide, 2,145 Clorazepate Dipotassium, 4 , 9 1 Cloxacillin Sodium, 4.113 Cycloserine, 1, 5 3 Cyclothiizide, 1,66 Dapsone, 5. 87 Dexamethazone, 2, 163; 4, 5 18 Diatrizoic Acid,4, 137,5 556 Diazepam, 1,79;4,517 Digitoxin, 3,149 Dioctyl Sodium Sulfosuccinate, 2,199 Diphenhydramine Hydrochloride, 3,173 DisuKiam, 4,168 Echothiophate Iodide, 3,233 Erthromycin Estolate, 1,101;2,573 Estradiol Valerate, 4, 192 Ethynodiol Diacetate, 3,253 Fiucytosine, 5, 115 Fludrocortisone Acetate, 3,281 Fluorouracil, 2,22 1 Fluphenazine Enanthate, 2,245; 4,523 Fluphenazine Hydrochloride, 2, 263; 4, 5 18 Gluthethimide, 5, 139 Halothane.1, 119;2,573 Hydroxyprogesterone Caproate, 4,209 Iodipamide, 3,333 Isocarboxazid, 2,295 Isopropamide, 2 , 3 15 Isosorbide Dinitrate, 4,225;5, 556 Levartereno1Bitartrate, 1,49; 2,573 Levallorphan Tartrate, 2,339 h o d o p a , 5, 189 Levothyroxine Sodium, 5. 225 Meperidine Hydrochloride, 1,175 Meprobamate, 1,209; 4,s 19 Methadone Hydrochloride, 3, 365; 4, 5 19 Methaqualone, 4, 245,519 Methotrexate, 5, 283 Methyclothiazide, 5. 307 Methyprylon, 2,363 Metronidazole,5, 327 Nitrofurantoin, 5, 345 Norethindrone, 4,268 Norgestrel, 4, 294 Nortriptyline Hydrochloride, 1,233; 2, 573 Oxazepam, 3,441 Phenazopyridine Hydrochloride, 3,465 Phenelzine Sulfate, 2, 383 Phenformin Hydrochloride, 4, 319;5. 429 Phenoxymethyl Penidllin Potassium, 1.249 Phenylephriie Hydrochloride, 3,483 Piperazine Estrone Sulfate, 5, 375 Rimidone, 2,409 Procainamide Hydrochloride, 4, 333 Rocarbazine Hydrochloride, 5,403 Promethazine Hydrochloride, 5. 429 PropiomazineHydrochloride, 2,439 Ropoxyphene Hydrochloride, 1,301; 4, 5 19 Reserpine, 4,384;5, 557 Rifampin, 5,467

559

CUMULATIVE INDEX

Secobarbital Sodium, I , 343 Spironolactone,4.43 1 Sulfamethoxazole,2, 467;4, 520 Sulfasalazhe,5, 5 15 Sulfisoxazole,2, 487 Testolactone, 5. 533 TestosteroneEnanthate, 4, 452 Theophylline,4,466 Tolbutamide, 3, 513;5, 557 Triamcinolone, I . 367;2, 571;4, 520,523 Triamcinolone Acetonide, I , 397; 2, 571; 4,520

Triamcinolone Diacetate, 1, 423 Triclobisonium Chloride, 2, 507 TriflupromazineHydrochloride, 2, 5 23; 4, 520;5, 557 TrimethaphanCamsylate, 3,545 TrimethobenzamideHydrochloride, 2, 55 1 Tropicamide, 3, 565 Tybamate, 4, 494 Vinblastine Sulfate, I , 443 Vincristine Sulfate, I , 463

A 8 C D

6 7 8 9

F 1

C 2 H 3 1 4
1 5

560