Professional Documents
Culture Documents
of
Drug Substances
Volume 18
Edited by
Klaus Florey
The Squibb Institute for Medical Research
New Brunswick, New Jersey
Contributing Editors
Abdullah A. Al-Badr
George A. Forcier
Harry G. Brittain
Lee T. Grady
San Diego
London
EDITORIAL BOARD
Abdullah A. Al-Badr
Gerald S. Brenner
Glenn A. Brewer
Hany G. Brittain
James E. Carter
George A. Forcier
Lee T. Grady
Eugene L. Inrnan
G. Williams Martin
John E. Zarembo
ISBN
0-12-260818-6
0099-5428
(alk. paper)
89-659072
AFFILIATIONS OF
EDITORS AND CONTRIBUTORS
vii
...
Vlll
Vijay K . Kapoor, Department of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India
J . Jantina Kettenes-van den Bosch, Faculty of Pharmacy, University of
Utrecht, 3511 GH Utrecht, The Netherlands
Leonard J . Kostek, Pfizer Incorporated, Central Research, Groton, Connecticut 06340
G. William Martin, Burroughs Wellcome Co., Research Triangle Park,
North Carolina 27709
Mohammad Saleem Mian, College of Pharmacy, King Saud University,
Riyadh 11451, Saudi Arabia
Neelofur Abdul Aziz Mian, College of Applied Medical Sciences, King
Saud University, Riyadh 11451, Saudi Arabia
Farid J . Muhtadi, College of Pharmacy, King Saud University, Riyadh
11451, Saudi Arabia
Gandharva Padmanabhan, Ciba-Geigy Corporation, Suffern, New York
10901
PREFACE
Ezzat M. Abdel-Moety
and
Hamad A . Al-Khamees
CONTENTS
1.
I NTRODUCT0R.Y
2.
DESCRIPTION
2-1. Name
2-2. Formulae
2-3. The Chemical Abstract Registry (CAS) Number
2-4. Appearance, C,olor, Odor and Taste
2-5. Physical Characteristics
2-6. Crystal Characteristics
2 - 7 . Spectral Characterization
3.
SYNTHESIS
4.
PHARMACOLOGY
5.
6.
TOXICOLOGY
7.
8.
PHARMACOKINETICS
8-1. Biotransformation
8-2. Absorption
8-3. Excretion
9.
METHODS OF ANALYSIS
9-1. Qualitative Methods
9-2. Quantitative Methods
ACKNOWLEDGEMENT
REFERENCES
AZINTAMIDE
1.
INTRODUCTORY
Azintamide is a true potent choleretic drug, which is
totally synthesized in 1 9 5 9 .
The drug has the
registered trade name OragallinB
In spite of the
potent choleretic activity, with moderate cholepoietic
action, and its wide therapeutic applications in
different conditions and countries, no detailed
informations about its physical, chemical, clinical,
and
bioavailability characteristics have been yet
collectively summarized in simple presentation.
The
present Analytical Profile is an effort in this
direction.
2.
DESCRIPTION
2-1.
Names
2.11.
Chemical:
thiol-N,N-diethylacetamide.
Other
chemical
names
2-[(6-Chloro-3-pyridazinyl)
are,
N,N-diethyl-2-[6-(3-
2.12.
Properioritg: Oragallin, Ora-gallin, and
ST 9067.
Azintamide has been registered under the trade name
Oragallin 8 for Osterreichische Stickstoffwerke, AG,
Linz/Donau - Austria.
2-2.
CI
[CioHi4ClN30S (259.77)]
2-3.
2-4.
Physical Characteristics
2-51.
Melting Range
90
Melting range
Mid-point
Literature
("C)
("C)
t 'C)
95.0-97.0
(2.0)
96.0
98-100
(2)
95.5-97.5
(2.0,
96.5
97-98
( ~ 3 )
*Sample from Bsterreichishe Stickstoffwerke, AG, Linz/DonauAustria, BN: 23540/524699 - all values ( ' C ) are uncorrected.
2-52. Differentional Thermal Scanning (DSC)
The DSC-curve was obtained on a DuPont TAto a data processing
unit. Figure 1 shows the DSC-curve of azintamide. The
running was between 50-150C at heating rate of
10"C.min-1.
The heat of activation and the purity of
the sample was determined using purity program.
9900 Thermal Analyzer attached
2-53.
Solubility
Sample : AZII~ITAMICJE-8
Size
: 4 . 6 0 mg
:lethod : DSC 50 TO 150
Comment:
@10 C/M.
DSC
Run Date:
02/11/88
19: 13
97.60
097.59
-1
-z
97.58
-u
97.97
-2-
\
x
D1
::
LL
4J
3
-3-
21
I
-4
0 1
Purity
:
Melting P t :
I
m
Oepresslan :
Oelta H
:
Corrcctlon :
I.101. I l e l g h t :
c e l l const :
-5 - O n s e t S l a p c :
97.55
100.01 Mole X
9 7 . 5 'C
-O.OOn'C
3 0 . 6 kJ/molB
1 . 0 7 x:
259.8 g / ~ o l e
50
',
60
Fiq. 1 :
2
I-
37.55
1.283
-7.90
mN/'C
, 10' .
70
37.54
T a t a l Ar.ea/Pdrt i d 1 A r e a
20
30
40
nn
-6.
a*
80
9b
id0
Temperature
110
50
120
140
('Cl
[he d i f f e r e n t i a - t Scanning
L L J I - V-cOSC)
~
60
140
37.33
120
PURITY V l . l . 4
of fizintnmide.
Optical Rotation
2-54.
Azintamide
species.
2-6.
is
an
optically-inactive
Crystal Characteristics
Crystallization
2-61.
acetone (1).
2-62.
Azintamide
Crystal Forms
Microscopic examination of the microcrystals of azintamide was carried out by using a Leitz
Camera Lucida (X = 4 0 ) attached to a Leitz projector.
shows the different crystal forms of
Figure 2
azintamide.
Fig.2:
AZINTAMIDE
2-63.
7.179
7.978
10.363
12.029
12.557
13.051
15.925
16.639
18.088
18.704
19.727
20.788
21.532
21.983
22.904
23.995
24.664
25.211
25.889
26.875
27.365
27.779
28.868
30.053
30.711
32.075
12.3132
11.0815
8.5357
7.3571
7.0488
6.7832
5.5650
5.3278
4.9041
4 7439
4.5003
4.2724
4.1269
4.0433
3.8827
3.7087
3.6094
3.5324
3.4414
3.3173
3.2591
3.2114
3.0927
2.9734
2.9112
2.7904
13.14
52.52
20.10
7.85
6.63
100.00
7.48
4.49
3.06
5.13
45.39
27.82
2.90
7.72
2.80
20.02
7.42
4.78
2.83
11.39
4.99
2.09
10.18
3.07
44.95
30.49
33.064
34.213
35.445
35,870
36.454
36.913
37.390
39.936
40.407
41.317
41.985
42.377
43.129
44.420
45.775
46.789
47.036
48.034
49.226
50.482
52.793
56.196
56.572
58.426
60.288
2.7092
2.6208
2.5324
2.5034
2.4647
2.4350
2.4051
2.2574
2.2322
2.1851
2.1519
2.1329
2.0974
2.0394
1.9821
1.9415
1.9319
1,8941
1.8510
1.8078
1.7340
1.6368
1.6268
1.5795
1.5351
2.52
12.79
2.67
4.91
4.18
2.77
4.28
6.63
2.19
2.87
2.91
15.98
8.66
6.11
2.70
2.63
2.08
3.67
8.22
5.36
2.99
4.13
2.16
5.00
5.06
60
55
50
45
40 35 30 2 5 20
2 0-Value
15
10
AZINTAMIDE
2-7.
Spectral Characterization
Ultraviolet ( U V ) Spectrum
2-71.
I\
0.0
200
L
,
,
250
3 00
350
Wavelength ( n m )
10
Table 3 :
Wavelength* (nm)
1 cm)
316
54.08
1.404 X 103
306
57.43
1.492 X l o 3
258
547.00
1 . 4 2 1 X 104
Assignment
2980 ( s ) , 2945 ( m )
CH-stretching in heteroaromatic
ring
C
= 0
1580 (m)
1500-1420 ( m )
1410 ( s )
860 (m)
(s)
1630-1636
~~
m = medium, s = strong
stretching, amide
N stretching
Fig. 5 :
i--l%,KBy).
12
2-73.
Mass Spectrum
r 42 I
I
5 -CH2 - co
145.5l
159.5
,
-558
CH2- CH3
1187.5
Base-line (100%) - k 7 2 - i
Az intamide
2-74.
'"1
-30
90
8ol
70
- 20
10
30
0
N
Lo
I:
M.2
SPEC i 3221002
AZI NTAMIOE
STEP MASS
= 10 , I /B/.S/C
: 1%
14
Table 5:
Measured mass*
Structural Assignment
262 ( 4 )
M t 2
c1
Formula
C i o H i 6 C 1 N 3 0s
187 ( 1 9 )
C6H3 C 1 N 2 0 S
159 ( 1 5 )
C5 H3 C l N 2 S
113 ( 5 0 )
C4HClNz
100 ( 5 0 )
85 ( 4 6 )
72 (100)
base-peak
71 (37)
,CH2 CHJCON,
CH2'
L
C2 H5
N'
'c2 H5
* CHCON,
I]
N:C2H5-
c4 H7 NO
CH3
C4 H6 N
CiHsN
c2 H5
58 ( 1 2 )
CH2 -CH3
57 (8)
CH3CON
CHCONHz
C2 H3 NO
56 ( 2 4 )
CHiCON
CH3
CCONH2
C2 Hz NO
44 ( 2 2 )
42 ( 2 5 )
C 5 H6 NO
'c2 H5
c2 H5
C2H6N
CH3
kY2#
CH2
H 2 N CH
y]
C2 H4 N
15
AZINTAMIDE
2-741.
CI
7.46
[d, 1 H ( 1 or 2 1 , aromatic]
7.37
[d, 1 H (1 or 2 1 , aromtic]
4.33
[ s , 2H ( 3 ) ,
3.52-3.41
1.32-1.25
[t, 3H ( 6 1 , CHzCH31'
1.18-1.11
[ t , 3H 1 7 1 , CHzCH31t
SCHzCOI
' '\
rotation about C
(5).
Fig. 7:
1 h e 200-MIiz
1 Ii-NMR
Spectui-m of_fizintam~dq.
17
AZINTAMIDE
*
+
12.85
14.28
[lc
34.10
[1C ( 5 1 , SCH2COl
40.76
42.52
(91, C.CH3It
127.39
[ l C (2), CH-aromatic]
128.16
153.60
161.21
[1C
166.01
(4), C-aromatic]
I O I '.E
' S Z 151
18
m
I
0
19
"
x
0
",I"".,
20
SYNTHESIS
Azintamide has been synthesized in 1959 by Schonbeck
( 6 ) among various
pyridazine derivatives having
Azintamde (ST
different choleretic activities ( 3 ) .
9067) was assigned and patented (German Patents:
1188604, Nov. 1965) to Lentia GmbH, Munich-F.R. Germany
(7).
the
synthetic
CI
CICH2CON(CH2CH3)2
( i n ethanol)
SH
.-c
8
CI
rc
<
SCH2CO N
F 2 H5
\
C2H5
Az in ta rnide
AZINTAMIDE
21
Azintamide,
[3-chloropyridazinyl-6-thiol-acetic
acid
diethylamide, could be obtained by reacting 7 . 3 parts
of 3-chloro-6-methylthiopyridazine (8)dissolved in 20
parts of 10% NaOH with a solution of 7 . 5 parts
chloroacetic acid diethylamide in 20 parts alcohol at
60'C for 30 min.
Crystallization can occur by cooling
to O'C and recrystallization from acetone { 3 ) .
4.
PHARMACOLOGY
The choleretic
activity of azintamide has been
predicted firstly by comparing with those of the 2-[(6chloro-3-pyridazinyl)thio]
acetic acid (ST 9024),
synthesized by Schonbeck (6). The substituted amide
showed the highest potency (3,6).
The choleretic
activity of azintamide has been carried out on rats
against dehydrocholic acid as reference substance (91,
then on human volunteers ( 1 0 - 1 3 ) .
Choleresis due to
azintamide in man with rapid onset was manifested
within a short administration time.
Azintamide is now
one of the most recommended choleretic references for
naturally occuring plant cholagogues ( 1 4 ) and other
(15).
synthetic choleretically active compounds
Azintamide, in identical doses, evokes a more powerful
choleresis than either of dehydrocholic acid and 1phenylpropanol ( 9 ) .
The elimination of bromosulfophthalene (BSP) is only influenced in the first 30minutes period following intradoudonal administration
of 50 mg.Kg-' dose. The same dose of dehydrocholic
acid, in every cases, retarded BSP-elimination over all
30 minutes periods.
The choleretic activity of the
drug can be demonstrated in selected patients by means
of Bartelheimer's double ballon tube.
An increase in
bile flow, depending on the dose, was stated after
This demonstrates
administration of 1 g azintamide.
the drug has true choleretic increasing secretion of
the components of the bile and not merely increasing
fluid volume, i.e. hydrocholeresis.
No increase of
serum bilirubin was observed after azintamide and the
maximum effect is reached within 20 to 40 minutes after
administration.
22
5.
Categoration
Contraindications
5-22.
de should be
and in cases
ithiasis, as
gall stones,
occlusion of
Dosage
TOXICOLOGY
Acute Toxicity (oral by mice):
(1.94-2.48) ( 9 , 10).
A-LDBO is 2.34
g.Kg-1
23
AZINTAMIDE
7.
8.
PHARMACOKINET1CS
8-1. Biotransformation
Through amide-linkage break,
[3-chloropyridazinyl-6-thiolacetic acid is identified in rats urine
Only small amount
as the main metabolic product ( 1 9 ) .
of the unchanged drug could be traced in plasma and
urine.
The principal metabolites, i.e. [3-chloropyridazinyl-6-thiolacetic acid and diethylamine, are
subject for further biotransformation such as soxidation or detoxification as sulfate and or glucuronate conjugates.
8-2.
Absorption
8-3.
Excretion
24
9.
METHODS OF ANALYSIS
9-1. Qualitative Methods
9-11. Elemental Composition
Element
C
H
c1
N
0
S
%-Composition
46.24
5.43
13.65
16.17
6.16
12.35
Reagent
2% anisaldehyde/conc.H3P04 t
CHJCOOH t CzH5OH (3:1:1,
v/v/v) ; heat.
Observation
Violet-red color (19)
* 0.5% p-dimethylaminobenzal-
* 0.5% PDAB/70%
* Vanilin/H+ : heat
Yellow-red (21).
White precipitate,
dissolves on boiling.
Blue-violet color to
turbidity (19).
25
AZINTAMIDE
Table 9
shows the PC-separation and
identification of azintamide.
Table 9:
Mobile phase
30% acetic acid
hRf
88
Visualization
0.5% PDAB in HCI
t CzH50H; heat at
Reference
(9)
70"C/5 min., or
under UV-light;
green-blue spots
appear
*hRf is the travelling rate (Rf) X 100.
Azintamide can be
chromatographed on
different thin-layers eithing by adopting the one- or
the two-dimensional techniques. Table 10 summarizes
the TLC-separation and characterization of azintamide.
26
Table 10:
Mobile phase
Layer
hRf
Visualization Reference
1.
One-dimensional TLC
WHCh t
CzHsOH
(100:5,
Silica
gel GFz54
v/v)
*CH30H t
conc. NHiOH
(100: 1.5,
v/v) ( 2 2 )
2.
Silica
gel GFz54
a. UV-light
( 2 5 0 nm)
b. Spray with
KMn04/0H- to
give yellow
spots on violet
background.
(2)
65-66
a. UV-light
( 2 5 4 nm)
b. 2% PDAB in
5% H2S04; gives
yellowish green
spots after ca.
30 min.
(21)
74
a. 2% anisalde
hyde in conc,
H 3 W 4 t CH3COOH t
CzHsOH ( 3 : 1: 1,
v/v/v); heat 1 2 0 " C /
2 0 min., to give
violet red spots
on white to light
rose background.
55
Two-dimensional TLC
Silica
gel G
*P1:
CH3COOCzH5 t
CH30H t
3-N( NH4 )2C03
DMF (12:2:1:1,
v/v/v/v 1
57
*PZ :
Cch
CH30H t
CH3 COOH
(77.5:20:2.5,
v/v/v 1
b. PDBA/H+ ; heat
12O"C/20 min, to
c. Vanilin/H+ ;
heat, to give blue
violet spots.
21
AZINTAMIDE
9-2.
Quantitative Methods
9-21.
Volumetry
9.211.
Titrimetric determination of
sulfer and chlorine contents
Instrumental Methods
9-221.
Colorimetry
ii-
Direct UV-measurement
Derivative sDectrophotometrY
28
PMR-sDectrophotometrp
El-Khateeb
and Abdel-Moety
described the application of proton-magneticresonance
spectrophotometry
for
quantitative
determination of azintamide in pure forms and in dosage
formulations.
The method involves comparing the
integral of both the multiplet centered at about 1 . 1 5
ppm and the sharp singlet at 4 . 3 0 ppm of azintamide
molecule to that of the sharp singlet signal at about
6 . 3 0 ppm of maleic acid which is chosen as internal
standard.
(24)
9-222.
SDectrofluorometry
Flow-injection analysis ( F I A ]
A single-manifold FIA-system f o r
quantitative determination
of
azintamide
via
spectrophotometric detection at 258 nm is investigated
The limit of quantificaby Abdel-Moety et al. (26).
tion and detection is about 5 pg.ml-1 of azintamide
AZlNTAMIDE
29
Gas-liquid chromatographs
( GLC )
High-Performanc Liquid
Chromatographs (HPLC)
30
9-23.
BioloRical Assap
Azintamide
can be quantified by the
measurement of its choleretic activity according to the
procedure described by Stormann ( 9 ) .
The biological
measurement is done against the activity of a well
known choleretic agent, such as dehydrocholic acid and
1-phenylpropanol, The measurement of the elimination
times of bromosulfophthalene (BSP) following suitable
intradoudenal doses of the drug in the first 30minutes.
9-3.
ACKNOWLEDGEMENT
The authors would like to thank Dr. R.R.
Abou-Shaaban
for DSC-investigation and Mr. T.A. Butt for typing the
manuscript.
31
AZINTAMIDE
REFERENCES
1.
2.
3.
E. Kloimstein, R.
Schonbeck,
Arzneim.-Forsch.; l4, 261 ( 1 9 6 4 ) .
4.
and H.
Stormann:
124, 730 ( 1 9 8 5 ) .
5.
6.
R. Schonbeck:
Reference 3.
7.
8.
9.
Mh.
Chem.; 90,
284 ( 1 9 5 9 ) ;
m;37,
through
Helv. Chim.
133 ( 1 9 5 4 ) .
H. Stormann:
10.
G.
11.
W. Neugebauer:
12.
H.L. Dierel:
13.
14.
R. Hansel:
15.
G.
Landarzt; 41, 33 ( 1 9 6 5 ) .
Wien-Med-Wochenschr.;
115, 613
(1965).
Ludwig:
32
16.
D.
Konstantinov
and L.
(1975).
17.
P. St.-Janiak:
18.
19.
Arzneim.-
20.
Scientific
21.
E.M.
22.
I . Sunshine:
23
24.
25.
26.
27.
28.
29.
30.
E.M.
Abdel-Moety:
Anal.
Lett.;
Sci. Pharm.;
Abdel-Moety:
Zentralbl. Pharm.
Laboratoriums-diagn.; 127, 137 ( 1 9 8 8 ) .
Pharmakother.
CHLOROTHIAZIDE
Harry G. Brittain
33
HARRY G.BRITTAIN
34
TABLE OF CONTENTS
1. Description
1.1 Name, Formula, and Molecular Weight
1.2 Appearance
1.3
History
2. Synthesis
3. Physical Properties
3.1
Infrared Spectrum
3.2
NMR Spectra
3.3 Mass Spectra
3.4 Crystallographic Properties
3.5 Hygroscopicity
3.6 Optical Activity
3.7 Melting Phenomena
3.8
Differential Scanning Calorimetry
3.9
Thermogravimetry
3.10 Ionization Constant
3.11 Solubility
3.12 Partition Coefficients
3.13 Ultraviolet Spectrum
3.14 Fluorescence Spectrum
4. Methods
4.1
4.2
4.3
of Analysis
Elemental
Spectrophotometric
Chromatographic
4.3.1 Thin-Layer
4.3.2 High Performance Liquid
4.4 Electrochemical
5. Stability
5.1 Solid Stability
5.2 Solution Stability
5.3 Incompatibilities
5.4 Stability in Biological Fluids
6. Drug Metabolism, Pharmacokinetics
7. Acknowledgement
8.
References
CHLOROTHIAZIDE
35
1. Description
1.1
C,H,C lN,O,S
M.W.
295.74
Appearance
HARRY G . BRITTAIN
36
Historv
1.3
Synthesis
CHLOROTHIAZIDE
31
3. Physical Properties
3.1
Infrared Spectrum
3.2
Mass Spectra
CrYstallosraRhic Properties
HARRY G. BRITTAIN
38
Figure 1.
%OOO
3600
3200
2300
2%0C
ZGOC
1500
1290
Energy (cm-I)
800
LL3C
CHLOROTHIAZIDE
39
Table I
'H and 13C Nuclear Magnetic Resonance Resonances
Observed in DMSO-d, Bolutions of Chlorothiazide
Resonance ~ Pm)
D
Assisnment
(b)
8.10
, 7.51
11-13
H at C1
aromatic H
H of the -NH group
c3
c5
C6
c7
C8
c9
c10
HARRY G . BRITTAIN
40
Hvaroscopicitv
3.6
Optical Activity
Meltina Phenomena
41
CHLOROTHIAZIDE
Figure 2.
10.0
15.0
20.0
Scattering Angle
25.0
30.0
(Degrees
2-e)
HARRY G. BRITTAIN
42
Table I1
Powder X-ray Diffraction Data Obtained for
Chlorothiazide: Scattering Angles, D-spacings, and
Relative Intensities
Scattering
Angle
ldecrrees 2-theta)
5.30
10.33
14.60
15.52
16.35
18.57
19.98
20.70
21.98
25.22
26.46
26.86
28.36
28.86
29.32
31.20
D-Spacing
Ancrstroms 1
16.66
8.56
6.06
5.70
5.42
4.77
4.44
4.29
4.04
3.53
3.37
3.32
3.14
3.09
3.04
2.86
Re1 at ive
Intensity
f I/IMX)
0.48
1.59
84.63
8.67
0.29
0.95
20.29
44.57
46.58
1.06
18.85
5.13
30.95
15.27
100.00
8.04
CHLOROTHIAZIDE
3.8
43
Thermoqravimetry
44
Figure 3.
HARRY G. BRI?TAIN
Temperature ("c)
CHLOROTHIAZIDE
45
basic solution
wavelength
maximum (nm)
280
294
E (1%, lcm)
580
630
HARRY G. BRITTAIN
46
Table I11
Solubility
Reference
water (pH=4)
16
water (pH=7)
0.65 g/L
16
ethanol
1.54 g/L
17
10 W L
acetone
17
diethyl ether
insoluble
17
chlorofo m
insoluble
17
benzene
insoluble
17
methanol
slightly soluble
18
pyridine
slightly soluble
18
dimethyl sulfoxide
freely soluble
18
dimethyl fomami.de
freely soluble
18
47
CHLOROTHIAZIDE
Figure 4.
240
260
300
Wavelength (nm)
HARRY G . BRITTAIN
48
4.
Methods of Analysis
4.1 Elemental Analysis
C
H
c1
N
S
28 43%
2.05%
11.99%
14.21%
21.64%
21.69%
28.38%
2.25%
12.12%
14.12%
21.57%
4.2 Spectrophotometric
CHLOROTHIAZIDE
49
Thin-Laver
HARRY G . BRImAIN
50
Table IV
Thin-Layer Chromatographic Systems and
Characteristic R, Values Obtained using Silica Gel
G as the Adsorbant
R,
Solvent System
Reference
0.17
26
0.70
27
0.85
27
0.26
27
0.64
27
0.42
27
chloroform, methanol
0.10
28
0.03
28
0.21
28
pure acetone
0.66
28
9O:lO
0.16
29
0.39
29
0.36
29
5 0 : 5 0 chloroform, acetone
0.31
29
51
CHLOROTHIAZIDE
Electrochemical
HARRY G. BFUTTAIN
52
5.
Stability
5.1 Solid Stability
Solution Stabilitv
6.
CHLOROTHIAZIDE
53
Acknowledqement
54
HARRY G . BRITTAIN
8.
1.
References
K.H.
2 0 , 410
(1977).
2.
X.H.
15
(1982).
3.
825-
830.
4.
5.
J . G.
6.
7.
Topliss, ltDiureticsl1,chapter 38 in
Medicinal Chemistry, 3rd edition, A. Burger,
ed., Wiley-Interscience, New York, 1 9 7 0 .
SOC., 7 9 , 2 0 2 8 ( 1 9 5 7 ) .
965,
8.
9.
970 ( 1 9 6 0 ) .
35,
2151 (1979).
247
(1987).
10.
11.
12.
C.W.
(1961).
13.
303 (1961).
CHLOROTHIAZIDE
55
14.
15.
16.
17.
18.
lvRemingtonts
Pharmaceutical Sciencesvv,A.R.
Gennaro, ed., 17thedition, Mack Publishing
Co., Easton, PA, 1985.
19.
20.
21.
22.
23.
25.
26.
27.
l9, 82 (1964).
28.
134 (1968).
HARRY G . BRITTAIN
56
29.
30.
31.
32.
33.
34.
35.
(1987)
36.
37.
38.
39.
40.
41.
42.
CLIOQUINOL
1.
Description
1.1 Name, Formula, Molecular Weight
1.2
2.
Appearance
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
3.
Synthesis
4.
Stability
4.1
4.2
Degradation
Solution
S o l i d State
57
58
6. Toxicity
7. Methods of Analysis
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
7.10
7.11
7.12
7.13
Identification
Elemental Analysis
Titrations
Phase-SolubilityAnalysis
Thin-Layer Chromatography
Gas Chromatography
High Pressure Liquid Chromatography
Infrared Absorption Spectrophotometry
Ultraviolet Absorption Spectrophotometry
Colorimetric Analysis
Polarography
X-Ray Fluorescience Analysis
Gravimetric Methods
References
1. DESCRIPTION
1.1
CgHsC1IN0
1.2
OH
Molecular Weight:
305.5
Appearance
CLIOQUlNOL
59
2.1
Assignment
785
1580, 1610
1205, 3140
Phenolic OH
Aromatic chloro
960
CI
OH
Hc
60
(D
0
1
0
0
co
0
0
s
2
0
0
0
0
P
0
01
v
s)z
8:
3
ze
o =
Ln
cu
0
0
0
0
0
Lo
0
0
61
0
0
(D
63
0
0
z
0
0
0 2
g$
0
0
0
N
cu
0
0
Lo
0
0
0
0
0
Lo
0
0
G
.E
PPM
Figure 3:
Proton NMR Spectrum of Clioquinol in Dimethyl Sulfoxide
CLIOQUINOL
63
Table I1 (continued)
Chemical
Shift
(ppm)
Number
Multiplicity
7.6-0.0
of
Protons
Assignment
Mu1tiplet
8.0
Singlet
8.4-8.1
Multiplet
8.9-9.1
Mu1tip1et
CI
OH
Chemical Shift, ppm
Assignment
78.845
119.400
123.383
125.663
10
132.959
134.929
R.j
65
CLIOQUINOL
Assignment
137.476
149.603
153.558
Fragment
305, 307
M+
277, 279
[M-CO ] -t
178, 180
[M-I]'
150, 152
[M-CO-I]-t
66
0.67
0.5-
0.4-
s 0.3n
a
0.2-
0.1
0.0
220
260
300
340
Wavelength, Nanometer
Figure 5:
Ultraviolet Absorption Spectrum of Clioquinol in 1:4 HCI
0.90-
0.800.70a,
0.60-
e
9a
0.50-
0.400.30
0.200.100.0-
200
220
240
260
280
300
320
340
360
380
400
Wavelength, Nanometer
Figure 6:
Ultraviolet Absorption Spectrum of Clioquinol in 0.1N Methanolic Sodium Hydroxide
68
1.0-
0.9-
0.8-
0.7-
0.6-
4? 0.5-
0.4-
0.3-
0.2-
0.1
0.0
200
250
300
350
400
69
CLlOQUINOL
9080
70
> 60.-+-.
cn
c
a)
.9
c
m
50-
2 403020
50
Figure 8:
Low Resolution Electron Impact Mass Spectrum of Clioquinol
70
Table IV (continued)
2.5
m/e
Fragment
123, 125
[ M-CO-I-HCN]
115
[M-CO-I-C1]
Optical Rotation
GANDHARVA PADMANABHAN E T A L .
72
Table V
Solvent
Temperature
Solubility, mg/mL
Water
Room Temperature
(0.01
Methano1
Room Temperature
1.9
Ethanol
Room Temperature
1.3
Ether
Room Temperature
5.4
Chloroform
Room Temperature
14.9
0.1N NaOH
Room Temperature
17.3
0.1N HC1
Room Temperature
Intestinal Fluid
37oc
Gastric Fluid
37oc
Acetonitrile
Room Temperature
Tetrahydrofuran
Room Temperature
Ethyl Acetate
Room Temperature
Carbon Disulfide
Room Temperature
>10 <20
Dimethyl Sulfoxide
Room Temperature
>20 (100
Dimethyl Acetamide
Room Temperature
>20 (100
0.02
(0.01
0.02
>1.7 (2.5
>20
<loo
>6.7 (10
Polymorphism
No polymorphism has been reported for clioquinol.
13
CLIOQUINOL
21 9
27 8
10
15
20
25
DegreesTwoTheta
Figure 10:
X-ray Powder DiffractionPatternof Clioquinol
30
35
Y .
74
2.12
P a r t i t i o n Coefficient
The f o l l o w i n g p a r t i t i o n c o e f f i c i e n t d a t a f o r c l i o q u i n o l
were o b t a i n e d when 25 mL of 0 . 1 mg/mL and 1 mg/mL s o l u t i o n s i n
t h e a p p r o p r i a t e o r g a n i c s o l v e n t s were p a r t i t i o n e d i n d i v i d u a l l y
w i t h 25 mL of t h e i n d i c a t e d aqueous s o l u t i o n s a t room
temperature.
Table V I
Aqueous
Phase
Organic
Phase
Partition
Coef f i c i e n t f c
0.1N H C 1
Chloroform
+ 0 1
pH 7 B u f f e r
Chloroform
+(XI
0.1N NaOH
Chloroform
0.36
0.1N H C 1
Ether
pH 7 Buffer
Ether
+a
0.1N NaOH
Ether
+o
pH 7 Phosphate
Saline Buffer
n-Decane
1750(2)
% o n c e n t r a t i o n i n o r g a n i c p h a s e / c o n c e n t r a t i o n i n aqueous
phase
2.13
D i s s o c i a t i o n Constant
C l i o q u i n o l can i o n i z e i n s o l u t i o n s b o t h a s a n a c i d and a s
a b a s e . The d i s s o c i a t i o n c o n s t a n t s 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 ( 2 ) based on t h e p a r t i t i o n of c l i o q u i n o l ( o r
t r i t i a t e d c l i o q u i n o l ) between n-decane and b u f f e r s of approp r i a t e pH v a l u e s and d e t e r m i n a t i o n o f c l i o q u i n o l by s p e c t r o photometry ( o r s c i n t i l l a t i o n c o u n t i n g ) . The b u f f e r s employed
were g e n e r a l l y 0.02 M i n phosphate and t h e y a l s o c o n t a i n e d
0 . 1 3 M sodium c h l o r i d e and 0 . 5 mM EDTA. The v a l u e s r e p o r t e d
f o r pKa were 3.17 f o r t h e d e p r o t o n a t i o n of t h e p r o t o n a t e d
n i t r o g e n and 8.01 f o r t h e d e p r o t o n a t i o n of t h e p h e n o l i c group.
The pKa v a l u e f o r t h e p h e n o l i c group has a l s o been r e p o r t e d t o
be 8 . 1 2 based on t h e p o t e q t i o m e t r i c ( 3 ) t i t r a t i o n a t 35'C w i t h
50:50 (v/u) e t h a n o l l w a t e r a s s o l v e n t .
CLIOQUINOL
75
2.14 Complexation
8-Hydroxyquinoline is
5, 6 ) and hence clioquinol
complexes with metal ions.
values f o r clioquinol have
(3)
Table VII
l o g K1
log Kz
log K
cu2+
8.85
6.95
15.80
Zn2'
6.99
5.48
12.47
Mn2'
5.48
4.00
9.48
Mg2+
4.95
3.60
8.55
Ca2+
4.85
3.28
8.13
Metals Ions
K1 = [MLi]/[M][Li]; K2 = [MLi2]/[MLil[Li];
K = K1K2[MLi2]/[Ml [LiI2
3. SYNTHESIS
NoI
+ NaIO3 + H202
in C2HsOH
OH
OH
76
4.
STABILITY-DEGRADATION
4.1
Solution
Time
% Clioquinol Remaining
Photolyzed
Refluxed
Refluxed
Acetonitrile
0.1N NaOH
4N HC1
Solution
100
100
100
2.5 Hours
N.D.
76
98
5 Hours
90
N.D.
98
1 Day
54
65
92
2 Days
32
34
80
3 Days
N.D.
N.D.
63
6 Days
N.D.
N.D.
39
CLIOQUINOL
71
250, 750 and 1500 mg of the drug in the powder form, no free
clioquinol was detected in urine samples ( 8 , 9). However,
evidence for the presence of less than 1% of conjugates of
6. TOXICITY
An oral LD50 value of 69 mglkg in male mice has been
reported for clioquinol (6). Other reported LDS0 values are:
>1.99 g/m3/4H for inhalation for male rates and >3.04 kg/kg
for dermal route for rabbits.
7. METHODS OF ANALYSIS
7.1
Identification
78
7.2
Elemental Analysis
Theory,
Carbon
35.38
35.4
Hydrogen
1.65
1.7
Nitrogen
4.59
4.6
Chlorine
11.60
11.3
Iodine
41.54
43.1
7.3
Found, %
Titrations
Non-aqueous Titration
Clioquinol can be titrated in glacial acetic acid with
perchloric acid in glacial acetic acid as titrant. The
titration can be carried out potentiometrically using a glass
calomel electrode containing lithium chloride saturated
glacial acetic acid. Clioquinol can also be titrated as an
acid in pyridine or dimethylformamide as solvents with methanolic sodium hydroxide as titrant.
7.3.1
CLIOQUINOL
System I
Adso rbent:
79
Mobile Phase:
Detection System:
Longwave W.
System I1
Adso rbent:
Mobile Phase:
Chloroform
Detection:
System I11
Adsorbent:
Mobile Phase:
Methano1
Detection System:
1. W at 266 MI
2. Pauly reagent spray
System IV
Adsorbent:
Silcia Gel
Mobile Phase:
Methanolfmethoxyethanolfhydrochloric
acid (88:10:2).
Developed three
times (12)
Detection
System V
Adsorbent:
Mobile Phase:
80
Detection:
Temperature:
Carrier:
Helium, 30 mL/minute
Detector:
Flame Ionization
System I1
The following systems have been employed for the determination of the drug substance in feed mixes (15).
Column:
Temperature:
Carrier:
Nitrogen, 80 mL/minute
Detector:
Flame Ionization; Hz
Air - 600 mL/minute
80 mL/minute,
81
CLIOQUINOL
System 111
Column:
Temperature:
Carrier:
Nitrogen, 60 mL/minute
Detector:
Flame Ionization
System IV
The following all glass system has been employed for the
analysis of active drug substance and related impurities (17)
Column:
Temperature:
Carrier:
Helium, 40 mL/minute
Detector:
Flame Ionization; H2
Air - 300 mL/minute
30 mL/minute,
82
System V
Column:
Temperature:
Carrier:
Nitrogen, 30 mL/minute
Detector:
Flame Ionization
System VI
The following system has been employed for the determination of the drug in urine and blood plasma samples (18).
Column:
Temperature:
Carrier:
Nitrogen, 30 mL/minute
Detector:
83
CLIOQUJNOL
System VII
The following system has been employed for the determination of clioquinol in biological samples (19).
Column:
Temperature:
Carrier:
Nitrogen, 30 mL/minute
Detector:
System VIII
The following system has been employed for the analysis
of clioquinol in human plasma (7).
Column:
Temperature :
Carrier:
Nitrogen, 50 mL/minute
Detector:
Internal Standard:
5,7-Dichloro-8-hydroxyquinoline
Sample :
84
System IX
The following procedure has been employed for the analysis of the active ingredient and formulations for both active
drug and related by-products (20).
Column:
Temperature:
290C.
Carrier:
Nitrogen, 45 mL/minute
Detector:
System X
The following procedure has been employed for the analysis of active ingredient (21, 22).
Column:
3 or 5% SE 30 on Varaport-30 (60-80
mesh)
Temperature:
Carrier:
Nitrogen
Detector:
Flame Ionization
gas
85
CLIOQUINOL
7.7
System I
The following system has been employed for the analysis
of clinquinol and the related impurities in the active drug
substance ( 6 ) .
Column:
Mobile Phase:
Acetonitrile/Water ( 6 0 : 4 0 )
Flow Rate/
Temperature:
Detection:
W at
Sample
Preparation:
240 nm
The above system has also been employed for the analysis
of active ingredient and several formulations with the following modifications: mobile phase - acetonitrile/water ( 8 0 : 2 0 ) ,
W detection at 260 nm and 1 mL/minute flow rate.
System I1
The following system has been employed for the analysis
of the drug in cream formulations and in the active substance
(12)
Column:
Mobile Phase:
Flow Rate:
Temperature :
Room Temperature
86
GANDHARVA PADMANABHAN ET AL
Internal
Standard :
Testasterone acetate
Detection
W at 254 nm
Sample:
To 5 mL of tetrahydrofuran extract
of sample containing -1.5 mg of drug
substance, 2 mL of 1:l pyridine/
acetic anhydride is added and heated
for 15 minutes at 6OOC. After
addition of internal standard and
evaporation of solvent at 4OoC, the
sample is redissolved in mobile phase
and injected into the column.
System I11
The following system has been employed for the analysis
of clioquinol in plasma (23) and cream and ointment formulations (24)
Column:
Mobile Phase:
( 1 ) 80% Methanol and 20% 0.05M phosphoric acid, (2) 70% Methanol and 30%
0.05M phosphoric acid
Internal
Standard:
Phenylsalicylate
Plow Rate/
Temperature:
1 . 0 mL/minute at 4OoC
Detection:
W at 256 nm
Sample:
System IV
The following high pressure liquid chromatographic
procedure for the analysis of the conjugates such as
glucoronide and sulfate of clioquinol in human urine has been
reported (25).
CLIOQUINOL
7.8
87
Column:
Mobile Phase:
Flow Rate/
Temperature:
Detection:
W at 254 nm
Sample :
88
7.11
Polarography
Gravimetric Methods
2.
3.
190-192.
4.
5.
(1976).
Merck and
23 (3), 456-458.
6.
7.
J. Pharm. Sci.,
62
1929-1932.
(1967).
CLIOQUINOL
89
10.
11.
12.
13.
14.
15.
16.
R e p o r t from Sub-Committee on M e d i c i n a l A d d i t i v e s i n
Animal F e e d s . ( 1 9 8 1 ) . A n a l y s t . 106, 105-113.
17.
18.
19.
Degen, P. H . , S c h n e i d e r , W . , V u i l l a r d , P . , G e i g e r , U. P.
and Reiss, W. ( 1 9 7 6 ) . J . Chromatogr., __
117, 407-413.
20.
Hartmann, Von V . ,
( 3 ) , 202-205.
21.
22.
23.
24.
25.
(1982). 3 . Pharm. S c i .
and Herrmann, W.
101, 167-173.
( 1 9 7 4 ) . Pharm. I n d . 36
90
26.
27.
28.
29.
30.
31.
1.
&.
If
3.
5.
Stability
6.
7.
9,
VIJAY K. KAPOOR
92
13..
Toxicity
CLOFAZIMINE
1.
93
Introduction
C1ofazi:nine ( ~ 6 3 3 ) 3
, piienazine d e r i v a t i v e , i s
one o f t h e r L o s t a c t i v e o f a s e r i e s of ccmpounds,
s y n t h e s i z e d by 3 a r r y :ind
callccl rimlxio-conpounds
his c 011cagties i n suppress i r 4 exi>ericlental tubmero ~ l o s i si n tlie mouse and guinea pig.
S t u d i e s Lave
s!ioim t h a t clofnziinitie is a l s o a c t i v e acainst o t h e r
mycobacterial i n f e c t i o f l s ; iLycobactesiu3 l e p r a e , i n
p a r t i c u l a r , seerns t o b e about ten times x o r e
m sc ep t i b 1e t o c l o f azirriirie thii 1A. tuberculosis.
--.
The c f f i c a c y o f clof'aaizine agaisst 1:uinan l e p r o s y ,
b o t h i n previmisly u i l t r e a t e d p a t i e n t s and i n
p a t i e n t s who lrave relapsed with dri s o n e - r e s i s t a n t
;A.
l c p a e , i s w e l l est,zl,lisfled.2-1'
2lofazii.iine
i s now roconuncnclcd a s a colllporicnt o f mciltiple-drcig
T:ic co:qmuncl i s also u s e f ' u l
therapy for l c p r o s y .
f o r tre:ttr.ieiit o f c h r o n i c s k i n a l c e r s produced hy
ulceraxis and i t fias some a c t i v i t y a g a i n s t t h e
1l. a v i u m - i n t r a c e l l u l a r e cot'iplcx.
The 5iol o g i c a l
and pharmacological a c t i v i t i e s o r c1ofazi:aine have
bccn d e s c r i b e d . 12-26
'
-::.
I
2.
9e script i o n
Cl
94
VlJAY K. KAPOOR
I'
2.2
almost odorless.
P h y s i c a l Properties
3.
3.1
Infrared Spectmm
Wavelength
9
10
11
12
13
04
15
.E
c
v)
2000
1500
1200
1000 900
Waven u m be r
8C0
700
3-
CLOFAZIMINE
3.2
95
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
The l i g h t a b s o r p t i o n , in t h e range 2 3 0 t o
350 n m , of a 1-cn l a y e r o f a 0.0002$ w/v solution
i n methyl a l c o h o l exhibits a maximum only a t 283 nm
( F i g u r e 2 ) ; 2 7 absorbance a t 223 nm is about 0.3. 28
The l i g h t a b s o r p t i o n i n t h e range 230 t o 600 mi of
a 0.001$ w/v s o l u t i o n i n O . O l H n e t l i a n o l i c hydroc h l o r i c a c i d e x h i b i t s two r u a x h i , at 283 nri~and1
4f37 nr11.~9 The absorbance a t 283 n m i s about 1.30
and at
Q,
u
C
In
225
250
275
300
325
35 0
Wavelength
Figure 2
U l t r a v i o l c t a b s o r p t i o n s2ectruio
of clofazimine
3.3
Mass Spectrun-
3.4
O p t i c a l ! i o t a t i o n-.
C l o f a z i m i n e exhibits no o p t i c a l a c t i v i t y .
VIJAY K. KAPOOR
96
C l o f a z i m i n e i s p r a c t i c a l L y insoluble in
w a t e r ; s o l t l b l e 1 i n 7 C O of e t l i r r n o l , 1 j.11 15 of
ciiloroforxi, and 1 i n lCUU o f e t J i e r ; s o l t l b l e in
d i l u t c a c e t i c CiC$d, d i ' i i e t ~ L ~ l [ ~ r ~ . ,clioxane
~ ~ ~ , i i ~and
e,
:nacro:ol
!$w.'7'- ,3' Ai c l e a r t r a n s p a r e n t lcmtcr
iiiisc i b l c L i c p i d p3iar:.mc m it i c a l voi Licle for c l o f azimine
c o n t n i n s 1)bironi.c i?- 127.3
'
CLOFAZIMINE
91
Atom
1?,4
el 18
c,
Qg
N
Ii
ia
C
C
C
C
C
0.1743
0.14'71
0.1628
0.'2253
0.2651
0.3195
0.3069
C
C
0.2496
0.2177
0.2761
cJ.2~00
0 . 1876
r=
C
C
0.2055
0.1145
U.Ut319
C
C
C
o.oou3
0.2874
0 3379
C
C
C
C
C
0.0249
0.0318
0. of396
0.3S77
0.3860
0 3365
0.21372
0.0677
C
C
0.0592
0.03'77
VIJAY K. KAFQOR
98
1.2055
6)
1.1903
1.29~3[16/
19
0.2202
0.0167(131
14)
0.4633
0.478511;/
1
1.1848 15
1.3O24( 14)
o.ooa3 1 1
0.1073( 12)
0.5590
16
004301 14)
CLOFAZIMINE
99
100
VUAY K. KAPOOR
Table
At oms
C
C
121 .8
117.0
114.1
120.0
113.4
126.6
122.7
118.3
122.2
119.4
120.1
120.9
121.5
119.1
119.4
122.8
117.6
119.6
117.a
122.6
119.6
118.9
N
N
C
C
C
C
C
C
C
C
C
N
N
C
C
N
N
N
C
C
C
N
N
N
N
N
N
C
C
C
C
C
tj
128.8 2
123.8
117.3
120.6
115.6
123.8
109,8
107.1
ij
2
2
'?I
3
2
2)
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
Tric linic
clofazimine
121.9
115.9
128.1
116.0
119.7
111.2
129.1
122.7
118.2
122.4
5
5
4
5
5
4
5
4
4
6
118.8 6
121.7
117.5
121.3
119.8
118.9
121.3
117.4
121.3
118.2
123.2
118.5
6
5
5
5
5
6
6
6
5
11a.0 5
123.a
118.2
121.5
114.1
122.4
5
5
5
5
5
CLOFAZIMINE
101
dl view o r t h c c l o f n z i t i i n c n o l c c c l c i n t h e iitorioclinic
C8
c7
Figure 3
View o f one molecule o f t h e monoc l i n i c f o m i or c l o f a z i m i n e . iitorilic thermal
ellipsoids are drawn a t t h e 2%: p r o b a b i l i t y
l e v e l ; atom X(3) i s shown as it sphere o f
a r b i t r a r y s i z e , and o.I;lier hydrogen atoms a r e
onittcd for clarity.
porpcnclicularity o f the 1 U-(E-c111orophenyl) and
dilydrophcnazinc rirq s y s t e n presumably s t c t l s from
unfavwrable i n t e r a c t i o n s between the hydrogen
atoms zt C ( 1 ) and C ( 9 ) arid t h o s e a t tlie pllenyl
o r t h o carbon a t o m s C ( 1 6 ) and C ( 2 0 ) and niny provide
s t r u c t u r a l basis f o r the observation33 that
c l o f a z i n i n e docs n o t i n t e r c a l a t e i n t o p o l y m c l c o t i d e s .
VIJAY K. KAPOOR
102
I n an c a r l i e r s t n r c t u r a l study34 c a r r i e d
o n clof'aziminc d i n e t i i y l f orcixiide t h e d a t a i s :
t r i c l i i i i c , sp5ce group rlT, w i 5 h a = 12.1135 4 ) ~ ,
11,
2 = lO.G24(4)A, o( = 1 1 1 . 7 4 3 ) 0 ,
arid ./= 9 0 . 9 0 ( 3 ) O ; d ( e x p t l ) = 1.29(2)
= 1.293 for Z = 2; it = O.ti82
f o r 2339
unique d a t a .
4.
Syntliesis
--
(27
CLOFAZIMINE
103
Cl
1
CI
Q
I
+
aN"
NHZ
Schcrne I
~~otozirninr
S y n t h e s i s o f Clofaziniirie
z2-
104
5.
VUAY K. KAPOOR
Stability
6.
6.1
Pharmacokinetics
1s
105
CLOFAZIMINE
of clol'azimine.
Six v o l u n t e e r s r e c e i v e d a s i n g l e
dose o f 200 m g t o g e t h e r w i t h food. A 200-1ng dose
was ad min iste re d i n three v o l u n t c e r s e i t h e r w i t h . or
without f o o d .
I n multiple-dosc experiments, t h r e e
v o l u n t e e r s were r e p e a t e d l y dosed w i t h 50 r n g p e r day
t o g e t h e r w it h f o o d f o r e i g h t days. Following a
s i n g l e o r a l dose of 200 mg, the mean peak plasma.
c o n c e n t r a t i o n o f clofaziraine was 861 pmol/g a f t e r
8 hours.
The mean t e r m i n a l h a l f - l i f e w a s 10.6 days.
Comparison o f the b i o a v a i l a b i l i t y o f clofazimine
ad min ister ed with or without food r e v e a l o d a 60.i;;
h i g h e r mean area under the curve ( N C ) and 3056 l u g h e r
m e a n m,wimtlm c o n c e n t r a t i o n ( C r n a x ) value w i t h fosd.
The medium of t i n e s t o peak (Tinax) was 8 hours with
I n m u l t i p l e dose
food and 12 hours without food.
sclidy, good agreement was found between t h e meat
experimental p l a s m a c o n c e n t r a t i o n v a l u e s and t h e
p l a s m a c o n c e n t r a t i o n p r o f i l e p r e d i c t e d from t h e
si n g le- d o se plmrmacokinotics.
The e l i m i n a t i o n h a l f l i f e c a l c u l a t e d from the te rmina l pkiase o f the
i n d i v i d u a l p r o f i l e s a f t e r the l a s t dose was 8.8 days.
The h a l f - l i f e obta ine d from t h e f i t t e d mean m u l t i p l e
dose p r o f i l e w a s 10.5 days. The s l o w e l i m i n a t i o n o f
clofaziiiine I n s i t s implications f o r the treatment
regimen In p a t i e n t s . To avoid t h e long lasting
accumulation toward t h e s te a dy state, h i g h e r d a i l y
l o ad in g doses a r e recoininended a t t h e beginning o f
t h e therapy rollowed by a d a i l y maintenance dose.
I n ;1 study done on a h e a l t h y vol u n t e e r plasma
clofazirnine l e v e l s f o l l o w i n p ; s i n g l e o r a l doses o f
200 t x g and 400 mg of the drug have bee determined
n s i n g d en sitome tric inethod (Figur e 4 ) . 34 ii peal<
clofazimin e c o n c e n t r a t i o n o f 70 ng/g w a s reached
e i g h t hours a f t e r a d r i i n i s t r a t i o n o f 200 mg o f
c l o f a z i n i n e , and one o f 162 n g / g f o u r hours a f t e r
the 400-mg dose. Pharrnacokinetics o f c l o f a z i m i n e
i n p a t i e n t s has a l s o been studied.55
106
\
0
C
VIJAY K . KAPOOR
200
150
100
H
Y
E,
50
0
Figure 4
P l a s m a l e v e l s o f c l o f a z i m i n c iii
a Iiealthy v o l u n t e e r following s i I q l e o r a l
doses o f 200 n~ (A- ) and 400 n i g (.----*)
of clofazimine af t o r on o v e r n i g h t f a s t .
Three l e p r o s y p a t i e n t s were staciiecl a t autopsy; a
slriii biopsy was s t u d i e d i n a fourth p a t i e n t .
Tissue
c o n c e n t r a t i o n s were analyzcci by a m o d i f i c a t i o n of
t h e c o l o r i a i e t r i c a s s a y of Barry ct s . 5 7 T i s s u e
c o n c e n t r a t i o n was observed i n t h e i n t e r n a l organs.
liiglies t c o n c e n t r a t i o n s of clofazimine were observed
i n t i s s u e w i t h h i g h f a t c o n t e n t and i n t h e b i l e .
T i s s u e s w i t h a r e t i c u l o e n d o t h e l i a l component o r
h i g h v a s c u l a r i t y also showed r e l a t i v e l y high
concerrtra t i o n s .
Iiowevcr, t h c r e l a t i v e l y h i g h
c o n c e n t r a t i o n of t h c drug i n t h e lcidney, which
could n o t depend upon t h e presence i n t h e organ
of a l a r g e component of r e t i c u l o e n d o t i i e l i a l t i s s u e
nor o f f a t s u g g e s t s t h a t t h e accucrulation of
clofazimine i n this organ i s r e l a t e d t o t h e E r i n a r y
excretion o f the drug. IUgli c o n c e n t r a t i o n o f t h e
drug i n b i l e and i n tlie gall bladder i s s u g g e s t i v e
o f t h e importance o f t h e b i l i a r y rollte o f e l i m i n a t i o n
of clofazimine f r o m tlie bod G1c A s k i d y c a r r i e d o u t
by Desilran and Ralalcrishnnn%; found c l o f a z i m i n e i n
a l l organs s t u d i e d b u t t h e b r a i n , i n d i c a t i n g that
i t d i d n o t c r o s s t h e blood-brain b a r r i e r .
6.2
i*tIet a b o l i s m
;.;etabollsrn o f clofaziLlirle i n l e p r o s y
107
CLOFAZIMINE
patients la been investigated by Feng and coworkers. 62*27 Based on mass, ultraviolet and
visible spectrometry, t h e metabolites from the
urine of %lie patients have been characterized as:
3-(~-liydroxyanilino)-lO-(~-chlorophenyl)-2,lOdihydro-2-isopropyliminophenazine (metabolite I),
Cl
CI
C l o t azi mine
CI
Q
CI
Metabolite I
Glucurcnidation
Hydro1y t ic
dehalogenotion
Metabolite I1
Figure 5
Proposed pathways of t h e
metabolite I and I1 formation in 1lu:nan.
3 - ( ~ - ~ - ~ ~ ~ c o p y r a n o s i d u r oacid)-lO-(pnic
chlorophenyl) -2,lO-dihydro-2-isopropylii~inopliemzine
(metabolite IX), and 3 - ( ~ - c h Z o r o a n i l i n o ) - 1 0 - ( p
chloropfienyl)-4,lO-dihydro-4 (D-D-glucopyranosiduroiiic acid)-2-isopropylir~iiiopllenazinc
( n c t a b o l i t e 111). It is suggested that t a e t a b o l i t e I
VIJAY K . KAPOOR
108
CI
CI
CI
Metabolite 111
FiL'Lzre 6
Proposcd patlivny o f tlic
n e t a b o l i t e III P o r i a t i o i i i n l u ~ . : ~ m i .
Tlierc a r c s e v e r a l r e p o r t s o c various
b i o c h e u i c a l e fP c c ts6 8 -8 1 o r c ! o f 3 7 i r i i ~ i e . ::ffect o f
c l o f a z i i . x h o o n tlic L1etabolisiii o f t h c o bher antilcprotlc drug depsone lias a l s o bceii rcported. i32
CLOFAZIMLNE
7.
109
8.
Toxicity
. have
~ c a3r r i e d o u t t o x i c i t y
Stenger et ~
s t u d i e s on clofaziinine. A f t e r a single oral dose
of clo fazimine t o mice, r a t s and gtiinea p i g s t h e
The
L D ~ oh a s been found t o be more than 4 rxg/kg.
r a b b i t i s somewhat inore s e n s i t i v c . l l a i l y o r a l doses
o r 50 o r 30 r:ig/l=, r e s p e c t i v e l y over a p e r i o d o f
s i x months a r e we ll t o l e r a t e d by rats and monkeys.
Reproduc tion t o x i c i t y e x p e r i n ~ e n t son mice, rats
and r a b b i t s have y i e l d e d no evidence f o r any
primary embryotoxic o r t e r a t o g e n i c a c t i o n o f
cl o fazimin e. I n a c u t e and c hroni c t o x i c i t y t e s t s
w i t h n i c e , r a t s and r a b b i t s clofazirnine qas found
t o be well t 0 l e r a t e d . 5 ~ A r e c e n t study8k has
shown t h a t clofazimine i s n o t mutagenic and i : 5 n o t
an in d u cer o f prophage
, and does n o t c l i n i n a t e
plasmids f r o m the appropriate ho st b a c t e r i a . C k O S S r e s i s tance between clofazimiiio, st r e p t o r i y c i n mcl
riaisii.cin could n o t be demonstrated.
VIJAY K. KAPOOR
110
9.
Methods of Analysis
9.1
Elemental Composition
i s a s foiiows85;
Per c e n t
Element
_
c
68.50
13
4.68
c1
14.98
11.83
3.2
I d e n t i f i c a t i o n C o l o r Tests
A n i n t e n s e v i o l e t c o l o r i s produced tvlien
ml of hydrocliloric a c i d i s added t o a s o l u t i o n
of 2 LIE o f clofazimine i n 3 1.11 of acetone. The
c o l o r c h a w e s t o orange-red o n a d d i t i o n o f 0.5 ml
of 52 sodiun hydroxide. 29 hp;>lication o f sn1rmA.c
a c i d d i r e c t l y t o the S E L J Al e o f clofaziz1iile a l s o
u n a d d i t i o n of a d r o p
produces a v i o l e t color.$7
of :;andelin's r e a g e n t , whic!l can b e prepared by
d i s s o l v i n g 0.5 g o f armonium vanadate ic 1.5 m l
o f water arid d i l u t i n g t o 100 1x1 w i t h s u l f a r i c a c i d
rollowed by f i l t r a t i o n tllrough g l a s s wool, c l o f a z i z i n e gives il yellow brown c o l 0 r . ~ 7 With Xarquis
reagent ( 1 volurae o f forrmldehyde s o l u t i o n and 9
volumes o f sn1f-ari.c a c i d ) c l o f a z i a i n e g i v e s a
v i o l e t c o l o r . 27
U.l
9.3
T i t r i i n e t r i c Analysis
9.4
Ltadiorne t r i c rinalysis
has been d e s c r i b e d
CLOFAZIMINE
111
--
-_I_
9.5
Spectrophotometric Analysis
9.51
Colorimetric
--
9.52
Densitometric
VIJAY K. KAPOOR
112
545 1:
15
-.
9.53
U 1traviole t
Ultraviolet spectrophotometry
coupled with high performance liquid chromatography
has been employed for deterruination of clofazimine
in serum.55990 The U V spectroph6tometry has also
been used in the characterization of the metabolites
of clofazimine.66,67
9.54
Fluorome tric
--
--
9.6
'9
9.7
Chromatographic Analysis
9.71
Paper Chromatography
9.72
Thin-Layer Chromatography
The following thin-layer chromatographic systems have been reconmended for the identification of clofaziminec
CLOFAZIMINE
113
-.
Plate
-- ----
--*
-_.
SiLicii ~ o G l,
(100: 1 . 5 )
dipped i n , o r
sprayed w i tit,
0. 124 KO11 i n
id^ t h a n o l
and
dried
250
0.70
P I I~~I ~ ~ C I C ,
Srride as above
0.57
Chlorof orm-frlethanol
( 9 0 : 10)
S u i e as above
0.59
Sthiyl a c e t a t e Benzene
0.33
(1o:go)
S i l i c a gel
tyiJe 1 p l a t e s
containing
0. l l i 1101!
(twb elutions)
1-Butnnol-BenzeneWater-ite t l u n o l
( 2 : 1 : 1 :1 . 2 5 )
Precoated TLC
p l a t e s , 250s i l i c a g e l &F
0.65
To luenc -tic e t i c
Yrecoated I?I?TC
S i l i c a g e l 60
p l a t e s (20 x
10 c u ) , LJiedeveloped in
chloroformmethanol ( 1 : l )
prior t o use
0.36
Cyclotiexane-TolueneL ) i e ttiy1ar.iine
(75: 15: 1 0 )
acid-water
( 5 0 : 50: 4 )
D e t e c t i o n of s p o t of c l o f a z i m i n e on t h e
has been a r r i e d o u t by a c i d i f i e d i o d o p l u t i n a t e
s o l u t i o n Z q or v i s u a l i z a t i o n by c o l o r and U V
a b s o r p t i o n . 66
9.73
Gas
---.-... Chromatograp&
.
-.
VIJWK. KAFQOR
114
9.74
10. References
1.
2. J . H . S .
Pettit and R . J . W .
2 , 391
(1966).
3. J.N.S.
23, 7 8 (1367).
28, 225 (1967).
22, 61 ( 1 9 6 8 ) .
A.G.
29
-9
CLOFAZIMINE
115
9 . L. Levy, C.C.
Med. Hyg.,
(1981).
499
. .
167 (1981).
13. W.
u u . Rev. R e s p i r a t . l ) i s e a s e s ,
e,764
Smith,
(1960).
G i l m o u r , Brit V e t . 3.
, 122, 517
16. W.A.
17.
W.A.
(1966).
1529 (1968)
m,
1979, P - 3 0 d *
Sancle, i i i G o o d m a n and
Gilrnans The Pharmacological B a s i s of
T h e r a p e u t i c s , Seventh Edn., A. Goodinan GiLman,
L.S. Goodman, T.W. R a l l and F. Murad, Eds.,
Kacmillan P u b l i s h i n g Go., New York, 1985,~.11213.
VIJM K. KAPOOR
116
26. D.M.
30. bhrtindale The Extra Pharmacopoeia, Twentyeighth Edn., J.E.F. Reynolds, Ed., The
Pharmaceutical P r e s s , London, 1 9 8 2 , p. 14&.
liychlewsla, FI.B.11.
and D.J. Hodgson, J.
4768 (1984).
Broom, D. S . Eggleston
Am.
Chen. S o c . ,
107,
-
- I
35.
V.C.
Boriy, J . G .
Belton, E.I.L.
Conalty and
CLOFAZIMINE
117
38
33.
4u.
41.
42.
43.
44
'45.
4('.
14-7.
48.
49
50
31.
-,
> 2 c .
33.
5't.
Z.
L-ayi
arid J . Y .
D u b o i s , J. Cilroe4:atogr.,
118
55
56.
57.
58.
Go.
61.
62.
63.
64.
65
66.
67.
68.
69.
70
VIJAY K. KAPOOR
CLOFAZIMINE
119
71
K. L a v r i j s e n , P. L a v r i j s e n and 12. L o n t i o ,
Biochem. Pharmacol., --2 6 , 1345 (1977).
72.
73.
74
75 *
2,
101,
c _
76
R.
77
A.J.
Van Rensburg and R. Anderson, Recent
ddv. Chemother., Proc. 1 4 t h I n t . C o n g r .
Chemother., 739 ( 1 9 8 5 ) , J. I s h i g a m i , Ed.,
U n i v e r s i t y of Tokyo P r e s s , Tokyo, J a p a n ;
C.A.,
1-05, 218377111(1986).
78
79
-,
80.
81.
82.
83
E.G.
84.
I
_
3324t ( 1388)
( 1988)
VUAY K. KAPOOR
120
85.
86.
B.G.
S.C.
87.
L.B. N e i f e t s , M.D. Iseliian and P.J. LindholmLevy, Drugs Exp. C l i n . Res., 12, 529 (1987);
C.A.,
88.
9,
34701 t
(1988).
2,
976 (1986)
89.
(1988).
90.
91.
Int.
ETOPOSIDE
Joost J.M. Holthuis, J. Jantina Kettenes-van den Bosich,
and Auke Bult
University of Utrecht
Department of Pharmaceutical Analysis
Utrecht, The Netherlands.
1. History
2. Description
2.1. Nomenclature, Formula, and Molecular Weight
2.2. Appearance, Odour, and Colour
3. Synthesis
4. Physical Properties
4.1. Ultraviolet Spectrum
4.2. Infrared Spectrum
4.3.
Fluorescence Emission Spectrum
4.4. Nuclear Magnetic Resonance Spectrum
4.5. Mass Spectrum
4.6. Melting Range
4.7. Differential Scanning Calorimetry
4.8. Optical Rotation
4.9. Dissociation Constant
4.10. Electrochemistry
5. Methods of Analysis
5.1. Thin Layer Chromatography
5.2. High Performance Liquid Chromatography
6. Stability and Degradation
6.1.
6.2.
7. Pharmacology
7.1. Mechanism of Action
7.2. Pharmacokinetics
7.3.
Clinical Activity
7.4. Clinical Toxicity
ANALYTICAL PROFILES OF DRUG SUBSTANCES
VOLUME 18
121
122
trices
8.1.
Etoposide
8.2. Etoposide Metabolites
9. References
123
ETOPOSIDE
1. HISTORY
Etoposide is a semi-synthetic derivative of epipodophlyllotoxin. It is used for the treatment of lung cancer, t.esticular cancer, lymphoma and several types of leukaemia, and
is one of the most active drugs against small cell lung cancer [l]. Etoposide was synthesized from podophyllotoxin in
1963, in the Sandoz Laboratories.
Podophyllotoxin is isolated from the dried roots and rhizomes of species of the genus Podophylein. The medicinal
properties of the ethanolic extracts of these roots and
rhizomes (podophyllin) have been known for more than 150
years.
Phodophyllin was used as a purgative, anthelmintic, chdoretic, and vesicant agent. Common sources of podophyllin are
the may apple or American mandrake (Podophylein pek%twn L.)
and P o d o p h y l h modi Wall. [ 2 1. Podophyllin contains
several podophyllotoxin derivatives among which podophyllotoxin proved to be the most active cytotoxic compound.
Several podophyllin components possess considerable
anti-tumour activity but are considered to be unaccepta.ble
for use in humans because of their severe side effects:
their toxicity prevents administration of doses high enough
to give sufficient therapeutic effect. In an attempt to find
compounds with an acceptable therapeutic index, a variety of
derivatives were synthesized from natural podophyllotoxin
[3-51. Etoposide proved to be one of the most promising cornpounds.
2. DESCRIPTION
2.1.
3.
124
H
OH
3 . SYNTHESIS OF ETOPOSIDE
The synthesis of etoposide from naturally occurring podophyllotoxin I (Scheme 1) is described in ref. C3-51. Podophyllotoxin is treated with HBr in 1,2-dichloroethane, resulting in 1 bromo-1-desoxyepipodophyllotoxin, which demethylates to l-bromo-4-demethylepipodophyllotoxin (11) when
the reaction mixture is kept at OC for about 24 hours. By
treatment of I1 with BaCO in an acetone/water mixture, the
3
bromine is replaced by a hydroxyl group, resulting in 4demethylepipodophyllotoxin (111) After protection of the
phenolic hydroxyl with benzyl chloroformate, the 4-OH group
is coupled with 2,3,4,6-tetra-O-acetyl-~-D-glucopyranose.
The sugar moiety probably enters from the less hindered
side, because glycosidation of podophyllotoxin itself also
results in an epi product [41.
The protecting group at the 4-OH is removed by
hydrogenolysis with H /Pd and the acyl groups by hydrolysis
2
with Zn(OACI2 in methanol. During the hydrolysis, about 30%
of the compound is converted into a mixture of the hydroxy
acid (by opening of the lactone ring) and the
lactone.
These products are easily removed by crystallization.
The last step in the synthesis is the reaction with
"
.:0
o , =
0
f
Q)
a
126
acetaldehyde
dimethyl acetal
in nitromethane, with
acid as a catalyst. Of the 0-4,6 cyclic
acetals, the isomer with the equatorial methyl group
predominates. Minor quantities of the axial isomer are
eliminated in the purification procedure [5].
-p-toluenesulfonic
4. PHYSICAL PROPERTIES
1Cm
-1
-1
4.2.
= 4245 l.mol
at 283 nm is 72.2
Infrared Spectrum
were used.
127
ETOPOSIDE
2bO
nm
300
350
4000
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
cm-'
800
650
128
300
350
400 nm
129
ETOPOSIDE
c H 3 Y 0 OH
OH
CHaO
g'
OCHI
3'\4'
OH
nl
I
I
5.00
4.80
4.60
4.40
4.20
4.00
3.80
3.60
3.40
3.20
3.00
I
i2.80
Proton NMR
etoposide.
spectrum
(2.70-5.10
p.p.m.)
Of
130
Table I. H
'
form.
NMR
chemical shift
6 (p.p.m.1
1.38
2.88
3.32
3.38
3.58
3.67
3.72
3.75
4.05
4.18
4.21
4.45
4.55
4.57
4.75
4.94
5.68
5.98
6.25
6.52
number of
protons
d
m
3
1
m
dd
t
s (br)
S
s (br)
dd
t
dd
d
assignment
(protons at
carbon number)
98
3
941 95
21 92
g6a
93
2
1
1
1
6
1
93-0h
OCH
g2-&
96e
11'
11
1
1
1
1
1
1
1
dld (AB)
2' ,6'
91
1
97
4
4'-0h
A
~~
g1192
92I93
93I 9 4
94I95
95I g6a
95 g6e
g6alg6e
97 I98
J(Hz)
4.9
14.0
3.4
10.5
8.5
8.5
-1
7.9
7.9
7.9
9.6
4.3
9.6
5.0
131
ETOPOSIDE
Table 111.
chemical shift
6 (p.p.m.)
19.9
37.2
40.6
43.2
55.9
65.8
67.4
67.8
72.1
72.9
74.3
79.4
99.1
100* 8
101.0
107.6
109.1
110.1
127.5
130.1
132.7
133.8
146.2
146.4
148.1
174.8
assignment
(carbon number)98
1
3
0ch3
95
4, 96
11
93
92
94
gl
97, A
2', 6'
8
5
4'
1'
8''
4' I
31, 5'
6, 7
13
132
111
180
160
140
120
100
80
60
40
20
ETOPOSIDE
133
+.
OH
in, e 4011 ( a 1
m/e 382 ( b )
m e 154 ( c )
H,CO
i w e 246 ( d )
OCH,
OH
m,e 400 ( a )
134
100-
50-
We
Optical Rotation
135
ETOPOSIDE
160
180
200
220
240
OC
Figure 7.
240
249
258
270
OC
136
-15 -
-10
PA
-5 -
0-
+5 -
+I0
+I5
+0.8
4.6
4.2
4.4
I
-0.2
ETOPOSIDE
137
+ 2e + H
H3C0
OCH,
H3CO
OCH,
OH
R
H&O
OCH,
HSCO
OCH,
OH
Q,
OCHj
H3C0
OCH,
+e
H3C0
0.
R
H$O
4
0.
OCH,
+ CH30H
0
H,CO
H3CO
OH
OH
138
aqueous solutions shows an overall transfer of two electrons. At pH values below 2.5, the oxidation proceeds in one
voltammetric, pH-independent oxidation step (1, Figure 10)
At pH values above 2.5, the oxidation proceeds in two voltammetric oxidation steps. The transfer of the first electron ( 3 , Figure 10) is reversible and is preceded by a proton transfer (2, Figure 10). The transfer of the second
electron (4) results in the formation of an unstable cation
which is converted rapidly into the 2-quinone of etoposide
(5). The 2-quinone is adsorbed at the electrode surface, and
is reduced in the kathodic scan (i ) to the corresponding
111
hydroquinone. The hydroquinone is oxidized in the second
anodic scan (i ) . Both the oxidation of the hydroquinone
111
and the reduction of the o-quinone are pH-dependent.
5. METHODS OF ANALYSIS
solvent (v/v)
compound
Rf
0.57
etoposide
Cid-etoposide
0.49
Cib-hydroxy acid
of etoposide
0.03
reference
11
silicagel chloroformetoposide
methanol (21:l)
12
butanol-glacial etoposide
acetic acidwater (3:l:l)
12
etoposide
12
cellulose
'I
II
ETOPOSIDE
139
mobile phase
detection
5um
(150X4.6 mm i.d.1
acetonitrileacetic acid
-water (27:1:72)
UV,230,254
and 286 nm
14
VBondapak
Phenyl 10 pm
(300~4.6mm i.d.)
0.02 M sodium
W1200-400
acetate buffer
pH 4-acetonitrile
(74:26 V/V)
13
pBondapak
Phenyl 10 pm
(300~4.6mm i.d.)
0.02 M sodium
W1200-400 nm
acetate buffer
p H 4-acetonitrile
(40:60 v/v)
13
pBondapak
Phenyl 10 pm
(300~4.6mm i.d.1
methanol-water
UV1254 and
(50:50 w/w) con280 nm
taining 0.5% (v/w)
0.5 M sodium phosphate buffer pH
6.5 and 0.5% (w/v)
tetrabutylammonium bromide solution (20% w/v)
10
FQ-81
reference
140
I
OH
Figure 11.
OH
111
I"
ETOPOSIDE
141
7. PHARMACOLOGY
7.1. Mechanism of Action
Etoposi.de differs in its biological action from its parent podophyllotoxin, which is a spindle poison.
Etoposide does not interact with the microtubule assembly
[15,161, but prevents cells from entering mitosis. In contrast, the precursor podophyllotoxin arrests cells in the
metaphase. Etoposide arrests cells in the late S or G2 phase
of the cell cycle and the cells accumulate in the G2 phase.
Cells treated with etoposide show a rapid decrease of the
mitotic index, with a simultaneous reduction of cell proliferation.
142
143
ETOPOSIDE
Figure 12.
The major metabolite sofar was found to be the glucuronide of etoposide [28,35,36]
(Pathway C) , the glucuronic acid
being attached to the phenol group at C-4' [35]. Glucuronides of etoposide were also isolated from bile of patients
[ 2 8 ] and from rat bile [38].
Sulphate conjugates of etoposide (pathway C) could not be detected in patients' urine
C28 1.
in
144
7 . 3 . Clinical Activity
8 . ANALYSIS OF
FLUIDS
8.1. Analysis of Etoposide
'3
ETOPOSIDE
145
146
Table VI. Published HPLC methods f o r the analysis o f etoposide i n b i o l o gical fluids.
matrix
flow
sample pretreatment
column
detection
plasma
Chloroform
extraction
Chloroform,
pre-extraction
d i isopropyl ether
Chloroform
extraction
Bondapak
C18, 10 m
Lichrosorb
RP 8, 5 m
UV
Bondapak
C18, 10 m
Fluorescence 215/
328 nm
plasma
plasma
plasma
ur ine
plasma
CSF
plasma
urine
plasma
urine
plasma
serum
plasma
plasma
plasma
determination
limit
500 ng/ml
reference
500 ng/ml
54
50 ng/ml
53
Chloroform extracti on
Ethyl acetate ext r a c t i o n a f t e r addit i o n o f (NH ) SO
4 2 4
Preconcentration on
PRP.l, post-column
e x t r a c t i o n w i t h 1,2dichloroethane
Chloroform extraction, wash step
w i t h buffer
1,2-dichloroethane
extraction
Solid-phase extract i o n C-18 c a r t r i d g e
Chloroform extraction
P a r t is i 1
uv
100 ng/ml
OD5
252 nm
Bondapak
ECW
20 ng/ml
Phenyl, 10 m + 0.85 V
vs Ag/AgCl
Lichrosorb
F1uores8 ng/ml
R? 18, 10 m cence 230
30 ng/ml
328 nm
55
1,2-dichloroethane
extraction
1,Z-di chl oroethane
extraction
Bondapak
Phenyl, 10
Radial -PAK
C18, 10 m
Chloroform extraction
Solid-phase extract i o n C-18 Bond E l u t
ODS Hypersil
UV
5 m
ODS Hypersil
229 nm
ECD
N.9
Lichrosorb
RP 18, 10 m
Bondapak
Phenyl 10
Bondapak
Phenyl, 10
Bondapak
CN, 10 m
11
254 nm
UV
254 nm
UV
280
MI
30 n g h l
50 ng/ml
ECD 0.50 V
2 ng/ml
m vs Ag/AgCl
UV
400 ng/ml
m 230 nm
10 ng/ml
ECO
+0.50 V vs
Ag/AgCl
ECD
31
50
57
6,56
58
59
5 nglml
60
unknown
51
500 nglml
52
500 ng/ml
61
m M.80 V
Mass spectrometry,
uv
plasma
urine
plasma
ETOPOSIDE
147
ACKNOWLEDGEMENT
The authors are gratefull to Mr. D. Seykens, Department
of Organic Chemistry, University of Utrecht, The Nethffleinds
for running the NMR spectra and providing relevant
C reference data.
148
REFERENCES
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(B.F. I s s e l , F.M. Muggia, and S.K. Carter, e d s ) . p. 1.
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ETOPOSIDE
149
150
38 Colombo, T.,
D'Incalci,
M.,
F a r i n a , P.,
R o s s i , C.,
G u a i t a n i , A., B a r t o s e k , I., B e n f e n a t i , E., F a n e l l i , R.,
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(1985). J. Chromatogr. 339, 419.
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J a r d i n e , I., a n d Colvin, M.
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Chromatogr. 222, 141.
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(1980). J. Pharm. S c i . 2, 1440.
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P i n e d o , H.M.,
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Van O o r t , W.J.
(1983). J. Pharm. B i o m e d . Anal. L, 89.
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11,
J.
E,
ETOPOSIDE
151
BY
154
Contents
1.
Description
Nomenclature
1 . 1 . 1 Chemical Names
1 . 1 . 2 Generic Names
1 . 2 Formulae
1 . 2 . 1 Empirical
1 . 2 . 2 Structural
1 . 2 . 3 CAS Registry Number
1 . 3 Molecular Weight
1 . 4 Elemental Composition
1 . 5 Appearance, Color, Odor and Taste
1.1
2.
Physical Properties
2.1
2.2
2.3
2.4
2.5
2.6
3.
Synthesis
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
3.10
3.11
4.
Melting Range
Solubility
pH
Stability
Crystal Structure
Spectral Properties
2 . 6 . 1 Ultraviolet Spectrum
2 . 6 . 2 Infrared Spectrum
2 . 6 . 3 Nuclear Magnetic Resonance Spectra
2 . 6 . 3 . 1 Proton Spectrum
2 . 6 . 3 . 2 13C NMR Spectra
2 . 6 . 4 Mass Spectrum
Route
Route
Route
Route
Route
Route
Route
Route
Route
Route
Route
1
2
3
4
5
6
7
8
9
10
11
FUROSEMIDE
5.
Methods of Analysis
5.1 Elemental Analysis
5.2 Identification Tests
5.3 Titrimetric Methods
5 . 4 Spectrophotometric Methods
5.4.1 Ultraviolet
5.4.2
Colorimetric Methods
5.4.3 Nuclear Magnetic Resonance
5.7 Chromatographic Methods
5.5.1 Column Chromatography
5.5.2 Gas Liquid Chromatography
5 . 5 . 3 Thin-Layer Chromatography
5.5.4 High Performance Liquid Chromatography
6.
Acknowledgements
7,
References
155
156
Furosemide
1.
Description
1.1
Nomenclature
1.1.1
Chemical Names
a)
5-(Aminosulfonyl)-4-chloro-2-[(2furanyl-methy1)amino)benzoic acid. ( 1 )
b)
4-Chloro-N-furfuryl-5-sulfamoylanthranilic acid. ( 2 ) .
c)
4-Cliloro-N-(2-Furylmethyl-5sulfamoylanthranilic acid. (3).
d)
4-Chloro-2-furfurylamino-5sulphamoyl benzoic acid. (4).
1.1.2
Generic Names
Frusemide,
Fursemide,
Aisemide ,
Beronald, Desdemin, Diural, Dryptal, Errolon, Frusemin,
Fulsix, Fuluromide,
Furosemide,
Mita,
Furosedon,
Katlex, Lasilix, Lasis, Lowpstron, Macasirool, Nicorol,
Profemin, Rosemide, Transit, Trofurit, Urosemide, Urex.
1.2
Formulae
Ciz H I 1 C l N 2 0 5 S
1.2.2
Structural
COOH
NH,SO, @NH-cH20
CI
157
FWROSEMIDE
GAS Registry
1.2.3
[54-31-91
1.3
Molecular Weight
CizHiiClNz05S = 3 3 0 . 7 7
4 3 . 5 7 % H , 3 . 3 5 % , C1 1 0 . 7 2 % N , 8 . 4 7 % , 0 , 24.19%,
S, 9.70%.
1.5
2.
Physical Properties
2.1
Melting Range
M.p.
2.2
206oC
Solubility
@ (4)
Stablity
158
manufacture.
Fursomide oral solution should be stored
at 15-30C and protected from light and freezing; once
opened unused portion should be discarded after 60
days.
Furosemide injections can usually be mixed with weakly
alkaline and neutral solutions having pH of 7-10, such
as 0 . 9 % sodium chloride injection or Ringer's injection
and some weakly acidic solutions having a low buffer
capacity.
The injection should not be mixed with
strongly acidic solutions (i.e. pH less than 5 . 5 ) such
as those containing ascorbic acid, tetracycline,
epinephrine, norepinephrine, because furosemide may be
precipitated. Other drugs which should not be mixed
with
furosemide injections include most salts of
organic bases including local anesthetics, alkaloids,
antihistamines, hypnotics, rneperidine, and morphine
(5).
2.5
Crystal Structure ( 6 )
159
FUROSEMIDE
F i g . 1:
160
b-c( A )
(abc
Position of c
Intramolecular
N(Z)-H,, ,,0(2)
2,11
2-3
101
x,y,e
Intramolecular
N(l)-H(l),, . 0 ( 3 )
3,OO
1.9
163
N(l)-H(2).o, 0 ( 3 )
2-91
2,3
114
x,y,z
c ( 1 I -c( 7 i
C(5)-s
S-N( 1)
N(2)-C(a)
C( 9)-C(10)
C(12)-0(5)
c(lo')-c(ll)
1.50(2)
l.18(1)
1.6211)
i041(2)
1&44(4)
1 35 (4)
1,61(5)
N( 1)-S-C(5)
O ( 3 ) -S-N(1)
O( 4)-S-( N( 1)
S-C( 5 1 4 6 )
Cl-C(4)-C(5)
C( ?)-C(1)-C(6)
C( l)-C(Z)-N(Z)
O ( 2)-C(1)-C(1 )
O(l)-C(T)-0(2)
C( 8 1 -C( 9)-O( 5 )
O( 5 ) -C( 9)-C( 10)
c~lo)-c(l~)-c(l2)
C(Z)-N(Z)
S-0(3)
C{?]-O(l)
c(a)-c(!)
C( lOl-C( 11)
C( 9 ) - O ( 5 ' )
C(Il)-C(lZ')
106,9(6)
105.8(1
108.6(7
ii~,a(3
121.8(3
116,3(9
l21,6(6
121(2
125( 1
124(2
94(2
113(3
1,40(1)
1.45(1)
1,29(2)
1,44(2)
1,25(4)
1.39 ( 3 )
1.31(4)
C(4)-Cl
S-0(41
c(?j-O(Z)
C( 9 ) - O ( 5 J
C( 11)-C(12)
C(9 )-C(10')
C(12')-0(5')
0(3)-s-0(4)
O( S)-S-C(5)
0[4) -S-C(5 j
S-C(5)-C(4)
c1-C(4)-C(3 )
C(?)-C(l)-C(Z)
C( 3)-C( 2)-N(2)
O(I)-C(7)-C(l)
N( 2)-C(8)-C( 9)
c( a )-c(9 1 -c( lo
C(9)-C( 10)-C(11)
C ( 11) -C( 12 ) -0I 5 1
1#?1(1J
1,40(1)
1,24{Z)
1,46(3)
1 . 2 6 ( 41
1.33( 5 )
1.40(5)
118,9(6)
108,0(6)
lO?.9( 6)
122,1(3)
118.1(3)
123 1 ( 9)
118.4(6)
114(2)
114(l)
129(2)
114(2)
I
106(3)
161
FUROSEMIDE
2.6
Spectral Properties
2.6.1
Ultraviolet Spectrum ( 7 )
max
Ethanol
nm
El%, 1 cm
288
Y45
256
336
226
273
336
388
144
1147
55i
133
( 95%)
0.1 N NaOH
2.6.2
Infrared Spectrum ( 7 a )
The
infrared absorption spectrum of
furosemide as hBr-disc was recorded on a Perkin Elmer
580 B Infrared Spectrophotometer ( F i g . 3 ) .
The
spectral assignments are listed in Table (4).
Table 4
Frequency cm-1
3350-3400
1671
Type of
vibration
NH
C-0
1596
NH
1322
-s=o
582
Assignment
Ci
C-NH
- COOH group
- NH2 group
-
SOz
group
c -
Cl
.
2 00
Fig. 2:
300
UV spectrum of furosemide in H 2 0 .
400
nm
163
I
n
0
0
r(
In
0
0
0
0
0
hl
0
M
0
0
0
0
164
2.6.3
N u c l e a r Magnetic e e s o n a n c e S p e c t r a
2.6.3.1
P r o t o n Spectrum ( 7 b )
The PMR s p e c t r u m of f u r o s e m i d e i n
DMSO-d6 ( F i g . 4 ) was r e c o r d e d on a Varian (FT 80 A ) .
NMR s p e c t r o p h o t o m e t e r u s i n g TMS as i n t e r n a l s t a n d a r d .
The PMR s p e c t r u m of f u r o s e m i d e is u n i q u e i n a way t h a t
a l l peaks a p p e a r as s h a r p s i n g l e t s .
Chemical s h i f t s
are shown i n T a b l e 5 .
Tabl e ( 5 ) .
PMR c h a r a c t e r i s t i c of Furosemide.
Group
Chemical S h i f t ( 6 ) PPm
4.5
-CH2
H3
6.41
and H4 o f t h e
furan ring.
H2
of t h e furan r i n g
7.24
H3
o f phenyl group
7.32
H6
of phenyl group
8.42
2.6.3.2
13C
NMR S p e c t r a ( 7 c )
Mass Spectrum ( 7 d )
.d
6 9s
9L-
166
..
v)
M
L
*-I
.
I
0
-0
' N
co
0
:.
-. t+
0
d
t+
:o
-N
:+-I
' 0
.LO
LO
Tr
167
1 - 3
.o
LO
. M
L O
:co
:o
-0
- M
. M
:o
y N
0
7 0
' M
:o
-co
: N
- 0
\o
N
.-
-N
-. 0
a,
a
a,
.rl
ln
rcc
rc:
5
k
e,
+J
!?
H
..
\o
M
.rl
LL
168
m/e
Relative intensity
%
251
20
96
14
81
100
64
38
53
22
48
15
--NHCH2$07
- so
169
FUROSEMIDE
3.
Synthesis
Furosemide can be s y n t h e s i z e d by d i f f e r e n t r o u t e s :
Route 1
Furfurylamine
5-sulfamoylbenzoate e s t e r
0
1
NH2CH2
heated for
1 h r a t 120'
1%hour a t
l0O0C
DMF
COOCH,
NaOH
COOH
Furosemide
170
Route 2
COOH
2,4-Dichloro-5-sulfamoyl
benzoic acid
10% HC1
Furfurylamine
COOH
Furosemide
Route 3
CI
mc'c,d
NH2S02
COOH
+
I
Furosemide
CHNH,
2
Furfuryl amin e
Route 4 (11)
Acid h y d r o l y s i s of c h l o r o t h i a z i d e and f u r f u r y l a l c o h o l .
172
Route 5 ( 1 2 )
2,4-Dichloro-5-sulfamoylbenzoic acid was treated with
N-furfurylaniline
and Renay Cu
in MeO(CHz )zOMe
(dimethoxy ethane) at 1 4 0 " .
The product dissolved in
dichloroethane, extracted with N NaOH, and precipitated
with AcOH-HC1. The precipitate in 10% aq. HC1 treated
with
NaNOz at 0 " and refluxed in NaOH to give
furosemide.
COOH
2,4-Dichloro-5-sulfarnoyl-
benzoic acid
N- Fur fury1a n i -
1ine
in Me0 (CH2) *0Me
Raney
cui
a t 14OoC
173
FUROSEMIDE
Route 6 (13)
Furosemide is prepared by saponification of corresponding nitriles with aq. NaOH at 1 5 " for 4 hours.
Route
7 (14)
Furosemide is obtained by treating 2 amino, 4 chloro-5sulfamoyl benzoate ester with furfuryl alcohol.
CH2OH
COOMe
2-Amino-4-chloro-5-sulfamoyl
benzoate ester.
Furfuryl alcohol
Refluxing f o r
2 hrs.
COOH
Furosemide
ABDULRAHMAN M. AL-OBAID E T A L
174
Route 8 (15)
The furosemide is prepared by alkaline saponification
of corresponding nitrile which is obtained by the
treatment of 3-sulfamoyl-4-chloro-6-chloro cyanobenzene, with furfurylamine.
Furfuryl amine
3-Sulfamoyl-4-chloro-6cyanoben z ene
2 hrs
-c
C'aNHC
NH2S02
COOH
Furosemide
175
FUROSEMIDE
Route 9 (16)
When a mixture of 3-sulfamoyl-4-chloro-6-flourobenzoic
acid and furfurylamine is heated f o r 3 hrs at 95C.
COOH
3-Sulfamoyl-4-chloro-6flourobenzoic acid
Furfuryl amine
c'mNHcHz-
NH2SO2
COOH
Furosemide
ABDULRAHMAN M . AL-OBAID E T U .
176
Route 10 ( 1 7 )
When 3-sulfamoyl-4,6-dichlorobenzo te ester is added
with stirring to furfurylamine at room temperature.
The mixture is heated at 110C with stirring f o r one
hour.
The obtained mixture is treated with CH3COOH to
give a crude reaction product. It is then treated with
5 N KOH and stirred for 1 hour at 55C.
F u r f u r y l amine
3-Sulfamyl-4,6-dichloro
benzoate e s t e r
100C s t i r r i n g f o r 1 hour
COOCH,
110-200c
COOH
Furosemide
177
FUROSEMIDE
Route 11
Furosemide is
bath
3-Sulfamoyl-4-chloro-6-amino
benzoic acid.
COOH
3-acetylsulfamoylC=O 4-chloro-6-acetyl
I
amino benzoic acid
CH3
stirring at
75OC f o r 3
hr .
scheme (18).
4NaOH
Fur f u r a 1
CH,
4-chloro-6-aminobenzoic acid.
COOH
3-acetylsulfamyl-4-chloro-6-furfuryl amino
benzoic acid
NH
COCH,
in 2N NaOH
NH,S 0 ,
reflux f o r 2 hrs
COOH
Furosemide
ABDULRAHMAN M.AL-OBAID E T A L
178
4.
FUROSEMIDE
179
therapy
is begun.
After I V administration of
furosemide, diuresis occurs wihtin 5 minutes, reaches a
maximum
wihtin
20-60 minutes,
and persists for
approximately 2 hours.
After I M administration, peak
plasma concentrations are attained within 30 minutes;
onset of diuresis occurs somewhat later than after I V
administration.
In patients with severely impaired
renal function, the diuretic response may be prolonged.
Only limited
information
is available on
the
distribution of furosemide.
The drug crosses the
placenta and is distributed into milk.
Furosemide is approximately 95% bound
to
proteins in both normal and azotemic patients.
plasma
ABDULRAHMAN M. AL-OBAID E T A L
180
5.
Methods of Analysis
5.1
Elemental Analysis
CizHiiClNz05S = 330.77
Element
C
43.57%
3.35%
c1
5.2
% Theoretical
10.72%
8.47%
24.19%
9.70%
Identification Tests
181
FWROSEMIDE
of barium
To
5.3
Titrimetric Method
(i)
Dissolve 0 . 5 g in 40 ml of dimethylformamide
and titrate with 0.1 N sodium hydroxide, using
bromothymol blue solution as indicator. Repeat the
operation without the frusemide, the difference between
the titrations represents the
sodium hydroxide
required. Each ml of sodium hydroxide is equivalent to
0.03308 g of CizHiiClNz05S ( 2 ) .
(ii)
Dissolve about
600 mg of furesemide,
accurately weighed in 50 ml of dimethylformamide to
which has been added 3 drops of bromothylmol blue and
which previously has been neutralized with 0.1 N sodium
hydroxide.
Titrate with 0.1 N sodium hydroxide to a
blue end point.
Each ml of 0.1 N sodium hydroxide is
equivalent to 33.07 mg CizHiiClNzOsS ( 3 ) .
(iii)
The titrimetric method for microdetermination of furosemide in pharmaceutical preparations
involves titration of a solution of 1 to 5 mg of drug
in 1 1 . 5 M HC1 with 0.02 M bromosuccinimide, with
methyl red as indication. For the cited range, the
1%(four determinations
coefficient of variation is <
at each level), and excipients do not interfere ( 1 9 ) .
_+
182
5.4
SDectrophotometric Methods
5.4.1
Ultraviolet
Colorimetric Method
FUROSEMIDE
183
Chromatographic Methods
5.5.1
Column Chromatography ( 2 6 )
(1 ml)
ABDULRAHMAN M. AL-OBAID ET AL
184
Acknowledgements
The authors
Department
Pharmacy,
assistance
manuscript
Table 7 :
Stationary phase
Mobile phase
Flow rate
Detection
Remarks
Bondapak Cia
corasi1
(1.8 X 2 mm)
0.02 M KC1-HCl
buffer pH 2 containing 24% and
26% acetonitrile
for urine and
plasma samples
respectively.
0.9 ml/min
UV at 280
p Bondapak Cia
(10 Pm)
Methanol-Hz0acetic acid ( 3 4 :
2 ml/min
Fluorimetrically
with the use of
Corning No. 7-60
primary filter
and Kodak No. 2 A
secondary filter
2 ml/min
U.V.
45:3)
p Bondapak Cia
reversed phase
50% methanol in
containing 0.5%
acetic acid
340 nm
Ref.
29
30
Levels of
frusemide down
to 0 . 1 pg/ml
can be
determined
33
Continued (Table 7)
Stationary phase
Mobile phase
l~ Bondapack C i s
Detection
Remarks
Fluorescent
detector
with the
excitation
wavelength
225 nm.
Method needs a
little quantity
of blood sample
i.e (200-300 111)
Lichrosorb RP8
(5 P I
Methylcyanidesod. acetate
mixture
U.V.
detection
at 275 nm
The limit of
detection is
10 ng/ml.
33
Varian CH-10
reversed phase
co1umn
0.5% CH3COOH in
CH~CN-HZO
(30:70)
Fluorescence
detector
N-benzyl-4chloro-5-sulamoylanthranilic
acid as internal
standard.
34
p Bondapack C i a
column
( 3 0 cn X 4 m m )
10 mM (NH4)HP04
in 25% methanol
U . V . 254 nm
Operated at room
temperature
35
reversed phase
Flow rate
2 ml/min
Ref.
32
Continued (Table 7 )
Stationary phase
p
Bondapack C i a
p Bondapack Cis
Mobile phase
Flow rate
Detection
Remarks
Ref.
Methanol-0.01 M
Na acetate ( 7 : 1 3 )
pH 5.0
2 ml/min
U . V . at 280 nm
Cephalothin sod.
is used as
internal standard
36
Acetonitrile0.01 M Na
acetate (1:3)
pH 5 . 0
2 ml/min
U.V.
Phenobarbitone
sod. is used as
as internal
standard
36
at 280 nm
4
W
Acetonitrile-
Fluorimetry at
(3:5)
Lichrosorb RP-8
( 5 pm)
Methanol-0.02 M
phosphate buffer
of pH 3.0
(1:l)
37
289 nm
0.5% H3P04
1 ml/min
Flourimetric
detection at
410 nm with
excitation at
275 nm.
All procedures
are carried out
under subdued
light
38
Continued (Table 7)
Stationary phase
Mobile phase
p Bondapack Ci8
Acetonitrile0 . 0 8 M H3P04
( 3 0 cm X 4 . 6 mm)
Flow rate
(3:5)
00
Radial Pak p
Bondapack Cis
reversed phase
column
Stainless steel
column packed
with a pellicular cation
exchange
resin
(305 cm X 1 ml)
50 mM phosphate
buffer pH 2 . 5
2.5 ml/min
0 . 3 3 ml/min
Detection
Remarks
Ref.
Fluorimetric
detection at
389 nm with
excitation at
2 3 3 nm
39
Fluorescence
detection with
exciation and
emission wavelengths of 3 3 0
and 415 nm.
40
Fluorescence
detection
Maintain the
column temperature at 7 4 C
41
Continued (Table 7 )
Stationary phase
Mobile phase
Flow rate
Detection
Zorbax ODS
0. 0 1 M-NaHz PO4
(pH 3 . 5 ) methanol ( 1 3 : 7 )
3 ml/min
Fluorimetric
detection at
389 nm (excitation at
235 nm)
42
2 ml/min
U.V. at 254 nm
43
( 5 w)
( 1 5 cm X 4 . 6 nm)
03
Remarks
Ref.
methanol ( 3 : 2 )
for urine
Bondapack Cis
( 3 0 cm X 4 mm)
M acetate
buffer (pH 5 . 3 )
containing 22%
of acetonitrile
Column packed
with ODS ( 5 pm)
Methanol-Hz0acetic acid
equipped with a
guard column of
Cis corasil
( 2 cm X 4 cm)
0.02
( 40:
57: 3 )
1.45 ml/min
Fluorescence
detection was
at 460 to 600
nm (excitation
at 330 to
400 nm)
Furosemide was
stable in
frozen plasma
and urine for
113 b 204 d a y s
respectively
44
ABDULRAHMAN M. AL-OBAID E T A L .
190
7.
References
1.
"The Merck Index", 9th ed. Merck and Co., Inc., Rahway,
N.J., U.S.A. 1976.
2.
3.
Soo.
191
FUROSEMIDE
Arch.
15.
16.
17.
20.
E.F.
21.
22.
u,
57(4),
Anal.
Vaughan, J. Pharm.
23. Dessouky,k Y.M. and Gad el Rub C.N. Pharm. kleekb) Sci,
Ed. 1980, 2 (4), 1128-1130.
24.
Sci.,=(1-4),
m t , J. Pharm.
192
26.
Lagerstrom Per-Olof.
Apple, 1 4 ( 2 ) 476-481,
27.
Lindstroem, B.
219-221 ( 1 9 7 4 ) .
28.
Heinrich,
E.
anbd
Geissler,
Monika;
Schaefer,
M u t s c h l e r , E r n s t . J . Chromatogr. 1 4 3 ( 6 ) , Biomed, Apple
1 ( 6 ) , 636-639 ( 1 9 7 7 ) .
Lindstroem, B. J . Chromat.,
189-191 ( 1 9 7 4
29.
J . Chromatogr.
1981.
and Molander,
M.
225(2);
Biomed.
J . Chromat. 1 0 1 ( 1 ) ,
m(l),
30.
31.
Roseboom,
75 *
32.
N a t i o n , Roger L . ; Peng, G e o f f r e y W . ,
J . Chromat., = ( 1 ) , 88-93 ( 1 9 7 9 ) .
33,
B r o y u a i r e , M , , M i t c h a r d , M.
(1979).
34 *
Meffin, P e t e r ,
J.,
Blaschke,
Swezey, S a r a h , E . ;
T e r r e n c e F . , J . Chromatogr., 1 7 4 ( 2 ) , 469-73 ( 1 9 7 9 ) .
35.
Ghanekar, A.G.;
Das Gupta, V . and Gibbs, C h a r l e s W . ,
j u n . J . Pharm. S c i . , 6 7 ( 6 ) , 808-811 ( 1 9 7 8 ) .
36,
37,
38.
Kerremans, A . L . M . ;
Tan, Y . ;
Van Ginneken, C.A.M.
and
Gribnav, F.W.J.
J . C h r o m a t o m . , 2 2 9 ( 1 ) , Biomed. A ~ p l . ,
1 8 ( 1 ) , 129-139 ( 1 9 8 2 ) .
39.
H . , S o r e l , R.H.A.
C.
Anal. L e t t . , B 1 1 ( 6 ) , 469Chiou,
Feuill. Biol.,
Wing,
L.
106, 99-103
FUROSEMIDE
193
40.
Kubo,
Hiroaki,
Li,
Haozhi,
Kobayashi,
Yoshie;
Kinoshita, Joshio.
Bunseki Kagaku, 3 5 ( 3 ) , 259-62
(1986).
41.
B l a i r , Andrew D . , F o r r e y , Arden, W . ; M e i j s e n , B. T e r i ;
and C u t l e r , Ralph E . J . Pharm. S c i . , 6 4 ( 8 1 , 1334-1339
(1975).
42.
43.
44
JI
A1
Dh. Muhammad U p p d Z u b h
&Ad&%&
Ce&u
PhVdUiioh,
81
Mahummad S d e e m Mian
AnnAinltan2 R u m c h e h ,
OepcvrRmed 06 Phmmace.u;ticd ChemhZhy, College 05
P h m a c y , King Saud U n i v e ~ L t y , Riyadh, Saudi Ahabia.
Cl
Neelo&u~AbdLLe A z i z Mian
Bio c hemdk,
Childhen
Mateh/ruzi.ty H a i i p i $ d , Riyadh, S a u d i A h a b i a .
196
1. D e s c r i p t i o n
1.1. Nomenclature
1.1.1. Chemical Names
1 . 1 . 2 . Generic Names
1 . 2 . Formulae
1 . 2 . 1 . Empirical
1.2.2. Structural
1 . 2 . 3 . Cas R e g i s t r y Number
1 . 3 . Molecular Weight
1 . 4 . Elemental Composition
1 . 5 . Appearance, C o l o r , Odor and T a s t e
2. Physical Properties
2.1.
2.2.
2.3.
2.4.
Melting Range
Solubility
Crystal Structure
Spectral Properties
2 . 4 . 1 . U l t r a v i o l e t Spectrum
2 . 4 . 2 . I n f r a r e d Spectrum
2 . 4 . 3 . Nuclear Magnetic Resonance Spectrum
2 . 4 . 3 . 1 . IH-NMR
Spectrum
2 . 4 . 3 . 2 . 13C-NMR Spectrum
2 . 4 . 4 . Mass Spectrum
3. S y n t h e s i s
4 . Metabolism
5. S t e r e o c h e m i s t r y
6. Thermal A n a l y s i s
7. Methods of A n a l y s i s
7 . 1 . Nalorphine Hydrobromide
7.1.1. Identification
7 . 1 . 2 . T i t r i m e t r i c Method
7 . 2 . Nalorphine Hydrochloride
7.2.1. Colorimetric
7.2.2. Spectrophotometric
7 . 2 . 3 . Thin-Layer Chromatography
197
NALORPHINE HYDROBROMIDE
1. Description
1.1. Nomenclature
1.1.1. Chemical Names
(a) N-Allylnormorphine hydrobromide (1,Z)
198
1 . 2 . 3 . CAS R e g i s t r y
1041-90-3 ( n a l o r p h i n e hydrobromide) (2).
57-29-4 ( n a l o r p h i n e h y d r o c h l o r i d e ) (2) .
62-67-9 ( n a l o r p h i n e ) ( 2 ) .
1.3. Molecular Weight
C1gH21N03HBr
392.3 ( 2 ) .
1 . 4 . Elemental Composition
C , 58.16%; H , 5.66;
2 . Physical Properties
2 . 1 . Melting Range
About 260' w i t h decomposition ( 2 ) .
decomposition ( 3 ) .
258'-259'
with
2.2. S o l u b i l i t y
I t i s s o l u b l e i n 24 p a r t s o f water and i n 35 p a r t s o f
95% e t h a n o l ( 1 ) . Aqueous s o l u t i o n s may d e p o s i t c r y s t a l s
o f t h e d i h y d r a t e . The h y d r a t e d form is r e a d i l y s o l u b l e
i n dehydrated e t h a n o l , and t h e s o l u t i o n s r a p i d l y g i v e
r i s e t o a d e p o s i t o f t h e anhydrous form ( 1 ) .
w,
They a l s o measured ( 5 ) i n t e n s i t i e s o f r e f l e c t i o n s on a
Nonius-CAD-4 d i f f r a c t o m e t e r w i t h g r a p h i t e monochromated
copper Ka r a d i a t i o n and a 8-29 s c a n .
NALORPHINE HYDROBROMIDE
Table 1:
199
8125 (1)
10395 (8)
3565 (6)
5852 (7)
7113(6)
6546(13)
5 1 6 3 (10)
4849 (10)
5874 (9)
7371 (8)
8024 (9)
8768 (9)
9534 (9)
10140(8)
9189 (10)
7599 (9)
7197 (7)
8143 (9)
9650 (7)
8316(10)
9016 (9)
11155 (10)
12620(11)
13027(15)
11069
3213
7500
682 7
4348
7 394
2
-
1 5 0 1 (1)
4475 (5)
6659 (7)
7192 (5)
9269 (4)
4714 (8)
5239 (8)
6052 (9)
6276 (7)
7282 (6)
8339 (6)
8174(6)
7249 (6)
5187( 6)
4618(7)
4965 (6)
5692 (6)
6174(5)
6339 (6)
5383(7)
4258 (8)
3376 (7)
3516 (8)
2853 (10)
4904
6999
10000
4165
4969
7354
1494 (1)
48 4(4)
1761 (4)
635 (3)
630 (4)
2415 (5)
2360 (6)
1798 (6)
1206 (5)
29 7(4)
649 (4)
1463 (5)
1599 (5)
1258 (4)
1889(5)
1819 (4)
1208 (4)
542 (4)
938 (4)
-198 (4)
18(5)
612 (5)
1030 (6)
1607 (8)
113
2292
625
2917
2 764
-350
B(X2)
5.52 (2)
3 . 8 6 (18)
6.23(21)
4. 76(17)
4 . 4 4 (16)
5.02(27)
5.13(28)
4.83(26)
3. 90(21)
3. 24(19)
3. 43 (19)
3.98 (21)
4. 14 (22)
3.19 (19)
3. 93(22)
3. 45 (19)
3 . 0 3 (17)
2 . 9 1 (17)
2 . 9 3 (18)
3.87(21)
4. 11 (23)
4. 51(24)
5 . 3 9 (28)
7.64 (40)
3. 71
5.99
4.33
5. 04
5.18
3.07
The atomic numbering system and t h e bond l e n g t h s of n a l o r p h i n e hydrobromide have a l s o been c a l c u l a t e d (5) and
a r e shown i n F i g u r e 1.
I t h a s been r e p o r t e d ( 5 ) t h a t most o f t h e bond l e n g t h s
and bond a n g l e s of n a l o r p h i n e hydrobromide a r e w i t h i n t h e
expected l i m i t s , however, t h e bond C ( 5 ) - 0 ( 2 ) h a s been
found t o be e n l a r g e d , p r o b a b l y because o f t h e s t r a i n it
e x p e r i e n c e s w h i l e l i n k i n g r i n g s A, C and D a s shown i n
Table 11.
200
Figure I .
NALORPHINE HYDROBROMIDE
Table 11:
Bond a n g l e s
201
(O)
Angle
C(9)-N-C(16)
C(9)-N-C(17)
C (16) -N-C (17)
C (4) - 0 ( 2 ) -C (5)
C(2) - C ( l ) - C ( l 1 )
c (1) -c ( 2 ) -c (3)
0(1)-C(3)-C(2)
O(l)-C(3)-C(4)
C(2)-C(3)-C(4)
0 (2) -C (4) -C (3)
0(2)-C(4)-C(l2)
c (3) -c (4) -c (12)
O(2) -C (5) -C [6)
O(2)-C ( 5 > - C ( l 3 )
C(6)-C(5)-C(13)
O(3) -C (6) -C (5)
0 ( 3 ) - C ( 6 ) -C(7)
C(5)-C(6)-C(7)
C (6) -C (7) -C (8)
C(7) -C (8) -C (14)
N-C(9) -C(10)
N-N(9) -C(14)
Angle
111.9(6)
1 1 5 . 7 (6)
109.0 ( 7 )
1 0 6 . 0 (6)
1 1 9 . 5 (8)
1 2 2 . 8 (9)
125.1(9)
1 1 7 . 6 (9)
117.2 (9)
126.2 (8)
112.5 ( 7 )
1 2 0 . 5 (8)
107.2 (6)
1 0 6 . 3 (6)
1 1 4 . 4 (6)
113.5 ( 7 )
113.7(6)
114.6 (6)
120.1(8)
120.0(8)
114.0(6)
1 0 4 . 6 (6)
116.9 (6)
113.2 ( 7 )
1 2 4 . 2 (8)
116.2(8)
1 1 9 . 0 (7)
123.2 ( 7 )
109.0(7)
126.9 ( 7 )
1 0 3 . 5 (6)
1 1 5 . 0 (6)
111.0(6)
106.0 (6)
113.5(6)
107.9(7)
114.0 (6)
108.5(6)
1 0 7 . 5 (6)
113.2 ( 7 )
109.5 (7)
1 1 3 . 0 (7)
1 2 1 . 6 (1 0)
S o l i d s t a t e c o n f o r m a t i o n o f n a l o r p h i n e hydrobroml; d e ,
morphine and n a l o x o n e h a s a l s o been compared by measuring
t h e i r t o r s i o n a n g l e s ( 5 ) . An O r t e p s t e r e o p a i r i s a l s o
shown i n F i g . 11. ( 5 ) .
F i g u r e 11: O r t e p s t e r e o p a i r o f n a l o r p h i n e H B r
While l i n k a g e o f n a l o r p h i n e m o l e c u l e s by hydrogen bond
between B r , 0 and N atoms, a l o n g w i t h t h e i r v a l v e s are
g i v e n i n T a b l e 111. (5)
202
Table 111:
D-H..
Hydrogen bonding d i s t a n c e s
.. A
O(1)-H(01). . . . B r
0(3)-H(03).
.Br
N-H(20). . 0 ( 3 )
..
F i g u r e 111.
(R)
and a n g l e s
Angle
D-H
__
H. . A
-
140.8
142.0
173.5
1.00
0.93
1.00
2.40
2.33
1.83
(O)
D. . A
3.246 (7)
3.119 (6)
2.829 (10)
P r o j e c t i o n i n t h e (001) p l a n e of n a l o r p h i n e H B r
s t r u c t u r e showing t h e packing and t h e hydrogenbond scheme. Large, medium and small c i r c l e s
r e p r e s e n t B r , 0 and N atoms r e s p e c t i v e l y ( 5 ) .
203
NALORPHINE HYDROBROMIDE
223.4
Figure IV.
,L,
2 85
300
204
2.4.2.
I n f r a r e d Suectrum
spectrum
PMR spectrum c h a r a c t e r i s t i c s of n a l o r p h i n e
hydrobromide
Assignment
6.50
H-1,
4.7 -4.86
H-5
5.26-5.62
CH = CH2 (m)
1 . 9 -4.40
2.4.3.2.
...
15
H-2 ( a r o m a t i c )
7 , 8, 9 , 1 0 , 15, 16-CH2
C-NMR Spectrum
Mass Spectrum
0
0
\D
0
03
0
.d
v)
0
-0
0
rl
Y
v)
cd
0
N
rl
.d
44
rd
F:
k
0
s
a
.d
a,
F:
a
x
a,
0
0
d
4
rl
0
0
\D
0
0
r(
co
0
0
0
N
c1
In
u.
.d
22
a,
k
>
44
d
cd
k
a
a,
a,
Ln
0
0
0
d.
0
0
8.0
Figure VI.
7.0
6.0
5.0
4.0
3.0
2.0
6'
1.0
NALORPHINE HYDROBROMIDE
Table V:
207
- TFA
Chemical s h i f t ( 6 ) ppm
Carbon No.
Nalorphine HC1
Nalorphine
c- 1
121.0
118.5
c-2
118.1
116.4
c- 3
138.1
138.5
c-4
165.1
146.2
c-5
90.1
9.5
C-6
66.0
66.4
c-7
133.1
133.4
C-8
124.9
128.4
c-9
58.5
56.0
c-10
20.8
21.1
c-11
122,6
125.4
c-12
128.8
131.0
C-13
42.2
43.5
C-14
38.5
40.5
C- 15
32.4
35.4
C-16
45.8
44.0
C-17
57.2
57.6
C-18
124.9
136.4
c-19
126.8
117.0
-.
Is
.o
.c\I
I
.o
,.- 0M
4 0
,- \ D
w
. N
- 0
N
(.- N
A 0
- 0
. N
r
0 0 0 0 0 0
O Q ) \ O d N
d
209
NALORPHINE HY DROBROMIDE
3. S y n t h e s i s
Scheme I (11,12)
Normorphine h a s been r e p o r t e d t o r e a c t r e a d i l y w i t h a l l y 1
bromide i n chloroform a t 110OC. I t s s t r u c t u r e was confirmed by c o n v e r t i n g it i n t o N - a l l y 1 n o r c o d e i n , according
t o Rodionov's method.
CH2Br
Normorphi.n e
HO
CHC 1
llOC
*-
Nalorphine HBr
Scheme I 1 (13-16)
I n i t i a l l y b o t h t h e hydroxyl groups o f morphine a r e a c e t y l a t e d w i t h a c e t i c anhydride. I t i s t h e n r e a c t e d with
cyanogen bromide t o remove t h e methyl group and c o n v e r t
it t o normorphine, which on r e a c t i o n w i t h a l l y l b r o m i d e a t
7OoC i s c o n v e r t e d t o a l lylnormorphine hydrobromide.
210
Scheme I1
Acetic
anhydride
0
Morphine
Ally1 bromide
Diacetyl
Morphine
NALORPHINE HYDROBROMIDE
21 1
Scheme I11
Morphine i s r e a c t e d with VOCCl i n d i c h l o r o e t h a n e t o a f f o r d
3 VOCl d e r i v a t i v e S e l e c t i v e N-VOC removal was e f f e c t e d
w i t h anhydrous hydrogen bromide i n e t h a n o l - e t h e r a t 25OC.
N - a l l y l a t i o n was achieved with a l l y 1 bromide and sodium
c a r b o n a t e i n e t h a n o l a t 7OoC f o r 6 h r s . The removal of
V O C l groups was e f f e c t e d with 1.7-N HC1 t o y i e l d n a l o r phine hydrobromide ( 1 5 ) .
5 Equav.
HO
voc
18-
Vinyl c h l o r o formate
(VOCC1)
W,N-CH
--z-
Morphine
-voc
voco
Anhydrous H B r
i n ethanolether
-H
HCl
1.7 N
l0O0C
CH2.HBr
HBr
212
Scheme IV
Morphine i s converted t o tri-voc-normorphine with VOCCl
r e a g e n t and i t s subsequent conversion t o di-vocnormorphine i s achieved with a mild a c i d o r by t i t r a t i o n
with B r 2 and subsequent d i l u t i o n with a l c o h o l (17). I t is
then r e a c t e d with a l l y 1 bromide and subsequent removal of
VOC groups a f f o r d s n a l o r p h i n e hydrobromide.
B r 2 in
alcohol
Morphine
carbonyl normorphine.
J(
voco
N-CH2-CH
CK2.HBr
0-0-di-voc-normorphine
hydrobromide
HO
Nalorphine H B r
213
NALORPHINE HYDROBROMIDE
4. Metabolism
Nalorphine h a s been r e p o r t e d t o b e c o n v e r t e d t o normorp h i n e by r a t s (18) and c a t s (19) i n vivo and by r a t l i v e r
microsomes i n v i t r o ( 2 0 ) . I t i s conjugated i n r a t s ( 2 1 ) ,
dogs ( 2 2 ) , r a b b i t s and c a t s ( 2 3 ) . Nalorphine-3-glucuronide
and n a l o r p h i n e 3 - e t h e r e a l s u l p h a t e have a l s o been
i s o l a t e d from t h e u r i n e o f c a t s and r a b b i t s (23). Recently
n a l o r p h i n e 6 - g l u c u r o n i d e h a s a l s o been i s o l a t e d from t h e
u r i n e o f c a t s and dogs ( 2 4 ) .
Nalorphine h a s been r e p o r t e d (2) t o be p o o r l y absorbed
when given o r a l l y ; however, on p a r e n t a l a d m i n i s t r a t i o n it
i s absorbed and r e a d i l y p a s s e s i n t o t h e b r a i n and a c r o s s
t h e p l a c e n t a . I t i s mostly m e t a b o l i s e d i n t h e l i v e r and
i s e x c r e t e d i n t h e u r i n e . Small amount o f unchanged
n a l o r p h i n e i s a l s o e x c r e t e d i n u r i n e . Nalorphine hydrobromide i s mainly used t o a n t a g o n i s e n a r c o t i c - i n d u c e d
r e s p i r a t o r y depression.
5. Stereochemistry
6 . Thermal A n a l y s i s (DSC)
A d i f f e r e n t i a l scanning c a l o r i m e t r y curve was o b t a i n e d
(Fig. IX) on a Perkin-Elmer DSC-2C d i f f e r e n t i a l c a l o r i meter (25 . Nitrogen was used a s t h e purge g a s . Scan was
performed a t a r a t e o f 40C/min from 80' t o 35OoC. The
DSC curve r e v e a l e d an endothermic m e l t i n g peak (Max
260.19OC)
followed by decomposition of t h e sample.
214
F i g u r e VIII.
Atomic model o f n a l o r p h i n e .
.rl
M
a,
a
0
0
-3
..
cd
k
a,
CI
0-l
4
. . .
rllnd
\ D M N
0 0 N N N
e m -
.. .. ..
B O W
O b a,
k v ,
.-
a,
i,
C
C
p.
v)
0
0
0
0
a,
.d
a
0
%k
9
M
ln
a
x
Jz
9
c
>
a,
rcl
0
cd
C
Jz
a
k
.rl
a,
0
N
0
0
0
m
N
0
0
\D
N
rl
0
0
0
*
0
0
0
3
0
0
03
216
7 . Methods of Analysis
Identification
a) A 0 . 0 1
bromide i n
350 nm and
extinction
b) A 0 . 0 1 p e r c e n t w/v s o l u t i o n , i n a 0 . 1 N sodium
hydroxide s o l u t i o n , i n 2-cm l a y e r c e l l e x h i b i t s a
maximum a t 298 nm and e x t i n c t i o n a t 298 nm is about
1 . 2 (1).
c) A drop o f d i l u t e ammonium hydroxide s o l u t i o n on
addition t o 5 m l , of a 3 percent s o l u t i o n of nalorphine hydrobromide, immediately produces a bulky
w h i t e p r e c i p i t a t e , which is s o l u b l e i n sodium
hydroxide s o l u t i o n ( 1 ) .
d) Addition o f a drop o f f e r r i c c h l o r i d e s o l u t i o n
t o 5 m l o f a 3 p e r c e n t w/v s o l u t i o n o f n a l o r p h i n e
hydrobromide g i v e s r i s e t o b l u e c o l o r ( 1 ) .
7.1.2.
T i t r i m e t r i c Method (1)
0.5 g of n a l o r p h i n e hydrobromide i s d i s s o l v e d i n
15 m l o f water and 5 m l o f d i l u t e ammonia s o l u t i o n
i s added. Nalorphine i s completely e x t r a c t e d i n t o a
mixture o f t h r e e volumes o f chloroform and one
volume o f i s o p r o p y l a l c o h o l , washing each e x t r a c t
with t h e same 10 m l o f w a t e r . The e x t r a c t s a r e
combined and t h e s o l v e n t is removed by e v a p o r a t i o n .
5 m l o f 95% a l c o h o l p r e v i o u s l y n e u t r a l i s e d t o methyl
r e d s o l u t i o n i s added t o t h e r e s i d u e , and i s l a t e r
removed by e v a p o r a t i o n , and t h e p r o c e s s i s r e p e a t e d .
Then d i s s o l v e t h e r e s i d u e i n 1 m l o f t h e n e u t r a l i s e d
95% e t h a n o l and a g a i n e v a p o r a t e . Dissolve t h e
r e s i d u e i n 1 m l o f n e u t r a l i s e d 95%. Alcohol, and
20 m l of 0.1-N h y d r o c h l o r i c a c i d and 10 m l o f water
and t h e mixture i s t i t r a t e d with 0 . 1 N sodium
hydroxide s o l u t i o n , u s i n g methyl r e d s o l u t i o n a s
i n d i c a t o r . Each m l o f 0.1-N h y d r o c h l o r i c a c i d is
e q u i v a l e n t t o 003923 g o f C1gH21N03, H B r .
NALORPHINE HYDROBROMIDE
217
7 . 2 . Nalorphine Hydrochloride
7.2.1. Colorimetric
S o l i d n a l o r p h i n e h y d r o c h l o r i d e o r a volume o f
i n j e c t i o n e q u i v a l e n t t o 0.010 g o f n a l o r p h i n e hydroc h l o r i d e i s d i l u t e d w i t h 0 . 1 I4 h y d r o c h l o r i c a c i d t o
100 m l . To 15.00 m l o f t h i s s o l u t i o n , a r e added
10 m l o f 0.1-M sodium n i t r i t e , and a f t e r 1 0 minutes
5 . 0 m l of 5-M ammonia s o l u t i o n i s added and i t s
volume is made up t o 50 m l w i t h w a t e r . A s t a n d a r d
s o l u t i o n c o n t a i n i n g 10 mg of n a l o r p h i n e h y d r o c h l o r i d e ,
and a b l a n k s o l u t i o n , i s s i m i l a r l y p r e p a r e d a s d e s c r i b e d above. The absorbance is measured a t 440 nm,
and t h e s t r e n g t h o f t h e unknown s o l u t i o n c a l c u l a t e d .
7.2.2.
Spectrophotometric
Nalorphine h y d r o c h l o r i d e as s o l i d o r a s an i n j e c t i o n
can be determined s p e c t r o p h o t o m e t r i c a l l y (26). About
25 mg o f n a l o r p h i n e h y d r o c h l o r i d e i s a c c u r a t e l y
weighed and t r a n s f e r r e d t o a 250 m l f l a s k . I t i s
d i s s o l v e d i n w a t e r and made up t o volume. I t s
absorbance a t 285 nm i s measured and compared w i t h a
s t a n d a r d sample s o l u t i o n .
7 . 2 . 3 . Thin-Layer Chromatography
T h i n - l a y e r chromatography h a s been a p p l i e d f o r t h e
d e t e r m i n a t i o n o f n a l o r p h i n e h y d r o c h l o r i d e (27).
50 g o f it were a p p l i e d t o a s i l i c a g e l G F254 t h i n
l a y e r p l a t e and e l u t e d i n a m i x t u r e o f , e t h a n o l dioxane-benzene-ammonia s o l u t i o n 25% (4 :8 :7 :1 ) . The
s p o t s were d e t e c t e d i n U . V . l i g h t (254 nm) and a l s o
by s p r a y i n g with Dragendorff r e a g e n t .
218
References
1.
2.
J.E.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
39,
305 (1926).
NALORPHINE HYDROBROMIDE
219
16.
17.
18.
19.
P a r t y Kay
3,
20.
21
- 22
142,
23.
24.
25.
26.
27.
NIFEDIPINE
SYED LAIK ALI
222
NIFEDIPIWE
SYED LAIK ALI
1.
2.
3.
3.1
3.2
4.
5.
5.1
5.2
5.3
5.4
5.5
5.6
5.7
5.8
5.9
5.10
5.11
5.12
5.13
5.14
5.15
5.16
5.17
5.18
6.
7.
History
Nomenclature
Description
Name, Formula, Molecular weight
Appearance, Colour, Odour
Synthesis
Physical Properties
Solubility
Loss on Drying
Melting Point
Sulfates
Chlorides
Sulphated Ash, Residue on Ignition
Heavy Metals
Dissociation Constant
Distribution Coeeficient
Light Sensitivity
Sensitivity to Temperature
Related Compounds
Impurities Titrable with Perchloric Acid
Crystal Structure
Ultraviolet Spectrum
Infrared Spectrum
Nuclear Magnetic Resonance Spectrum
13C-NMR-Spectrum
Colour and Identification Reactions
Stabilitv and Deqradation
NIFEDIPINE
8.
8.1
8.2
8.3
8.4
8.5
8.5.1
8.5.2
8.5.3
8.5.4
9.
223
10.
Methods o f Analysis
Ti trimetry
Polarography
UV Spectrophotometry
Fluorimetry
Chromatographic Methods
Thin Layer Chromatography
Gas L i q u i d Chromatography
High Performance Liquid Chromatography
Gas Chromatography-Mass Spectrometry
Radioactive Measurements of C-14 Label led Nifedipine
in Bodv Fluids
In vitro Dissolution
11.
12.
Acknowledqements
13.
References
224
N i f edi pine
1. History
2. Nomenclature
225
NIFEDIPINE
Fig. 1
Nifedipine S t r u c t u r a l Formula
226
3 . Description
4. Synthesis
NIFEDIPINE
227
228
Fig. 2 (6 1
Nifedipine Snthesis Scheme
NIFEDIPINE
5.
229
Physical Properties
171-175C
230
Maximum 10 ppm
NIFEDIPINE
231
a)
than 0.2 %
b)
232
The test is to be performed through thin layer chromatography on silicagel plates with diisopropyl ether as the
solvent using corresponding USP reference substances.
NIFEDIPINE
233
0.1
N NaOH
nm
235 nm
338 nm
340 nm
238 nrn
238 nm
145
165
165
624
595
592
5010
5740
5740
2 1590
20600
20510
Absorption Maximum
340
A1%
1cm
0.1 N HC1
5 . 1 6 Infrared
Spectrum
234
O!
0.
0.:
0.f
E lcm
0.4
0.3
0.2
0.1
Fig.3 ( 1 4 )
- Methanol
N - HC1
-- 0.1
---
0.1 N
- NaOH
UV S p e c t r a o f N i f e d i p i n e i n d i f f e r e n t S o l v e n t s
r-
236
Frequency c d
3331
3102
2931, 2842
1689
1679
1625
1574
1530
1433
1380
1227
1121
Ass i qnments
NH stretching
vibrations
CH-aromatic
CH-a1 iphatic
C=O ester
-C=C-aromatic
-C-CH3
-C-0-ester
231
NIFEDIPINE
Nifedipine
P
6
H3COOC
ti
i'
S t r u c t u r a l A s s i g n m e n t o f NMR
Fig. 5
12
Signals
ul
.
r
E
0
al
k
+
V
Q
v)
k
a,
Y
3
NIFEDIPINE
H-Atom in
Fia.5
239
Chemical Shiff
PPm
H-1
2.31
H-2
5.74
H-3
3.60
H-5
7.68
H-6
7.26
H-7
H-8
H-13
7.50
7.51
6.56
Mu1 t ip l icity
Number of H
Atoms
singulett
s i ngu 1ett
si ngulett
doub 1ett
mu1 tiplett
dou b 1ett
multiplett
broad singulett
6
1
6
1
1
1
1
1
5.18 13C-NMR-Spectrum
Q)
.d
S
.Fl
240
a-
C
'4
Q
-4
a,
D
%
rc
..
c)
Q,
v)
24 1
0
m
N
242
C-Atoms in
Fiq. 5
Chemical Shift
c-1
c-2
c-3
c-4
c-5
C-6
c-7
C-8
c-9
c-10
c-11
c-12
Multiplicity
Number of C
Atoms
quadruplett
doub lett
quadruplett
s i ngu 1 ett
doub 1 ett
doublett
doublett
doublett
singulett
s ingu lett
s ingulett
s ingulett
2
1
2
2
1
1
1
1
1
2
1
DPm
19.10
34.51
50.87
103.24
123.72
126.93
130.95
132.69
142.16
145.23
147.74
167.62
NIFEDIPINE
100
Fig.9
Mass Spectrum o f N i f e d i p i n e ,
V a r i a n MAT 311 Mass S p e c t r o m e t e r
243
244
Mass number
346
329
315
298
284
270
268
254
224
192
6.
S tru ctura 1
assi Qnment
Molecular peak M
M-OH
M-OCH3
M-OCH3.OH
M-OCH3 .OCH3
M-CO2CH3*OH
M-OCH3-NOz.H
M-OCH3.OCH3. NO
M-CgHqNO2
M-C6HqN02*OH CH3
C17H18N206
The substances (50 mg) is dissolved in a solution of equalvolumes of alcohol and 3 N hydrochloric acid (10 ml).
About 0.5 g of zinc granules are added and the solution is
allowed to stand for 5 minutes with occasional swirling.
After filtering the solution 5 ml of 1 % sodium nitrite
solution is added and the mixture is allowed to stand for
2 minutes. Subsequently 2 ml of 5 % ammonium sulfamate are
added, shaken vigorously and finally 2 ml of a 0.5 % solution of N-( 1-naphthy1)ethylenediamine dihydrochlor de solution is added. An intense red colour develops wh ch persists for not less than 5 minutes.
The nitro group of nifedipine could be reduced to hydroxylamine which forms with benzoyl chloride hydroxamic acid
derivative. The derivative reacts with iron (111) chloride
solution to give a deep red coloured solution.
NIFEDIPINE
7.
245
NIFEDIPINE
241
248
(min-1)
t50
(min)
t90
(min)
spectrotest (xenon
lamp) 700-372 nm
0.198
3.5
0.5
spectrotest (xenon
lamp) 700-417 nm
0.040
17
2.5
45
135
20
Light source
light bulb, 40 W
0.005
1.5
I
I
I
I
I
Decomposition o f N i f e d i p i n e , d i s s o l v e d i n a b s o l u t e e t h a n o l , i n R e l a t i o n t o t h e
L i g h t Source
M d i f f u s e d a y l i g h t November
-light
b u l b 40 Watt, 1 1 cm d i s t a n c e
250
NIFEDIPINE
blished. A
showed 50 %
whereas a
sed to 50 %
25 1
decomposition
product
Dirnethyl-4-(2-nitrosophenyl)
2,6-dimethyl-pyridine-3,5-dicarboxylate is formed in normal day light and limited upto 0.2 % in nifedipine by USP
X X I . The other product Dimethyl 4-(2-nitrophenyl)-2,6-dirnethylpyridine-3,5-dicarboxylate is considered to be syn-
252
NIFEDIPINE
8. Methods o f Analysis
8.1 Titrimetry
253
254
8.2 Polaroqraphy
Direct current and alternating current polarographic methods were developed to determine the content uniformity
and photostability o f nifedipine in pharmaceuticals. Form
and position of the polarographic curves are pH-dependent.
At pH 1.65 and a voltage of -0.75 V versus Ag/AgCl electrode a suitable diffusion current is obtained. Nifedpine
receives 4 electrons per molecule during cathodic reduction. A mixture of 0.1 N HC1 + ethanol, 1:l was used as
supporting electrolyte. With alternating current polarography nifedpine peak appears at -0.710 V which disappears
in course of photolytic decomposition and a new peak
appears at -1.22 V. This allows a specific determination
of nifedipine and its decomposition product. The standard deviation of the method is 0.28 % in the concentration range of lo-4~. The detection limit is given as
5x10-7M (24).
NIFEDIPINE
255
8.4 Fluorimetrv
methyl-5-methoxycarbonyl-6-methyl-pyridine-3-carboxylic
256
NIFEDIPINE
251
258
In course of pharmacokinetic studies of nifedipine a gaschromatographic method with electron capture detection has
been applied. A DB 1.5 m column operated at 160C column
temperatureI 250C injector temperature and 300C detector
temperature was used with helium as carrier gas. The oven
temperature was programmed from 160 to 270C with increament of 10"C/min (29,26). For the determination of nifedipine in biological fluids several gas chromatographic methods have been described, with either electron-capture
NIFEDIPINE
detection or selective ion monitoring through mass spectrometry. Kleinbloessem (30) and coworkers report that the
thermostability of nifedipine under the gas chromatographic conditions applied (230-250C) represents a serious
problem, since nitroderivative of nifedpine is formed in
non-reproducible amounts. Therefore Kondo et al. (31) and
Higuchi and Shiobara (32) oxidised nifedipine prior to GC
analysis, despite the loss of selectivity. The nifedipine
content o f plasma samples was analysed through capillary
gas chromatography with EC detector (33). In another GLC
method with ECD upto 2 ng/ml nifedipine could be detected in plasma (34). Nifedipine and its nitropyridine derivative in human plasma were analysed through GLC. A 63
Nickel electron-capture detector and a glass 1.83 m x 2.0
mm ID column packed with 2 % OV 17 on gas-chrom Q (100-120
mesh) and argon-methane (95:5) carrier gas with a flowrate 32 ml/min were used. Injection port temperature was
maintained at 245"C, column temperature at 235C and detector temperature at 280C (35). Due to the thermoinstability of nifedipine at higher temperatures a controversy
exists in the literature concerning the detection and
quantification of this substance in complex matrices. The
authors maintain that the possibility o f thermal degradation of nifedipine to its nitroderivative during the
chromatographic process cannot be ruled out with GLC at
higher temperatures (230-250C). This procedure is appl icable to the simultaneous determination of nifedpine,
n i tropyridine and ni trosopyidine derivatives. Linearity of
the procedure was established for 10-200 ng nifedipinel ml
plasma and 10-50 ng nitropyridine derivative/ml plasma.
The detection limit is given as 1 ng/ml. The relative
259
260
NIFEDIPINE
261
Nifedipine has been separated on a Bondapack C18-NH2 column, 30 cm x 3.9 mm using chloroform as mobile phase at a
flow rate o f 1.5 ml/min and detected at 254 nm. Nitrosopyridine derivative and nifedipine eluted at retention
times of 4.30 and 7.30 minutes respectively ( 1 5 ) . Nifedipine and the related substances nitroso- and nitropyridine
derivatives in nifedipine can be analysed through HPLC on
a reversed phase Nucleosil C18, 5 m column with a mobile
phase acetonitrile + methanol + water, 9:36:55. The flow-
262
pl
NIFEDIPINE
263
264
length was set at 320 nm. The calibration curve was linear
between 180-1800 ng/ml (43).
The assay of nifedipine in human plasma using an automatic
HPLC procedure through a column switching technique with
only one step dilution i s described. Because of its sensitivity of 2-3 ng/ml and its good reproducibility the method is suitable for in vivo drug level monitoring and
pharmacokinetic studies (44). A hyperchrome column, 125 x
4.6 mm, filled with Shandon Hypersil ODS 5,5 m connected
P
with a precolumn, 40 x 4.6 mm filled with perisorb RP 18
was used. The mobile phase consisted of a 0.01 M disodium
hydrogen phosphate buffer adjusted to pH 6.1 with phosphoric acid and methanol, 53:50; the flow-rate was 1.0 ml/min
and the UV detection was set at 236 nm. The flow-rate of
the wash-liquid (0.01 M disoidum-hydrogen phosphate, pH
6.1) in the column-switching technique was kept at
2.0 ml/min (44).
HPLC of nifedipine, its metabolites and photochemical degradation products was performed on a 300 mm x 4.0 mm in U
Bondapak C18 column, l o p , at room temperature. A precolumn of Bondapak C18 corasil was used for the analysis
of animal plasma. The flow-rate was 2 ml/min and UV detection was done at 254 nm. The mobile phase consisted of
0.01 M disodium hydrogen phosphate buffer, adjusted to pH
6.1 with 50 % phosphoric acid and methanol, 45:55. Nifedipine, its metabolites in plasma and its photochemical degradation products were well separated. This mobile phase
a1 lowed the separation of drugs from endogenous plasma
substances or from the components of the pharmaceutical
preparations as well as the use of 4-dimethyl-aminoben-
NIFEDIPINE
265
zaldehyde as a suitable internal standard. The minimum detectability of all compounds with the described procedure
was 10 ng. Precision over a 30-day period using different
plasma samples was 2 4,l % (45).
Determination of nifedipine in human plasma by HPLC with
electrochemical detection is reported. Chromatography was
performed on a Unisil pack C18 column, 5 p m , 150 x 4.6 mm
I.D. using the mobile phase methanol-tetrahydrofuran-0.05
M phosphate buffer (pH 3 . 0 ) , 660:10:330 at a flow-rate of
0.8 ml/min and at 20C. An electrochemical detector model
VMD 501 (Kyoto, Japan) was set at the potential 0.95 V
versus Ag/AgCl reference electrode. The detection 1 imit
for a standard solution injected at 0.95 V was approximately 40 pg. A toluene extract o f an alkalinised plasma
sample was chromatographed using diethyl 1.4-dihydro-2,6dimethyl-4- (2-nitrophenyl )pyridine-3 ,5-dicarboxylate as an
internal standard. The recovery of nifedipine from plasma
was about 100 % and detection limit in plasma was 2 ng/mlusing 0.5 ml o f sample. The assay gave linear response
over the concentration range 5-400 ng/ml in plasma. Photodegradation products and metabolites of nifedipine did not
interfere in the analysis (46).
266
GC-MS
Higuchi and Shiobara have also reported a gas chromatography - mass spectrometric method of determination of
nifedipine in plasma samples (32).
NIFEDIPINE
267
Body Fluids
Experimental studies in animals on pharmacokinetics and
b iotransformation of radioactive 1 abel 1 ed n i fedipine were
carried on ( 4 7 ) . In another study clinical investigations
on the pharmacokinetics of radioactive labelled nifedipine
in human test persons were performed (48).
Following
intravenous administration of C-14 labelled nifedipine in
rats and dogs the C-14 radioactivity eliminated in urine
and faeces was measured in a liquid szintillation counter.
Solid samples were burnt in oxygen and residue taken in a
10 ml szintillator consisting of 850 ml toluene, 150 ml
methanol + 8 g butyl PBD. For liquid samples a szintillation mixture of toluene t dioxane + ethanol, 1:l:l to
which 8 g butyl PBD had been added, was used. Human body
fluids were also treated likewise and C-14 activity was
measured with liquid szintillation counter (46).
A radioreceptor assay of nifedipine has also
been reported
(49).
10. In Vitro-Dissolution
The specifications of the manufacturer Bayer, Germany demand that nifedipine sustained-release tablets should
liberate in in vitro dissolution test after 2 hours between 40-70 % of the drug and after 6 hours at least 65 %.
In case of tablets 70 % drug should have been dissolved
after 30 minutes (50).
268
In vitro dissolution behaviour of two nifedipine sustained-release preparations was studied. A dissotest CE 6
(Sotax, Base1 , Switzerland) dissolution apparatus according to the flow-through method of Langenbucher was used.
The dissolution medium for the first hour consisted of
0.08 N HC1, pH 1.2 and for the hours 2 to 8 it comprised
of 0.05 M phosphate buffer solution pH 6.8. The flowthrough rate was set at 1000 ml per hour. Nifedipine in
both formulations liberated at a uniform rate and reached
the value of 80 % after 8 hours ( 4 3 ) .
An automatic system for the dissolution of slightly soluble drug substances is described. Aqueous dissolution
medium is pumped through a flow-through cell of variable
design. The drug substance in the cell is thus partially
dissolved. In a separate extraction compartment the dissolved amount is extracted into the chloroform phase and
the two phases are separated subsequently. The chloroform
phase serves as a sink for the slightly soluble substance
and will be used for the spectrophotometric determination
of the dissolved substance. The system was successfully
used for the dissolution of nifedipine. Because of the bad
substance wettability flow-through cells with stirrer produced better results than those without stirrer. Nifedi-
NIFEDIPINE
Nifedipine will be completely absorbed in gastro-intestinal tract by healthy test persons as well as patients.
After sublingual administration resorption takes place
already in mouth mucosa. The rate of resorption depends
upon the type of formulation administered (48). After
peroraler application of nifedipine capsules the drug is
detectable in plasma after 15 minutes. In sublingual mode
of administration nifedipine is present in plasma after
5-10 minutes. The maximum plasma concentrations are reached between 60-120 min (53,54). Fig. 12 shows typical
concentration-time curves of nifedipine in human plasma
after administration of 10 mg Adalat capsule and 10 mg
Adalat tablet T 10 (54a). I n case of sustained-release
formulations the drug is already present after 15 to 30
minutes and maximum plasma concentrations are obtained
between 2-4 hours (54) and therapeutic active levels of
about 11 ng/ml are present sill after 12 hours.
Foster and coworkers (55) have reported the results of
unchanged nifedipine concentrations in plasma of 25
healthy test persons after administration of nifedipine
capsules,
sustained-release tablets and intravenous
application. The persons received I mg intravenously or
10 mg by the intestinal route. Due to the low dosage ra-
269
.+
NIFEDIPINE
27 1
272
COOC
Ii3C
COOCH,
I
H
Nifedipine
(only i n plasma)
M e t a b o l i t e 111
( i n plasma and u r i n e )
Fig. 13 (39)
Metabolism o f N i f e d i p i n e
Loctonform
( i n p l a s m a and u r i n e )
NIFEDIPINE
273
274
Sublingual administration of 10 mg nifedipine was performed in four healthy volunteers. They were instructed to
bite the capsule apart and keep the substance in the mouth
for 5 minutes before swallowing. Plasma concentrations of
nifedipine as well as the nitropyridine product as main
metabolite were determined. Two persons had a fast absorption with maximum concentrations of 70-100 ng/ml nifedipine, reached within 30-60 minutes and two persons had a
slower absorption with maximum
concentrations
of
20-40 ng/ml reached within 90-120 minutes (28). Plasma
concentrations of nifedipine in patients with renal failure as well as in those with normal renal function were
determined. Whereas the presence of renal failure did not
influence the plasma concentrations, lower concentrations
were found in the patients on haemodialysis than in the
other groups (59).
NIFEDIPINE
The pharmacokinetic behaviour of new nifedipine preparations in humans was studied. Following a single dose of a
20 mg nifedipine sustained-release tablet to 12 heal thy
volunteers in a randomized two-fold cross-over trial, mato
ximum plasma concentrations in the range of 21.5
73.9 ng/ml were found between 45 minutes and 2 hours.
Minimum therapeutic plasma level of 10-15 ng/ml was maintained upto 8 to 10 hours. The relative bioavailabilities
and important pharrnacokinet ic parameters were calculated.
Terminal el imination half-lives were calculated to be 5
and 8 hours respectively (60).
Bioavailabil ity of two preparations o f soft gelatine capsules containing 10 mg nifedipine was performed. Mean peak
47.3
and 104.5 2
plasma concentrations o f 121.2
32.9 ng/ml were reached after about 30 minutes. Both plasma level curves were practically identical with the exception of the obtained absolute peak plasma concentrations.
Some important pharmacokinetic parameters and the relative
bioavailabilities of the preparations were calculated and
compared statistically (61). I n a randomized three-way
crossover study with twelve volunteers the bioavailability
and main pharmacokinetic parameters of three different
galenic formulations of nifedipine (hard gelatine capsules
with pellets, soft gelatine capsules with liquid nifedipine and a sustained-release tablet) were determined. Statistically significant differences for AUC, Cmax and tmaxvalue were found between three preparations. Cmax-values
lie between 40-201 ng/ml and tmax-value between 0.48 1.96 hours. No accumulation of nifedipine in plasma occured following multiple dosing (62). In another investigation the bioavailability parameters of different nife-
215
216
A bioequivalence study of 4 commercial nifedipine formulations was carried on. No therapeutic significant differences were found from the evaluated parameters AUC, Cmax,
tmax among the commercial preparations (63). A review of
nifedipine with focus on pharmacokinetic characteristics,
available analytical methodologies and selected aspects o f
clinical pharmacology is presented (64). The pharmacokinetics of a sustained-action oral preparation of nifedipine (Adalat retard) following a single dose of 20 mg
tablet was studied in 9 male volunteers and in one subject
nifedipine was compared to the conventional capsule. Compared to conventional nifedipine capsule there was delay
in peak concentration and much smaller range of peak plasma levels were observed (65).
The effects of Atenolol and Metoprolol on nifedipine plasma concentrations and the effects of nifedipine on beta
blocker plasma levels were evaluated in 8 female subjects.
No pharmacokinetic interaction between nifedipine and
betablockers was found (66). Nifedipine and its metabol ites were analysed in plasma of 6 volunteers after receiving both an i.v. injection (3.5 mg infusion over 4 min)
and 2 new oral tablet formulations (20 and 30 mg). Oral
administration of nifedipine was associated with a bioavailability of 0:43 and presence of its nitropyridine
metabolite in the plasma. The nitropyridine metabolite was
present only in trace amounts in plasma samples taken from
subjects following i.v. administration. The peak plasma
NIFEDIPINE
277
concentration and AUC-time curve suggest that the nitropyridine analog is a major, first-pass metabolite of nifedipine (58).
The pharmacokinetics and pharmacological effects of nifedipine by i.v. injection of 0.015 mg/kg, 20 mg capsule and
20 mg sustained-action tablet were compared in 8 healthy
subjects in a crossover design. After i.v. injection the
elimination half-life was 1.7 2 0.5 h, systemic clearance was 26.7 2 5.4 l/h and volume of distribution was
0.8 2 0.2 l/kg. After capsule administration plasma concentration rose rapidly to a maximum of 117 2 15 ng/ml at
a tmax of 1.4 2 0.5 h. After the sustained-action tablet
tmax was 4.2 2 0.7 h and Cmax was 26 2 10 ng/ml. Bioavailability of nifedipine was 56 2 25 % after capsules and 32
-+ 8 % after tablets. There was a hyperbolic correlation
between nifedipine plasma concentrations and changes in
diastolic blood pressure, with a minimal effective concentration of about 15 ng/ml. It i s concluded that nifedipine pharmacokinetic correlates directly with the effect
on blood pressure and heart rate. Side effects from a high
plasma concentration can be avoided with the tablet (67).
The pharmacokinetics of nifedipine was determined in 12
healthy adults after single doses of 10 mg orally and one
mg i .v. administration. Nifedipine was eliminated after
i.v. dosis with a half-life of 1.77 2 0.25 h and the total
clearance was calculated to be 0.62 2 0.09 l/kg/h. With
oral nifedipine dosage the elimination half-time was twice
as long. Withing the group of subjects fast and s l o w absorption profiles for nifedipine were obtained. Bioavailability was ascertained with 0.45 2 0.08 (55).
278
Clinical eff cacy and bioavailability of a sustained release nifedip ne formulation containing 20 mg was studied.
The tablets were administered twice daily to 30 patients
suffering from coronary diseases and or hypertension. The
product under study showed a satisfactory bioavailability
when compared to the reference preparation (68). Bioavailability and bioequivalence of a new nifedipine preparation was tested in comparison to a reference preparation
following a single dose administration of 5 mg soft gelatine capsule of each preparation t o 12 healthy volunteers
in a randomized, 2-fold cross-over trial. About 0.5 hour
after administration peak plasma concentrations of about
110-120 ng/ml were reached, the mean terminal half-1 ife
being 1.5 h. The AUCs were, except for the negligible
different peak concentrations, virtually identical. Both
preparations were considered to be bioequivalent (69).
The effect of food on the bioavailability of nifedipine,
10 mg capsules was studied under fasting, after a low-fat
meal and after a high-fat diet. The results indicated that
the rate, but not the extent of nifedipine oral absorption
is altered by food. The Cmax- and tmax-values for fasting,
low-fat and high-fat meals were 78.9, 42.2 and 58.7 ng/ml
and 0.97, 1.89 and 1.07 hours respectively (70).
The plasma pharmacokinetics of nifedipine and the formation of its metabolites have been studied in volunteers
under conditions which would affect the activity of the
cytochrome p-450-system. Smoking does not contribute significantly to the variability in nifedipine pharmacokinetics. However the interaction between nifedipine and cimetidine, but not raniti'dine, may be of clinical impor-
NLFEDIPINE
219
280
NIFEDIPINE
281
References
1)
2) U.
3)
(1982)
4)
2 4003
(1949)
5 ) F.
6)
7 ) F.
(1971)
17 956
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12) R.
Triggle, J.
Med.
14) H.W.
123 2003
Ebel,
H.
Schutz
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A.
Arzneim. -Forsch./Drug Res. 28 2188 (1978)
16) S.
(1983)
Hornitschek,
R. Ziegler,
C1 inical pharmacology in treatment of cardiovascular
diseases by Adalat, edited by R. Krebs, Schattauer
Verlag, Stuttgart, New York 1986
17)
47 207
47
(1985)
319 (1985)
NIFEDIPINE
19)
20) V.
21) M.
22)
23) F.A.
24) K.
27) H.
28)
29)
283
284
K. Yamamoto, K. Akimoto, K .
Takahashi, N.Awata and J. Sugimoto, Chem. Pharm. Bull.
28 1 (1980)
32) S. Higuchi and J. Shiobara, Biomed. Mass spectrom.,
220 (1978)
33) S.R. Hamann and R.G. McAllister Jr., Clin. Chem.
158 (1983)
11
18 (1983)
39) K.D. Ramsch and R. Ziegler, 6 t h International Adalat
Symposium, Geneva 18-20 April 1985. Monography published by Excerpta Medica, Amsterdam 23 (1986)
NIFEDIPINE
285
Levy, Biopharm.
Bayer, Leverkusen, W.
Germany
42)
T.
219 (1984)
43)
44)
516 (1981)
46)
48) F.A. Horster, B. Duhm, W. Maul, H. Medenwald, K. Patzschke and L.A. Wegner, Arzneim.-Forsch./Drug Res. 22
330 (1972)
49) R.J.
Gould, K.M.
33 2665
(1983)
286
51)
Ind. 47 77
4 55 (1981)
tablets, Experts Expertise on the relative bioavailability of a new nifedipine formulations product-report (R 3049) , Bayer
AG, 1984
56)
57) E.M.
Sorkin,
182 (1985)
58) D.G.
59) M.G.
C.F.
NIFEDIPINE
287
60) D. Lutz, G. Pabst, W. Dahmen, K.-H. Molz and H. Jaeger , Arzneim. -Forsch./Drug Res. 35 1840 (1985)
61) W . Dahmen, G. Pabst, K.-H. Molz, D. Lutz and H. Jaeger , Arzneim. -Forsch. /Drug Res. 3 1842 (1985)
62) G. Pabst, D. Lutz, K.-H. Molz, W. Dahmen and H. Jaeger, Arzneim.-Forsch./Drug Res. 36 256 (1986)
6 3 ) K.G.
67) C.H.
Kleinbloesem, P. Van Brummelen, J.A. Van de
Linde, P.Y. Voogd and D.D. Breimer, Clin. Pharmacol.
Ther. , 35 742 (1984)
68) C. Piovetta, Arzneim.-Forsch./Drug Res. 37 832 (1987)
69) G.
Achtert, R. Wisotzki, D. Lutz and H. Jaeger,
Arzneim. -Forsch. /Drug Res. 37 1296 (1987)
Zinny,
288
Scholtan,
77) F.A.
ANALYTICAL PROFILE OF
PHYSOSTIGMINE SALICYLATE
Department of Pharmacognosy,
College of Pharmacy, King Saud U n i v e r s i t y ,
Riyadh, S a u d i A r a b i a .
290
PHYSOSTIGMINE SALICYLATE
CONTENTS
1. Description
1.1 N o m e n c l a t u r e
1 . 2 Formulae
1 . 3 M o l e c u l a r Weight
1 . 4 Elemental Composition
Physical Properties
2 . 1 M e l t i n g Range
2 . 2 E u t e c t i c Temperature
2.3 Specific Optical Activity
2.4 Solubility
2 . 5 D i s s o c i a t i o n Constant
2.6 Crystal Structure
2 . 7 X-Ray
Powder D i f f r a c t i o n
2.8.2
I R Spectrum
2 . 8 . 3 Nuclear M a g n e t i c i l e s o n a n c e S p e c t r a
2 . 8 . 4 Mass s p e c t r u m
PHYSOSTIGMINE SALICYLATE
3. Isolation of Physostigmine
4. Synthesis of Physostigmine
5. Biosynthesis of Physostigmine
8.4.2 UV Determination
8.4.3 Spectrofluorimetric
8.4.4 Phosphorescent Analysis
8.5 ihzymat ic Methods
8.6.4GLC
9. Therapeutic Uses
29 1
292
1.
Description
1.1 Nomenclature
1.1.1
Chemical Names
(3aS-cis)-l,2,3,3a,8,8a-Hexahydro-lJ3a,8trimethylpyrrolo [2,3-b] indol-5-01 methylcarbamate (ester).
Generic Names
Physostigmine Salicylate; Eserine Salicylate;
Physostol Salicylate.
1.1.3
1.2
Formulae
1.2.1
Empirical
C15H21N302
(Physostigmine)
C15H21N302. C,H603
(Physostigmine Salicylate)
Structural
Physostigmine is an indole alkaloid, having
a urethane side chain.
PHYSOSTIGMINE SALICYLATE
293
1.2.4
1.2.5
Absolute Confieuration
The absolute configuration has been deduced
from combinations of chemical degradations
and correlations (9), O.R.D. studies of
(-)-physostigmine with several related ( - ) substances (9) and by the use of the nuclear
Overhauser effect (NOE) in NMR spectroscopy
(10) together with the determination of the
crystal structure by measuring the threedimensional intensity data on a computercontrolled four-circle diffractometer (11).
The absolute structure o f (-) -physostigmine
is shown above and a perspective view of it,
294
(Physostigmine)
(Physostigmine salicy 1at e)
(Physostigmine sulfate)
C,
PHYSOSTIGMINE SALICYLATE
295
Physical Properties
2.1
Melting Range
The following melting points are reported:
Physostigmine : 105-106' (12); not lower than
103' ( 1 3 ) ;
Physostigmine salicylate : 185-187' (12); at
about 184' (13);
Physostigmine sulfate : 140' (after drying at 100')
(12); at about 143' ( 1 3 ) .
2.2
141'
139'
Sal.
156'
156'
Benzan.
2.3
2.4
Solubility
Physostigmine is slightly soluble in water, freely
soluble in alcohol, benzene, very soluble in chloroform, it is soluble in fixed oils ( 1 2 , 1 3 ) .
Physostigmine salicylate is soluble in 90 parts of
water and in 25 parts of ethanol ( 9 6 % ) , very slightly
296
s o l u b l e i n e t h e r ( 1 4 ) ; 1 g i s s o l u b l e i n 75 m l w a t e r ,
16 m l a l c o h o l , 6 m l chloroform and about 250 m l e t h e r
(12,13).
One gram of physostigmine s u l f a t e i s s o l u b l e i n 4 m l
of w a t e r , 0 . 4 m l a l c o h o l and about 1200 m l e t h e r
(12,13).
2.5
D i s s o c i a t i o n Constant
(13).
Crystal Structure
C r y s t a l s of physostigmine a r e orthorhombic, space
group P212121 with a= 1458(1) , b= 1435(1) , c = 7 2 7 . 1
( 6 ) pm.
The v a l u e s of R are 0.060 o v e r t h e 1,520 s i g n i f i c a n t
r e f l e x i o n s and 0.097 over a l l 2,531 d a t a (11).
The conformation o f t h e molecule i s shown i n F i g . 1 ,
t h e a b s o l u t e c o n f i g u r a t i o n corresponds t o t h a t of
t h e n a t u r a l a l k a l o i d . The two p y r r o l i d i n e r i n g s a r e
c i s - f u s e d , and t h a t a d j a c e n t t o t h e a r o m a t i c r i n g i s
p r a c t i c a l l y f l a t , while t h e o t h e r i s i n t h e h a l f c h a i r (C2) conformation w i t h t h e two-fold a x i s p a s s i n g through C g and t h e c e n t e r o f t h e Cg-Cio bond.
Both r i n g n i t r o g e n atoms a r e t e t r a h e d r a l , b u t Ng
is r a t h e r f l a t t e n e d and h a s more s p 2 c h a r a c t e r , judgi n g from t h e bond a n g l e s . The C18 methyl group i s
c i s t o t h e hydrogen on C6. The lone p a i r on N 7 i s
c i s t o t h e hydrogen atom on Cg. The carbamate group
i s p l a n a r , w i t h t h e hydrogen on N16 trans t o t h e
k e t o oxygen. The t o r s i o n a n g l e C12-C1-O13-C14
ranges
2.7
X-ray Powder D i f f r a c t i o n
The X-ray d i f f r a c t i o n p a t t e r n of physostigmine s a l i c y l a t e was determined on a P h i l i p s X-ray d i f f r a c t i o n
spectrogoniometer f i t t e d w i t h PW 1730 g e n e r a t o r .
R a d i a t i o n was provided by a copper t a r g e t (Cu anode
2000 w). High i n t e n s i t y X-ray t u b e o p e r a t e d a t 40 KV
and 30 MV was used. The monochromator was a s i n g l e
PHYSOSTIGMINE SALICYLATE
297
12.55
9.7
4.45
19.7
3.15
12.4
10.04
10.4
4.35
19.0
3.07
18.3
9.65
15.8
4.25
11.5
2.83
12.0
7.42
47.1
3.90
31.5
2.75
10.8
7.02
19.7
3.76
37.4
2.74
10.3
5.96
32.2
3.69
20.6
2.69 51
11.9
5.54
33.5
3.64
19.8
5.31
18.1
3.49
33.4
4.99
15.8
3.46
49.0
4.84
17.0
3.39
10.5
4.68
56.5
3.26
15.2
4.55
100.0
3.17
11.8
d = i n t e r p l a n n e r d i s t a n c e , 1/10 = r e l a t i v e i n t e n s i t y
(based on h i g h e s t i n t e n s i t y of 100).
298
Spectral Properties
2.8.1
Ultraviolet Spectrum
The UV spectrum of physostigmine salicylate
in methanol (Fig. 3) was scanned from 200 to
400 nm using a Pye-Unicum SP 8-100 Spectrometer.
Physostigmine salicylate exhibited the f o l l o w ing UV data (Table 2).
1OgE
A(l%, lcm)
239
4.09
297
252
4.04
266
303
3.78
146
299
PHYSOSTIGMINE SALICYLATE
2 00
Fig. 3 :
300
400
300
0.2NH2S04
246 (E
lo6 ,I
cm=390) ,
(16)
Phys0s tigmine
Salicylate
2.8.2
Methano 1
253(~12700)
310(~3010).
(8)
Methano 1
240(~25100)
300(~11200).
(8)
Infrared Spectrum
NH stretch
CH stretch
0
I1
1745
-C-0-(aromatic ester)
1634
HN-C-
1595
C=C (aromatic)
(amide)
1489,1465
CH bending vibrations
1089,1056,1030
C-N vibration
755
(a1iphatic)
Monosubstituted
aromatics
E,
-.
302
The IR of physostigmine exhibited the following other characteristic absorption bands:1385, 1326, 1285, 1245, 1202, 1185, 1152, 1141,
1010, 994, 945, 920, 880, 860, 808, 705, 670,
570, 540 and 525 cm-1.
Clarke (16) reported the following principal
peaks or physostigmine base in KBr disc:1200, 1243, 1495 and 1724 cm-l.
Other IR data f o r both physostigmine and its
salicylate salt have also been reported (8).
2.8.3 Nuclear Magnetic Resonance
2.8.3.1
'H-NMR Spectrum
1
The H-NMR spectra of physostigmine
salicylate in DMSO-D6 (Fig. 5) were
recorded on a Varian FT 80A, 80 MHz
NMR spectrophotometer using TMS as
an internal reference.
The following structural assignments
have been made (Table 4).
11
NH
CH,
I
0
Table 4. 'H-NMR Characteristics of Physostigmine.
Chemical Shift
(PPmI
Assignment
Multiplicity
4.15
1H
singlet
2.56
2H (CH2)
triplet
1.81
3H (CH2)
triplet
PHYSOSTIGMINE SALICYLATE
Chemical S h i f t
(PPm)
305
Assignment
Mu 1t i p 1i c i t y
singlet
A-B p a i r o f d o u b l e t s
66.43,6.81
6.85
6.77
6.49
6H
7.52
7H (NH)
2.73
8H (N-CH3)
2.90
9H (N-CH3)
2.46
10H (N-CH3)
singlet
1.35
1 1 H (C-CH3)
singlet
(aromatics)
q u a r t e t (D20
exchangeable)
doub 1et (sharpened
i n t o s i n g l e t upon D20)
singlet
1
Other H-NMR d a t a f o r physostigmine have been
reported (8,10,17).
2.8.3.2
I3C-NMR
Spectra
306
.s
307
308
Chemical S h i f t s
[PPml
Carbon
Chemical S h i f t s
[PPml
97.18 (d)
106.0
52.50 ( t )
c10
142.83 (s)
40.47 ( t )
137.16 (s)
116.15 ( d )
38.40 (q)
c11
51.90 (s)
36.38 (9)
26.96 (q)
13
155.66 ( s )
14
149.10 (s)
15
120.28 (d)
(d)
26.96 (q)
s = s i n g l e t ; d = doublet; t = t r i p l e t ;
q = quartet.
Other C-NMR
2.8.4
Mass Spectrum
The mass spectrum of physostigmine i s p r e s e n t e d i n Fig. 8. T h i s spectrum was o b t a i n e d
by e l e c t r o n impact i o n i z a t i o n on an E . I .
Finnigan model 300 (70 eV i o n i z a t i o n p o t e n t i a l )
with INCOS d a t a system.
The spectrum was scanned t o t h e mass 300 amu.
I t shows a m o l e c u l a r i o n peak M+ a t m / e 275.
The b a s e peak i s a t m/e 160.
The most prominent fragments and t h e i r r e l a t i v e i n t e n s i t i e s (RI) a r e l i s t e d i n t a b l e 6.
Table 6 .
m/e
RI
m/e
RI
275
218
217
203
8.8
53.4
13.2
9.2
188
18.6
175
174
10.9
9.2
21.6
87.1
m/ e
173
162
161
160
RI
17.1
21.0
80.2
100. 0
310
m/ e
159
146
145
144
RI
15.2
15.9
11.6
8.2
m/ e
RI
132
17.5
11.9
10.3
10.0
131
117
96
ml e
RI
77
58
57
8.3
19.6
15.3
CH3
m / e 174
31 1
PHYSOSTIGMINE SALICYLATE
HO
n*,HO
CH3
CH3
I
m/e 160
CH3
CH3
m/e 161
I
CH3
312
3.
I s o l a t i o n o f Physostigmine
(2).
P r e p a r a t i o n of Physostigmine S a l i c y l a t e
The s a l i c y l a t e s a l t i s p r e p a r e d by adding 2
p a r t s of physostigmine t o a s o l u t i o n o f 1 p a r t of
s a l i c y l i c a c i d i n 35 p a r t s of b o i l i n g d i s t i l l e d
w a t e r and a l l o w i n g t h e s a l t t o c r y s t a l l i z e on c o o l i n g
(241.
I n a n o t h e r procedure, an aqueous s o l u t i o n of
physostigmine s u l f a t e i s added t o a s a t u r a t e d s o l u t i o n of sodium s a l i c y l a t e , thereupon c r y s t a l l i n e
physostigmine s a l i c y l a t e i s p r e c i p i t a t e d o u t , c o l l e c t e d and d r i e d ( 2 3 ) .
4.
S y n t h e s i s of Physostigmine
4.1
P a r t i a l Svnthesis
(-) E s e r o l i n e can b e c o n v e r t e d i n t o (-)-physostigmine
by t r e a t m e n t of t h e former with methyl i s o c y a n a t e
(25).
4.2
313
Total Synthesis
The f i r s t t o t a l synthesis o f physostigmine was
achieved i n 2935 by Julian and PikZ ( 3 ) , since then
several syntheses have been reported (4-71. The
f i r s t synthesis i s presented i n scheme I .
N-methylphenetidine [ I ] was condensed with a-bromopropionyl bromide to give the amide adduct which
when treated with aluminium chloride underwent cyclization, accompanied by loss of the ethyl group to
give 1,3-dimethyl-5-hydroxyoxindole [ I I ] . This was
ethylated by treatment with ethyl sulfate and condensed with chloroacetonitrile in the presence of
sodium ethoxide afforded the nitrile 1,3-dimethyl-5e t h o x y o x i n d o l y l - 3 - a c e t o n i t r i l e [ I I I ] . Catalytic
hydrogenation of [111] furnished the primary amine
1,3-dimethyl-5-ethoxy-3~-aminoethyloxindole [ I V ] .
3 14
315
PHYSOSTIGMINE SALICYLATE
[V I
Phy s 0 s t i gmine [ V I I ]
CH3
CH3
316
(?)-eserethole has earlier been resolved and converted into (-) -physostigmine [VII] ( 25 ),this method constituted a brief formal total synthesis of the natural
a1kaloid.
A further synthetic approach t o the physostigmine
ring system has been developed ( 5 ) . This approach is
presented i n scheme III ( 28) and constitutes the
third synthesis of physostigmine.
Ethyl-2-formylbutyrate unsyrn-N-methylphenylhydrazone
[I] was indolized under Fischer conditions ( 29 ) to
give [11] which was converted into [111] by alkaline
hydrolysis. [111] was treated in methanol with methylamine at S O 0 to afford [IV]. The latter was reduced
with lithium aluminium hydride to give (+)-desoxyeseroline [V] .
If [V] can be converted into (2)-eseroline [VI] by
oxidation, (-)-physostigmine can then be produced
as previously described.
One of the most recent syntheses of the physostigmine ring system has been accomplished by Ikeda
e t a1 ( 6 ) . This procedure i s presented i n scheme I V
and constitutes the fourth t o t a l synthesis o f physostigmine.
The methoxy derivative of ethyl-Z-cyano-1,Z-dihydroquinoline-1-carboxylate (Reissert compound) [I] was
irradiated (30) to give the methoxy derivative of
ethyl-l-cyano-l,la,2,6b-tetrahydrocycloprop
[b] indol2-carboxylate [ 111 (photochemical synthesis) . This
cycloprop [b] indole [11] was treated with 10% potassium hydroxide and heated in a sealed tube at 12013OoC for 3.5 h. to produce the methoxy derivative
of 3,3a,8,8a-tetrahydro-3a-methylfuro [2,3-b] indol2-one [III].
[111] was treated with methyl iodide in acetone and
heated in a sealed tube at 60'C for 4.5 h. to give
3,3a,8,8a-tetrahydro-5-methoxy-3a, 8-dimethylfuro
[2,3-b]-indol-2-one [IV]. This was reacted with
methylamine in ethanol to afford 3,3a,8,8a-tetrahydro5-methoxy-1,3a,8-trimethylpyrrolo [2,3-b] indol-2one [V]. The resulting pyrroloindole [V] was reduced
with a solution of lithium aluminium hydride in dry
tetrahydrofuran to yield (+)-esermethol (eseroline
methyl ether) [VI] in quantitative yield (identical
with an authentic sample prepared from physostigmine).
Esermethole was then demethylated into (5)-eseroline
[VII] which can be converted into physostigmine [VIII]
by the standard procedure ( 25 ) .
PHYSOSTIGMINE SALICYLATE
317
I
CH
N-N
//
c =o
I
-.-
OC 2HS
I
CH3
[I1
rIVI
Physostigmine
WII
";m
CH3
\.
[VI 1
CH3
I
318
C02Et
[111
[I1
C02Et
CH3
CH3
CH3
CH3
KOH
CH3
\N
CH3
[VI
[VI I I]
CH3
P I I1
CH3
PHYSOSTIGMINE SALICYLATE
319
dinone
[ IV]
(R=H), or 1,4-dimethyl-4-(2-nitro5-ethoxyphenyl)-5-hydroxy-2-pyrrolidinone [IV] (R=
OEt). This was hydrogenated over 10% Pd/C where
cyclization occurred t o produce 1,3a-dimethyl-8formyl-3,3a,8,8a-tetrahydropyrrolo [2,3-b] indol-2one [V] (R=H), or 1,3a-dimethyl-5-ethoxy-8-formyl-3,
3a,8,8a-tetrahydropyrrolo [2,3-b] indol-2-one [V]
(R=OEt). Desoxyeseroline [VI] (R=H) was then produced by the reduction of [V] (R=H) with LAH, or
eserethole [VI] (R=OEt) was obtained from [V] (R=
OEt) with the same treatment.
320
R = H Desoxyeseroline
R = OEt Eserethole
PHYSOSTIGMINE SALICYLATE
5.
321
Biosynthesis of Physostigmine
Postulation of the biosynthetic pathway of physostigmine started in 1 9 2 5 with the suggestion of Robinson to
Barger et a1 ( 2 , 3 ) that this alkaloid might be built up
by the plant from oxytryptophan in a manner indicated by
the formulas [I]
[IV] .
0;)COOH
CH2-CH-NH2
I
070cH2-cH2-NH2
L
oxidation
decarboxyl.
a*)
[IVI
CH3
CH3
[I11
Methyl.
red.
o=c-o.o&,
YH
/
CH3
H I
CH3
CH3
1
[111]
322
6.1 Absorption
Excretion
Although the excretion of physostigmine is not
completely understood, it is known that very small
amounts ofits are excreted in the urine (34).
PHYSOSTIGMINE SALICYLATE
6.5
323
Half-life
Physostigmine is relatively short acting, possessing a half-life o f 1 to 2 hours ( 38 ) . Other
study showed that in several healthy individuals,
a terminal elimination half-life of 15-40 minutes has
been reported (34).
6.6 Onset
Duration may be less than 1 hour requiring repeated doses at 30 to 60 minute intervals ( 40 ) o r as
long as 3 to 4 hour intervals (41).
The duration of action of physostigmine as an
anticholinergic antidote is 0.5 to 4 hours (42).
When used as a miotic drug in the treatment of
glaucoma, it has a duration of action of 12 to 36
hours (43).
324
7. Drug Stability
Physostigmine and salts acquire a red tint on contact
with metals o r after long exposure to heat, light and air
( 34 ) . They should be kept in well closed containers
protected from light (14).
In aqueous solutions, particularly in the presence of an
alkali, physostigmine and its salts undergo a progressive
change in color, at first becoming red, then blue and
finally brown ( 33). This decomposition of physostigmine
involves first hydrolytic cleavage of its methyl carbamyl
group with l o s s of methylamine and carbon dioxide and formation ofthe hydroxy derivative esero1ine;this compound is
then oxidized to a dioxy derivative mbreserine,so named
because of its red color; further degradation results in
the formation of the colored derivatives eserine bZue and
eserine brom. Both rubreserine and eserine blue inhibit
destruction of acetylcholine by cholinesterase, hut both
are considerably weaker than physostigmine (44). Eseroline exerted practically no effect in inhibiting cholinesterase ( 33). This studies indicate that coloration of
physostigmine solutions always evidences some loss of
potency and eventually the solution become therapeutically
worthless ( 33). The decomposition may be retarded by
previously rinsing out the bottle with diluted hydrochloric
acid, and subsequently with distilled water, or by coating
the inner surface of the dispensing vial with paraffin (33).
Preservation of physostigmine solutions for 6 months by
addition of 1:lOOO of ascorbic acid has been claimed (45 ) .
Potassium metabisulfite o r sodium bisulfite, in concentrations of 0.1% have been also reported as satisfactory preservatives for physostigmine salicylate solutions (34).
The soZution o f physostigmine saZicyZate should
prefeYIabZy be freshly prepared.
This solution should not be sterilized by heat and should
be storted in tight, light-resistant containers at a temperature less than 4OoC, preferably between 15-30C; freezing should be avoided ( 34 ) . Commercially available
physostigmine salicylate injection has an expiration date
of 2 years following the date of manufacture (34).
PHYSOSTIGMINE SALICYLATE
8.
325
Methods o f A n a l y s i s
Identification
8.1
326
8.2
M i c r o c r y s t a l Formation
The m i c r o c r y s t a l s of physostigmine were p e r f o r med on a s o l u t i o n o f physostigmine s a l i c y l a t e i n
water ( 1 mg i n 1 ml). 1 t o 2 drops of t h i s s o l u t i o n
were t r e a t e d w i t h an equal q u a n t i t y of t h e s p e c i f i c
r e a g e n t on a m i c r o s c o p i c a l s l i d e . After a s p e c i f i c
time, t h e c r y s t a l s s o formed were m i c r o s c o p i c a l l y
examined ( 4 8 ) . Shapes of t h e c r y s t a l s a r e p r e s e n t e d i n t a b l e 7.
M i c r o c r y s t a l s of Physostigmine
Table 7
~~
Plate
~~~
~~
Reagent (16)
Time o f
formation
minute
Shapes of C r y s t a l s
Picric acid
Radiating i r r i g u l a r
plates.
Lead i o d i d e
2-3
S e r r a t e d b l a d e s (16)
Gold c h l o r i d e
R a d i a t i n g small rods.
Mayer I s
3-4
Small r e c t a n g u l a r
plates.
Wagner I s
1-2
Sharp r a d i a t i n g n e e d l e s .
Potassium p e r manganate
8.3
immediate
Long s o l i t a r y n e e d l e s .
Titrimetric Methods
8.3.1
Non-aqueous T i t r a t i o n
A non aqueous t i t r a t i o n procedure or t h e
d e t e r m i n a t i o n o f physostigmine s a l i c y l a t e
was d e s c r i b e d i n t h e E . P . 1973 ( 4 9 ) . Dissolve
about 0.25 g a c c u r a t e l y weighed physostigmine
s a l i c y l a t e i n 25 m l CHC13, add 25 ml g l a c i a l
a c e t i c a c i d and t i t r a t e with 0.02N p e r c h l o r i c
a c i d i n dioxane. Each 1 m l o f 0.02N p e r c h l o r i c
a c i d = 0.00827 g o f physostigmine s a l i c y l a t e .
PHYSOSTIGMINE SALICYLATE
PLATE
327
: r i I C R O C R Y S T A L S OF P H Y S O S T I G M I N E
WITH PlCRIC
PLATE
2 :
M I C R O C R Y S T A L S OF P H Y S O S T I G M I N E
#ITH
PLATE
3 :
ACID,
LEAD I O D I D E ,
f ' l I C R O C R Y S T A L S OF P H Y S O S T I G E I I N E
\.!ITti GoLD C H L O R I D E ,
328
~~
PLATE
: n I C R O C R Y S T A L S OF P H Y S O S T I G M I N E
WITH MAYER'S
REAGENT,
PLATE
5 :
R I C R O C R Y S T A L S OF P H Y S O S T I G M I N E
WITH
PLATE
6 :
I*!AGNER'S
REAGENT,
M I C R O C R Y S T A L S OF P H Y S O S T I G M I N E
W I T H P O T , PERMANGANATE.
PHYSOSTIGMINE SALICYLATE
329
end p o i n t i s determined p o t e n t i o m e t r i c a l l y .
Each 1 m l of 0 . 1 M p e r c h l o r i c a c i d 3 0.04135 g
o f physostigmine s a l i c y l a t e .
The USP XX ( 5 0 ) d e s c r i b e d an a s s a y proc e d u r e f o r physostigmine and physostigmine
s a l i c y l a t e u s i n g 175 mg and 250 mg r e s p e c t i v e l y . The weighed amount, d i s s o l v e d i n 25ml
CHC13, 25 ml g l a c i a l a c e t i c a c i d added and t h e
s o l u t i o n was t i t r a t e d with 0.02 N p e r c h l o r i c
a c i d i n dioxane VS. The end p o i n t was d e t e r mined p o t e n t i o m e t r i c a l l y .
Each 1 m l o f 0.02N
p e r c h l o r i c a c i d i s e q u i v a l e n t t o 5.507 mg
physostigmine and 8.270 mg physostigmine s a l i c y l a t e . F o r physostigmine s u l f a t e , 200 mg
i n 25 ml of w a t e r are t o be used. The s o l u t i o n rendered a l k a l i n e by t h e a d d i t i o n of
about 1 g o f sodium b i c a r b o n a t e and e x t r a c t e d
w i t h one 25 m l and f i v e 10-ml p o r t i o n s o f
C H C 1 3 , each time shaking v i g o r o u s l y f o r 1 min.
To t h e f i l t e r e d combined e x t r a c t , 15 m l of
g l a c i a l a c e t i c a c i d and 10 m l o f a c e t i c anhyd r i d e a r e added and t h e amount of p h y s o s t i g mine s u l f a t e determined by non aqueous t i t r a t i o n w i t h 0 . 0 2 N p e r c h l o r i c a c i d as above.
Each m l o f 0 . 0 2 N p e r c h l o r i c a c i d i s equival e n t t o 6.488 mg of physostigmine s u l f a t e .
S o l u t i o n of 0.03-0.1% physostigmine s a l i c y l a t e
was analyzed by a d j u s t i n g a sample of i n j e c t i o n s o l u t i o n t o pH 8-9 w i t h NaHC03, e x t r a c t i n g 3 times w i t h CHC13, f i l t e r i n g t h e e x t r a c t s
and t i t r a t i n g i n t h e p r e s e n c e of dimethyl
yellow by 0.005 N p - t o l u e n e s u l f o n i c a c i d i n
dioxane ( 5 1 ) .
Another method u s i n g a r y l s u l f o n i c a c i d s
i n q u a n t i t a t i v e a n a l y s i s o f a l k a l o i d s i n non
aqueous media was r e p o r t e d ( 5 2 ) . As t i t r a n t s
were used 0.005 N dioxane s o l u t i o n s of t h e
following a c i d s : naphthalene-sulfonic a c i d ,
naphthalene-2-sulfonic a c i d , S-nitronaphthal e n e s u l f o n i c ( I ) , 2 - n a p h t h o l - 6 - s u l f o n i c (11),
2-methoxynaphthalene 6 - s u l f o n i c and 2-propoxynaphtha 1ene - 6 - s u 1f o n i c ( I I I)
The t i t r a n t s c o n t a i n e d I % p h e n o l a n d were
standardized against atropine o r brucine diss o l v e d i n C H C 1 3 u s i n g 0.1% d i m e t h y l yellow a s
i n d i c a t o r . For t h e d e t e r m i n a t i o n , 5 m l of a
s o l u t i o n t o be analyzed were t a k e n c o n t a i n i n g
330
- 5 mg a l k a l o i d s a l t , i t s pH a d j u s t e d t o 8-9
w i t h s a t u r a t e d NaHC03 s o l u t i o n o r 5% NaOH,
and t h e s o l u t i o n was e x t r a c t e d 4-5 times w i t h
5-10 m l C H C 1 3 each. The combined e x t r a c t s
were f i l t e r e d and t i t r a t e d . Many a l k a l o i d s
i n c l u d i n g physostigmine were t h u s determined
with t h e e r r o r ( + 1%. Best r e s u l t s were o b t a i ned w i t h 1-111 a s t i t r a n t s .
8.3.2
Pot en t iomet r ic T i tr a t i on
A p o t e n t i o m e t r i c t i t r a t i o n method u s i n g
2.5% Na t e t r a p h e n y l b o r a t e and valinomycin
ion s e l e c t i v e e l e c t r o d e f o r d e t e r m i n a t i o n of
physostigmine s a l i c y l a t e was r e p o r t e d (53).
8.3.3. Iodometric
Determination
A method was d e s c r i b e d f o r i o d o m e t r i c
d e t e r m i n a t i o n of physostigmine s a l i c y l a t e .
(10-20 h r . r e a c t i o n a t room temperature i n
H20-HOAC and H 2 0 - N a C 1 mineral a c i d systems)
(54).
8.4
Spectrophotometric Methods
8.4.1
C o l o r i m e t r i c Determination
A method based upon t h e c o l o r i m e t r i c
d e t e r m i n a t i o n f o r r u b r e s e r i n e formed by hydrol y s i s and o x i d a t i o n of physostigmine i n aqueous
s o l u t i o n was r e p o r t e d ( 5 5 ) .
This method was employed by a n o t h e r a u t h o r
( 5 6 ) who r e p o r t e d t h a t under t h e same conditions a s i m i l a r solution containing ascorbic
a c i d 0.1% i n p l a c e of sodium m e t a b i s u l f i t e
l o s t 25% of i t s c o n t e n t of physostigmine.
I n v e s t i g a t i o n s of t h e e f f e c t s o f pH and tempe r a t u r e on t h e s t a b i l i t y of physostigmine
have been f a c i l i t a t e d by t h e development of
s p e c i f i c a n a l y t i c a l methods f o r t h e determinat i o n of t h e drug i n t h e p r e s e n c e of i t s d e g r a d a t i o n p r o d u c t s . F l e t c h e r and Davies ( 57 )
and Smith ( 5 8 ) have used m o d i f i c a t i o n s o f
t h e method o f Haugas and M i t c h e l l ( 5 9 ) ,
based upon t h e r e a c t i o n o f physostigmine
with n i t r o u s a c i d t o form a yellow compound
t h a t can b e e x t r a c t e d with CHC13 and d e t e r m i ned c o l o r i m e t r i c a l l y . In a s t u d y o f s t a b i l i t y
PHYSOSTIGMINE SALICYLATE
331
8.4.2
UV Determination
Determination o f physostigmine by UV
spectrophotometry a f t e r s e p a r a t i o n of t h e
i n t a c t drug from i t s d e g r a d a t i o n p r o d u c t s by
e x t r a c t i o n with cyclohexane - amyl a l c o h o l (4: 1)
was r e p o r t e d [62).
Another method envolving s e p a r a t i o n of
physostigmine from i t s d e g r a d a t i o n p r o d u c t s
by TLC on alumina, w i t h CHC13 - a c e t o n e (5:4)
as the solvent,was presented ( 6 3 ) . Physostigmine was e l u t e d w i t h methanolic HC1 and d e t e r mined by UV spectrophotometry. Two methods
were used f o r t h e c o r r e c t i o n of i r r e l e v a n t
absorbance. A d i f f e r e n t i a l method i n which
absorbance measurements were made a t t h r e e
wavelengths, and a method i n which o r t h o l o g i c a l f u n c t i o n s were a p p l i e d t o absorbance
measurements a t a set of n i n e wavelengths.
The r e p r o d u c i b i l i t y of t h e e l u t i o n method
a p p e a r s t o be s l i g h t l y b e t t e r t h a n t h a t o f t h e
d i r e c t r e f l e c t a n c e method ( c o e f f i c i e n t of
v a r i a t i o n , 5.81%) b u t t h e t e c h n i q u e of e l u t i o n
i s l a b o r i o u s and slow. Both methods g i v e more
r e p r o d u c i b l e r e s u l t s t h a n t h e g a s chromatog r a p h i c method of T e a r e and B o r s t f o r physost i g m i n e s a l i c y l a t e ( c o e f f i c i e n t of v a r i a t i o n ,
332
~ j .
PHYSOSTIGMINE SALICYLATE
333
Assay p r e p a r a t i o n - T r a n s f e r t o a s e p a r a t o r
a weighed q u a n t i t y o f physostigmine s u l f a t e
ophthalmic ointment, e q u i v a l e n t t o about 1 2 mg
o f physostigmine s u l f a t e . Add 100 m l o f e t h e r ,
and shake u n t i l t h e ointment base d i s s o l v e s .
E x t r a c t with f o u r 30-ml p o r t i o n s of w a t e r ,
c o l l e c t t h e aqueous e x t r a c t s i n a 200-ml volum e t r i c f l a s k , add w a t e r t o volume and mix.
Procedure - Concomitantly determine t h e
absorbances o f both s o l u t i o n s i n 1-cm c e l l s
a t t h e wavelength o f maximum absorbance a t
about 305 nm, w i t h a s u i t a b l e s p e c t r o p h o t o meter, u s i n g water as t h e blank. C a l c u l a t e
t h e q u a n t i t y , i n mg of physostigmine s u l f a t e
i n t h e p o r t i o n o f t h e ointment t a k e n by t h e
formula 200C (Au/As), i n which C i s t h e conc e n t r a t i o n , i n mg/ml, of USP physostigmine
s u l f a t e RS i n t h e s t a n d a r d p r e p a r a t i o n , and
Au and A s a r e t h e absorbances of t h e Assay
p r e p a r a t i o n and t h e s t a n d a r d p r e p a r a t i o n ,
respectively
8.4.3
Suectrofluorimetric
A procedure f o r s p e c t r o f l u o r e m e t r i c
d e t e r m i n a t i o n of physostigmine was r e p o r t e d
(64).
8.4.4
Phosphorescent A n a l y s i s
A phosphorescent p r i n c i p a l was adopted
f o r t h e 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 physostigmine (65).
8.5
Enzymatic Methods
A q u a n t i t a t i v e enzymatic a s s a y f o r measuring
physostigmine i n human whole blood was d e s c r i b e d ( 6 6 )
The a u t h o r s s t a t e d t h a t s i n c e t h e drug i s given t o
p a t i e n t s i n low doses (2 mg) and i t s metabolism i n
t h e body i s q u i t e r a p i d , only nanogram l e v e l s o f t h e
334
PHYSOSTICMINE SALICYLATE
8.6
335
ChromatoaraDhic Methods
8.6.1
Paper Chromatography
C l a r k e (16) d e s c r i b e d t h e f o l l o w i n g t e c h n i q u e which was r e p o r t e d f o r t h e a n a l y s i s of
n i t r o g e n o u s b a s e s i n c l u d i n g physostigmine.
Whatman No.1 s h e e t s were b u f f e r e d by d i p p i n g i n
a 5% s o l u t i o n o f sodium dihydrogen c i t r a t e and
a s o l v e n t composed of 4 . 8 g c i t r i c a c i d i n a
m i x t u r e of 130 m l water and 870 m l of n - b u t a n o l
was used. An Rf v a l u e of 0.43 was r e p o r t e d
f o r physostigmine ( 6 8 , 6 9 ) .
The f o l l o w i n g systems were a l s o employed
f o r t h e i d e n t i f i c a t i o n of physostigmine.
S o l v e n t system
Paper
Ref.
-
impregnated
w i t h formamide + ( 7 0 )
8% ammonium
format e
Chloroform
Chloroform m i x t u r e w i t h benzene,
toluene-xylene
impregnated
w i t h formamide
8.6.2
(71)
336
A l k a l o i d s were e x t r a c t e d from b i o l o g i c a l
m a t e r i a l w i t h 0.02 N H2S04 a t pH 2.5-3.0.
The e x t r a c t was a l k a l i n i z e d w i t h 20% NaOH
t o pH 8-9, e x t r a c t e d with CHC13 and chromatographed on TLC of s i l i c a g e l KSK. CHC13 MezCO-EtzHN (50:30:2); E t 2 0 - Me2CO - NH3
(40:20:2) and CHC13 - Me2CO-NH3 (30:30:2)
were t h e s o l v e n t systems. The Rf of physost i g m i n e i n t h r e e d i f f e r e n t s o l v e n t systems
was r e p o r t e d ( 76) :0.55 i n MeOH - s t r o n g ammo n i a (100 :1 . 5 ) ,O .12 i n Cyclohexane - t o l u e n e d i e t h y l a m i n (75:15:10) and0.36in C H C 1 3 - MeOH
( 9 0 : 10) a c i d i f i e d potassium permanganate s o l u t i o n was used f o r l o c a t i o n of t h e s p o t s .
8.6.3
PHYSOSTIGMINE SALICYLATE
331
1
l
!
!
8 1 2 ' 18
~
2 mIImin
1
~!
22
26
l~ l
30 min
F i g . 10 HPLC of pilocarpine, physostigmine and preservatives. Peak: 1 = pilocapine; 2 = salicylate; 3 = methyl p-hydroxybenzoak; 4 = physostigmine; 5
tion: UV. 235 nm. ( 7 7 ) .
338
1
1
1
1
1
0
4
8 min
F i g . 11 HPLC of an aged physostigmine solution (left) and a rubrcserine standard solution (righl).
Peaks: 1 Rubrcscrine; 2 = salicylate; 3 = physosligmine. Detection: UV, 292 nm. ( 7 7 ) .
E
PHYSOSTlGMINE SALlCYLATE
339
340
PHYSOSTIGMINE SALICYLATE
341
342
8.7
Other Methods
8.7.1
PolaroeraDhic Methods
O s c i l l o g r a p h i c polarography h a s been employed
t o determine t h e s t a b i l i t y of physostigmine
ophthalmic s o l u t i o n s . Such s o l u t i o n s were
found t o be most s t a b l e a t pH v a l u e between
2 . 0 and 4 . 5 (82,83).
8.7.2
Coulometry
Physostigmine s u l f a t e was determined by a
c o u l o m e t r i c method, a p r e c i s i o n o f 1.0 CV
was r e p o r t e d ( 8 4 ) .
9.
Therapeutic Uses
Physostigmine i s long well known a s an a n t i c h o l i n e s t e r a s e
i n h i b i t o r i . e . i t i s a choZinergic drug.
As p h y s o s t i g m i n e i s a t e r i a r y amine a l k a l o i d , i t h a s t h e
a b i l i t y t o p e n e t r a t e t h e blood b r a i n b a r r i e r , while neos t i g m i n e ( a s y n t h e t i c r e l a t e d d e r i v a t i v e ) which i s a
q u a t e r n a r y ammonium i o n , i s n o t c a p a b l e of c r o s s i n g t h e
b a r r i e r (37, 8 5 ) .
Physostigmine i s a r e v e r s i b l e a n t i c h o l i n e s t e r a s e which
e f f e c t i v e l y i n c r e a s e s t h e c o n c e n t r a t i o n of a c e t y l c h o l i n e
a t t h e s i t e s of c h o l i n e r g i c t r a n s m i s s i o n , it t h e r e f o r e
i n h i b i t s t h e d e s t r u c t i o n o f a c e t y l c h o l i n e s t e r a s e and
t h e r e b y p r o l o n g s and e x a g g r a t e s t h e e f f e c t of a c e t y l c h o l i n e ( 8 5 ) . I t can r e v e r s e b o t h c e n t r a l and p e r i p h e r a l
a n t i cho 1i n e r g i a .
I t i s t h e r e f o r e used t o r e v e r s e t h e e f f e c t upon t h e c e n t r a l
nervous system, caused by c l i n i c a l o r t o x i c dosages o f
drugs c a p a b l e of producing t h e a n t i c h o l i n e r g i c syndrome (85).
Physostigmine can r e v e r s e such syndrome a s d e l i r i u m and
h a l l u c i n a t i o n r e s u l t i n g from b e l l a d o n n a a l k a l o i d overdose
(86,87) ; a c u t e t r i c y c l i c a n t i d e p r e s s a n t poisoning (88) ;
i n t o x i c a t i o n from asthma powders (stramonium) and s l e e p i n g p r e p a r a t i o n s (89,90) ; p h e n o t h i a z i n e and diazepaminduced come (91); a n t i h i s t a m i n e - i n d u c e d excitement and
d e p r e s s i o n (37) and r e v e r s a l o f a m i t r i p t y l i n e i n t o x i c a t i o n
(411 *
Since physostigmine is rapidZy hydroZyzed by cholinestrase
(92), repeated therapeutic doses may be necessary at 30
to 60 minute intervaZs (37).
343
PHYSOSTIGMINE SALICYLATE
Ocular therapy
A n t i c h o l i n e s t e r a s e a g e n t s p a r t i c u l a r l y physostigmine ( a s
t h e s a l i c y l a t e s a l t ) a r e o f g r e a t v a l u e i n t h e management
of t h e primary t y p e glaucoma a s well as c e r t a i n c a t e g o r i e s
of t h e secondary t y p e ( 9 3 ) .
Physostigmine produces a f a l l i n i n t r a o c u l a r p r e s s u r e i n
primary t y p e glaucoma ( 9 3 ) .
Low c o n c e n t r a t i o n s of physostigmine a r e r e p o r t e d t o d e c r e ase t h e b l u r r e d v i s i o n and p a i n a s s o c i a t e d w i t h t h i s cond i t i o n (94).
In a l t e r n a t i o n w i t h a m y d r i a t i c drug such a s a t r o p i n e ,
physostigmine h a s proven u s e f u l f o r t h e b r e a k i n g of adhesi o n s between t h e i r i s and t h e l e n s o r c o r n e a (93,95,96).
Alzheimer's disease
A d e f i c i e n c y of f u n c t i o n a l c h o l i n e r g i c neurons, has been
observed by s e v e r a l groups i n p a t i e n t s w i t h p r o g r e s s i v e
dementia of Alizheimer t y p e . Physostigmine h a s been
employed i n t h e e a r l i e r s t a g e s o f t h e d i s e a s e t o improve
memory. R e s u l t s have been v a r i a b l e , a l t h o u g h some i n v e s t i g a t o r s have employed dosage s c h e d u l e s t h a t appear t o c a u s e
t r a n s i e n t improvement (97).
I n Myasthenia gravis
Physostigmine h a s been used i n Myasthenia g r a v i s ( 9 8 ) ,
b u t f o r t h i s purpose i t i s now l a r g e l y r e p l a c e d by n e o s t i g mine ( 9 3 ) .
O t h e r a r t i c l e s and reviews d e a l i n g w i t h t h e pharmacology
and t h e r a p e u t i c u s e s o f p h y s o s t i g n i n e have been r e p o r t e d
(99-103).
ACKNOWLEDGEMENT
The a u t h o r s would l i k e t o thank M r . Uday C .
Sharma, Department o f Pharmacognosy, C o l l e g e
o f Pharmacy f o r h i s v a l u a b l e h e l p i n t y p i n g
t h e manuscript.
344
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Prazosin Hydrochloride
Leonard J. Kostek
Pfizer Inc. Central Research
Groton, Connecticut 06340
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Introduction
Description
Synthesis
Physical properties
4.1
Infrared Spectrum
4.2
Ultraviolet Spectrum
Proton Nuclear Magnetic Resonance Spectrum
4.3
Carbon-1 3 Nuclear Magnetic Resonance Spectrum
4.4
4.5
Mass Spectrum
Elemental Analysis
Solubility
Polymorphic and Solvated Forms
Identification
8.1 Thin-Layer Chromatography
8.2
Infrared Absorption Spectroscopy
8.3 Ultraviolet Absorption Spectroscopy
Methods of Analysis
9.1
HPLC
9.2
Potentiometric Titration
9.3
Thin-Layer Chromatography
Pharmacokinetics and Metabolism
Determination in Biological Fluids
Bibliography
LEONARD J . KOSTEK
352
1.
Introduction
Prazosin hydrochloride was synthesized and developed by the
Medicinal Chemistry Laboratories of Pfizer Inc.1 Prazosin
hydrochloride was introduced as a novel antihypertensive agent w i th
pharmacological activity attributed t o arteriolar vasodilation. The
antihypertensive activity was initially reported by Scriabine et. al.2 A
description of the hypotensive action o f prazosin and the
pharmacology of the drug was first explained by Constantine et. al.3 A
further review o f the pharmacology and therapeutic efficacy of
prazosin hydrochloride is given by Brogden et. al.4 This review was
updated in 1983 by Stanaszek et. al.5 The anhydrous alpha form is the
preferred polymorphic form of prazosin hydrochloride due t o i t s nonhygroscopic characteristic leading t o ease o f handling and
formulation. The prazosin hydrochloride alpha polymorphic form was
used throughout the clinical efficacy studies. Clinical observations of
prazosin hydrochloride in ambulatory patients was reported by
Cohen.6 This preliminary study was conducted using a crossover
protocol wit h prazosin hydrochloride, polythiazide, prazosin hydrochloride/polythiazide combination and a placebo.
2.
Description
2.1
Nomenclature
Chemical Name
1-(4-Ami no-6.7-di methoxy-2-quinazol inyl)-4-(2furoy1)piperazine monohydrochloride
Generic Name
Prazosin Hydrochloride
Laboratorv Code
CP-12,299-1
Trade Names
353
PRAZOSIN HYDROCHLORIDE
2.2
HCI
2.3
Synthesis
The synthetic process for prazosin hydrochloride has been
documented in U.S. Patent #3,511,836.1 The schematic representation
of the synthetic process for prazosin hydrochloride is shown in Figure
(1). 2-Amino-4,5-dimethoxybenzoic acid is reacted with potassium
cyanate while in acetic acid forming an intermediate which is cyclized
with the addition of sodium hydroxide. The reaction mixture i s
acidified with hydrochloric acid forming 6,7-dimethoxy-2-4-(1H,3H)quinazolinedione crystals. The dione product i s refluxed with
phosphorous oxychloride in N, N-di methylani Iine to produce 2.4dichloro-6,7-dimethoxyquinazoline. Amination of the dichloro
compound in tetrahydrofuran with anhydrous ammonia produces one
of the intermediates 2-chloro-4-amino-6,7-dimethoxyquinazoline
needed in the final coupling step to synthesize prazosin hydrochloride. The second coup1ing intermediate, 1-(2-furoyl)piperazine, is
produced by a reaction of piperazine with hydrobromic acid and 2furoyl chloride in an aqueous-ethanol solvent. The two immediate
precursors 2-chloro-4-amino-6,7-dimethoxyquinazol ine and 142furoyl) piperazine are coupled by refluxing i n isoamyl alcohol t o
produce the desired product, prazosin hydrochloride.
CI-c
-42
355
PRAZOSIN HYDROCHLORIDE
Physical Properties
4.1
Infrared Spectrum
The infrared absorption spectrum of prazosin hydrochloride
was obtained as a KBr pellet using a Perkin-Elmer model 21
infrared spectrophotometer. The infrared spectrum of prazosin
hydrochloride is shown in Figure (2). Assignments for characteristic infrared absorption bands for prazosin hydrochloride in
potassium bromides have been listed in Table (1).
Table (1).
4.2
Wavelengths
Microns
Assi gnment
6.26
6.12
6.75
7.02
8.08
9.02
10.02
Amide stretching
C = N quinazoline stretching
C = C aromatic ring stretching
hydrogen bending in piperazine ring
methoxy group vibrations
C-0-Csingle bond stretching in the furan ring
methoxy group vibrations
Ultraviolet Spectrum
The ultraviolet absorption spectrum of prazosin
hydrochloride is shown in Figure (3) a t a concentration of 0.0056
mg/ml. The spectrum was obtained on a Hewett-Packard model
8450. This prazosin hydrochloride spectrum in methanolic 0.01 N
hydrochloric acid is characterized by two well defined maxima at
246 nrn and 329 nm. The molar absorptivity values for a bulk
sample of prazosin hydrochloride when determined in
methanolic 0.01N hydrochloric acid were 137 + I- 3 at 246 nm and
27.6 +/- 0.3 at 329 nrn. The ultraviolet absorption spectrum of
cl
PRAZOSIN HYDROCHLORIDE
351
HYDROCHLORIDE
PRFIZOSIN
1.0
0.90
0.80
0.70
0.60
a
m
0.50
p:
0.40
a
0.30
0.20
0. I D
0.0
O
N
WFIVELENGTH
Figure(3)
C n m 3
U l t r a v i o l e t Absorption Spectrum of P r a z o s i n H y d r o c h l o r i d e a t a
Concentration of Approximately 0.0056 mg/ml i n M e t h a n o l i c
0.01 N H y d r o c h l o r i c Acid
358
LEONARD J . KOSTEK
Chemical Shift
Proton Assiqnment
proton in furan ring at C-5"
aromatic proton a t C-5
amide protons
proton in furan ring a t C-3"
aromatic proton at C-8
proton in furan ring at C-4"
piperazine protons
methoxyl protons
4.4
multiplet
singlet
singIets
doublet
sing let
quartet
m u Iti plet
doublet
7.905
7.779
8.685, 8.990
7.096
7.648
6.664
4.002
3.876
PRAZOSIN HYDROCHLORIDE
r.
R
.r
<
<
359
YI
0
(D
0
P
2
Is
! : o
r2
tI:
P=.
PRAZOSIN HYDROCHLORIDE
361
NH2
HCI
Chemical Shift
0
161.23
158.48
155.21
151.39
146.78
144.92
136.35
116.01
1 1 1.33
105.07
101.72
99.28
56.00
56.21
44.39
43.30
Assi q n ment
nonprotonated quinazoline carbon l a
nonprotonated carbon C-2 or carbonyl
phenyl C-7
nonprotonated carbon C-2 or carbonyl
phenyl C-6
furan carbon C-5"
quinazoline carbon at amide linkage C-4
furan carbon C-3"
furan carbon C-4"
protonated phenyl carbon C-5
nonprotonated quinazoline carbon C-4a
protonated phenyl carbon C-8
methoxyl carbon at C-6 linkage
methoxyl carbon at C-7 linkage
piperazine carbons at C-2', 3', S', 6' w i th
some unexplained
line broadening at the lower chemical shift
362
LEONARD J. KOSTEK
4.5
Massspectrum
Elemental Analvsis
A reference sample o f prazosin hydrochloride was analyzed for
carbon, hydrogen, nitrogen, chloride and methoxyl content. The
analysis for C, H, N was conducted using a Perkin-Elmer model 2400
elemental analyzer. The chloride content was determined using a
Schronigher combustion technique followed by titration w i t h a
standardized silver nitrate solution. The methoxyl content was
determined by refluxing prazosin hydrochloride in the presence of
hydroiodic acid, treating the refluxed solution w i t h bromine and
titrating wit h standardized sodium thiosulfate solution. The results
from these determinations are given in Table (2). These data show
excellent correlation w i th the theoretical calculated values.
Table (2).
6.
Element
% Calculated
% Found
C
H
N
54.35
5.28
16.68
54.34
5.26
16.49
CI
8.44
8.42
Methoxyl
14.78
14.70
Solubility
The following solubility data for prazosin hydrochloride (Table 3)
have been obtained at ambient temperature.9
PRAZOSIN HYDROCHLORIDE
363
95.
98.
85.
88.
X.
a.
65.
Figure(6)
364
Figure(7)
LEONARD J. KOSTEK
PRAZOSIN HYDROCHLORIDE
Table (3).
365
Solvent
acetone
methanol
ethanol
dimethylformamide
dimethylacetamide
water (pH = 3.5)
chloroform
Approximate Solubility
(milligramlmilliliter)
0.0072
6.4
0.84
1.3
1.2
1.4
0.041
7.
LEONARD J . KOSTEK
366
J
A
5000 3000
1500
4000 2000
1000
900
Frequency (CM-')
Fiaure(8)
800
I,,,rId.........l.........I
1000
800 700
900
Frequency (CM-')
PRAZOSIN HYDROCHLORIDE
Fiqure(9)
367
Powder X-ray D i f f r a c t i o n P a t e r n s f o r P r a z o s i n
R y d r o c h l o r i d e Polyrnorphs and Solvates
LEONARD J. KOSTEK
368
characterize each of the solvated forms are listed i n Table (4). The
characteristic powder x-ray diffraction peaks for each prazosin
hydrochloride solvated form are listed in Table (5).
8.
Identification
8.1 Thin-Layer Chromatoqraphy
The thin-layer chromatographic system used for identification of prazosin hydrochloride separates prazosin from
potential contaminates. The system as descri bed in the USP
XXIlO consists of silica gel GF plates, ethyl acetate:diethylamine
(19.1, v/v) asdeveloping solvent and 254 nm ultraviolet light as
the detection method. The identity test is conducted with 5.0
mg/ml solutions of prazosin hydrochloride reference standard
and sample. An aliquot, 0,100 ml of reference solution,
approximately 2.5 inches long isapplied t o one half of the origin
line. Similarly, 0.100 ml of the sample solution i s applied t o the
other half of the origin line. The thin-layer plate i s developed
using an ascending technique until the solvent front reaches one
inch of the top of the plate. The thin-layer plate is air dried, then
examined under 254 nm ultraviolet light. Prazosinappearsas a
blue band on a yellow-green fluorescent background.
8.2 Infrared Absorption Spectroscopy
Table 4.
Characteristics Infrared Absorption Bands for Prazosin Hydrochloride Polymorphic and Solvated Forms
Characteristic Bands
cm-1
microns
band shape
alpha
795
12.6
sharp
beta
770
13
sharp
gamma
770
743
13
13.4
doublet
doublet
polyhydrate*
1260
755
1000
7.95
13.3
10
sharp
broad
doublet
anhydrate
1260
755
1005
7.95
13.3
9.95
sharp
broad
triplet
870
11.5
sharp
Polymorphic Form
Solvated Form
methanolate
Table 5.
Characteristics Powder X-rav diffraction peaks for Prazosin Hvdrochloride Polvmorphic and Solvated Forms
Polymorphic Form
Characteristic Peak
alpha
1 :3:3: 1 quartet centered at 23 degrees sharp bands at 93 degrees and 23.6 degrees
beta
gamma
Solvated Form
polyhyd rate
anhyd rate
same as monohydrate form sharp bands at 10.5 degrees, 12.0 degrees and 16.9 degrees
doublets at 24.5 degrees, 26.5 degrees
methanolate
sharp bonds at 10.5 degrees, 17.3 degrees, 23.9 degrees, and 25.5 degrees
PRAZOSIN HYDROCHLORIDE
371
XXI' 1.
9.
Methods of Analysis
9.1
9.2
Potentiometric Titration
Prazosin hydrochloride is assayed potentiometrically by
dissolving thesample in ethanol:water(l:l, v/v) and titrating
with standard aqueous sodium hydroxide and a combination pH
electrode i s used for the potentiometric system. The theoretical
neutral equivalent for prazosin hydrochloride i s 419.87. When a
reference standard of prazosin hydrochloride was assayed by this
potentiometric procedure, a value o f 420.9 was obtained for the
neutral equivalent. The pKa value for this reference standard
determined in ethanol:water ( 7 :1, v/v) was determined t o be
6.54. A typical potentiometric curve for prazosin hydrochloride
obtained on a Metrohm model E636 autotitrator is presented in
Figure (10).
9.3
Thin-Laver Chromatoqraphy
Prazosin hydrochloride i s analyzed for potential impurities by
a thin-layer chromatographic procedure asdescribed in the USP
XXIl2 and USP XXI Supplement 713. The analysis involves three
thin-layer chromatographic systems and t w o detection methods.
The method is capable o f separating prazosin hydrochloride from
small amounts o f five potential impurities. Also, the procedure
can establish the level o f these potential impurities, if present, i n
t he prazosin hydrochloride sample. The chromatographic procedure uses silica gel GF (Merck-Darmstadt) plates as the solid
phase adsorbent. The three developing solvents and the t w o
detection methods are listed in Table (6). The prazosin hydrochloride sample is prepared at a concentration of approximately
u
W
-1-
=a
\ -
- %
Figure(l0)
PRAZOSIN HYDROCHLORIDE
373
Table 6.
Relative Mobilities in
Solvent Systems*
Detection
Method
Compound
(a)
(b)
-
(1) 2-Chloro-4-amino-6,7-
0.27
0.65
0.40
0.00
0.33
0.70
0.15
0.00
0.25
2
1
0.00
0.25
0.05
0.18
0.00
0.73
0.75
0.80
0.48
1
1
(2)
(3)
(4)
(5)
(6)
dimethoxyqui nazoline
1-(2-Furoyl)piperazine
1,4-Bis-(4-amin0-6,7-dimethoxy-2qui nazol iny1)-piperazi ne
4-Amino-6,7-dimethoxy-2-(1pi perazi nyl)-qui nazol i ne
di hydrochloride tri hydrade
1,4-Bis-(2-furoyl)piperazine
Prazosin Hydrochloride
(C)
PRAZOSIN HYDROCHLORIDE
10.
315
Pharmacokineticsand Metabolism
The pharmacokineticsand metabolism of prazosin hydrochloride
have been studied by numerous investigators. Early studies by
Wood14 using normotensive volunteers (six male and four female)
given a single ( 5 mg) dose after overnight fasting found peak plasma
levels at 2-3 hours after oral administration. Plasma concentrations
followed first order kinetics with a half-life of 3.9 hours. Likewise,
Collins15 found significant first pass liver metabolism of prazosin
hydrochloride. Five hypertensive volunteers were given a single dose
of between 2 mg and 5 mg of prazosin hydrochloride after fasting.
Plasma half-life was determined to be a t 3.28 hours and mean liver
extraction t o be approximately 29.9%.
Taylor et. a1.16 performed radiolabeled prazosin hydrochloride
studies in rats and dogs. Within 30 minutes prazosin was found
distributed into the lung, heart and vascular tissues of the dog.
Urinary excretion of prazosin by rats and dogs was low with the major
route of elimination by biliary secretion through the feces.
Biaavailability of prazosin in beagle dogs was found t o be dosedependent by Baughman et. d.17 Metabolism in the rat and dog was
found t o occur primarily by 6- or 7-0-dealkylation with subsequent
glucuronide formation. To a lesser extent, N-dealkylation was
observed with the piperazine ring opening. Studies in man with
unlabeled prazosin hydrochloride produced similar metabolites t o
those found in the rat and dog. A further review of prazosin
pharmacokinetics has been summarized by Siedmanl8 along with the
effects of an alpha-adrenoceptor blockade, with special reference t o
prazosin, in the treatment of hypertension.
Bateman et. af.19 studied the pharmacokinetic effects of prazosin
hydrochloride after oral and intravenous dosing using erect and
supine volunteers. Intravenous and oral dosing each produced a mean
terminal (beta) half-life of 2.9 hours with oral bioavailability a t 56.9%.
There was a greater effect in blood pressure after intravenous rather
than oral dosing plus a significant correlation between blood pressure
lowering and drug plasma concentration after intravenous administration. Other prazosin hydrochloride studies have been conducted
t o describe pharmacokinetics in man by Hobbs et. al.20, the effects of
food on prazosin hydrochloride intake by Verbesselt et. al.27 and
plasma levels, blood pressure and heart rate by Wood et. al.22 An
additional study by Hobbs and Twomey23 investigated the protein
binding properties of prazosin. They found that approximately 92
percent of the prazosin was bound t o serum proteins whether
prazosin was administered separately or in conjunction with other
frequently administered drugs.
11.
316
LEONARD J . KOSTEK
PRAZOSIN HYDROCHLORIDE
12. Biblioaraphv
US Patent #3,511,836 t o Dr. Hans-Jurgen Hess, Pfizer Inc., May
1970.
Scriabine,A., Constantine, J. W., Hess, H. -J., and McShane, W.
K. "Pharmacological Studies w i th Some New Antihypertensive
Aminoquinazolines." Experientia, 24, 1968, p. 1 150.
Constantine, J. W., McShane, W. K., Scriabine,A. and Hess, H. J. Hypertension: Mechanisms and Manaqement. Editors:
Onesti, G., Kim, K. E. and Moyer, J. H.,p. 429. New York:
Grune and Stratton, Inc., 1973.
Brogden, R. N., Heel, R. C., Speight, T. M., Avery, G. S.
"Prazosin: A Review of i t s pharmacological properties and
therapeutic efficacy i n hypertension." Druqs, 14, 1977,P. 163.
Stanaszek, W. F., Kellerman, D., Brogden, R. N. and
Romankiewicz, J. A,, "Prazosin Update: A review of its
pharmacological properties and therapeutic use i n
hypertension and congestive heart failure." Druqs, 25, 1983,
333-384.
Cohen, B. M., "Prazosin hydrochloride (CP-12,299-l), and oral
anti-hypertensive agent: preliminary clinical observations in
ambulatory patients."l. Clinical PharmacoloqK, 1970, 10, p.
408.
Honkanen, E.,Taskinen, J., Koivisto, M. and Nore, P., "New
practical synthesis of prazosin." 1. Het. Chem., 17, June 1980,
p. 797-798.
Finegan, R. L. and Forcier, G.A., Pfizer Inc., personnel
communication, 1972.
Honkanen, E., Taskinen, J., Koivisto, M. and Nore, P., "Mass
spectrometric fragmentation of prazosin and synthesis of i t s
deuterium labelled derivation." Finn. Chem. Lett., 1, 1979,
p.20-22.
USP XXI, 1985, p. 868-869.
USP XXI, 1985, P. 868.
USP XXI, 1985, P. 869.
USP XXI, Supplement 7, 1988, P. 2820.
Wood, A. J., "Pharmacokinetics o f prazosin i n man." Clin. and
EXP. Pharmacol. and Phvsiol., 2, 1975, p. 446.
Collins, I. S., "Pharmacokinetics of prazosin, a new
antihypertensive compound." C/in. and Exp. Pharmacol. and
Phvsiol. 2, 1975, p. 445.
Taylor, J. A., Twomey, T. M . and Wittenau, M. 5 . V., "The
metabolic fate o f prazosin." Xenobiotica, 7, No. 6, 1977, p.
357-364.
Baughman, R. A. Jr., Sorgel, F., Mico, B. A. and Benet, L. Z.,
"Dose-dependent bioavailability of prazosin in beagle dogs."
J. o f Cardiovascularfharm., 5, 1983, p. 613-617.
371
LEONARD J. KOSTEK
378
(18)
Seideman, P., "Pharmacokinetic and dynamic aspects of alphaadrenoceptor blockade in hypertension." Acta Med. Scand.,
wl.,
1982,665, p. 61-66.
Bateman, D. N., Hobbs, D. C., Twomey, T. M., Stevens, E. A. and
Rawlins, M. D., "Prazosin, pharmacokineticsand
concentration effect." Eur. 1. Clin. Pharmacol. 16, 1979, p. 177181.
Hobbs, D. C., Twomey, T. M. and Palmer, R. F.,
"Pharmacokinetics of prazosin in man." 1. Clin. Pharmacol.,
18, 1978, p. 402-406.
Verbessett. R., Mul1ie.A.. Tjandramage, T. B., DeSchepper, P.J.
and Dessain, P., "The effect of food intake on the plasma
kineticsand toleration of prazosin." Acta rher., 2, 1976, p. 27.
Wood, A. J., Bolli, 6. and Simpson., F. O., "Prazosin in normal
subjects: plasma levels, blood pressure and heart rate."
Clin. Pharmacol., 1, 1976, p. 199.
Hobbs, D. C. and Twomey, T. M.,"Protein binding properties
of prazosin." Chem. Path. andPharmaco/.,25, No. 1, 1978, p.
188- 192.
Yee, Y. G., Rubin, P. C. and Meffin, P., "Prazosin determination
by high-pressureliquid chromatography using fluorescence
detection." 1. Chrom., 172, 1979, p. 313-318.
Reece, P. A., "Quantification of prazosin in plasma by highperformance liquid chormatography." 1. Chrom., 221, 1980, p.
188- 192.
Dokladalova, J., Coco. 5. J., Lernke, P. R., Quercia, G. T. and
Korst, 1. J., "Determination of polythiazide and prazosin in
human plasma by high-performance liquid chromatography. ''
1. Chrom., 224, 1981, p. 33-41.
Bhamra, R. K., Flanagan, R. J. and Holt, D. W., "Highperformance liquid chromatographic measurement of
prazosin and terazosin in biological fluids." J. of Chrom., 380,
1986, p. 216-221.
Piotroskii, V. K., Belolipetskaya, V. G., EI'Man, A. R. and
Metelitsa, V. I.,"lon-exchange high-performance liquid
chromatography in drug assay in biological fluids." 1.Chrom.,
278, 1983, p. 469-474.
TETRACAINE HYDROCHLORIDE
380
MOHAMMAD RIAZ
1. DescriDtion
1.1 memica1 Name
m eNam
Pontocai ne hydrochloride, Anestaron(21,
Anethaine(3)
Structural
. HCl
381
TETRACAINE HYDROCHLORIDE
Molecular Weiaht
300.83
3. Armearance. Color. Odor(2)
59.89 sb
9.31 74
C1
0
11.78 %
10.64 W
8.38%
5. Physical ProDerties
5.1 Loss On Drying(5)
When dried t o a constant weight at 100 to 105 OC,
the drug loses no more than 1 % of i t s weight.
5.2 Solubilitv(6)
The drug is soluble at 20 OC in 7.5 parts of water,
40 parts of alcohol and 30 parts of chloroform; practically
insoluble in ether and acetone.
5.3 Aciditv
A one percent solution has a pH of 4.5 t o 6.5(6).A
MOHAMMAD RIAZ
382
5.9
TETRACAINE HYDROCHLORIDE
5.E
SCAN RATE.
mg
10.W d.g/min
383
MOHAMMAD RIA2
384
Table 1
\max
So1vent
E( 1Sb, 1cm)
H$l
469,876
14108,
26352
509,55,76*
---
308
----
(nm1
225,310
CHC13*
--- --#
7586,
295 12
27542
not given.
Assignment
1610
TETRACAINE HYDROCHLORIDE
385
386
MOHAMMAD RIAZ
TETRACAINE HYDROCHLORIDE
387
1687
2966
3422
1.95
2.15
2.5 1
2.66
2.80
2.96
3.12
3.30
3.48
3.56
4
4
5
5
7
6
10
10
17
8
3.78
3.97
4.22
4.43
4.83
5.05
5.42
6.28
8.38
12.55
10
11
20
12
67
8
12
42
100
MOHAMMAD W
388
r9
96*c;-H2c
-H2N
-,
5
H- N-CH2-CH2
Proton
posit ion
Chemical Shift
(PPm)
4
N CCH3
H 34
-CH2 -CH3
Number of
protons
Multiplicity
triplet
2
sixtet
1.4937
2
quintet
2 . ~ ~ 9
6
s1ngl et
3.0456
2
triplet
3.5285
2
mu1tiplet
4.5180
2
multiplet
6.6282
2
doublet
7.7641
2
doub1et
4.7422(residua\protons present in Dfl)
0.8443
1.3051
MOHAMMAD RIAZ
390
10
H- N-
Carbon atom
Chemical shift
(PPm)
O f f resonance
multlpliclty
1
2
15.616
22.094
quartet
3
4
32.849
44.652
5
6
7
0
9
10
11
12
45.506
58.340
60.903
113.631
117.205
134.095
155.730
169.556
5.15
triolet
triplet
quartet
triplet
triplet
triplet
doublet
singlet
doublet
singlet
sing1et
Mass SDectrurn
fI 4
L
Fig 5 Proton decoupled FT-13C NMR spectrum
ll
392
f
G
--_
-H
MOHAMMAD RIA2
394
were used.
Electron Impact
Sample introduction:
Direct probe
Electron energy: 70 electron volt
50 OC
Sample temperature:
Ion source temperature: 200 OC
Chemical ionizat ion
Sample introduction:
Direct probe
Electron energy: 200 electron volt
Reagent gas:
(i) isobutane
(ii) ammonia
Pressure i n ion source region: 10'4 mbar
100 OC
Sample temperature:
Ion source temperature: 200 OC
The molecular ion peak at 264 m/e i s absent in electron impact
mass spectrum(See Fig 7).But protonated molecular ion
peak(M+ 1 or M+H) at 265 m/e i s the base peak in chemical
ionization mass pectra(See Fig 8 & 9).The assigment of m/e
values t o positive ions i s given i n Table 6
r 72
m/e
Ion
265
266
194
[ mCH2CH2N(CH& i s lost]
395
TETRACAINE HYDROCHLORIDE
193
+OCOHC~H~NHC&J
176
150
72
71
58
44
42
Polymeric Form
(m. Dt.1
130- 136 OC
139 OC
Refractive Indices
1.497
1.584
> 1.740
1.697
6. Synthesis
MOHAMMAD RIAZ
396
I*
n
9.
8.
n
m.
a.
0.
5.
Y.
4s.
Y.
X.
11.
x.
a.
IS.
I I
IN.
TETRACAINE HYDROCHLORIDE
0.
u.
0.
S
(I.
a.
I.
1.
8
397
MOHAMMAD RIAZ
398
111
%.
9,
..
6,
5.
m.
0.
a.
5.
PI.
(I.
9.
I.
3.
?i
a.
IS.
II.
5.
I-
. ' '
.I
I.
N
TETRACAINE HYDROCHLORIDE
399
400
MOHAMMAD R I M
7.2.6
Thirty to f i f t y mg of the drug are dissolved
in 30 ml of water and after the addition of 1.0 g potassium
bromide, 3 ml of 30 W nitric acid and one drop of 4ethoxychrysoidine solution, the mixture i s titrated w i t h 0.1 N
potassium bromate to a color change from red to yellow.
Alternatively the end-point can be determined by an
amperometric method.
401
TETRACAINE HYDROCHLORIDE
Precision:
As
10-20 mg
Potent 1omet rlc, P t
indicator vs
Ag/AgCI
reference
0.2 % relative standard
deviation
MOHAMMAD RIM
402
TETRACAINE HYDROCHLORIDE
403
MOHAMMAD RIA2
System A(31)
Column: Column (6ft x 4 mm) packed with 3.8 W
slllcone rubber UC W 98 on Dlatoport 5,
80/ 100 mesh (Hewlett Packard).
Column temperature:
200 OC
Detector temperature: 230 OC
Injection port temperature:
200 OC
64
A t tenuat ion:
50 cc/mln
Carrler gas(nitrogen)flow rate:
Hydrogen flow rate:
35 cc/rnin
A i r flow rate:
300 cc/min
Detector:
Flame ionization
Interna1 standard( I.S.
1:
Procaine
hydrochloride( 10
mg/ml in water)
Retention time:
1 1 min. (tetracaine
hydrochloride), 5
min.(procaine hydrochloride)
Sample:
Inject 3 ~1 chloroform extract
The concentration of tetracaine hydrochloride was
determined by the following formula.
Concentration(mg/ml) =(Area of sample peak/Area of
internal standard peak) X (Area of 1.5. peak/Area of R.5. peak) x
(Conc. of R. S. i n mg/ Volume i n ml o f sample used)
where R. S. i s reference standard. It was
tetracaine hydrochloride( 10 mg/ml in water).
System B(32)
Column:
290 OC
Injection port temperature:
265 OC
Attentuation :
20 or 50 x 102
405
TETRACAINE HYDROCHLORIDE
Chart speed:
t a r r l er gas:
Nltrogen
Carrier gas flow rate:
50 ml/mln
Butacaine sul phat e
Internal Standard:
Sample: inject 8 ul of ether extract
A calibration curve of tetracaine/butacaine peak r a t i o
against tetracaine hydrochloride concentration( 10 t o 50 mg)
was made. The method was used for the determination of
tetracaine hydrochloride in eye drop preparations.
7.5.3 ah
iJi
. .
Column:
MOHAMMAD RIA2
406
Solvent system:
TETRACAINE HYDROCHLORIDE
407 t
8. Stabllltv
Tetracaine hydrochlride powder should be stored in air
tight containers, protected from Ilght(3). The Preparations
should be stored at 2-8 OC and also be protected from light
(39).In aqueous solution, tetracaine hydrochloride hydrolyze4to
n-bu t y laminobenzolc acid and 2-dime thy laminoethanol;
decarboxyl at ion of n- but y 1 am inobenzoic acid to but y lani 1ine
occurs and the butylaniline oxidizes t o form various co^_lored
products. The hydrolysis of the drug is catalysed by both
hydrogen and hydroxyl ions. At 25 OC, solutions are most stable
at about pH 3.33).The degradation product, p-nbutylaminobenzoic acid is only sparingly soluble in water and
potentially can cause crystal depoisition in amethocaine
hydrochloride preparations such as injections, eye drops,
topical solution and sterile solution(39).
The addition of alkali hydroxides or carbonates to
tetracaine hydrochloride solutions precipates tetracaine base
as an oily liquid(39). The stability of the drug appear to be
unaffected by the addition of dextrose(40). The stability of
408
MOHAMMAD RIM
Lh(intravenous in mice)
LDm(subcutaneous i n mice)
13 mg/Kg
35 mg/Kg
1 1. DOSaQe Forms
( 1) U.S.P. HI(12)
TETRACAINE HYDROCHLORIDE
409
MOHAMMAD RIAZ
410
TETRACAINE HYDROCHLORIDE
411
BY
4 14
Contents
1.
Description
1.1 Nomenclature
1.2 Formulae
1.3 Molecular Weight
1.4 Elemental Composition
1.5 Appearance, Color, Odor and Taste
1.6 Occurance.
2.
Physical Properties
2.1
2.2
2.3
2.4
Melting Range
Solubility
pH and Stability
Spectral Properties
3.
Synthesis
4.
Metabolism
5.
Vitamin Deficiency
6.
Human Requirements
7.
Methods of Analysis
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.0
7.9
7.10
7.11
Elemental Analysis
Identification
Titrimetric Method
Gravimetric Method
Volumetric Method
Colorimetric Method
Spectrophotometric Method
Polarographic Method
Fluorimetric determination
Atomic Absorption Spectroscopy
Chromatographic Methods
8.
Acknowledgement
9.
References
THIAMINE HYDROCHLORIDE
415
Thiamine Hydrochloride
1.
Description
1.1
Nomenclature
1.1.1
Chemical Names
a)
3-[(4-Amino-2-methyl-5-pyrimidinyl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium
chloride
monohydrochloride. (1)
b) 3-(4-Amino-2-methylpyrimidin-5-methyl)5-2-hydroxyethyl)-4-methylthiazolium
chloride hydro-
chloride. ( 2 )
1.1.2
Generic Names
Thiamine Hydrochloride, Vitamin Bi.
1.1.3
Trade Names
Vitamin B Hydrochloride, Thiamine chloridehydrochloride, Aneurine hydrochloride, Thiaminiumchloride Hydrochloride, Bedome, Belgiolan, Benerva,
Bequin, Betabion hydrochloride, Betalin S, Betraxin,
Bethiaxine, Bevitex, Bewon, Biuno, Bivatin, Bivita,
Clotiamina, Metabolin, Thiadoxine, Thiavit, Tiamidon,
Tiaminal, Vitaneuron, Thiaminii chloridum, Thiamini
Hydrochloride,
Aneurine
chloride Hydrochloride,
Thiaminium chloride, Thiamin Hydrochloride, Thiamine
chloride.
1.2
Formulae
1.2.1
Empirical
Ci z H i aClzN40S.
416
1.2.2
Structural
1.2.3
1.2.4
Molecular Weight
337.25
1.4
Elemental Composition
C 42.73%
0 4.74%
1.5
H 5.38%
S 9.51%
C1 21.03%
N 16.61%
Occurance
THlAMINE HYDROCHLORIDE
2.
Physical ProDerties
2.1
Melting Range
248" with decomposition. ( 1 )
2.2
Solubility
pH and Stability
418
Spectral Properties
2.4.1
Ultraviolet Spectrum
Infrared Spectrum
Frequency cm-
Assignment
3420, 3495
-NH2
3255
-OH
3000
Aromatic -CH
2930
Aliphatic -CH
1650
C-N
1600
Aromatic C = C
0
0
Ln
0
0
0
cv
0
0
0
?
0
0
u!
0
0
c
3:
0
(D
.rl
0
00
m
.d
.rl
..
.d
a,
-0
0
k
c
0
r(
a,
m
0
N
m
0
0
m
cv
0
(D
(v
0
(v
cv
cv
0
0
k
a,
$
>
B
0
0
U
0
(D
0
OD
0
0
2
0
s
0
I
0
E!
N
0
0
0
0
0
U
N
0
a3
0
r-
:.I
0
0
m
42 1
THIAMINE HYDROCHLORIDE
2.4.3
Proton Spectrum
Group
-CH3
2.53
-CH3
2.61
-CHZ
3.17
-CHZ
3.85
N-CHZ
5.55
s = singlet, d = doublet, m
= multiplet
9.8.
2.4.3.2
13C-NMR Spectra
Peak listing
No.
-
ppm
2.53
2.61
3.17
3.19
3.85
3.86
3.88
2
3
/I
4
5
6
7
8
9
10
>,
10
Fig. 3:
4.80
5.55
8.01
423
a,
425
THlAMINE HYDROCHLORIDE
Table ( 3 ) .
m/e
Relative intensity
Fragment
0
143
30
122
42
112
100
85
30
45
26
36
34
3.
+ CH 2CH 2 0 H
I
Synthesis
The synthesis of
different ways.
Scheme 1 ( 3 )
It is possible to synthesize the pyrimidine nucleus and
the thiazole nucleus separately and afterwards to
connect both parts.
It is also possible to synthesize
one of the nuclei with an extra side branch and
afterwards to connect both parts.
KHALID A . M . AL-RASHOOD E T A L .
426
b)
The thiazol moiety may be synthesized in several
ways.
The method of Buchman consists in condensing
thioformamide with bromoacetopropanol.
By heating the hydrobromide of the pyrimidine compound
with the thiazol compound, the thiamine hydrobormide is
formed.
The bromide hydrobromide may be converted into the
chloride hydrochloirde by treating with AgCl or by
precipitating the practically
insoluble thiamine
picrate and dissolving it in hydrochloric acid.
Ethyl formate
COOC~H,
Ethyl-B-Hydroxy
propionate
COOC,H,
amidine
427
THIAMINE HYDROCHLORIDE
Scheme I (Continued)
NH2
I
CH
II
S
Br
+CH3COChCH,CH,OH
-+
C H,C H,O H
Broacetopropanol
T h i a z o l e compound
Thioformamide
N=C-NHrHBr
iCH,Br
CH3-I
N-CH
T h i a z o l e compound
Heating
Pyrimidine n u c l e u s
N=C -NH,-HBr
CH3-C
C-CH,-N
II II
N-CH
Thiamine Hydrochloride
428
Scheme I I
(4)
429
THIAMINE HYDROCHLORIDE
(4
Scheme I1
Ethyl acrylate
C2H50Na
2-Methyl-5-ethoxymethoyl
6-hydroxy pyrimidine
NH
Acetamidine
c'cYH3
- H2N
C2H50CH,
CHOCH,
2 5
2-Methyl-5-ethoxymethyl
6-chloropyrimidine
430
(9
Scheme I1 c o n t i n u e d
O=C-CH,
O= C- CH,
CPP ._+ AHC/O
CH2COOC2H,
Ethylene o x i d e
Ethyl a c e t o a c e t a t e
0
I
CH 2Ck
Acety 1-but rryl l a c t o n
J
CI
I
CH$OCHCH,CH,OH
Chloro-acetopropanol
(Thio
O=C.CH,CI
-c02
Chloroacetylbutyro1a c t one
NH2
I
1 )
CH2CH,
CHC
Heated w i t h
HC 1
formamide
4-Methyl-5(0-hydroxyethyl)thiazole
S0Cl2
AgCl
Bromo-Pyrimidine
Thiamine Hydrochloride
THIAMINE HYDROCHLORIDE
43 I
silver
Metabolism
Thiamine hydrochloride is usually given by mouth.
Parenteral administration is unnecessary except in
patients with impaired absorption or cardiac failure in
beriberi.
Thiamine is absorbed from the gastro-intestinal tract
It is
and is widely distributed to most body tissues.
not stored to any appreciable extent in the body and
amounts in excess of the bodys requirements are
excreted in the urine.
About 1 mg of thiamine is
metabolised in the body daily and numerous metabolites
are also excreted in the urine. ( 2 )
Thiamine is fundamentally associated with carbohydrate
metabolism.
By combining with the pyrophosphoric acid
in nucleated cells particularly in the liver, kidneys,
and white blood cells, it is converted in to its
pyrophosphate which acts as a coenzyme
in such
reactions, as the decarboxylation of a-ketoacids,
particularly of pyruvate and a-keto-glutarate.
In the
presence of thiamine deficiency pyruvic and lactic
acids accumulate
in
the
tissues.
Thiamine
pyrophosphate also acts as a co-enzyme in the direct
oxidative pathway of glucose metabolism. ( 2 )
The pyruvic acid concentration in newborn infants after
prolonged labour had been reported to be elevated
probably due to disturbed carbohydrate metabolism in
the
tissues leading
to anoxia.
The pyruvate
concentration was reduced, though not to normal values,
in infants born to mothers given 100 mg of thiamine
hydrochloride intramuscularly during labour. Maternal
pyruvate concentrations were not effected. ( 2 )
Following oral administration of small doses, thiamine
hydrochloride is readily absorbed, however, absorption
is an active process and the total amount absorbed
following oral administration of a large dose is
limited to about 4-8 mg.
G1 absorption of thamine is
decreased in alcohoics and in patients with cirrhosis
or malabsorption.
The rate but not the extent of G1
432
5.
Vitamine Deficiency
Deficiency of thiamine results in fatigue, anorexia,
gastro-intestinal disturbances, trachycardia, and
irritability, and where there is reason to assume that
this syndrom may be of dietry origin thiamine may be
usefully employed.
It may also be employed with
benefit as a supplement to the diet in conditions in
which thiamine deficiency may be caused by interference
with its ingestion, absorption and utilization, or by
increasing its destruction or excretion. Thus, its use
is advisable in patients on restricted diets and those
suffering from gastro-intestinal diseases.
Extensive
burns, diabetes, impaired kidney or liver function,
hyperthyroidism, mental disease, alcoholism, and drug
addiction and
in those receiving antibiotics or
sulphonamides over prolonged periods.
There is some evidence that alcoholic neuritis and
neuritis associated with pregnancy and the neuritis of
THIAMINE HYDROCHLORIDE
433
434
Human Reauirements
Owing to the fact that thiamine is not stored in the
body and is rapidly lost from the tissues during short
periods of deficiency, normal health cannot be
maintained unless the diet regularly contains an
adequate amount of the vitamin.
The requirement is
directly related to the carbohydrate intake and the
metabolic rate. A daily intake of 200 pg per 1000 Kcal
is usually adequate to satisfy minimum requirements and
tissue saturation occurs with intakes of more than 330
pg per 1000 Kcal.
The requirement is increased during
periods of active growth or heavy muscular work, during
pregnancy and lactation, in pathological conditions
such as fevers and hyperthyroidism, and in other
conditions causing increased metabolism or diuresis.
The basic recommended intake of thiamine was 400 pg per
1000 Kcal of diet, so that men who took a diet of 3200
Kcal daily required 1-3 mg of thiamine and women who
took a diet of 2300 Kcal daily required 900 pg of
The US National Research Council made
thiamine. ( 2 )
daily dietry
the following recommendations for
allowances of thiamine:
435
THIAMINE HYDROCHLORIDE
Infants upto
2 months
2-6 months
6-12 months
Children:
1-3
3-4
4-6
6-8
8- 10
years
years
years
years
years
600 Pg
700 Pg
800 Pg
1 mg
1 . 1 mg
10-12
12-14
14-18
18-35
35-55
55
over
years
years
years
years
years
years
1.3
1.4
1.5
1.4
1.3
1.2
mg
mg
mg
10-12
12-18
18-35
35
years
years
years
years
1.1
1.2
1
900
mg
mg
mg
Pg
Males:
Females:
over
200 llg
400 Pg
500
mg
mg
mg
(Adverse effects)
Thiamine is usually
non-toxic even
following
administration of large doses however feelings of
warmth, tingling, pruritus, pain, urticana, weakness
sweating, nausea, restlessness, tightness of the
throat, angioedema, respiratory distress, cyanosis,
pulmonary edema, GI bleeding transient vasodilation and
hypotension, vascular collapse, and death have
occurred, mainly following IV administration of the
drug.
In animals very large parenteral doses of
thiamine have produced neuromuscular and ganglionic
blockade. ( 5 )
7.
Methods of Analysis
7.1
Elemental Analysis
C
42.73%
C1 21.03%
0
4.74%
H
N
S
5.38%
16.61%
9.51%
436
7.2
Identification
A.
To a volume equivalent to 20 mg of thiamine
hydrochloride diluted if necessary to 10 ml of H20 for
injection Or
Dissolve a quantity of powdered tablets equivalent to
20 mg of thiamine hydrochloride as completely as
possible in 10 ml of HzO, and then process for both
tablets and injections as: added 2 a1 of dil. CHBCOOH
and filter add 1.6 ml of N NaOH and heat in a water
bath for 30 minutes.
Cool and add 5 ml NaOH solution,
10 ml of pot. ferricynide solution and 10 m l of n-butyl
The alcohol
alcohol, shake vigrously for 2 minutes.
layer shows an intense light blue
fluorescence,
especially on exposure to U.V. light. Repeat the test
adding 0.9 ml of N NaOH and 0 . 2 g of sod. sulphite
No fluorescence is
instead of 1.6 ml of N NaOH.
produced. ( 8 )
B.
THIAMINE HYDROCHLORIDE
437
(D)
The identification of vit B i is based on its
reaction with picrolonic acid to form an insoluble ppt
in HzO.
Fan-shaped crystals with two ends back in a
form, specific for vit.
Bi are observed at a
mangification of 400.
Other B vitamins do not react
with picrolonic acid.
The sensitivity of the reaction
is 0.0062 pg/pl and dilution limit is 1: 150,000.
The
reaction takes place in the molar ratio of the two
reactants. (9)
(E) 10 ml of trinitrophenol solution is add to 5 ml of
thiamine solution containing 50 mg of thiamine
hydrochloride.
The ppt is manipulated gently with a
glass rod and the mix. is allowed to stand f o r about 20
minutes.
The ppt is dried on a porous earthenware and
then at 105'C for 30 minutes its MP" is found to be
207C with darkening and decomposition for sintering at
200'C.
(10)
aq.
438
Gravimetric Method
The method is based upon the reaction of silicotungstic acid solution with thiamine hydrochloride
gravimetrically. An accurately measured assay solution
corresponding to 50 mg of vitamin B i is diluted to 50
ml; HzO, 2 m l of HC1 is added and solution is heated to
THIAMINE HYDROCHLORIDE
439
Volumetric Method
(I)
Silicotungstic acid solution is standardized
3 9 7 . 2 ) or
against strychnine nitrate (mol. wt.
anhydrous thiamine hydrochloride (mol. wt. 3 3 7 . 3 ) which
has previously been dried at 105C for 2 hrs before
A standard solution of thiamine hydrochloride
use.
containing 1 mg/ml of vitamin Bi is prepared and is
titerated with silicotungstic acid solution using 0 . 5
ml of metanil yellow as an indicator. The end point is
marked when the yellow ppt changes to reddish violet
through yellowish red.
440
Colorimetric Method
THIAMINE HYDROCHLORIDE
441
above procedure.
The amount of vitamin B i from the
assay solution is determined by reference to the
calibration curve.
Ascorbic acid interferes and is
oxidized with iodine before assay. ( 1 5 )
(11) A measured volume containing 3-60 pg of thiamine
hydrochloride is diluted to 25 ml with HzO and adjusted
pH 5 with 10% acetic acid. 5 ml of diazo reagent [sod.
nitrite solution t trichloroacetate of ethyl paminobenzoate solution (1:1)] is added and allowed to
stand for 2-3 minutes before it is made alkaline with
1N NaOH, again allowed to stand for 2 minutes so that
5 ml of
maximum intensity of colour is developed.
isoamyl alcohol is added, the mixture is shaken
thoroughly and two layers are allowed to separate. The
alcoholic layer is removed, dried over anhydrous sod.
sulphate and the absorbance is measured with
colorimeter. A calibration curve is prepared by taking
different volumes of standard thiamine solution and by
following the above procedure.
The amount of vit B i
from the assay solution is determined by reference to
the calibration curve. ( 1 6 )
7.7
Spectrophotometric Method
Visible Absorption Spectroscopy
0.2
uv
Absorvtion SDectroscoDy
THIAMINE HYDROCHLORIDE
443
Polarographic Method
444
Fluorimetric determination
1.
The thiochrom method for determination of th amine
in pharmaceutical preparations was adapted to a
contineous flow system based on the flow inj ction
principle.
The sample volume required for an analysis
is about 150 111 and
for routine purposes a
concentrations ranges of 3 X lo-* - 6 X 1 0 - 4 mg/ml is
used. Results obtained with the system agree well with
the results obtained manually.
The consumption of
organic phase is 2-3 ml/sample and the sampling rate is
30/h.
A sampling rate of 70/h is easily attained if
necessary, the relative standard deviation is about 1%.
(26).
3.
An improved method for electrophoretic separation
and fluorometric (or radiometric) determination of
thiamine and thiamine mono-, and triphosphates in
animal tissues (liver, small intestine, kidney, heart,
The amount of total thiamine
brain) is described.
handled was 1.5-3.5 pg.
The procedure includes, acid
extraction of
the compounds form
the tissue,
deproteinization with TCA, purification of the extract,
on partially, deactivated charcoal elution with 10%
Propanol in 0.1N
formic acid, lyophilization,,
electrophoresis on gelatinized cellulose acetate,
strips elution of the thiamine bands with 50% ethyl
THIAMINE HYDROCHLORIDE
445
Spectroscopy
in pharmaceutical
The determination of Bi
preparations using a lead ion selective electrode and
atomic absorption spectroscopy based on its reaction
0.02
M alk. plumbile, under absorption
with
spectroscopy (217 nm) or by titration with EDTA (pH
4.5) with Pb ion.
Selective electrode and Gran's plot
KOH is the preferred alkali in the desulfurization
reaction.
The results obtained by this method
compared favourably with those obtained by
USP
fluorimetric method. Other vitamins and excipients did
not interfere with the electrode or absorption
spectroscopy procedures. Recoveries averaged 99.1% and
99% for the spectroscopy and electrode methods
respectively and standard deviations were 0.8% and 0.7%
respectively. (29)
7.11 Chromatographic Methods
446
Gas Chromatography
Gas chromatographic methods have been used for the
determination of thiamine hydrochloride and are
surnmarised in the Table ( 4 ) .
Thin-layer Chromatography (TLC)
A summary of some of the TLC systems investigated
for the analysis of thiamine hydrochloride are given in
the Table ( 5 ) .
Acknowledgment
The authors would like to thank Mr. Tanvir A. Butt,
College of Pharmacy, King Saud University, Riyadh,
Saudi Arabia for typing the manuscript.
9.
References
3.
2b.
S . Harris.
Mesh
Temp.
0.325% (w/w)
80-100
75C
80-100
150C
EGA AW H1
Chromosorb
5% OV-17
Chromosorb
WAW DMCS
Flow rate
Sample
Carrier gas
Helium
60 ml/min
H2 50 ml/min
Plasma
18 ml/min
Ref.
31
32
Developing Solvent
Detection
Extd. Solvent
Rf
Ref.
Silica gel G
0.25 m m thick
Me2 Co-MeOH-CsHs
(1:2:8)
Densitometer
33
Silica gel G
CHC13-EtOH-H20
(50:25: 1)
Spectrophotometer
UV 246 nm
2N HC1
34
Silica gel
Densitometer
0.057+0.01
35
0.56
36
GF2 5 4
Silica gel
GF2 5 4
Diethanolamine:
Methanol: formic
acid: basic Na.
phosphate
(1: 15: 1.5:5)
Continued (Table 5)
Plate
Developing Solvent
Detection
Extd, Solvent
Po 1yam ide
layers
Iodoplatinate
or
Iodine vapor
Ferligfolien
D. H z 0
Expose dried
Chromatogram
to C1. Then
spray o tolidine KI reagent
UV 254
P
P
a
Rf
Ref.
38
Table ( 6 ) .
Column
Zipax SCX.
Mobile phase
Flow rate
Retention
time
Sample
Detection
0 . 0 5 M NazHPOlr
Capsule
extract
U V photometer
0 . 2 M NaHZP0.I
0 . 5 ml/min
DeprotiSpectro40
nized
fluorometer
blood
supernatent
adjusted to
pH 4 . 5 with
NaOAc.
CH3CN : Hz 0
(70:30)
Blood
plasma
Fluorimetry
0.5 ml/min
Nervous
tissues
Spectro42
fluorometer
R0
p-Ekmdapack
C18
Shimadzu
0.7 M sod. aceISA-07/52504 tate
LC column
( 2 5 mm X
0 . 4 mm) ID
Ref.
39
41
Continued (Table 6)
Column
Mobile phase
Flow rate
Retention
time
Sample
Detection
3 ml/min
Acidified
urine
Flourescence
43
Column
37% methanol
(10 cm X
0.1M phosphate
18 mm) ID
buffer (pH 7 . 0 )
Radial-PAK
Cs (10 urn)
and guard
column of
Bondapak Ci s /
Porasi 1
1 5 or
3 ml/min
Food
Flourimetric
530 nm
44
Nucleosil
Methanol: H2O
(19:l)
Bloodor
urine
Flourimetric
45
Cis ( 5
urn)
Ref.
Continued (Table 6 )
Column
Mobile phase
Flow rate
1.0 ml/min
8 p Bondapak 3 to 8 mM Na
hexanesulphonate
C18
in aq. 25%
methanol containing
1% of acetic
acid
u
P
l
N
Retention
time
Sample
Detection
Powdered
tablets
o r injections
254 nm
Ref.
46
LiChroaorb
RP-8
Methano1-acetonit rile-isobutyl
alcohol (8:l:l)
Food
Spectro47
fluorimetric
425 nm.
( 3 0 cm X
0-80% of methanol
in Hz0
2.0 ml/min
Multivitamin
tablets
280 nm
48
0.2M acetate
buffer with 5 mM
heptane sulphonic
acid
1.0 ml/min
Food
UV 250 nm
49
3.9 mm) of
p Ebndapak
phenyl
( 1 0 Ilm)
(30 cm X
4 mm) of
p Bondapak
c18
Continued (Table 6)
Column
W
w
Stainless
steel column
(50 cm X
2.1) mm
packed with
Spherisorb
silica
Mobile phase
Flow rate
Retention
time
Sample
Detection
Ref.
CH3C1: methanol
(9:l)
1.0 ml/min
or
0.8 ml/min
Meat
Flourimetry
367
50
Methanol-aq. 5 mM
hexanesulphonate
containing 1% of
acetic acid
(1:3)
0.5 ml/min
Multivitamin
UV 270 nm
51
(20 pm)
Stainless
steel column
(30 cm X
4 m m ) packed
with p Bondapak Cia
( 1 0 pm)
Continued (Table 6)
Column
Two columns
(50 cm X
21 m m ) connected in
series and
packed with
HS Pellionex
SCX
Mobile phase
0.1M phosphate
Flow rate
10-20 ml/hr
Retention
time
Sample
Detect ion
Multivitamin
tablets
UV 254 nm
or
280 nm
Ref.
52
455
THIAMINE HYDROCHLORIDE
Hosp tal
5.
6.
Sushko,
7.
8.
9.
L.I.;
Lukienko, P.I.,
Farmakol Toksikol,
Neurol, 9 ( 4 ) ,
10.
11.
J.
Am.
334-9
Pharm. ASSOC.,
40, 609 ( 1 9 5 1 ) .
12.
13.
u,
277
(1968).
489
(1953).
14.
H. Wachsmuth.
15.
16.
E.R.
Kirch and 0. Bergeim.
(1942).
17.
Tuschhoff.
Anal.
Chem.,
25,
1198 (1968).
18.
A.M. Aliev.
19.
A.M.
(1971).
20.
Pharm. Sci.
57,
456
21.
22.
Wokes.
J.
Pharm.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
Vitam.
Chim.
Nutr.
Acta,
Res.
Chromatogr., 76(1),
Mikrochim. Acta,
Analyst (London
M.C
451
THIAMINE HYDROCHLORIDE
36.
37.
H.P.
Chuang, H.C.
Chiang and K.T.
Chromatogr., 41, 487 (1969).
38.
39.
40.
41.
42.
Miekokimura,
Tomiofujita, Shigeki, Nishida and
Yoshinori, Itonawa, J. of Chromatography 188, 417-419,
Elseveir Scientific Publishing Co. Amsterdam. Printed
in Netherland, 1980.
43.
44.
45.
46.
47.
48.
49.
Skurray, Geoffre, R.
1968).
Wang
Int.
J.
J.
J.
458
50.
51.
Kirchmeier, R.L.;
and Upton,
67(10), 1444-1446, 1978.
52.
L.
R.P.,
J.
A.J.
Pharm. Sci.,
Chromatographia,
7(11),
ANALYTICAL PROFILE
OF
THIORIDAZINE
THIORIDAZINE HYDROCHLORIDE
460
CONTENTS
1. INTRODUCTORY
2. DESCRIPTION
2.1
2.2
2.3
2.4
Nomenclature
Formulae and Molecular Weight
Appearance, Color, Odor and Taste
The Three-Dimensional Structure
3. PHYSICAL CHARACTERISTICS
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
Elemental Composition
Acidity (pH)
Ionization Constant (pKa)
Melting Range and Boiling Point
Thermal Behavior
Solubility
Crystallographic Data
Spectroscopic Data
3.81
3.82
3.83
3.84
4. SYNTHESIS
4.1 Manufacturing
4.2 Partial Synthesis
4.3 Cyclization
5. PHARMACOKINETICS
5.1 Absorption
5.2 Distribution
5.3 Biotransformation
5.4 Drug Concentration Levels
5.5 Elimination
6 . THERAPEUTIC CATEGORATION
6.1 Pharmacology
6.2
6.3
6.4
6.5
6.6
6.7
Uses
Drug-Drug Interactions
Toxicology
Cautions
Administration and Dosage
Pharmaceutical Preparations
6 . 8 Stability
6.9 Laboratory Test Interferences
7. ANALYTICAL METHODS
7 . 1 Qualitative
7.2 Quantitative
7.21 Determination in Bulk Materials
7.22 Determination in Pharmaceutical Formulations
7.23 Determination in Tissues and Biological Fluids
ACKNOWLEDGEMENT
REFERENCES
46 1
462
1.
INTRODUCTORY
Much attention has been given to a group of psychotropic
drugs because of the increasing abuse of these drug
substances for suicides and use a s narcotics. The
antipsychotic phenothiazine group has a variety of
derivatives to which our drug thioridazine belongs.
Thioridazine is an alkylpiperidine derivative of the
prototype phenothiazine. The drug was synthesized
firstly in the year 1958 and has been patented (U.S.
Patent : 3.239.514) t o Sandoz Ltd. Basel-Switzerland.
Since then huge numbers of publications have appeared
concerning the clinical (therapeutic and pharmacokinetic) and chemical characteristics of the drug. This
work is a trial to summarize and integrate the net
findings of such investigations collectively in a useful
profiling way; any gap is not intended of course.
2.
DESCRIPTION
2.1 Nomenclature
2.11 Systemic Name
Thioridizine : lo-[ 2-( 1-Methyl-2-piperidy1)e thyl]2-(methy1thio)phenothiazine (1).
Thioridazine Hydrochloride : 10-[2-(1-Methyl-2piperidyl)ethyl]-2-(methylthio)phenothiazine
monohydrochloride (1).
2.12 Other Chemical Names
Thi o ridazine is 2-me thylmercapt0-1 0-[ 2-( N-methyl-2piperidyl)ethyl]phenothiazine; 3-methylmercapto-N[2'-(N'-methyl-2-piperidyl)ethyl]phenothiazine;
1methyl-2-[2-(2-methylthiophenothiazine-lO-y~)ethyl]
piperidine (2) ; o r 10- [ 2-( l-Methylpiperid-2yl)ethyl]-2-methylthiophenothiazine
(3).
2.13 Pharmacopoeias
Thiordazine in N0rd.P. and U.S.P.
Thioridazine.HC1 in B.P., Cz.P.,
and U.S.P.
Jug. P.
N0rd.P.
463
464
Fig.1
3.
PHYSICAL CHARACTERISTICS
3.1 Elemental comDosition
Elemental
Thioridazine
Thioridazine.HC1
(X)
-
(%>
-
68.06
7 -07
61.96
6.44
8.96
6.88
15.76
c1
7.56
17.31
N
S
72-74
Thioridazine
hydrochloride
157-163 (1,5)
159-163 (2,3)
158-160(4).
(3-5)
230 (0.02
mmHg) (3,4)
465
3 . 5 Thermal B e h a v i o r
The d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y (DSC) t h e r m a l
curve f o r t h i o r i d a z i n e hydrochloride is given i n Figure
2. The s c a n n i n g h a s b e e n r u n a t a r a t e o f 10C.min-l
f r o m 50 t o 20OoC. The h y d r o c h l o r i d e s a l t of t h i o r i d a z i n e
m e l t s a t 1 6 6 . 8 O C , t h e A H - v a l u e i s 44.2 J . m o l e - l
for
95.84 m o l e % p u r i t y . A DuPont TA-9900 Thermal A n a l y z e r
a t t a c h e d t o a DuPont Data U n i t e were used f o r t h e DSCscanning.
,--
U a,
z
urn
rnzbi
>.I
0
UUl
r
i m
u=rm
m
a
.ri
d
0
ran
k 0
c, 0
a, k
wn.4
.ri
D O \
u m v
..
E d
I - \
U N
.... ..
a,
L u
ou
m a
U Q
cd
dOIL
o c
6,
N
- n a
D1 (d
uou
d d
.ri ri
d k
d 0
cd d
cn
m e,
t
l
cd 0
.ri
c, a,
z >
a,
k
a, u
w
\
V
w +
s:IDI
.ri
cd
a, a,
?c c
d::
b e,
..
$
N
2
Z
W P
E 0
N K I
S
dP
bl V
N *Vl
.ri
V N O
., .. .. ..
U
u' n c
o u
PUS E
r(
E N U E
m - e o
VlUlIU
LL
466
3 . 6 Solubility
3.71
Crystallization
The free base thioridazine a s well as monohydrochloride salt crystallize from acetone ( 4 ) .
3 . 7 2 Crystal Forms
467
3.73 X-Rav D i f f r a c t i o n P a t t e r n
T h i s p a t t e r n w a s o b t a i n e d on a P h i l i p s PW-1710
D i f f r a c t o m e t e r w i t h s i n g l e c r y s t a l monochromator
a n d c o p p e r K,
radiation.
The x - r a y
powder
d i f f r a c t i o n p a t t e r n s were r e c o r d e d on a P h i l i p s PM8 2 1 0 p r i n t i n g r e c o r d e r . The t a b l e s o f 2 8 , ds p a c i n g ( A 1, a n d c o u n t s w e r e a u t o m a t i c a l l y
o b t a i n e d on a P h i l i p s D i g i t a l p r i n t e r .
T a b l e 1: The X-ray d i f f r a c t i o n a l p r i n c i p a l l i n e s of
t h i o r i d a z i n e hydrochloride.
28
d(A )
[I/IoxlOO]
28
5.104
7.367
10.073
13.253
15.638
15.914
16.375
17.267
17.891
18.488
19.550
19.964
21.178
21.613
23.555
24.260
24.876
25.330
26.044
26.904
27.770
28.491
29.302
30.495
30.866
31.514
32.292
17.3150
11.999
8.781
6.680
5.666
5.568
5.413
5.135
4.957
4.799
4.540
4.447
4.195
4.111
3.776
3.668
3.579
3.516
3.421
3.313
3.212
3.132
3.047
2.931
2.896
2.838
2.772
100
32.611
33.063
33.474
34.582
36.541
37.035
38.369
39.127
39.465
40.404
41.090
42.119
42.767
44.247
44.578
45.535
46.144
46.465
48.306
49.298
51.336
53.138
53.687
54.121
55.438
56.916
24.4
15.2
46.4
46.9
30.7
19.4
36.2
19.9
7.1
19.1
40.8
35.5
8.0
12.8
10.4
45.2
35.3
10.6
17.1
10.8
8.6
17.6
6.8
6.5
7.6
7.8
d(& )
2.745
2.709
2.676
2.593
2.459
2.427
2.346
2.302
2.283
2.232
2.196
2.145
2.114
2.047
2.032
1.992
1.967
1.954
1.884
1.848
1.779
1.723
1.707
1.694
6.657
1.617
[I/Io x 1001
10.8
6.8
7.4
10.8
10.8
8.1
4.1
9.4
7.1
4.7
7.1
7.4
11.0
6.6
8.6
4.3
5.3
4.6
4.8
4.8
5.9
4.1
4.5
4.4
4.1
3.6
468
(nm)
Solvent
Water
--
95% ethanol
263
314
230
263
313
0.1N Acid
0.1N Alkali
Thioridazine.HC1
A(l%,lcm) E
--
--
1030
124
565
1240
141
38172
4595
20939
45954
5226
(nm) A(l%,lcm)
262
310
264
310
264
305
263
1028
79
1022
80
1041
135
4.52
E
41842
3215
41598
3256
42371
5495
18392
F i g . 4 : C h a r a c t e r i s t i c p r i n c i p a l l i n e s of t h e X-ray powder
d i f i r a c t ion of- thior-i _
d a-z-i n e h y d r o c h l o r i d e .
~
470
472
Group assignment
Remarks
>CH2-CH3, CH stretching
+HN<, stretching
broad due t o pre+HNC ,
sence of overtone
CH2, -CH3, CH-deformation
bands.
S-CH3, S-C stretching.
CH2, rocking.
473
Emperical
Structure
Mass /charge
R a t i o (m/e>
[I011
(%)I
Fragment
ion
C2H4N
42
CH =CH-NH
2
CH3S
47
CH3 S
gH4
76
22
C6H12N
98
100
sHlg N
126
16.4
C8H16N2
140
3.1
H3C
H3C
CHZ- CHZ
3CH - C H z
II
NH
Nos
4
7 H6NS2
167
-cH3
t a b l e 4 contd...
474
SH11NS2
185
19
1 3H9NS
211
12
C14H12NS
226
12
1 3H10NS
244
10.9
1 5H13NS2
271
3.1
@ D S C H 3
QyOSC
H3CH2c
CH2
C20H23N2S
323
2.1
416
Drug
Thioridazine
Thioridazine aromatic,1,3-8(7)
CH3N & CH3S;2,16(6)
hydrochloride.
CH2; 11-15(9)
CH2; lO(2)
; 9(2)
+NH( 1 )
(ppm, TMS)
7.30-6.70
2.50-2.30
1.90-1.30
3.90-3.50
4.40-4.10
7.20-6.80
2.49-2.46
2.30-1.20
3.50-2.80
4.20-3.80
11.78
Multiplicity
m
S
m
m
m
m
S
m
m
m
s ,broad
H-NMR
S p e c t r u m of T h i o r i d a z i n e H y d r o c h l o r i d e .
478
3.842 13C-NMR
Spectrum
The 13C-nuclear m a g n e t i c r e s o n a n c e s p e c t r u m
o f t h i o r i d a z i n e h y d r o c h l o r i d e was o b t a i n e d i n
CDC13 a t ambient t e m p e r a t u r e u s i n g TMS as t h e
i n t e r n a l s t a n d a r d on a V a r i a n X L - 2 0 0
S p e c t r o m e t e r . The c h e m i c a l s h i f t s , m u l t i p l i c i t i e s and s p e c t r a l a s s i g n m e n t s are g i v e n i n
T b l e 6, while F i g u r e 9 shows t h e o b t a i n e d
'C-NMR
spectrum. The DEPT and APT s p e c t r a of
thioridazine hydrochloride are given i n
F i g u r e s 10 a n d 11 r e s p e c t i v e l y .
T a b l e 6: Chemical s h i f t s and s p e c t r a l a s s i g n m e n t s of 13CNMR of t h i o r i d a z i n e and t h i o r i d a z i n e h y d r o c h l o x d e .
.ci
1
S -C%
Carbon p o s i t i o n
c1
c2
c3
c4
c5
'6
c7
'8
C9
Assignment7
Chemical s h i f t
( 6 ,ppm)
16.08
40.90
22.37
56.77
63.60
22.97
28.51*
27.68'
43.19
t a b l e 6 contd.
1 1 1 I N CDCLT,k.S.U.
LirECTRPL LINES FOR 1W
NFL=
1bl.l
RFP=
INDEX
< t
FREU
7315.0
1!2
03
7 49.6
1246.4
114
69bI.5
h9W.4
a.27.3
115
I>L
c07
6423.9
U8
A.19.1
09
b 50."
10
&2,2.5
I 1
I2
b1m.h
61.49.2
boBB. b
m5.5
,I
14
I5
16
17
18
5839.5
5757.1
57a7.e
39F.L
FFn
INTENSITY
44.015
24.40a
4s.755
107.297
37.249
127.756
122.192
127.AE9
172.9I3
137.5YI
173.ove
12b.238
52.173
I 2 3 . 2 ~ 9 11s.229
145.403
1.4.102
144.039
138.3711
138.15.
123.212
44.111
122.627
121.0x
116.390
116.07a
48.778
94.-
114.434
114.m
18.070
!V
3896.2
77.445
20
5863.7
3197.7
-2.4
-.2
2sav. 3
2213.9
76.-
21
I 2
23
24
25
26
27
3
30
31
32
33
14
35
Sb
37
38
21A9.9
2057.6
Lsn.2
1431.s
lSEB.8
1758.3
1149.3
1121.8
io11.1
962.0
814.1
@?.I
0
17.64
0
L3.5.5,
152.503
39.119
132.w2
37.811
64. 7M . W1
M.P.8
1Sb.02,
17. I 8 9
98.726
24.083
21.94b
74.793
43.132
40.900
1m.4.8
a
.
2
8
0 m.ov6
28.4s
93.375
27.405
42.689
2S.011
25.796
22.845
94.408
22.297
9&-3
59.hao
56.733
51.4-
44.m
m.m
m . o
19.121
I6.18S
16.083
19.017
S9.030
140.789
35.m
7
! I
v)
48I
482
10
C11
5 2
1 3
14
1 5
16
17
c18
19
c20
c21
-CHC-a romatic
CH-aromatic
CH
7 9
H - Y ,
CH - I f
H - Y Y
C-aromatic
CH - 9 ,
CH
9 ,
CH
, I
114.40 i
138. IS
116.37
121.00
123.27
+
)121.55-127.73
)
145.40 X
144.10
)
) 138.38
4. SYNTHESIS
There are different synthetic methods f o r preparation of
thioridazine starting from various materials.
483
piperidyl-2')-l-chloro-ethane
i n 40 p a r t s of a b s o l u t e
xylene i s t h e n added dropwise i n t h e c o u r s e of 1-1/2 h r
A f t e r f u r t h e r h e a t i n g f o r 3 h r s . , t h e r e a c t i o n mixture
i s cooled and, a f t e r t h e a d d i t i o n of 5 p a r t s of ammonium
c h l o r i d e , i s s h a k e n 3 t i m e s w i t h w a t e r . The x y l e n e
s o l u t i o n i s e x t r a c t e d once w i t h 35 p a r t s of 3N a c e t i c
a c i d and t h e n 3 t i m e s , each time w i t h 15 p a r t s of a c e t i c
a c i d , a f t e r which t h e a c e t i c a c i d e x t r a c t i s washed w i t h
6 0 p a r t s of e t h e r and i s t h e n made p h e n o l p h t h a l e i n a l k a l i n e by means of 25 p a r t s of c o n c e n t r a t e d a q u e o u s
c a u s t i c soda s o l u t i o n .
The p r e c i p i t a t e d o i l y base i s t a k e n up i n a t o t a l of 100
The benzene l a y e r , d r i e d over K2C03
p a r t s of benzene.
i s f i l t e r e d and t h e n e v a p o r a t e d under reduced p r e s s u r e .
The r e s i d u e f r o m e v a p o r a t i o n i s d i s t i l l e d i n a h i g h
vacuum; a f t e r s e p a r a t i n g a p r e l i m i n a r y d i s t i l l a t e which
p a s s e s o v e r up t o 2 2 8 O C under a p r e s s u r e of 0.92 mm Hg.
t h e p r i n c i p a l f r a c t i o n , 2-methylmer~apto-l0-[2~-(Nmethyl-piperidyl-2")-ethyl-l]-phenothiazine,
which
d i s t i l l s o v e r a t 228OC t o 232OC u n d e r t h e m e n t i o n e d
pressure, is collected.
484
SCHEME 1
485
SCHEME 2
486
4 . 3 Cyclization
SCHEME 3
5. PHARMACOKINETICS
5.1 Absorption
Thioridazine like other phenothiazines is generally well
absorbed from the GI tract and from parenteral routes,
however, absorption may be erratic, particularly
following oral intake. Many interindividual variations
in peak plasma contractions may be attributed to genetic
differences in the rate of metabolism of the drug during
absorption in the GI-mucosa and pass through the liver
( 5 , B ) . Following oral administration of thioridazine,
the drug is detectable in serum after 1 hr post
administration (16). Following multiple daily dosing,
accumulation of the drug occurs within 3 to 4 days and
serum levels may be maintained for 100 to 120 hrs after
withdrawal of the drug ( 1 7 ) .
5.2 Distribution
Thioridazine and its metabolites are distributed into
most body tissues and fluids, with high concentrations
being distributed into the cerebrospinal fluid (CSF),
lungs, liver, kidneys and spleen. In the same mode of
distribution of phenothiazines, thioridazine is highly
bound to plasma proteins. The drug can readily cross the
placenta. It is not known if the drug is distributed
487
5.3 Biotransformation
Thioridazine, like other phenothiazines , undergoes a
series of metabolic transformationsin the organism.
Several non-conjugated metabolites were identified, when
radioactively-labelled thioridazine was given to rats
(18). Among those metabolites are side-chain sulf oxide
[la] , ring sulfoxide [ lb] , side-chain sulfones [lc] ,
ring sulfones [ Id ] , disulfoxides [ l e I and disulfones as
well as the corresponding demethylated compounds [2a-e 1
(18,19). Scheme 4 demonstrates the formation pathways of
different metabolites of thioridazine.
SCHEME 4
489
(TG)
The mean s e r u m T % i n 2 0 p a t i e n t s t r e a t e d w i t h
t h i o r i d a z i n e o n l y was 16.1 h r s , b u t 1 7 . 1 h r s i n
p a t i e n t s who r e c e i v e d a l s o a d d i t i o n a l m e d i c a t i o n s
( 3 5 ) . The T7/2 -value i s recorded a l s o t o be 24 h r s
i n some s t u d i e s (36). The i n t e r i n d i v i d u a l v a r i a t i o n
of t h e e l i m i n a t i o n of t h i o r i d a z i n e from serum was
t h u s of t h e same magnitude as t h e v a r i a t i o n of t h e
serum c o n c e n t r a t i o n . I n e l d e r l y p a t i e n t s , a
d e c r e a s i n g a b i l i t y t o e l i m i n a t e t h i o r i d a z i n e , i. e . ,
490
d i u r e n a l d e c r e a s e , was o b s e r v e d ( 3 5 ) ; t h i s means
t h a t a g i v e n d o s e of t h i o r i d a z i n e w i l l g i v e a
h i g h e r serum c o n c e n t r a t i o n i n e l d e r l y p a t i e n t s than
i n younger ones.
5.53 E f f e c t of Other Medications on Serum l e v e l and
Elimination
A x e l s s o n and M a r t e n s s o n ( 3 5 ) o b s e r v e d t h a t
alcoholics eliminated thioridazine f a s t e r than t h e
o t h e r p a t i e n t s even when were r e c e i v i n g i n c r e a s i n g
d o s e s . T h i s was e x p l a i n e d by i n t e r i n d i v i d u a l
d i f f e r e n c e s i n metabolism of a l c o h o l i c s , whom l i v e r
enzymes may be induced by a l c o h o l . It was r e p o r t e d
t h a t t h e a d d i t i o n of p r o p r a n o l o l , a l i p i d - s o l u b l e
B-adrenergic r e c e p t o r a n t a g o n i s t , commonly presc r i b e d f o r h y p e r t e n s i o n , t o t h e pharmacotherapy of
p a t i e n t s r e c e i v i n g t h i o r i d a z i n e i n c r e a s e d t h e serum
l e v e l s of t h i o r i d a z i n e i n t o t h e p o t e n t i a l l y t o x i c
range (37-39).
It i s known t h a t t h e a d v e r s e
r e a c t i o n s a r e more l i k e l y t o be r e l a t e d t o t h e
c o n c e n t r a t i o n of t h i o r i d a z i n e i n t h e body t h a n t o
t h e d o s e i n g e s t e d , t h e c l i n i c i a n should a l e r t t o
i n i t i a t e t h e c o r r e c t i v e measures i n each i n d i v i d u a l
case.
5.55 E x c r e t i o n
Thioridazine i s extensively metabolized, princip a l l y i n t h e 1ivc.r v i a h y d r o x y l a t i o n , o x i d a t i o n ,
d e m e t h y l a t i o n , s u l f o x i d e f o r m a t i o n a nd c o n j u g a t i o n
with glucuronic a c i d ; metabolic a l t e r a t i o n s i n the
s i d e - c h a i n may a l s o o c c u r . A t l e a s t two m e t a b o l i t e s
of t h i o r i d a z 1 n e ,Ir c p h a m a co 1og i c a 11y a c t i v e ; wh i 1e
m o s t o f t h e m r i ~ t i n a c t i v e . T h i o r i d a z i n e a n d i t s
metabolites a r c txcreted i n u r i n e and f e a c e s ; t h e
excretory pat tel ns have not been f u l l y charact e r i z e d . The d r u g i s e x c r e t e d i n f e a c e s
biliary
e l i m i n a t i o n , p r i n c i p a l l y as m e t a b o l i t e s , a n d a l s o
a p p e a r s t o u n d r 1 go e n t e r o h e p a t i c c i r c u l a t i o n .
C e r t a i n m e t a b o l i t e s and o n l y s m a l l amounts of t h e
u n c h a n g e d d r u g h a v e b e e n d e t e c t e d i n u r i n e i n some
p a t i e n t s f o r u p t o 6 m o n t h s f o l l o w i n g s t o p p i n g of
therapy w i t h t h e drug ( 4 0 , 4 1 ) .
6. THERAPEUTIC CATEGORATION
6.1 Pharmacology
Like the other phvnothiazines antipsychotic agents,
t h i o r i d a z i n e h a s a l s o b e e ? d e s c r i b e d as n e u r o l e p t i c
agents because of its a c t i v i t y i n inducing the
n e u r o l e p t i c syndromr, ( i . e . , depressed i n i t i a t i v e , decreased e f f e c t , disinttrest i n s u r r o u n d i n g s , s u p r e s s i o n
of complex b e h a v i o r ,inn s p o n t a n e o u s movements, d e c r e a s e d
a g r e s s i v e n e s s and i m p u l s i v i t y , e x t r a p y r a m i d a l a c t i o n ) .
P h e n o t h i a z i n e , t h e 5 t r r i c t i i r a l p r o t o t y p e of t h e p h e n o t h i a z i n e s , i s n o t more i n u s e a s u r i n a r y t r a c t
a n t i s e p t i c d u e t o i t 5 t o x i c i t y , b u t s t i l l u s e d as a n
a n t h e l m i n t i c i n vettrinary medicine and a s an
i n s e c t i c i d e ( 4 2 ) . The d e v e l o p m e n t of p h e n o t h i a z i n e s a s
psychopharmacologica 1 agents r e s u l t e d from t h e observed
s e d a t i o n a c t i v i t y of c e r t a i n a n t i h i s t a m i n i c p h e n o t h i a z i n e compounds. I n a n a t t e m p t t o enhance the
s e d a t i v e e f f e c t s o f s u c h g r o u p o f d r u g s , some a n a l o g u e s
were s y n t h e s i z e d .
T h e p h a r m a c o l o g y o f t h i o r i d a z i n e a s a l l o t h e r phe not h i a z i n e s i s c o m p l e x , arid d u e t o i t s a c t i v i t y on t h e
c e n t r a l a n d au t o n o mi c- n e r v o u s s y s t e m s , t h e d r u g a f f e c t s
many d i f f e r e n t s i t e . , i i i t h e body.
492
493
On t h e w e i g h t b a s i s , t h i o r i d a z i n e i s a b o u t a s
potent as chlorpromazine, but has s t r o n g
a n t i c h o l i n e r g i c and s e d a t i v e e f f e c t s a n d weak
extrapyramidal e f f e c t s . Thioridazine has l i t t e a n t i emetic a c t i v i t y , which would b e m e d i a t e d % a d i r e c t
e f f e c t o n t h e m e d u l l a r y c h e m o r e c e p t o r t r i g g e r zone
(CTZ) , a p p a r e n t l y by b l o c k i n g dopamine r e c e p t o r s i n
t h e CTZ.
T h i o r i d a z i n e i n h i b i t s t h e c e n t r a l and
p e r i p h e r a l e f f e c t s of apomorphine and e r g o t
a l k a l o i d s (42).
6.12 E f f e c t s on C a r d i o v a s c u l a r System (CVS)
T h i o r i d a z i n e h a s d i r e c t and i n d i r e c t a c t i o n s o n t h e
h e a r t and v a s c u l a t u r e making t h e c a r d i o v a s c u l a r
e f f e c t c o m p l e x . The d r u g i n h i b i t s p e r i p h e r a l a adrenergic blocking a c t i v i t y and c a u s e s v a s o d i l a t i o n l e a d i n g t o o r t h o s t a t i c h y p o t e n s i o n . The d r u g
may i n c r e a s e t h e c o r o n a r y b l o o d f l o w a s a r e s u l t of
increased heart rate. Transient antiarrhythmic
e f f e c t s h a v e b e e n o b s e r v e d i n some p a t i e n t s a t
h i g h e r d o s a g e s . T h i s may r e s u l t f r o m e i t h e r a
d i r e c t quinidine-like properties or probable l o c a l
a n a e s t h e t i c e f f e c t of t h e d r u g .
6.13 E f f e c t s on E n d o c r i n e s
T h i o r i d a z i n e may i n d u c e s e c r e t i o n of p r o l a c t i n from
t h e a n t e r i o r p i t u i t a r y by i n h i b i t i n g d o p a m i n e
r e c e p t o r s i n t h e p i t u i t a r y and hypothalmus d u r i n g
long-term a d m i n i s t r a t i o n . P r o l a c t i n s e c r e t i o n may
be a c c o m p a n i e d a l s o w i t h a m e n o r r h e a , g y n e c o m a s t i a
and impotence. D e c r e a s e s of u r i n a r y c o n c e n t r a t i o n s
of g o n a d o t r o p i n p r o g e s t i n s may be o b s e r v e d i n some
p a t i e n t s , and may be v a s o p r e s s i n and c o r t i c o t r o p i n
i n some o t h e r p a t i e n t s ( 4 2 ) .
6.14 O t h e r E f f e c t s
T h i o r i d a z i n e may h a s a n t i i n f l a m m a t o r y a n d / o r
a n t i p r u r i t i c e f f e c t s , r e s u l t i n g from a n t a g o n i s m of
v a r i o u s m e d i a t o r s u b s t a n c e s , s u c h as s e r o t o n i n and
histamine.
494
6.2 Uses
Thioridazine i s mostly p r e s c r i b e d f o r t h e symptomatic
management of p s y c h o t i c d i s o r d e r s . The d r u g i s a l s o used
f o r t h e short-term treatment of a d u l t s w i t h m a j o r
d e p r e s s i o n who h a v e v a r y i n g d e g r e e s of a s s o c i a t e d
anxiety ( 4 0 ) .
6 . 3 Drug-Drug I n t e r a c t i o n s
6 . 3 1 CNS D e p r e s s a n t s
S i n c e t h i o r i d a z i n e may b e a d d i t i v e w i t h , o r may
p o t e n t i a t e t h e a c t i o n o f , o t h e r CNS d e p r e s s a n t s
such a s o p i a t e s or other a n a l g e s i c s , b a r b i t u r a t e s
or other sedatives, general anaesthetics, o r
a l c o h o l . Caution should be t a k e n t o avoid probable
e x c e s s i v e s e d a t i o n o r CNS d e p r e s s i o n ( 4 2 ) .
6 . 3 2 Lithium
P a t i e n t s r e c e i v i n g combined t h e r a p y of l i t h i u m and
t h i o r i d a z i n e may e x e r t a c u t e e n c e p h a 1o p a t h i c
s y n d r o m e s o c c a s i o n a l l y o c c u r i n g e s p e c i a l l y when
h i g h e r serum l i t h i u m c o n c e n t r a t i o n s are p r e s e n t .
S u c h p a t i e n t s s h o u l d b e o b s e r v e d f o r e v i d e n c e of
a d v e r s e n e u r o l o g i c e f f e c t s and t r e a t m e n t s h o u l d b e
r a p i d l y d i s c o n t i n u e d i f t h o s e s i g n s o r symptoms
appear ( 4 0 , 4 3 , 4 4 ) .
6 . 3 3 Metrizamide
The c o n c u r r e n t u s e of m e t r i z a m i d e w i t h t h i o r i d a zine, a drug lowers the s e i z u r e threshold, an
i n c r e a s e d r i s k o f s e i z u r e s c a n b e e x p e c t e d . The
m a n u f a c t u r e r s s t a t e t h a t phenothiazines should not
be u s e d i n p a t i e n t s r e c e i v i n g m e t r i z a m i d e a n d f o r
t h e c o n t r o l of m e t r i z a m i d e - i n d u c e d n a u s e a a n d
vomiting ( 4 2 , 4 4 ) .
6.34 Anticonvulsants
Because of t h e s e i z u r e - t h r e s h o l d l o w e r i n g e f f e c t of
t h i o r i d a z i n e , d o s a g e a d j u s t m e n t of a n t i c o n v u l s a n t s
may b e n e c e s s a r y when t h e y a r e p r e s c r i b e d w i t h
t h i o r i d a z i n e . The CNS d e p r e s s a n t e f f e c t s o f
495
--
496
d e p r e s s i o n p r o g r e s s i n g t o coma w i t h a r e f l e x i a may
o c c u r , t h i s c a n be accompanied by t a c h y c a r d i a , ECGchanges and c a r d i a c a r r h y t h m i a s , h y p o t h e r m i a ,
m i o s i s , t r e m o r , muscle t w i t c h i n g , spasm o r
r i g i d i t y , s e i z u r e s , muscular hypotonia, i l e u s , d r y
mouth, d i f f i c u l t y i n swallowing or breathing,
cyanosis, and r e s p i r a t o r y and/or v a s o m o t o r
c o l l a p s e , e v e n w i t h a b r u p t apnea ( 4 9 - 5 1 ) .
(4,52).
6.43 T r e a t m e n t
T r e a t m e n t o f t h i o r i d a z i n e overdosage involves
s y m p t o m a t i c and s u p p o r t i v e c a r e . T h e r e a r e n o
s p e c i f i c a n t i d o t e s f o r thioridazine intoxication;
however, a n t i c h o l i n e r g i c a n t i p a r k i n s o n i a n d r u g s may
b e u s e f u l i n management of e x t r a p y r a m i d a l
r e a c t i o n s accompanied by t h i o r i d a z i n e o v e r d o s a g e .
The s t o m a c h s h o u l d b e e m p t i e d by g a s t r i c l a v a g e
f o l l o w i n g a c u t e i n g e s t i o n of t h i o r i d a z i n e . I f t h e
p a t i e n t i s comatose, h a v i n g s e i z u r e s o r a d y s t o n i c
r e a c t i o n , g a s t r i c l a v a g e may be p e r f o r m e d w i t h a n
e n d o t r a c h e a l t u b e with cuff i n f l a t e d t o avoid
a s p i r a t i o n of g a s t r i c c o n t e n t s . Due t o g r e a t
r e d u c t i o n o f GI-mo t i 1i t y f o 11owing o v e r d o s a g e of
t h i o r i d a z i n e , g a s t r i c l a v a g e may be u s e f u l e v e n
several hours a f t e r the drug ingestion.
A d m i n i s t r a t i o n of a s a l i n e c a t h a r t i c may b e
b e n e f i c i a l i n e n h a n c i n g e v a c u a t i o n of t h e d r u g from
t h e GI-tract. S u i t a b l e therapy should be i n s t i t u t e d
i f h y p o t e n s i o n o c c u r s ; e p h e d r i n e s h o u l d be a v o i d e d
( 4 2 ) . I n a c u t e t o x i c i t y e x c h a n g e t r a n s f u s i o n s may
b e b e n e f i c i a l , b u t h e m o d i a l y s i s i s of l i t t l e v a l u e
f o r r a p i d e l i m i n a t i o n of t h e drug.
6.5 C a u t i o n s
Care s h o u l d be t a k e n t o a v o i d s k i n c o n t a c t w i t h
t h i o r i d a z i ne o r t h i o r i d a z i n e h y d r o c h l o r i d e , s i n c e
c o n t a c t d e r m a t i s i s has been observed. T h i o r i d a z i n e
a p p e a r e d t o b l o c k u s e of f e e d b a c k i n f o r m a t i o n , t h i s
o b s e r v a t i o n was o b t a i n e d f r o m a n e v a l u a t i o n o f
j u d g m e n t ' s p e r f o r m a n c e i n some s c h i z o p h r e n i c p a t i e n t s
(53).
497
6 . 6 A d m i n i s t r a t i o n and Dosage
6.61 A d m i n i s t r a t i o n
T h i o r i d a z i n e and t h i o r i d a z i n e h y d r o c h l o r i d e a r e
a d m i n i s t e r e d o r a l l y , b u t when t h i o r i d a z i n e
hydrochloride o r a l concentration s o l u t i o n i s u s e d ,
t h e d o s e s h o u l d be d i l u t e d ( w i t h w a t e r o r f r u i t
j u i c e ) j u s t before administration (40,54).
6.62 Dosage
498
6.7 Pharmaceutical P r e p a r a t i o n s
Thioridazine
O r a l S u s p e n s i o n - e q u i v a l e n t t o t h i o r i d a z i n e hydroc h l o r i d e 25 o r 100 mg/5 m l . Meuaril-S, Sandoz.
T h i o r i d a z i n e Hydrochloride
O r a l S o l u t i o n , c o n c e n t r a t i o n - 3 0 mg/ml [ M e l l a r i l
C o n c e n t r a t e ( w i t h a l c o h o l 3% and p a r a b e n s ) , S a n d o z ;
T h i o r i d a z i n e H C 1 I n t e n s o l , Roxane.]
100 mg/ml - M e l l a r i C o n c e n t r a t e ( w i t h a l c o h o l 4.2% and
p a r a b e n s ) , Sandoz; Thioridazine.HC1 I n t e n s o l , Roxane.
T a b l e t s - 10 mg, 15 mg, 25 mg, 150 mg and 200 mg
M e l l a r i l ( w i t h parabens and povidone).
Sandoz 50 mg M e l l a r i l , Sandoz. 100 mg M e l l a r i l ( w i t h
povidone), Sandoz.
T a b l e t s , film-coated
10 mg, 15 mg, 25 mg, 50 mg, 100 mg and 200 mg ( a v a i l a b l e
by n o n p r o p r i e t a r y name) (40).
Other P r o p r i e t a r y Names:
&.-Meleril;
Be1g.-Mellerettes;
Canada.-Mellaril,
Novoridazine, T h i o r i l ; Ger.-Melleretten;
Ita1.Mellerette;
M e l l e r e t t e n ; S p a i n - M e l e r i l ; Swed.M a l l o r o l ; S w i t z . - M e l l e r e t t e n ; USA-Mellaril.,
TP21,
Sonapax, (Also a v a i l a b l e a s M e l l e r i l i n A u s t r a l . , Belg.,
E.,
S. Afr., Switz.)
(3,498).
m-
m.,
s.,
w.,m.,w.,
499
6.8 Stability
7. ANALYTICAL METHODS
7.1 Qualitative
7.11 PharmacoDoeial Identifications
USP X X I (1) specifies the comparison of the IRspectrum o f the sample w i t h a USP-reference
standard of thioridazine base or its HC1-salt, in
addition to the test for chloride in case of the
salt.
BP 1 9 8 0 (2) recommends the concordance o f the
sample with corresponding IR-spectrum of the
reference drug substance. Light absorption scanning
(350-230 nm) of ethanolic solution of the drug is
also recommended beside the blue coloration with
sulfuric acid and also the reactions characteristics of chloride in case of the HC1-salt.
500
Reagent
Color
Sulfuric acidformaldehyde
Ammonium molybdate.
Ammonium Vandate
Vitalis test
Sensitivity Reference
--
Purple-red
blue
green violet (in case
of mesoridazine).
Deep blue
green
--- purple
0.1 mg
0.25 mg
3,55
3,55
--
55
Ref e rence
Pale pink
Urine
deeping to blue.
3,55
S pec imen
Color
2% Ferric chloride/
30% sulfuric acid.
Gold-cyanide
solution.
Krauts
reagent
mg
Screening Test
Reagent
Reagent
3,55
0.1
Deep blue
Blue-purple/purplebrown/yellow brown.
Forrests reagent Blue
Red (in case of
mesoridazine).
7.13
0.1 mg
55
Crystals
Sensitivity Reference
Branching needles
(overnight )
Orange-red
precipitation
1 in 200
1 in 50
7.15
50 1
Chromatography
7.151 Zone Electrophoresis
502
hR,r
Thioridazine
1N sodium acetate
1N sodium formate-npropanol (90:lO)
Formamide + ammonium
formate
Cyclohexane-benzene
(90:l o )
Kerosene/ethanol-waterammonia (60:38:2).
Kerosene/ethanol-waterammonia (75:23:2).
Tributyritd0.2 M acetate
buffer pH 4.58, at
95OC
24
40
value*
Mesoridazine
54
61
Reference
62
62
43
57
16
57
35
58,59
19
60
503
Detection: For d e t e c t i o n of t h e d r u g i t i s
p o s s i b l e t o u s e UV-light, e s p e c i a l l y i n t h e
s h o r t region, where fluorescence o r
a b s o r p t i o n c a n be o b s e r v e d ( 6 1 ) . Because o f
t h e p h o t o o x i d a t i o n of t h i o r i d a z i n e and i t s
h y d r o c h l o r i d e s a l t , i t i s recommended t o
record the fluorescence or a b s o r p t i o n
i m m e d i a t e l y a f t e r t a k i n g t h e chromatogram o u t
of t h e j a r . The c o l o r of t h i o r i d a z i n e s p o t s
c a n a l s o be i n f l u e n c e d by t h e rests of t h e
s o l v e n t s y s t e m used.
According t o M e l l i n g e r
and Keeler ( 6 2 ) t h e f l u o r e s c e n c e is m a i n l y
i n f l u e n c e d by t h e c h e m i c a l s t r u c t u r e o f t h e
p h e n o t h i a z i n e s . The main r o l e i n t h i s r e s p e c t
i s due t o t h e s u b s t i t u e n t on t h e C-2 c a r b o n
i n t h e p h e n o t h i a z i n e r i n g ( 6 1 ) . The
s u b s t i t u t i o n by a n a l k y l m e r c a p t o - g r o u p i n
thioridazine results in bluish yellow
f l u o r e s c e n c e o r b l u e f l u o r e s c e n c e . On a
f l u o r e s c e n t l a y e r of s i l i c a g e l t h i o r i d a z i n e
like other phenothiazines appear mostly a s
q u e n c h i n g s p o t s . As f o r b a s i c d r u g s
Dragendorff's r e a g e n t , o r d e t e c t i o n w i t h
i o d o p l a t i n a t e a r e t h e most common c h e m i c a l
methods f o r d e t e c t i o n of t h i o r i d a z i n e . B e s i d e
these reagents, s u l f u r i c acid i n various
modifications is usually used, i t has the
a d v a n t a g e of d i s t i n g u i s h i n g t h e d i f f e r e n t
p h e n o t h i a z i n e s by c o l o r . I t i s p o s s i b l e t o
s p r a y w i t h a q u e o u s 20-50% o r 10% e t h a n o l i c
s o l u t i o n s of s u l f u r i c a c i d . S u l f o x i d e s r e a c t
w i t h t h e s e r e a g e n t more s l o w l y t h a n t h e
original drug (63). Sulfuric acid with
anhydrous sodium s u l f a t e ( 4 : l v/w) ( 6 0 ) , and
f o r m a l d e h y d e - s u l f u r i c a c i d (Marqui's r e a g e n t )
a r e a l s o m o d i f i c a t i o n s ( 6 1 ) . Some a u t h o r s
recommend s p r a y i n g w i t h a 0 . 5 % s o l u t i o n o f
p a l l a d i u m c h l o r i d e , with about 5 ugs e n s i t i v i t y l i m i t , which is s e l e c t i v e f o r
phenothiazines in general (58,59,61).
Thioridazine r e a c t s w i t h t h i s reagent with
t h e f o r m a t i o n of d a r k r e d s p o t s . S i m i l a r
r e a g e n t s are f e r r i c c h l o r i d e , g o l d c h l o r i d e
and c e r r i c s u l f a t e ( 5 8 , 5 9 ) ; t h e f i r s t r e a g e n t
(aqeuous 2%) i s a u s e f u l d i f f e r e n t i a t i n g
reagent f o r phenothiazines.
504
505
.;
Adsorbent
Solvent System
ReferhRf -value
Thiori- Mesori- ence.
dazine dazine
-
24
13
32
62
66
67
71
46*
65
39
13
62
45
17
70
506
i) Aqueous Titration
Thioridazine is alkylated by treatment with
iodome thane in methanolic medium at 4O-5O0C
f o r 3 0 min.; the resulting quaternary
ammonium compound is titrated with ammonium
thiocyanate ( 7 5 ) .
ii) Non-aqueous Titration
The USP X X I ( 1 ) recommends the determination
of thioridazine base o r the hydrochloride
salt by titrating the drug solutions in equal
parts of glacial acetic acid and acetic
anhydride against standard solution of 0.1 M
acetous perchloric using potentiometry for
end points detection. The B P 1 9 8 0 ( 2 )
describes another non-aqueous procedure by
titrating the drug solution in acetone
containing about 7% mercuric acetate solution
against 0.1N acetous perchloric acid standard
using saturated solution of methyl orange in
acetone as indicator.
7.212 Electrochemistry
i) Controlled-potential coulometry
Merkel and Discher ( 7 6 ) described an accurate
controlled potential coulometric technique
507
f o r q u a n t i f i c a t i o n of t h i o r i d a z i n e b y
a d o p t i n g two d i f f e r e n t s u p p o r t i n g e l e c t r o l y t e s but using platinum a s the working
e l e c t r o d e . Table 9 summarizes t h e o v e r a l l
a n a l y t i c a l c o n d i t i o n s and r e s u l t s .
Table 9 : Applications of Controlled Potential M a n e t r y f o r Qmtification of Woridazine using P l a t i m Working Electrode (77).
Supporting electrolyte
12-N H2S04 i n 30%
ethanol (v/v).
1 - N H2SO4
Control
potential,
V vs. SCE Reaction
sample
weight,
mg
Refer* ence
-
Precision
+ 0.55
oxidn
10-40
40.17 mg
76
+ 0.75
oxidn
40
M.4 mg
76
or coefficient
of variation ( X ) as reported.
+ Date from mre than one sample (3 replicates) or from different
weight ranges *re p l e d t o obtain the standard deviation
(n-value is 1-2).
i i ) Voltammetry
A p p l i c a t i o n of m i c r o l i t e r v e s s e l s i n
v o l t a m m e t r i c q u a n t i f i c a t i o n of s m a l l sample
volumes of t h i o r i d a z i n e i s d e s c r i b e d by E b e l
et a1 ( 7 8 ) .
O x i d a t i v e voltammetry of
t h i o r i d a z i n e a t 3-mm v i t r e o u s - c a r b o n ; i . e . ,
s t a t i o n a r y e l e c t r o d e (sample volume 80 u l ) ,
o r a r o t a t i n g - d i s c e l e c t r o d e (sample volume 1
ml) u s i n g d r o p p i n g mercury e l e c t r o d e a r e
a p p l i e d f o r q u a n t i f i c a t i o n of t h e drug.
7.213 S p e c t rophotome t r y
Ramappa a n d B a s a v a i a h ( 7 9 ) d e s c r i b e d a
c o l o r i m e t r i c procedure f o r q u a n t i f i c a t i o n of
five phenothiazines including thioridazine
h y d r o c h l o r i d e i n p u r e and i n some d o s a g e
f o r m u l a t io n s
508
7.214 Chromatography
i) Combination of PC with Spectrophotometry
Densitometric evaluation of the spots of
phenothiazines, including thioridazine,
separated by PC can be made after the
r e a c t i o n w i t h specific reagents, e.g.
palladium chloride. Spectrophotometric
determination after PC-separation is also
recommended by using short-wave UV-light.
Presentation of the drug on an ion exchanger,
such as Amberlite IRC 50 and elution with
citric acid-phosphate buffer pH4, then
addition of 0.2 ml of 2% ghomma ghatti and
0.1 ml of iodoplatinate reagent followed by
measuring the resulting color at 610 nm (61).
ii) Combination of TLC with Spectrophotometry
Extraction of the separated spots of the drug
o n T L C w i t h m e t h a n o l o r e t h a n o l and
spectrophotometric determination at 260 nm
(61,80,
81). Bulenkov ( 8 2 ) recommends the
extraction of the drug from thin layers by
ether-isopropanol ( 2 8 : 2 , v/v), followed by
extraction into an acetate buffer and then
colorimetric determination using palladium
chloride reagent.
iii) Combination of PC and TLC with other
Chromatographic Methods
T h e s e p a r a t i o n of t h i o r i d a z i n e with
p romaz i ne, c h lorpromazine, together with
promethazine is achieved on thin layers, and
the additional separation by GC-column is
realized successfully. Rf -values calculated
from elution data in centripetal TLC coincide
with those for classical linear TLC. The
c o m b i n a t i o n of PC and TLC with other
chromatographic methods seems to have good
p r o s p e c t s for q u a l i t a t i v e as well a s
quantitative analysis i n pharmaceutical
substances and related interest (61).
7.22
509
Spectrometry
(HPLC)
Mehta (84) described a liquid chromatographic
method for determination of thioridazine
hydrochloride i n s y r u p s , injections or
tablets. Solutions of the drug (in water or
methanol) were analyzed on a column ( 2 5 cm x
5 mm) of Hypersil O D S ( 1 0 pm) with aqueous
9 0 % methanol containing 0.2% of ethanolamine
as mobile phase and 2 6 5 nm detection; 0.2%
c i n c h o c a i n e hydrochloride was used as
internal standard. Coefficient of variation
was < 2X and results were in good agreement
with those obtained by B.P.
or B.P.C.
methods. For identity, assay and content
uniformity of t h i o r i d a z i n e , a l i q u i d
chromatographic method is described by
5 10
51 I
7.23 D e t e r m i n a t i o n i n T i s s u e s and B i o l o g i c a l F l u i d s
7.231 S p e c t r o p h o t o m e t r y and S p e c t r o f l u o r o m e try
T h i o r i d a z i n e c a n be s p e c t r o p h o t ome t r i c a l l y
d e t e r m i n e d i n u r i n e a f t e r e x t r a c t i o n (87,881.
A non s p e c i f i c s p e c t r e f l u o r o m e t r i c method f o r
d e t e r m i n a t i o n of t h i o r i d a z i n e and some o t h e r
phenothiazine drugs i n plasma i s described
(89). Some of t h e non-conjugated m e t a b o l i t e s
of t h i o r i d a z i n e , c h l o r p r o m a z i n e , and t r i f l u o r o p e r a z i n e c a n be d e t e c t e d and d e t e r m i n e d
too. The d r u g i s e x t r a c t e d w i t h h e p t a n e , ree x t r a c t e d i n t o a c e t i c a c i d and t h e n o x i d i z e d
w i t h hydrogen p e r o x i d e t o the corresponding
f l u o r o p h o r e s . The measurement i s c a r r i e d o u t
a g a i n s t a b l a n k o f plasma; f o r t h i o r i d a z i n e
EX: 355 nm, EM: 430 nm, and a n a l y s i s a t 3704 8 0 nm. T h i s p r o c e d u r e d i s t i n g u i s h e s between
t h e common p h e n o t h i a z i n e s b u t n o t b e t w e e n a
d r u g and i t s m e t a b o l i t e s i n a l l c a s e s . Pacha
(90) described a s i m i l a r s p e c t r o f l u o r i m e t r i c
method f o r q u a n t i f i c a t i o n of t h i o r i d a z i n e and
mesoridazine i n plasma and, u r i n e a f t e r
t r e a t m e n t w i t h 0.2-N H2S04 and 0.1% KMn04; EX
: 355 nm and EM : 440 nm.
7.232 Chromatography
i ) Thin-Layer Chromatography (TLC)
Tewari ( 9 1 ) i n v e s t i g a t e d t h e a p p l i c a b i l i t y of
TLC u s i n g 18 s y s t e m s f o r d e t e c t i o n a n d
determination of 2 2 d i f f e r e n t p s y c h o t r o p i c
drugs including thioridazine i n toxicological
screening.
ii) Gas-Liquid
Chromatography (TLC)
et a1 ( 9 2 ) d e s c r i b e d a GLC-procedure
Dinovo f o r t h e d e t e c t i o n a n d d e t e r m i n a t i o n of
t h i o r i d a z i n e and i t s m a j o r m e t a b o l i t e s i n
p l a s m a s p e c i m e n s . A f t e r a l k a l i n i z a t i o n of
plasma, i t i s e x t r a c t e d w i t h o r g a n i c s o l v e n t
m i x t u r e f o l l o w e d by s e v e r a l c l e a n - u p s t e p s
512
Specimen
Temp.
(OC)
Thioridazine
Standard
2% SE-30 on chromosorb
W(100-120 mesh); chlorpromazine.
220
Thioridazine
Plasma
260
240
Thioridazine
Plasma
Non-conjugated urine
metabolites
&
Rt
(min.)
Detection
Reference.
FID
95
N-FID
93
FID
19
FID
12.0
270
260
3% OV-17 on Chromosorb Q
(100-120 mesh);chlropromazine
275
3.6
8.3
12.8
3.8
8.7
20.9
22.8
FID
35,94
92
5 14
T a b l e I 1 : IIP1.C
D c t e r m l n ~ t l o n of T h l o r l d a r l n e
l l l o r l d a z l n e (1)
P l m
~ r t l d o r f d s r l r c(IE)
T-2-suIfar ( s u l f o r l d a r l m )
T-2sulIoxide (nxsorldsrlnc)
T-hulfoxlde
NT-2sdfbdde
2,2,4-Trlncthylpntane
(YbZ), Zamlnoprnpinc
(0.7621, a c e t m l t r l l e (2.42)
ard etlvlml 0.482) a t
1.14 m l . d n -
lhlorldazlne (1)
1-2sulfoxlde
tktbml (Gm)nnd I2
T r l f l u q r r a z l n e ocetlc ocld
cmtalnlng ncld s I i m s a l c
(342) a t 2 nl.rnln-
1.2
nnd t l c t a b o I l t c s .
3 1 5 ng;ul-'
(2% Inn)
74
1.6
2.2
2.7
Fluorescence
M:36S/DI:440 m.
3-15
(Injected)
ng;ul-'
W (261 m)
0.25-10 ng;"l-l
9a
99
4.0
5.7
Plasm
(dole
8.5
1.9
9.
Ddoddazlnc (T)
Plasm
T-2-suUodde
T-24ulfare
Fluorescrncr a l t e r 2 W
xemn mrnrry
ltq~
0.5 ng
(Injected)
Irrndlatlon.
sulfate a t I ml.dn
l l d o r l d a z l (TI
T-2sul f oxlde
T-2sulf0ne
Plasm
l l l o r l d a z l n e (T)
I-2sdfoxide
T-2-sulfooe
PIam
n ~ l o r l d a r l n e(T)
T-2-sulloxlde
T-2sulfar
Urlne.plamu,
blle.
-50-120 p6
(0.01-4 w>ul-')
(injected)
103
0.052 ng..d-'
(0.035 ng.,uI-1
derectnblc).
101
W (265 nn)
4-45 "g.pl-1
102
Fluorencence Ec:J45/
m:425 m
2-20 ng(1njectcd)
-3.5 ng is deteccoble In sem).
103
p t -
Qrtm L l S s W S .
T a b l e 11 c o n t d ...
5 c l u l t l v lt y
nilorldazlre (T)
lbrthlorldarlne (M)
T-2 or S-sulfoxide
T-2auUonc
T-hulfoxldc d l s
s t e r e o l s m r l c palrs
nllorlddne (T)
T-2-sulfaxlde
Plasm
% alllra eel
vlth
precolum f l l t e r s .
rimm
b urine
PIasrm
s;um
T-Z-sulforr
I - h u l f o x l d e (2 I s m r s )
nzlne.llc1.
Plasm
(Mule
blood)
W (154 rm)
IIM Slllwlnrblellt
a f t e r T E separarlon on
xNt2 c o l m 4 t h elution
v l t h ethyl acetate (85%)
nrd p r o p a r r 2 a l (15.2).
IE $ I C ~ I ~pIa r t i c l e
slllw nid prccolvm s o l l d e
w r e l a t 2SoC. Trllupnmr
lhlorldarlne ( T )
bbrthlorldarlne (M)
T-2-sulfoxlde
1-S-rulfoxlde
Reference
32 on cyambrded
rrwned phase mlum
anblent. Cl\lorpmrurlne.
107
517
518
REFERENCES
1.
9,
10 (1967).
12. J., Renz., J.P., Bourquin, G., Gamboni and G., Schwarb;
U . S . Patents: 3,239,514, assigned to Sandoz Ltd.,
Switzerland.
5 19
13. M .
14. J.P.
Textbook of Pharmaceutical
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Press, London, 1969, p . 674.
16. T.J.
17. DRUGDEX @
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5,
2, 297
(1971).
1,2 3 0
Cubeddu; J. Clin.
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463
520
27. M.L. Rao, W.A. Brown and R. Wagner; Ther. Drug Monit.,
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28. C.D. Kilts, R.B. Mailman, E. Hudson, and G.R.
Fed. Proc., 40, 238 (1981).
Breese;
26,
298 (1968).
19, 242
Katz;
521
2, 561
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I,771
21,
889 (1986).
47. C.D. Sands, J.D. Robinson, R.B. Salem, R.B. Stewart, and
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2, 224
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18, 185
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55. Clarkes Isolation and Identification of Drugs in
Pharmaceuticals, Body Fluids, and Post-Mortem Materials,
2nd edn., A.C. Moffat (Editor), The Pharmaceutical
Press, London, 1986, p. 2 5 8 . , p. 134.
522
56.
26,
283-286
(1977).
57. K. Macek, J. Vecerkova, and J.
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19, 312
17,22
(1962).
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65.J.
66.W.
Lugas; J.
(1962).
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31, 405
23
16,236
(1967).
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Chromatogr.,
2,
523
72. J.
Lucas; J.
73. J.A.F.
27,
2,
2,
223
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78.S.
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117
2,
321
14,
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-
5280e (1964).
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111,
524
87. I.S. Forrest, F.M. Forrest and S.L. Kanter, J.E. Sperco,
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93. D. Debruyne,
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.
J. Pharm. Sci.,
40, 3D49 (1981).
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DeLeenheer; J. Chromatogr.,
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Chromatogr., 53, 413 (1970).
97. P.N. Kaul, M.W.
(1970).
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674
339 (1973).
Cimbura; J.
and G.J.
Clark, Nature;
21,
2,
226,
372
105 (1979).
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Brinkman and R.W.
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23,
&
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W e l l s , E.C.
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a n d W.B.
J.
Frei;
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31,
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H a w e s , and K.K.
J a w o r s k i , and
THIOTHIXENE
Dorothy K . Wyatt, Lee T. Grady
528
1.
Introduction
2.
Description
2.1
2.2
3.
Synthesis
4.
Physical Properties
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
4.10
5.
Infrared Spectrum
Nuclear Magnetic Resonance Spectra
Ultraviolet Spectrum
Mass Spectrum
Me1ting Range
Solubi1ity
Moisture Content
Isomerism
X-Ray Crystallography
Fluorescence Spectroscopy
Methods of Analysis
5.1
5.2
5.3
5.4
5.5
5.6
5.7
5.8
Elemental Analysis
Color Tests
Spectrophotometric Analysis
Fluorescence Analysis
Paper Chromatography
Thin-Layer Chromatography
Gas-Liquid Chromatography
High Performance Liquid Chromatography
Degradation-Stability
Pharmacokinetics
7.1
Absorption
7.2
Distribution
7.3
Metabolism
7.4
Excretion
8. Determination in Biological Fluids
8.1
Plasma
8.2
Blood
8.3
Urine
8.4
Other
9. Determination in Pharmaceuticals
10. References
6.
7.
THIOTHIXENE
1.
529
Introduction
thiothixene
thioproperazine
530
KN
CH C H ,-CH
SO,-~(CH,X
Thiothixene
2 H2gN302S2
mo?ecular weight: 443.62
CAS Registry No. : 5591-45-7
(2-)
3313-26-6
THIOTHIXENE
2.2
531
Synonyms
( dimethylsulfonamide th ioxanthene
r 23,251
3.
Synthesis
See following pages.
4.
Physical Properties
6.1
7.5
8.7
12.
4.2
assignment
C=C
502
502
vinyl CH
3.2
& N ~ N H
S%N(C
22
a lky la t i n 9
agents
2-Thlothlxcnc
6)
0
I
W
4
c
0
*
c
l.8I
NI
533
--
z,
0
E)
5?*
i-
6)
535
THIOTHIXENE
Multiplicity
of Proton
Characteristic
Reference
7.81
7.60
singlet
doublet
doublet
doublet
doublet
triplet
triplet
triplet
singlet
quartet
triplet
mu1tiplet
singlet
singlet
singlet
mu1tiplet
mu1tiplet
mu1tiplet
1 -H
3-H
4-H
8-H
5-H
6-H
7-H
14-H
( CH3 1SO2-CH2CH=
-CH2Npiperazine
CH3NCHQN( CH-))S0214-H
aromatic CH
1-H
aromatic CH
14-H
aromatic CH
31
7.57
7.48
7.35
7.31
7.24
6.01
2.73
2.62
2-54
2.52, 2.45
2.27
2.28
2.72
6.03
72-7.7
7.86
7.3-7.8
6
9.3-8.4
28
32
32"
0 0 ' 0 s OO'SZ
33NtlLlIUSNUYl
00'001 O O ' S L
00'
>
3
>
I
:
2
3
a.
jn
23
owz
D
..aJ
x
F:
a,
.A
50
.?I
a,
rn
a
a,
c,
&
.d
c,
0
0
O
N
0
O
0
0
N
0
m
0
0
1.
L4
537
THIOTHIXENE
Fig. 3.
downfield signals.
THIOTHIXENE
539
140.1
137.7
135.1
134.2
133.4
11
12
13
9
2
14
127.5
127.2
127.0
6
7
4
3
8
5
125.9
125.8
125.8
~~~
Carbon Number
132.1
130.4
127.7
10
1
540
Table IV
Ultraviolet Wavelengths and Absorptivity of Thiothixene
Solvent
Wavelength (nm)
methano1
230
methanol
methanol
0.1 N sulfuric
acid-
0.2
sulfuric
acid
strongly basic
0.1 M hydrochloric acid
alkaline pH
Reference
22
307
228
260
310
228
260
310
4.6
4.3
3.9
4.6
4.2
3.9
228
23
28
25
257
308
228
concentrated
sulfuric acid
Absorptivity
308
257
286
387
489
227
850"
140"
33
32
30
307
309
229
310
30
34
120"
33
8.
THIOTHIXENE
541
azm
E 18
h8
Fig. 6.
-.-
Conditions
Reference
Class I
22
22,28
24,26
542
544
4.6 Solubility
The approximate solubilities are reported in Table
VI.
Table VI
Solubilities of Thiothixene
Solvent
Solubility
water
alcohol
alcohol
chloroform
acetone
methanol
carbon tetrachloride
Reference
practically insoluble
solub1e
slightly soluble
ver; soiuble
slightly soluble
slightly soluble
slightly soluble
24,26
24
26
24,26
24,26
24
26
below.
Cis-trans Isomerism
Thiothixene exists in the two isomeric forms shown
Cis- ( 2 - ) Thiothixene
THIOTHIXENE
545
4.9
Crystal Structure
ANLP
A'N
A'NLP
4.10
4.9
7.4
7.9
6.1
6.8
Angle I AA'
142"
Z-Coordinates
ZAN
1.2
ZANLp
ZA'N
0.8
2.5
ZA'NLp 2 . 1
Fluorescence Spectroscopy
The excitation and fluorescence maxima f o r thiothixene are 335 nm and 385 nm, respectively as measured using
a Baird Atomic SF 100E spectrofluorometer fitted with a 150-w
xenon source. Excitation and emission slits were fixed to
provide a spectral bandpass of 6 nm [421. The fluorescence
maxima has also been reported as 380 nm using a Bearn
546
Fig. 9.
u
3.76
Mt
7.97
THIOTHIXENE
547
5. Methods of Analysis
percent
62.27
carbon
hydrogen
nitrogen
oxygen
sulfur
6.59
9.47
7.21
14.46
Color
Sensitivity
acid-formaldehyde
red
molybdate
red
vanadate
red
test
red-faint green
acid
orange
0.1 ?Jg
0.1 pi3
0.25 U-R0.1 p g
Reference
25
25
25
25
32
548
1451.
5.5
THIOTHIXENE
549
550
THIOTHIXENE
55 I
552
6.
Degradation-Stability
either addition of a
in an intermediate
which then collapses
formation of a charge
THIOTHIXENE
553
7.
Pharmacokinetics
7.1
Absorption
1291.
554
7.2 Distribution
Thiothixene is widely and rapidly distributed in
the tissues of rats [29]. Distribution after a single dose
(8mg/kg of thiothixene-26 ) is given in Table VIII for
tissues examined 4 hours a%ter dosing. At 4 hours after
intraperitoneal dosing, all tissues examined had higher
levels of radioactivity.
Table VIII
Tissue Levels
(pg
Intraperitoneal
24 hour
4 hour
heart
lung
liver
kidney
stomach
skin
muscle
brain
1.12,1.50
4.75,2.05
11.43,7.91
1.50,0.46
23.50,10.69
1.651.31
1.20,1.25
0.23,O .09
0.14,O.Og
0.70,0.80
4.42,5.91
0.61,0.38
0.23,0.46
0.23,O. 32
0.09,O. 14
0.02,0.04
4 hour
Oral
-
0.21 ,O. 13
0.85,O.90
4.96,7.17
0.47,0.68
24 hour
0.14,0.05
0.09,o. 10
6.60,5.71
0.42,O. 19
9.12,14.35 0.04,O.05
0.28 2 0.01
0.04,O. 05
0.04,O.09 0.03,O.02
0.04,O. 02
0*01,0.01
555
THIOTHIXENE
Table IX
Tissue Levels ( p g Thiothixene Equivalent/gm) in Rats
Days After Last Dose
heart
lung
liver
kidney
stomach
muscle
brain
1.07
2.08
21.35
0.61
0.81
3.58
3.37
0.98
0.06
1.67
1.87
15.62
1.77
3.05
0.23
0.03
12.32
2.96
2.84
0.66
0.21
0.72
0.66
0.54
0.43
0.60
0.59
0.73
556
Levels of thiothixene in the eye remained stationary for several days and then declined. The rate of decline
was slower than that of the liver. Thiothixene accumulates
in the pigmented choroid which is 8% of the total eye weight
so that concentration in the choroid may actually be quite
high relative to the liver concentration. Thiothixene has
some affinity for melanin and, hence, pigmented areas o f the
body such as the skin of rats or the choroid in the eye would
have a higher concentration of thiothixene.
7.3 Metabolism
The liver is the major site of thiothixene metabolism. Thiothixene is rapidly metabolized with little of the
drug excreted unchanged [ 2 9 ] . Some adverse effects on hepatic excretory function have been reported in rats [51. Bile
samples obtained within 0.5 hours of drug administration
contain the same range of metabolites as later urine and bile
samples.
The major metabolite of thiothixene in man [35,50],
and rats and dogs [291 has been identified as N-dimethylthiothixene. The thioxanthene moiety is believed to be excreted
unchanged [351. With the exception of possible !-methyl
fragments, thiothixene does not appear to be incorporated
into normal tissue constituents [44].
&Q
Jz
N-Demethylthiothixene
7.4 Excretion
Thiothixene is excreted mainly in the bile of both
rats and dogs studied although dogs did excrete a greater
fraction in the urine [29]. A 5:2 ratio of biliary to
urinary excretion was obtained for dogs 1291. Little to no
thiothixene is excreted unchanged with no discernible changes
in the pattern of metabolites during the course of excretion
[291.
THIOTHIXENE
8.
557
8.1 Plasma
A GC/EIMS procedure using a 1% Pentisil TM-350
column has been developed f o r the determination of thiothixene in plasma (sections 4.4, 5.7) [351. The plasma samples
were prepared by adding 80 ng of the internal standard, trideuterothiothixene, to 4 mL of plasma. The solution was made
alkaline by adding 4 drops of 2 sodium hydroxide and then
extracted first with diethyl ether and then with 5 mL of
ether-hexane (3:l). The combined organic extracts were dried
over sodium sulfate and the solvent evaporated under dry
nitrogen. The residue was dissolved in 0.1 mL of 0.01
methanolic acetic acid and 0.05 mL aliquots were analyzed by
G U M S using m/e 113 (thiothixene) and m/e 116 (trideuterothiothixene) fragments for quantitation [35]. Sensitivity
was determined to be less than 1 ng/mL of plasma. The ratio
of the peaks at the two masses together with the known concentration of internal standard added to the plasma samples
were used to calculate the concentration of thiothixene
present.
A GC/CIMS-SIMS procedure was developed for the
determination of thiothixene in plasma using a 3% SP-2250-DB
column (sections 4.4, 5.7). The mass fragment at 113 was
used for quantitation. As above, trideuterothiothixene was
used as the internal standard. The percent of the inactive
trans-isomer can also be determined using this procedure.
The trans-isomer exhibits a mass fragment at m/e 447 which
was not observed for the --isomer.
Plasma samples were
prepared by centrifugation of blood in a heparinized vacutainer. Four mL aliquots of plasma were taken, internal
standard added, and the pH adjusted to pH 10.5-11.5 by dropwise addition of 1 !sodium hydroxide. The samples were then
extracted with 6 mL of ether-hexane (3:1, v/v) followed by
two 4 mL extractions using the ether-hexane mixture. The
organic phases were combined and evaporated at 30C under
nitrogen. The residue was dissolved in 10 uL of methanol;
5 pL was used for the determination of thiothixene content in
plasma. The detection limit was less than 1 ng/mL of plasma
[361.
558
THIOTHIXENE
559
Blood
560
8.4 Other
A gas chromatographic procedure was used in the
analysis of liver samples [49] as described in section 5.7.
Thirty grams of liver were homogenized in 20 mL of water for
30 seconds. After the addition of 110 mL of ethanol the
solution was homogenized for 2 minutes. After centrifugation, the weight of the separated supernatant was adjusted to
150 g by addition of ethanol and the extract was filtered.
To 2.5 g of extract (0.5 g of wet liver tissue), 200 pL of
methanol solution was added containing 0.05 mg/mL of cyclizine and 0.1 mg/mL of mesoridazine as internal standards.
The solution is evaporated to near dryness under a stream of
nitrogen. The residue is reconstituted in 0.5 mL of Tris
buffer yielding a final pH of 9.0. The mixture was shaken
for 1 minute after the addition of 1 mL of butyl acetate.
The butyl acetate layer is removed after centrifugation and
injected into the gas chromatograph.
Thiothixene can be analyzed in brain tissue by the
fluorescence assay used in plasma determinations [63] after
treatment. The brain tissue is homogenized by hand in a
mixture of 2 mL of 1 y sodium carbonate and 2 mL of 1
sodium bicarbonate. The homogenate is extracted by shaking
with 10 mL of 1,2-dichloroethane f o r 15 minutes. After
centrifugation for 20 minutes, 5 mL of the dichloroethane
phase was withdrawn The remaining material was homogenized
again and re-extracted with 10 mL of 1,2-dichloroethane for
15 minutes. The phases were again separated by centrifugation and 10 mL of the dichloroethane solution was removed and
combined with the previous 5 mL aliquot. The thiothixene was
then extracted into 1.5 mL of 0.1
sulfuric acid by shaking
for 15 minutes and determined by the fluorescence assay
described previously.
9.
Determination in Pharmaceuticals
THIOTHIXENE
561
562
References
1.
2.
3.
4.
90,
153
(1974).
5.
6.
7.
8.
9.
10.
9(1),
10 (1967).
90,
298
(1967).
8,
400
(1968).
11.
12.
13.
263,
338
(1976).
14.
70,
310 (1965).
15.
16.
17.
Iversen, L.L.,
a,
188, 1084 (1975).
-9
THIOTHIXENE
563
18.
19.
20.
21.
22.
23.
24.
25.
w,
3, 425
1021.
26.
27.
28.
29.
30.
32.
564
13,
34.
35.
36.
-1,
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
11, 512
114,42
(1972).
29,
THIOTHIXENE
565
., 76(3),
231 ( 1987).
2, 166 (1981).
U, 9, 169 (1981).
9,
568
Contents
1.
Description
1.1
1.2
1.3
1.4
2.
Nomenclature
Formulae
Molecular Weight
Elemental Composition
Physical Properties
2.1
2.2
2.3
2.4
2.5
3.
Synthes is
4.
Biosynthesis
5.
Stab i 1i ty
6.
Methods of Analysis
6.1
6.2
6.3
6.4
6.5
6.6
7.
Titrimetric Methods
Spectrophotometric Methods
Photographic Methods
Differential Thermal Analysis
Chromatographic Methods
Biological Methods
Pharmacokinetics
Acknowledgment
References
5 69
CYCLOSERINE
1.
Description
1.1
Nomenclature
1.1.1
Chemical Names
D-4-Amino-3-isoxazolidinone
D-4-Amino-3-isoxazolidone
1.1.2
Generic Names
Far-
Trade Names
Formulae
1.2.1
Empirical
C3H6N20 2
1.2.2
Structural
-NH
4
Is
0'
\;/
$0
1.2.3
1.2.4
T50MVTJ DZ *DX ( 3 )
570
1.3
Molecular Weight
102.09 (1)
1.4
Elemental Composition
C 35.29%, H 5.92%, N 27.44%, 0 31.34% (1).
2.
Physical Properties
2.1
Melting Range
155-156C [with decomposition] (lY3).
2.3
Solubility
Optical Rotation
23
[a]D
2.5
Spectral Properties
2.5.1
The UV
absorption spectrum of Dcycloserine in neutral methanol is obtained on a Cary
219 spectrophotometer. The spectrumy shown on Figure
1, is characterized by a maximum at 215 nrn. Reported
UV maxima are 226 nm E(l%, lcm) 402 in water (1,3), 217
nm in 0.1 N HC1 ( 3 ) and 222 nm in 0.1 N NaOH ( 3 ) .
2.5.2
The
IR absorption spectrum of Dcycloserine is obtained from a potassium bromide diper-
571
CYCLOSERINE
200
(nm) 300
400
in neutral methanol.
572
Freauency ( cm-1 )
Assignment
3300-2100
1630
1600-1500
800-650
500
Tortional (-NH3) N
oscillation
2.5.3
-H
100,
4000
3ooo
2500
WAVE NUM BE R ( C M - 1 )
1400
1000 800
from
600
K B r disc
400
200
5 74
ru
0
0
a,
.-
aJ
.-C
m
-0U
V
I
n
0
E
L
aJ
a
m
..
Z
1
I
m
.-(7,
LL
Dioxane
i'l
CH2'u
CH
u)
0
LL)
,c=o
\
Figure L :
'3CNMR
dioxane
spectrum of
D-cycloserine
as internal reference.
in
D20 w i t h
576
2.5.5
Mass Spectrum
H
L
- co
HG?
NH2
:cJt
m / e 102
NH,
L
m / e 74
II
C
7'
H-0
N -CH2=CHNH 2
N'
.'\o
*O
m / e 102
m / e 59
CYCLOSERINE
M I E 50
5 77
60
70
80
90
F i g u r e 5: M a s s s p e c t r u m
100
of
110
D-cycloserine
120
578
2.5.6
Thermal Analysis
The x-ray diffraction pattern of Dcycloserine was determined with a Philips Pull
automated X-ray Diffraction Spectrogoniometer equipped
with PW 1730/10 generator. Radiation was provided by a
copper target (Cu anode 2000 W,y = 1.5480 A ) and high
The
intensity x-ray tube operated at 40 KV and 35 MA.
monochromator was a curved single crystal one
(PW1752/00) Divergance slit and the receiving slit wire
1 and 0.1" respectively. The scanning speed of the
goniometer (PW1050/81) used was 0.02-28 per second.
The instrument is combined with Philips PM 8210 printing recorder with both analogue recorder and digital
printer.
The goniometer was aligned using silicon
sample before use.
The x-ray pattern of D-cycloserine is presented in
Figure 7.
The interplanner distance dA and relative
intensity 1/10 are shown in Table 2.
100
110
120
130
140
150 160
Temperature CC.1
170
180
190
200
580
28
Figure 7 : The X-ray
diffraction pattern
Of
58 1
CYCLOSERINE
Table 2.
16.53
9.10
6.21
5.67
5.10
4.71
4.55
2.24
4.00
3.84
3.71
3.44
3.39
3.34
3.25
3.10
3.02
2.82
2.69
2.67
2.62
2.54
2.49
2.46
2.43
2.40
2.35
2.27
2.26
2.22
2.20
20.25
18.48
3.95
3.30
2.98
100
57.80
70.16
37.82
12.17
23.23
11.90
6.08
6.27
8.37
7.74
43.76
28.46
33.73
14.14
3.00
3.24
5.45
9.59
10.27
8.58
7.32
5.54
4.43
3.66
3.30
2.18
2.15
2.13
2.06
2.02
2.00
1.95
1.92
1.89
1.84
1.82
1.77
1.77
1.73
1.72
1.69
1.66
1.64
1.64
1.60
1.58
1.57
1.55
1.52
1.49
1.48
1.45
1.43
1.42
1.40
3.39
2.91
5.36
2.01
4.15
4.13
3.79
5.26
3.75
3.05
2.02
3.07
3.46
2.33
2.97
4.60
2.54
3.47
4.01
2.64
3.22
2.76
2.22
2.46
1.98
1.83
2.03
2.56
2.23
2.19
Synthesis
D-Cycloserine can be chemically synthesized by the
method of Evan ( 1 0 ) in which DL-serine is converted to
582
its methyl ester. The ester is treated with triphenylmethyl chloride and methane sulfonyl chloride to give
the substituted ethylene amine ( I , Scheme I ) . The latter (I) can be converted into the hydroxamic acid (11).
Reaction of (11) with hydrochloric acid yielded D-aamino-B-chloro-N-hydroxypropionamide (111) which upon
treatment with a basic ion exchanged cyclizes to Dcycloserine
Scheme I.
-*
- COOH
30-CH2-CH
I
I
- COOCH3
HO-CH2-CH
I
I
NH2
NH2
CONHOH
Ph-
I - Ph
dCOOCH3
I
Ph-
- Ph
Ph
Ph
C1-CH -CH-NH2
2 l
CONH-OH
(1111
H
D-Cycloserine
CYCLOSERINE
4.
583
Biosynthesis
D-Cycloserine is produced by various Streptomyces.
Harned et al. (11) isolated the antibiotic from culture
filtrates by adsorption onto a strong base anion exchange resin and elution with HzS04. The antibiotic is
then converted to a water-insoluble silver salt.
The
isolated pure salt is then decomposed with HC1 to give
cycloserine which is crystallized from the filtrate
with alcohol or acetone.
A more recent method of isolation of crystalline Dcycloserine from fermentation broth filtrates is
reported by Yakhontova et al. (12).
The method includes sorption of cycloserine in the cationic form by
a strong cross-linked sulfo-cation exchange resin (on
the basis of styrene and divinylbenzol) in a series of
columns.
Desorption is accomplished using an aqueous
ammonia solution. A product of high purity is claimed
to be obtained from the diluates after their clarification, evaboration and dilution of the concentrate with
ethyl alcohol.
Cai et al. (13) presented evidence for the presence of
plasmids in Streptomyces and their possible control
over the biosynthesis of antibiotics. They carried out
curing studies with acridine orang and indicated that
the production of antibiotics by three strains of
Streptomyces was
plasmid-determined, with curing
frequency of 5-10%. Curing of Streptomyces strain 2286
with acridine orange caused loss of the ability of synthesizing cycloserine, accompanied by the disappearance
of the intermediate 0-carbamyl-D-serine.
Using incubation experiments, Svensson and Gatenbeck
(14) proposed a pathway for the biosynthesis of Dcycloserine in Streptomyces garyphalus. The incubation
experiments carried out with washed cells and toluenetreated cells of S. garyphalus, showed that 0-acetyl-Lserine and hydroxyurea are intermediates in the biosynthesis of D-cycloserine.
The formation of [I4C]Oureidoserine from 0-acetyl-L-serine and hydroxyurea was
demonstiated by enzymic incubations using 14C-labeled
substrates. Desalted cell-free extracts catalysed the
conversion of 0-ureido-D-serine to D-cycloserine in an
ATP- and Mg++- requiring reaction. The proposed path-
584
- -
2 1 COOH
It
CH -C-O-CH2-CH - NH
--
I
COOH
0-Acetyl-L-serine
H2N-CONHOH
Hydroxy
urea
0-Ureido-L-serine
T2- T -
NH2
0-Ureido-D-serine
NH2
D-Cycloserine
CYCLOSERINE
5.
585
Stability
Cycloserine deteriorates upon absorbing water and is
destroyed at neutral or acidic pH.
D-cycloserine capsules should be stored in tight containers at less than
40'C, preferably between 15-3O'C ( 4 ) .
Absorbed moisture from the air was found to affect the
stability of cycloserine preparations in the metal
packed capsules produced by various companies after
testing for more than one year ( 1 5 ) .
Ciestak et al. ( 1 6 ) studied the effect of acidity on
the stability of cycloserine during its recovery from
filtered culture broth, When an aqueous solution of
cycloserine adjusted to pH 1 . 2 6 - 3 . 6 0 , the degree of
decomposition of the drug was 50% after 22 hrs of
standing at 2 5 " , and 25% at 4 " , irrespective of the
concentration or type of acid used.
The product of
decomposition was identified as l3-aminoxyalanine which
is believed to be subsequently converted to serine.
The stability of samples of aluminium foil sealed
cycloserine capsules produced by different companies
were tested at room temperature, 37' amd 38" with
saturated humidity.
Samples from the company kept under the last condition, decomposed after one month
storage ( 1 7 ) .
Kartseva et al. ( 1 8 ) studied the stability of acycloserine samples containing various amounts of moisture and kept in sealed glass tubes with silica gel at
one end.
The samples were analysed when the controls
without silica gel reached 20% decomposition. At 40"
the greater the extent of decomposition. Little decomHowever, at
position was observed at 0 . 1 5 % moisture.
1 . 3 % a sharp drop was observed with increasing distance
from the silica. When the powder with 1.1% moisture
was initially stored for 12 days at 5-8" or 18-20' and
then at 40', little decomposition was observed.
586
6.
Methods of Analysis
6.1
Titrimetric Methods
6.1.1
Non-Aaueous Titration
Cycloserine was determined (19) by nonaqueous titration using the following procedure:
Dissolve 0 . 1 g of active substance, o r the equivalent
amount of powdered tablet, in 15 ml of conc. acetic
acid, add 30 ml of dioxan and 4 drops of 1% methanolic
mentanil yellow ((2.1. Acid yellow 3 6 ) , and titrate with
0.1 N perchloric acid in dioxan to the color change
from yellow to red-violet; 1 ml of acid = 10.2 mg of
cycloserine.
6.1.2
Potentiometric Methods
Braibante et al. (20) studied the equilibrium of D-cycloserine with protons and cobalt (11),
nickel (11), copper (11) zinc (11) aqueous ions in
solutions, the equivalent of D-cycloserine (HL) with
the ions of H, Co, Ni, Cu and Zn were studied potentiometrically at 25' and 0.1 mol/dm3KC1.
The protonation constants are log K = 7.346(5) (-NH3+) and log Kz
= 4.388(6) (-OH); the corresponding entalpy changes are
- 32.25(15) and - 14.52(15) KJ/mol respectively. The
metal ions form the complexes M(HL)z+, MLC and ML2.
Stability contents are given.
6.2
SDectroDhotometric Methods
CYCLOSERINE
587
588
Photographic Methods
Chemiluminescence determination of micro amounts of organic reductants (drugs including cycloserine) by reaction involving vanadate (30).
The method involves
reduction of 0 . 5 m M-NaV03 to V1v by on organic reductant in 0.05 N - HzS04 medium. The reaction mixture is
heated for 10 to 40 minutes and the VlV is determined
photographically by its catalysis of the chemiluminescence reaction is carried out with 20 @-luminol
is
sodium carbonate-sodium bicarbonate buffer solution of
pH 11. The method was used for determining 0.5 to 3 pg
in 5 ml solution of cycloserine. The coefficient of
variation were < 30%.
Mohamed and Tawakkol (32) have reported a quantitative differential thermal analysis (DTA) of
cycloserine. The drug was detected in bulk powder and
tablets by a DTA method based on the electric voltage
generated by a thermocouple due to a difference in temperature (AT) between the sample and a reference
material for the time interval during which the phase
change occurs as the system is heated.
An empty
aluminium crucible was the reference material and the
heating rate maintained at 5% minutes. Areas under the
CYCLOSERINE
589
AT-time
the peak
1-5 mg/
accuracy
6.5.
ChromatograDhic Methods
6.5.1
Paper ElectroDhoresis
590
electrophortic mobility decreased with increasing ammonia concentration. A development time of 2 hours allowed the separation of a mixture of the six antibiotics.
6.5.3
Biological Methods
Cycloserine was detected in food and was estimated by a simplified zone inhibition method.
The
bioassay cycloserine and other antibiotics in food was
carried out using bacteria ( 3 8 ) .
The calculation of the kinetics of extraction of
cycloserine and other antibiotics from the native solutions by a fluidized-bed ion-exchange method, have been
A mathematical formula and a nanogram
reported (39).
for the kinetics of extraction of antibiotics from native solutions by the title method were suggested. The
CYCLOSERINE
591
calculated results were in good agreement with experimental data e.g. on the extraction of cycloserine
and of Kanamycin.
The microbiological turbidimetric potency assay for
cycloserine and other antibiotics is modified ( 4 0 )
under the federal food drug and cosmetic act, to
provide for developing a standard curve with concentrations of 6 4 , 80, 100, 125 and 156% of the reference
concentration of the assay. More accurate potency concentration estimates are obtained when samples are
diluted to a concentration in the 80-125% range.
The
modification are for tests and methods of assay of
antibiotics and antibiotic-containing drugs; revised
standard response line concentrations.
indicator method of determining tuberculostatic
drugs (including cycloserine) in the urine was pubAn indicator containing 1 part of sodium
lished ( 4 1 ) .
pentacyanamino-ferronate and 5 parts
lactose was
devised for determining tuberculostatic drugs in urine.
The method was sensitive to 10 pg/ml and took only 2-3
minutes.
Cycloserine was indicate by a blue green
color.
An
7.
Phareacokinetics
7.1
Absorption
592
Distribution
CYCLOSERINE
593
7.3
Elimination
The plasma half-life of cycloserine is approximately 10 hrs., in patients with normal renal function.
In patients with impaired renal function, plasma concentrations are higher and the half-life is prolonged.
When an oral dose of cycloserine was given to patients
with normal renal function, 60-70% of the dose was excreted unchanged in urine by glumerular filtration
within 72 hrs. Small amounts of the drug were also excreted in feces.
The remainder of the dose is thought
to be biodegraded to unidentified metabolites ( 4 2 ) .
The pharmacokinetics comparative study of Zitkova and
Tousek ( 4 4 ) on cycloserine and terizidone has showed
that the excreted quantity of terizidone in urine was
higher, but the differences as compared with excreted
cycloserine were not
statistically significant.
Patients with higher age average showed slower excretion rate in urine.
Coletsos ( 4 8 ) studied the elimination of cycloserine,
given S.C. or in to guinea pigs, rabbits and chicken.
The drug was almost completely eliminated in several
594
2.
"Index Nominum: International Drug Directory", Compiled by the Scientific Documentation Centre of the
Swiss Pharmaceutical Society, Zurich, 1987.
3.
4.
5.
6.
7.
C. Stammer and J .
(1965).
McKinney,
SOC.,
77,
2345
CYCLOSERINE
595
8.
K . Fukushima and T.
107 (1979).
9.
10.
27(2),
1965.
11.
12.
13.
14.
M.L.
Microbiol.,
1 3 1 ( 2 ) , 129 (1982).
15.
16.
I.
Cieslak, N.
Antibiotiki,
12, 410
(1967).
17.
18.
19.
20.
21.
M.
19(12),
734 (1968).
71(5-6),
V.D.
Chem.,
223-233 (1981).
307 (1965).
22
Through Analyti-
596
23.
V.S.
Svinchuk, V.F.
Krdarenko
Probl. Tuberk, 6, 64-66 (1979).
24.
V.S.
Svinchuk, V.P.
Kramarenko and
Farm. Zh., 1, 74-75 (1980).
25.
V.S.
26.
27.
R.C.
46,
and M.M.
Orlinskii,
M.M.
Orlinskii,
6 , 46-49 (1982).
19 (1984).
E. El-Sayed, Z.H.
Analyst,
Wahbi,
28.
29.
30.
N.M.
Lukovskaya and E.V.
Mitropolitska,
Khim., 30(5), 985-998 (1975).
31.
V.S.
32.
33.
K.C.
34.
(1970).
109, 388
Zh.
6 , 46-49 (1982).
Sci.,
King
(1967).
Maa Bared,
J.
Chromatogr.,
120-123 (1968).
35.
analit
36(1),
33,
36.
L. David, F.E.
Gainer and H.J.
S c i . , 62(8), 1344-1346 (1973).
37.
D.G.
Musson, S.M. M a g l i e t t o , S.S. Hwang, D. G r a v e l l e s e
and W. F. Bayne, J. Chromatogr. Biomed. A p p l . , 414, 121
Wasselman,
J.
Pharm.
(1987).
38.
H. Murakami, M.
Kanzaki, C. Fujimoto and M. Haruta,
Shokuhin Eiseigaku Zasski, l2(2), 86-94 (1971).
CYCLOSERINE
597
39.
40.
41.
42.
43.
44.
45.
46.
K.G.S.
Nair, I . G . Epstein, H. Baron and M . G . Mulinos,
A n t i b i o t i c s Annual, 136-140, 1955-1956.
47.
48.
P. Coletsos, Scand.
(1970).
9(6),
(1974).
113 (1979).
J.
Resp.
Dis.
Suppl.,
7l,
31,
40
600
CONTENTS
1.
Therapeutic function.
2.
Description.
3.
Physical Properties.
4.
Spectral Properties.
4.1
4.2
4.3
4.4
4.5
4.6
Ultraviolet Spectrum.
Infrared Spectrum.
Mass Spectra.
Nuclear Magnetic Resonance Spectra.
Thermal Analysis.
X-Ray Powder Diffraction.
5. Chemical Properties.
6.
Synthesis.
7.
Metabolism.
8.
Pharmacokinetics.
9.
Clinical Toxicity.
Colorimetry.
Ultraviolet Spectrometry.
Infrared Spectrometry.
Flourine-19-NMR.
Mass Spectrometry.
Fluorometry.
Papaer Chromatography.
Thin-Layer Chromatography.
High-pressure Liquid Chromatography.
60 I
FLUOROURACIL
602
5-FLUOROURACIL
1.
THERAPEUTIC FUNCTION
5-Fluorouracil has been used in the treatment of cancer
or more than two decades. It is a fluorinated
antimetabolite of the pyrimidine uracil. It slows tumour
cell growth by inhibiting thymidine formation, thereby
inhibit protein synthesis by incorporating into RNA.
2. DESCRIPTION
2.1 Nomenclature
2.1.1
Chemical Names
5-Fluoro-2,4(1H,3H)-pyrimidinedione,
2,4-Dioxo-5-fluoro pyrimidine,
2,4(1H,3H)-pyrimidinedione, 5-fluoro.
2.1.2
Generic Names
Trade Names
Structural
9"
603
FLUOROURACIL
H:2.32%,
F:14.61%,
N:21.54%,
0:24.60%.
3.
PHYSICAL PROPERTIES
3.1 Melting P o i n t
Melting p o i n t l i e s between 282 t o 283OC w i t h decompos i t i o n (3).
604
~~
~~
~~
Constants
Values
Melting Point
(deg. Kelvin)
551.85
Sol. i n mter
(ml/L)
4.711537E-04
Heat of fusion
8800
Sol.in glycerol
~ml/L)
6.7037963-04
19694.43
Sol. i n FG
1.109421E-04
28494.43
Sol.in nrethanol
(ml/L)
8.535111E-05
493.3872
Sol.in ethanol
(ml/L>
1.856792E-05
4569.321
Sol.in propanol
5.508214E46
5543.161
Sol.in acetone
(mol/L)
2.174898E-07
Heat of Dissolution
i n water (cal/ml)
9293.387
Sol& dioxane
(mom)
3.011725E-09
Heat of Dissolution
i n octanol (cal/ml)
13369.32
Heat of Dissolution in
chloroform (cal/ml)
14343.16
9.3742983-08
4.849687E-07
(cal./ml)
constants
Values
(mom
(mol/L)
Log PC n-octanol
-2.987449
kg Pc chloroform
-3.701223
containers.
605
FLUOROURACIL
3.4
Caution
SPECTRAL PROPERTIES
4.1 Ultraviolet Spectrum
Infrared Spectrum
Assignment
3122
1718 and 1655
1425
1243
812
NH Stretch
C = 0, C =
N- stretch
CH in plane
CH out plane.
606
u
z
OH
m
L1:
I n d
_~
1
3
0
w
0
u
0
0
0
0
s
0
co
z
5:
0
N
60
609
FLUOROURACIL
Proton NMR
Proton assignment
(DMSO-d6)
b (HDO)
C
2.49
3.43
7.80
d
e
10.80
11.45
4.4.2
Carbon-13 NMR
1000
r3228
50.C
15261 68
I. ' ' ' I '
' I .
.-
88 97 108
I
"
- ' 1 ' .n
611
613
FLUOROURACIL
OH
Carbon assignment
a (DMSO-d6)
b (Keto-enol)
c
(Keto-enol)
d
e
f
;::p
137.57
142.08
150.08
158.00
158.21
0.
282 *O
-2.
-281.5
.
E
3
LL
Purity
!
Melting pt
:
Depression :
:
Delta H
Correction :
Mol.weight :
C e l l const :
Onset slope:
,A
-6.
a
a,
I -8
$7 f3
,
I
-1 0-
-1 2
oz
h
-281.0
3 -4
E
!?
100.800Moleo/o
278.7C
-0.55c
36.9kJ I mole
20.00/0
130.1 g l M o l e
I . 282
-7.90 m w / c'
-280.5
-280.0
i 60
5I
,
I
180
-279.5
30
l
35
,
,
I
200
220
240
260
280
300
Temperature
aJ
I-
-279.0
Total Area / partial Area
I0I l 15l , 20
25
l
,
l
;;i
L
aJ
Q
E
(c")
Figure 7 : Thermal c u r v e of 5 - F l u o r o u r a c i l .
FLUOROURACIL
615
1/10
7.94
6.87
6.64
5.60
5.47
4.97
4.69
4.50
4.32
4.07
3.96
3.90
3.74
3.60
3.51
3.45
3.29
3.19
3.11
2.87
2.80
2.73
2.70
2.639
2.571
2.47
0.218
0.268
0.190
0.44
0.461
0.25
0.50
0.37
0.725
1.09
1.17
0.58
0.68
0.83
1.24
0.53
0.49
3.27
100
2.30
0.96
0.48
0.48
0.31
0.44
0.42
1/10
2.27
2.39
2.29
2.19
2.14
2.10
2.04
1.98
1.95
1.94
1.91
1.861
1.84
1.83
1.81
1.79
1.77
1.75
1.70
1.68
1.61
1.59
0.33
0.23
0.21
0.15
0.19
0.23
0.18
0.19
0.18
0.22
0.24
0.17
0.15
0.17
0.24
0.29
0.24
0.18
0.25
0.35
0.18
0.35
1.55
1.15
1.52
1.50
1.38
0.22
0.37
0.12
d = i n t e r p l a n a r d i s t a n c e 1/10 = r e l a t i v e i n t e n s i t y
based on h i g h e s t i n t e n s i t y of 100.
616
10-
9876-
70 65
55
45
35
20
25
15
617
FLUOROURACIL
5. CHEMICAL PROPERTIES
5.1 Effect of Flourine
10
I'
H+
SYNTHESIS
a) Potassium fluoroacetate (I) is reacted with methyl
bromide to form methyl fluoroacetate (11) which is then
subjected to Claisen condensation with methyl formate
and sodium ethoxide to produce the potassium enolate of
the methyl ester o f fluoromalonaldehyde (111).
Cyclization of I11 is effected through condensation
under anhydrous conditions with S-benzylisothiourea
618
li
II
FCH,C-OK +. CH,Br
I
FCH,C-0-CH,
NaO C,H
F O
K O C ~ - ' I . . -e-O-CH,
111
H
H
H
VI
FLUOROURACIL
619
c) 5-Fluorouracil was prepared by the procedure described in Scheme 1 using ethylfuoroacetate as starting
material and 2-ethyl-2-thiopseudourea hydrobromide
instead of benzylisothiourea (1).
7. METABOLISM
5-Flourouracil is metabolized extensively in the liver
and its concentration decline rapidly to undetectable
level within 2 hours. As plasma 5-f luorouracil
concentration decline, concentrations of its major
metabolites, 5,6-dihydro-5-fluorouracil (f luorouracilH2), W - f luoro-p-ureido-propionic acid (FUPA) and tX fluoro-j3-guanido-propionic
acid (FABL) increase (9).
Fluorouracil-H2 is detectable within 5-minutes of
administration of 5-f luorouracil with peak plasma conenrations of 23.7 pmol/L occuring after 1 hour (10). It
was reported that fluorouracil-H2 is an important active
fluoropyrimidine catabolite (11).
The liver converts fluorouracil-H2 to f luorouracil-PA
and FBAL by a dose-dependent saturable system.
Fluorouracil-PA and FBAL peak serum concent rations are
detectable approximately 90 minutes after infusion (12).
Inactivation of 5-fluorouracil by the liver during
continuous regional and systemic infusion in pigs was
et a1 (13).
reported by Almersjo -Fluorouracil is converted intracellularly to 5-f luoro2-deoxyuridylate (FdUMP) by a series of enzymatic
reactions. Initially, 5-monophosphate nucleotide (FUMP)
is formed either by orotate phosphoribosyl transf erase
in the presence of 5-phosphoribosyl-1-pyrophosphate
(PRPP), or by the action of uridine phosphorylase and
then uridine kinase (14) (Figure 9). The FUMP is further
metabolized to diphosphate (FUDP) and t h e n t o
riphosphate (FUTP) which can be incorporated into RNA
thus producing a fraudulent RNA. However, the primary
activation steps of fluorouracil involves the formation
of the deoxymonophosphate (FdUMP) by the reduction with
ribonucleotide reductase to FdUDP and then by the action
of the phosphorylase to FdUMP.
620
FUDP /
FUTP
FUMPI
5FU
\\
FUR
uridine
FUdR
/
-
FdUDP
RNA
FdUMP
PHARMACOKINETICS
8 . 1 Absorption
5-Fluorouracil
i s most
commonly
administered
FLUOROURACIL
62 1
4.
8.3 Elimination
Urinary exfietion of intravenously injected 5-f luorouracil-2- C , given as a single dose, amounts to only
11% in 24 hours; however, during this period, 63% of the
radioactivity is expired as carbon dioxide. Given by
continuous intravenous infusion for 24 hours, plasma
concentration in the range of 0.5 to 3.0 pM are obtained
and the uri ary excretion of 5-fluorouracil is only 4%,
while the C02 excretion rises to 90% (25).
5-Flourouracil-HZ represents 1% of total metabolites
eliminated by the kidney, while FBAL accounts for more
than 70%. Only minor amounts of 5-fluorouracil and
fluorouracil-PA are detected in the urine.
622
CLINICAL TOXICITY ( 6 )
The clinical manifestations of toxicity caused by
fluorouracil and floxuridine are similar and may be
difficult to anticipate because of their delayed
appearance. The earliest untoward symptoms during a
course of therapy are anorexia and nausea; these are
followed shortly after by stomatitis and diarrhea, which
constitute reliable warning signs that a sufficient dose
has been administered. Stomatitis is manifested by
formation of a white patchy membrane that ulcerates and
becomes necrotic. The occurrence of similar lesions in
the stoma of colostomies and at post-mortem examination
of the gastrointestinal tract, as well as complaints of
dysphagia, retrosternal burning, and proctitis, indicates that enteric injury may occur at any level. The
m a j o r t o x i c e f f e c t s , however, result from the
myelosuppressive action of these drugs; clinically, the
effects are most frequently manifested as leukopenia,
the nadir of which is usually between the ninth and
fourteenth day after the first injection of drug.
Thrombocytopenia and anemia may complicate the picture.
Loss of hair, occasionally progressing to total alopecia, nail changes, dermatitis, and increased pigmentation and atrophy o f the skin may be encountered.
Neurological manifestations, including an acute cerebellar syndrome, have been reported, and myelopathy has
been observed after the intrathecal administration of
fluorouracil. The low therapeutic indices of these
agents emphasize the need for very skillful supervision
by physicians familiary with the action of the
fluorinated pyrimidines and the possible hazards of
chemotherapy ( 6 ) .
623
FLUOROURACIL
A: The i n f r a r e d a b s o r p t i o n s p e c t r u m of a m i n e r a l o i l
d i s p e r s i o n of i t e x h i b i t s maxima o n l y a t t h e same
w a v e l e n g t h s a s t h a t of a s i m i l a r p r e p a r a t i o n of USP
F l u o r o u r a c i l RS.
B: T h e u l t r a v i o l e t a b s o r p t i o n s p e c t r u m of a 1 i n
100,000 s o l u t i o n i n a pH 4.7 a c e t a t e b u f f e r ( p r e p a r e d
from 8.4 g o f sodium a c e t a t e and 3.35 mL of g l a c i a l
a c e t i c a c i d mixed w i t h w a t e r t o make 1000mL) e x h i b i t s
maxima and minima a t t h e same wavelengths as t h a t of a
s i m i l a r s o l u t i o n of USP F l u o r o u r a c i l RS, c o n c o m i t a n t l y
measured, and t h e r e s p e c t i v e a b s o r p t i v i t i e s , c a l c u l a t e d
on t h e d r i e d b a s i s , a t t h e wavelength of maximum absorbance a t about 266 nm do n o t d i f f e r by more t h a n 3%.
To 5 mL of a s o l u t i o n ( 1 i n 100) and 1 mL of bromine
water TS: t h e bromine c o l o r i s discharged.
C:
10.2 F l u o r i n e Content
US Pharmacopeia 1985 ( 3 ) d e s c r i b e s t h e a s s a y of f l u o r i n e
5 - f l u o r o u r a c i l a s follows:
F l u o r i n e c o n t e n t - - [ N o t e - A l l l a b o r a t o r y u t e n s i l s used
i n t h i s procedure should be s c r u p u l o u s l y c l e a n and f r e e
f r o m e v e n t r a c e a m o u n t s o f f l u o r i d e . The u s e of
p l a s t i c w a r e , wherever p o s s i b l e , i n t h e p r e p a r a t i o n and
s t o r a g e of s o l u t i o n s and f o r measurement of p o t e n t i a l s
is recommended].
I s o p r o p y l a l c o h o l s o l u t i o n - D i l u t e 295mL o f
i s o p r o p y l a l c o h o l w i t h water t o 500 mL.
Buffer solution
To 55 g of sodium c h l o r i d e i n a 1l i t e r v o l u m e t r i c f l a s k add 500 mg of sodium e i t r a t e , 255
g of sodium a c e t a t e and 300 mL of w a t e r . Shake t o
d i s s o l v e , and add 115 mL of g l a c i a l a c e t i c a c i d . Cool t o
room t e m p e r a t u r e , add 30 mL of i s o p r o p y l a l c o h o l , d i l u t e
w e i t h water t o volume, and mix. The pH of the r e s u l t i n g
s o l u t i o n i s between 5.0 and 5.5.
624
R e a g e n t b l a n k - P i p e t 15 mL of 1,2-dimethoxyethane
i n t o a f l a t - b o t t o m , g l a s s - j o i n t , 500-mL f l a s k , and
p r o c e e d a s d i r e c t e d u n d e r T e s t p r e p a r a t i o n , beginning
w i t h "add t h e c o n t e n t s of a 15-mL v i a l 0s s o d i u m
b i p heny 1 s o 1u t ion.
Modified calomel r e f e r e n c e e l e c t r o d e - Mix 70 mL of
a f r e s h l y prepared s a t u r a t e d potassium c h l o r i d e s o l u t i o n
with 30 mL of i s o p r o p y l a l c o h o l , f i l l t h e e l e c t r o d e with
t h e c l e a r s u p e r n a t a n t l i q u i d , and a l l o w t h e e l e c t r o d e t o
soak i n t h e remainder of t h e s o l u t i o n f o r a minimum of 2
hours b e f o r e using. S t o r e t h e e l e c t r o d e immersed i n t h e
p o t a s s i u m c h l o r i d e - i s o p r o p y l a l c o h o l s o l u t i o n when not
i n use.
Standard s t o c k s o l u t i o n - Weigh a c c u r a t e l y 2.211 g
of sodium f l u o r i d e , p r e v i o u s l y d r i e d a t 150' f o r 4
h o u r s , i n t o a 1 - l i t e r v o l u m e t r i c f l a s k , and d i s s o l v e i n
a b o u t 200 mL of w a t e r . Add 1 mL of sodium h y d r o x i d e
s o l u t i o n ( 1 i n 2 5 ) , d i l u t e w i t h w a t e r t o volume, and
mix. S t o r e t h i s s o l t u i o n i n p l a s t i c c o n t a i n e r s . One mL
is e q u i v a l e n t t o 1 mg of f l u o r i d e .
Standard curve - D i l u t e 10.0 mL of S t a n d a r d S t o c k
s o l u t i o n with w a t e r t o 100 mL. I n t o each of f o u r 100-mL
v o l u m e t r i c f l a s k s p i p e t 0.8, 1 . 0 ,
1.2 and 1.6 mL,
r e s p e c t i v e l y , o f t h e r e s u l t i n g s o l u t i o n . To each f l a s k
add 15 mL of Reagent blank, d i l u t e w i t h B u f f e r s o l u t i o n
t o volume, and mix. Use t h e s e d i l u t i o n s , c o n t a i n i n g ,
r e s p e c t i v e l y , 0.8, 1.0, 1 . 2 and 1.6 ug p e r mL, t o
c o n s t r u c t t h e s t a n d a r d curve a s follows. Determine t h e
p o t e n t i a l s of each s o l u t i o n a s d i r e c t e d under Procedure.
P l o t t h e r e s u l t s of f l u o r i n e c o n c e n t r a t i o n a s t h e
a b s c i s s a , i n mg p e r 100 mL v e r s u s t h e p o t e n t i a l , as t h e
o r d i n a t e , on s e m i l o g a r i t h m i c graph paper, f o r each of
t h e s t a n d a r d s . Draw t h e b e s t s t r a i g h t l i n e through t h e
plotted points.
Test p r e p a r a t i o n
P l a c e 200 mg of F l u o r o u r a c i l ,
a c c u r a t e l y weighed, i n a 250-mL v o l u m e t r i c f l a s k , add
about 150 mL of 1,2-dimethoxyethane, shake by mechanical
means t o d i s s o l v e , d i l u t e w i t h t h e same s o l v e n t t o
volume, and mix. P i p e t 1 5 mL of t h i s s o l u t i o n i n t o a
flat-bottom, g l a s s - j o i n t , 500-mL f l a s k , add t h e c o n t e n t s
of a 15-mL v i a l of sodium biphenyl s o l u t i o n through a
long-stem f u n n e l t o prevent s p l a t t e r i n g , s w i r l t h e f l a s k
g e n t l y , and cover with a watch c r y s t a l . Allow t o s t a n d
FLUOROURACIL
625
Assay - 0.1 N T e t r a b u t y l a m m o n i u m h y d r o x i d e i n
methanol - D i l u t e w i t h methanol a commercially a v a i l a b l e
s o l u t i o n of tetrabutylammonium h y d r o x i d e i n m e t h a n o l ,
and s t a n d a r d i z e a s d i r e c t e d u n d e r Tetrabutylammonium
Hydroxide, Tenth-Normal (0.1 N).
Procedure - T r a n s f e r about 400 mg of F l u o r o u r a c i l ,
a c c u r a t e l y weigh t o a 250-mL c o n i c a l f l a s k , add 80 mL of
dimethylformide and warm g e n t l y t o d i s s o l v e . Cool, add 5
d r o p s of 1 i n 1 0 0 s o l u t i o n o f thymol b l u e i n
dimethylformamide, and t i t r a t e w i t h 0.1 N T e t r a b u t y l ammonium h y d r o x i d e i n m e t h a n o l t o a b l u e e n d - p o i n t ,
t a k i n g p r e c a u t i o n s t o prevent a b s o r p t i o n of atmospheric
626
Colorimetry
Hassib ( 2 7 ) r e p o r t e d a q u a l i t a t i v e and q u a n t i t a t i v e
a n a l y s i s of two u r a c i l a n t i c a n c e r drugs i n c l u d i n g 5f l u o r o u r a c i l . The drugs were s e l e c t i v e l y i d e n t i f i e d
and e s t i m a t e d i n p u r e and i n dosage forms by means
of c o l o r r e a c t i o n s . 5 - F l u o r o u r a c i l was t r e a t e d with
bromine w a t e r i n borox medium, and t h e n w i t h 2,4dimitrophenylhydrazine i n a c i d i c medium t o g i v e a n
o r a n g e - r e d p r e c i p i t a t e -7hich produces a d i s t i n c t l y
v i o l e t s o l u t i o n when t r e a t e d w i t h p o t a s s i u m
hydroxide s o l u t i o n . From 100 gm sample c o n t a i n i n g 30
ug of 5 - f l u o r o u r a c i l , 29 p g were r e c o v e r e d by t h i s
method.
L i ( 2 8 ) r e p o r t e d t h e s e p a r a t i o n of 5 - f l u o r o u r a c i l
from 5 - f l u o r o c y t o s i n e . The m i x t u r e was s t i r r e d i n
1 0 % h y d r o c h l o r i c a c i d a t 5-10'
f o r one hour t o
p r e c i p i t a t e 5-f l u o r o u r a c i l . The f i l t r a t e was made
a l k a l i n e w i t h 20% N H 4 0 H t o p r e c i p i t a t e 5-fluorocyt o s i n e . The l a t t e r was d e c o l o r e d by a c t i v e c a r b o n
and washed w i t h a c e t o n e t o g i v e 5 - f l u o r o c y t o s i n e
w i t h 99% p u r i t y .
10.3.2
U l t r a v i o l e t Spectrometry
Gaussian a n a l y s i s of a b s o r p t i o n s p e c t r a f o r a 0.01 N
sodium h y d r o x i d e s o l u t i o n Containing 5-f l u o r o u r a c i l
a t 270 and 300 nm have been r e p o r t e d by Tikhvinskaya
and E g e r t s ( 2 9 ) . I t showed t h e m o l a r e x t i n c t i o n
c o e f f i c i e n t s t o be 4570 and 3000 r e s p e c t i v e l y .
D e t e r m i n a t i o n of t h e d i f f e r e n c e i n e x t i n c t i o n
c o e f f i c i e n t s f o r t h e drug i s s u g g e s t e d f o r q u a n t i t a t i v e a n a l y s i s of t h e drug i n t h e presence of t h e
others.
Borodavkin e t a 1 ( 3 0 ) have s t u d i e d t h e a b s o r p t i o n
u l t r a v i o l e t s p e c t r o s c o p y and e l e c t r o n i c s t r u c t u r e of
some 5 - s u b s t i t u t e d analogs of pyrimidines.
621
FLUOROURACIL
10.3.3
I n f r a r e d Spectrometry
Fluorine-19-Nuclear
Magnetic Resonance
Mass Spectrometry
Marunaka ( 3 4 ) r e p o r t e d t h e e l e c t r o n i m p a c t mass
s p e c t r a f o r 5-f l u o r o u r a c i l and some N - s u b s t i t u t e d
d e r i v a t i v e s . C h a r a c t e r i s t i c fragment i o n s were
produced by r e t r o - D i e l - A l d e r d e c o m p o s i t i o n of t h e
f l u o r o u r a c i l skeleton.
628
Thin-Layer Chromatography
FLUOROURACIL
629
630
FLUOROURACIL
63 I
632
F'LUOROURACIL
633
634
FLUOROURACIL
635
11. ELECTROCHEMISTRY
Abou-Shaaban R.R.A.
Personal Communication.
page 4092,
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KirKwood, W. E n s m i n-g e r .- a n d A . R o s o w s k y . N .
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S h i r a s a k a and S.
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Farmakoter,
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m.
1 9 . T. T a g u c h i ,
K l i n . F a r m a k o t e r , 1 2 , 205 ( 1 9 8 3 ) .
2 0 . W.L. Washtien, Cancer R e s . 4 4 ( 3 ) , 9 0 y ( 1 9 8 4 ) .
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S c h u t t and H.W.
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Johns,
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Kamano. I n Kimura e t a l . ( E d s ) F l u r o p y r i m i d i n e s i n
c a n c e r t h e r a p y , pp. 229-241, Tokyo ( 1 9 8 4 ) .
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D i a s i o , H.L.
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LaBudde, R.F. Mayol and
Browder, EKsp. K l i n . Farmakoter.
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Hassib, Talanta,
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A n a l . P r i b a l t . Resp. 2.
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ERRATA
SPIRONOLACTONE-Volume
4, p. 431
641
CUMULATIVE INDEX
Cefazolin, 4, 1
Cefotaxime, 11, 139
Cefoxitin, sodium, 11, 169
Cephalexin, 4, 21
Cephalothin sodium, 1, 319
Cephradine, 5, 21
Chloral hydrate, 2, 85
Chlorambucil, 16, 85
Chloramphenicol, 4,47, 518; 15, 701
Chlordiazepoxide, 1, 15
Chlordiazepoxide hydrochloride, 1, 39; 4, 518
Chloroquine, 13, 95
Chloroquine phosphate, 5, 61
Chlorothiazide, 18, 33
Chloropheniramine maleate, 7, 43
Chlorprothixene, 2, 63
Chlortetracycline hydrochloride, 8, 101
Chlorthalidone, 14, 1
Chlorzoxazone, 16, 119
Cholecalciferol, see Vitamin D,
Cimetidine, W , 127; 17, 797
Cisplatin, 14, 77; 15, 796
Clidinium bromide, 2, 145
Clindamycin hydrochloride, 10, 75
Clioquinol, 18, 57
Clofazamine, 18, 91
Clofibrate, 11, 197
Clonazepam, 6,61
Clorazepate dipotassium, 4, 91
Clotrimazole, 11, 225
Cloxacillin sodium, 4, 113
Cocaine hydrochloride, 15, 151
Codeine phosphate, 10, 93
Colchicine, 10, 139
Cyanocobalamin, 10, 183
Cyclizine, 6, 83; 7, 502
Cyclobenzaprine hydrochloride, 17,41
Cycloserine, 1, 53; 18, 567
Cyclosporine, 16, 145
643
Cyclothiazide, 1, 66
Cypropheptadine, 9, 155
Dapsone, 5, 87
Dexamethasone, 2, 163; 4, 519
Diatrizoic acid, 4, 137; 5, 556
Diazepam, 1,79; 4,518
Dibenzepin hydrochloride, 9, 181
Dibucaine and dibucaine hydrochloride, 12,
105
Diflunisal, 14, 491
Digitoxin, 3, 149
Digoxin, 9, 207
Dihydrcergotoxine methanesulfonate, 7, 81
Dioctyl sodium sulfosuccinate, 2, 199; 12, 7113
Diperodon, 6, 99
Diphenhydramine hydrochloride, 3, 173
Diphenoxylate hydrochloride, 7, 149
Disopyramide phosphate, 13, 183
Disulfiram, 4, 168
Dobutamine hydrochloride, 8, 139
Dopamine hydrochloride, 11, 257
Doxorubicine, 9, 245
Droperidol, 7, 171
Echothiophate iodide, 3, 233
Emetine hydrochloride, 10, 289
Enalapril maleate, 16, 207
Ephedrine hydrochloride, 15, 233
Epinephnne, 7, 193
Ergonovine maleate, 11, 273
Ergotamine tartrate, 6, 113
Erythromycin, 8, 159
Erythromycin estolate, 1, 101; 2, 573
Estradiol, 15, 283
Estradiol valerate, 4, 192
Estrone, 12, 135
Ethambutol hydrochloride, 7, 231
Ethynodiol diacetate, 3, 253
Etomidate, 12, 191
Etoposide, 18, 121
Fenoprofen calcium, 6, 161
Flucytosine, 5, 115
Fludrocortisone acetate, 3, 281
Flufenamic acid, 11, 313
Fluorouracil, 2, 221; 18, 599
Fluoxymesterone, 7, 251
Fluphenazine decanote, 9, 275; 10, 730
Fluphenazine enanthate, 2, 245; 4, 524
Fluphenazine hydrochloride, 2, 263; 4, 519
Flurazepam hydrochloride, 3, 307
Furosemide, 18, 153
Gentamicin sulfate, 9, 295; 10, 731
Glibenclamide, 10, 337
Gluthethimide, 5 , 139
Gramicidin, 8, 179
Griseofulvin, 8, 219; 9, 583
Guanabenz acetate, 15, 319
Halcinonide, 8, 251
Haloperidol, 9, 341
Halothane, 1, 119; 2, 573; 14, 597
Heparin sodium, 12, 215
Heroin, 10, 357
Hexestrol, 11, 347
Hexetidine, 7, 277
Homatropine hydrobromide, 16, 245
Hydralazine hydrochloride, 8, 283
Hydrochlorothiazide, 10, 405
Hydrocortisone, 12, 277
Hydroflumethiazide, 7, 297
Hydroxyprogesterone caproate, 4, 209
Hydroxyzine dihydrochloride, 7, 319
Impenem, 17, 73
Imiprarnine hydrochloride, 14, 37
Indomethacin, 13,211
lodamide, 15, 337
Iodipamide, 2, 333
Iopamidol, 17, 115
Iopanoic acid, 14, 181
Isocarboxazid, 2, 295
Isoniazide, 6, 183
Isopropamide, 2, 315; 12, 721
Isoproterenol, 14, 391
Isosorbide dinitrate, 4, 225; 5, 556
Ivermectin, 17, 155
Kanamycin sulfate, 6, 259
Ketamine, 6, 297
Ketoprofen, 10, 443
Ketotifen, 13, 239
Khellin, 9, 371
Leucovorin calcium, 8, 315
Levallorphan tartrate, 2, 339
Levarterenol bitartrate, 1, 49; 2, 573; 11, 555
Levodopa, 5, 189
Levothyroxine sodium, 5, 225
Lidocaine base and hydrochloride, 14, 207; 15,
761
Lithium carbonate, 15, 367
Lorazepam, 9, 397
Maprotiline hydrochloride, 15, 393
Mebendazole, 16, 291
Mefloquine hydrochloride, 14, 157
Melphalan, 13, 265
Meperidine hydrochloride, 1, 175
Meprobamate, 1, 209; 4, 520; ll, 587
6-Mercaptopurine, 7, 343
644
646