ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1995, p. 7986 0066-4804/95/$04.

00 0 Copyright 1995, American Society for Microbiology

Vol. 39, No. 1

Structure-Activity and Structure-Selectivity Studies on Diaminoquinazolines and Other Inhibitors of Pneumocystis carinii and Toxoplasma gondii Dihydrofolate Reductase
ANDRE ROSOWSKY,1* JOHN B. HYNES,2
AND

SHERRY F. QUEENER3

Dana-Farber Cancer Institute and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 021151; Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina 294252; and Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 462023
Received 21 July 1994/Returned for modication 21 September 1994/Accepted 24 October 1994

Twenty-eight 2,4-diaminoquinazolines with alkyl, halogen, or alkoxy groups at the 5-, 6-, and/or 7-position, eight 2,4-diaminopteridines with alkyl and aralkyl groups at the 6- and 7-positions, ve 1,3-diamino-7,8,9,10tetrahydropyrimido[4,5-c]isoquinolines with an alkyl, alkylthio, or aryl group at the 6-position, and nine 4,6-diamino-1,2-dihydro-s-triazines with one or two alkyl groups at the 2-position and a substituted phenyl or naphthyl group at the 1-position were evaluated as inhibitors of dihydrofolate reductase enzymes from Pneumocystis carinii, Toxoplasma gondii, and rat liver. Halogen substitution at the 5- or 6-position of 2,4diaminoquinazoline favored selective binding to the P. carinii enzyme but not the T. gondii enzyme. For example, the 50% inhibitory concentrations of 2,4-diamino-6-chloroquinazoline as an inhibitor of P. carinii, T. gondii, and rat liver dihydrofolate reductase were 3.6, 14, and 29 M, respectively, corresponding to 12-fold selectivity for the P. carinii enzyme but only marginal selectivity for the T. gondii enzyme. Greater than vefold selectivity for P. carinii but not T. gondii dihydrofolate reductase was also observed for the 2,4-diaminoquinazolines with 5-methyl, 5-uoro, 5- and 6-bromo, 6-chloro, and 5-chloro-6-bromo substitution. In contrast, alkyl and aralkyl substitution at the 6- and 7-positions of 2,4-diaminopteridines was found to be a favorable feature for selective inhibition of the T. gondii enzyme and, in two cases, for both enzymes. Nine of the fty-one compounds tested against P. carinii dihydrofolate reductase and four of the thirty compounds tested against T. gondii dihydrofolate reductase displayed vefold or greater selectivity for the microbial enzyme versus the rat liver enzyme. The most selective against both enzymes was 2,4-diamino-6,7-bis(cyclohexylmethyl)pteridine, with a selectivity ratio 2 orders of magnitude greater than the value reported for trimetrexate and piritrexim. Since substitution at the 7-position is generally considered to be detrimental to the binding of 2,4-diaminopteridines and related compounds to mammalian dihydrofolate reductase, the selectivity observed in this study with the 6,7-bis(cyclohexylmethyl) analog may represent a useful approach to enhancing selective inhibition of the enzyme from nonmammalian species. Infection by Pneumocystis carinii and Toxoplasma gondii, two opportunistic organisms that ordinarily pose little or no hazard to individuals with an intact immune system, has been found to be profoundly disabling and often fatal in patients with AIDS (9, 11, 25). Though apparently less severe in patients with other immune disorders (22), these infections can nonetheless cause major clinical problems during immunosuppressive cancer chemotherapy or when immunosuppressant drugs are administered as part of a bone marrow transplantation program (4, 33). Several classes of compounds have found use in the treatment and/or prophylaxis of P. carinii and T. gondii infections, but none to date has been entirely successful. Standard agents currently approved for the treatment of P. carinii pneumonia are trimethoprim-sulfamethoxazole (12) and aerosolized pentamidine (28). Standard treatment of toxoplasmosis, on the other hand, utilizes the combination pyrimethamine-sulfadiazine (24). Newer drug combinations which have shown promise for prophylaxis or treatment include pyrimethamine-sulfadoxine (42), trimethoprim-dapsone (23), clindamycin-primaquine (41), and pyrimethamine-clindamycin (19). Atovaquone (566C80), an antimalarial hydroxyquinone with a mechanism of action unlike that of any of the above drugs, has also recently been reported to have signicant activity in laboratory animals (14) and humans (15). A number of other interesting compounds are reportedly active against one or the other organism in experimental systems. Among these may be mentioned the traditional Chinese herbal medicines quinghasosu (artemisinin) (27, 29) and the sesquiterpene bilobalide from ginkgo leaves (3), some plant alkaloids of the acridone family (32), members of the pneumocandin family of lipophilic cyclic peptide cell wall biosynthesis inhibitors (43), the iron chelator deferoxamine (46), and several macrolide antibiotics (2). While dihydrofolate reductase (DHFR) inhibitors like pyrimethamine or trimethoprim in combination with dihydropteroate synthetase inhibitors like sulfamethoxazole or sulfadiazine continue to be the rst-line treatment of both P. carinii and T. gondii infections, they leave much to be desired, the main problem being that these regimens frequently produce adverse reactions requiring either dosage adjustment or cessation of therapy (25). In addition, chronic use of an antifolate or combination of antifolates favors eventual colonization of the host by antifolate-resistant mutants. For this reason, there have been vigorous efforts to develop newer therapeutic approaches, a notable example of which has been the use of two potent lipid-soluble DHFR inhibitors originally developed as
79

* Corresponding author.

80

ROSOWSKY ET AL.

ANTIMICROB. AGENTS CHEMOTHER.

were competitive inhibitors of all three enzymes whereas TMQ yielded noncompetitive kinetics typical of pseudoirreversible inhibitors (data not shown).

anticancer drugs, trimetrexate (TMQ) and piritrexim (PTX) (reviewed in reference 5), in combination with leucovorin (5formyl-5,6,7,8-tetrahydrofolate) to selectively protect host tissues (1, 20, 21, 26). The rationale for this strategy is that P. carinii and T. gondii both have the ability to synthesize reduced folate cofactors de novo (20) but lack the ability to take up leucovorin by active transport and are therefore impervious to its protective effects. Since TMQ and PTX both have higher afnity for mammalian DHFR than for the P. carinii and T. gondii enzymes (1, 21), these drugs cannot be used clinically at high doses without coadministration of leucovorin. On the other hand, if a DHFR inhibitor with preferential afnity for P. carinii or T. gondii enzyme could be found, there would be no need to use leucovorin. This could result in less expensive, and possibly safer, therapy. As part of a broader investigation aimed at identifying structural features that might favor the desired binding selectivity, a diverse group of previously synthesized diaminoquinazolines, diaminopteridines, diaminopyrido[4,5-c]isoquinolines, and diamino-s-triazines were assayed as inhibitors of puried DHFR from P. carinii, T. gondii, and rat liver as described earlier (6, 8). A number of the compounds were found to have greater selectivity than TMQ or PTX for the P. carinii or T. gondii enzyme and thus offered potentially useful structureactivity and structure-selectivity guidelines for analog synthesis.

RESULTS Diaminoquinazolines. The activity of a panel of simple 5-, 6-, and 7-substituted 2,4-diaminoquinazolines (1618, 37) as inhibitors of P. carinii and rat liver DHFR is presented in Table 1, along with the selectivity ratio for each compound. The IC50s of the 5-substituted compounds ranged from 0.28 M (5-Br, entry 5) to 82 M (5-CF3, entry 2) against P. carinii DHFR and from 0.12 M (5-OC3H7, entry 9) to 215 M (5-CF3) against the mammalian enzyme. The CF3 group was particularly unfavorable where both enzymes were concerned. Overall, the most active subgroup against the P. carinii enzyme was the halides, whereas the most active against the mammalian enzyme was the alkoxy compounds. The most favorable halogens against the P. carinii enzyme was Br, and the least favorable was I. Against the mammalian enzyme, the most favorable halogen was again Br; however, the least favorable was F. The best alkoxy substituent against both enzymes was OC3H7. Replacement of H atoms by F atoms in the alkoxy group had substantial but inconsistent effects; for example, the OCH2CF3 analog (entry 10) was less active than the OCH2CH3 analog (entry 8) against P. carinii enzyme but more active against the rat liver enzyme. Fluorination of the longer alkoxy groups was unfavorable for binding to both enzymes. Replacement of OCH3 by SCH3 at the 5-position (entry 13) also increased binding to both enzymes. The compound with the greatest selectivity for P. carinii DHFR was the uoride (entry 3), with the chloride (entry 4) and bromide (entry 5) being only a little less selective. The IC50s of the 6-substituted 2,4-diaminoquinazolines ranged from 1.6 M (6-Br, entry 18) to 119 M (6-F, entry 16) against P. carinii DHFR and from 4.1 M (6-CF3, entry 15) to 452 M (6-F) against the rat enzyme. Activity increased in the order F Cl Br I against both enzymes, with the uoride being substantially less active than the other halides. In general, all the 6-substituted compounds were substantially less active than the corresponding 5-substituted compounds. Additionally, the CF3 analog was found to be much more active than the F analog in the 6-substituted series, whereas in the 5-substituted series the inverse had been observed (see above). However, two compounds in this group, the 6-chloro and 6-bromo derivatives, were approximately as selective as their 5-substituted counterparts. The 7-substituted 2,4-diaminoquinazolines were also less active as a group than the 5-substituted compounds, and 7-bromo substitution was again more favorable than 7-uoro or 7-chloro substitution; however, selectivity was poor. Two compounds substituted at both the 5and 6-positions were also tested. One of them, the 5-chloro-6bromo derivative, was more active than either the 5-chloro or 6-bromo analog and displayed moderate selectivity. Interestingly, the 5,6-dichloro derivative was less active than either the 5-chloro or 6-chloro analog against P. carinii DHFR and was also much less selective. A small subgroup of the 2,4-diaminoquinazolines was tested against T. gondii DHFR. Their IC50s and selectivity ratios relative to the mammalian enzyme are given in Table 2. The best inhibitors were found to be the 5-OCH3 and 5-OC3H7 derivatives, each of which was somewhat more active and more selective against this enzyme than against P. carinii DHFR. However, most of the other compounds were less active, and none had a selectivity ratio greater than 3.0. Diaminopteridines. Of the rst eight symmetrically 6,7-disubstituted pteridines tested in this pilot series (Table 3), the

MATERIALS AND METHODS

Chemicals. The 2,4-diaminoquinazolines used in this work were archival samples synthesized from substituted 2-aminobenzonitriles and cyanamide, cyanoguanidine, or guanidine (37) or from substituted 2-uorobenzonitriles and guanidine (1618); the 2,4-diaminopteridines were synthesized from 2,4,5,6-tetraaminopyrimidine and -diketones or substituted thiophen-4-ol-3-one 1,1-dioxides (masked -diketones) (34); the 1,3-diamino-7,8,9,10-tetrahydropyrimido [4,5-c]isoquinolines were synthesized from 3-chloro-4-cyano-5,6,7,8-tetrahydroisoquinolines and guanidine (40); and the 1-aryl-4,6-diamino-1,2-dihydro-striazines were synthesized from arylamines, carbonyl compounds, and cyanoguanidine (35, 39). 9,11-Diaminonaphtho[1,2-g]pteridine was synthesized from 2,4,6triamino-5-nitrosopyrimidine and 2-naphthol (10). Stock solutions for testing were made up in dimethyl sulfoxide and were diluted into water to provide the desired range of concentrations of drugs and a nal dimethyl sulfoxide concentration of 1%. Diluted solutions were either used the same day or stored in a desiccator at 70 C until needed. DHFR assay. The isolation and purication of DHFR from rat liver, P. carinii, and T. gondii was carried out as previously described (6, 8). Rats are routinely used as an animal model for P. carinii infection (31). Moreover, rat liver DHFR can be viewed as a reasonable surrogate for human DHFR on the basis of studies showing similar patterns of selective inhibition of the two enzymes by pyrimethamine and trimethoprim (7). Spectrophotometric assays of enzyme activity were carried out according to a standard procedure which has been found to be highly reliable (6, 8). Pyrimethamine was used routinely as a positive control; 50% inhibitory concentrations (IC50s) (mean standard error) obtained for this reference compound over the past 5 years have been 1.52 0.32 M for rat liver DHFR and 2.39 0.42 M for P. carinii DHFR. The standard reaction mixture for assays using rat liver and P. carinii enzyme contained 0.092 mM dihydrofolate, 0.117 mM NADPH, 8.9 mM 2-mercaptoethanol, and 3.7 IU of enzyme (1 IU 0.005 optical density unit/min) in 40.7 mM sodium phosphate buffer, pH 7.4, in a 1-ml cuvette. Assay mixtures used to measure T. gondii enzyme activity also contained 150 mM KCl. Reactions were carried out at 37 C and were initiated by adding dihydrofolate to the mixture 0.5 min after the enzyme. Activity was linear over a fourfold enzyme concentration range. Inhibitors were added over a range of concentrations sufcient to produce 10 to 90% inhibition. Semilog plots showing the normal sigmoidal pattern were linearized by converting percent inhibition to probit values, which were replotted as a function of the log of inhibitor concentration and tted to a straight line by least-square regression. The IC50 for each inhibitor was dened as the concentration for which the probit value was 5.0. Selectivity of P. carinii DHFR versus rat liver DHFR inhibition, and of T. gondii DHFR versus rat liver DHFR inhibition, was expressed as the ratio of the respective IC50s. Where there was enough test compound to allow the results of replicate assays to be analyzed statistically, the standard error of the mean was less than 20% in almost all cases. Although it was not practical to analyze the kinetics of inhibition for the compounds in this study, such experiments had been done earlier with standard inhibitors; as expected from the literature (7), trimethoprim and pyrimethamine

VOL. 39, 1995

INHIBITORS OF P. CARINII AND T. GONDII DHFR

81

TABLE 1. Inhibition of rat liver and P. carinii DHFR by 2,4-diaminoquinazolines with small substituents at the 5-, 6-, or 7-position

Entry

R1

R2

R3

Sourcea Rat liver

IC50 ( M)b P. carinii

Selectivity ratioc

5-Substituted 1 2 3 4 5 6 7 8 9 10 11 12 13 6-Substituted 14 15 16 17 18 19 20 7-Substituted 21 22 23 24 25 5,6-Disubstituted 26 27 6,7-Disubstituted 28

a b

CH3 CF3 F Cl Br I OCH3 OC2H5 OC3H7 OCH2CF3 OCH2C2F5 OCH2C3F7 SCH3 H H H H H H H H H H H H Cl Cl H

H H H H H H H H H H H H H CH3 CF3 F Cl Br I OCH3 H H H H H Cl Br CH3

H H H H H H H H H H H H H H H H H H H H CH3 CF3 F Cl Br H H CH3

A, B B B B B B A, B A A B B B B A B B A, B B B A A, B B B A, B B B B A

7.4 2.8 (n 6)d 215 5.0 3.9 2.3 2.4 1.2 (n 5) 11 0.12 0.14 0.41 6.1 0.54 55 4.1 452 29 (n 14 5.9 46 42 (n 63 189 53 (n 19 3.5 0.8 7.9

1.5

0.6 (n 3)d 82 0.41 0.42 0.28 0.52 4.5 (n 2) 4.7 0.96 13 54 71 1.1

5.0 2.6 12 9.3 8.2 4.6 0.27 2.3 0.13 0.011 0.007 0.086 0.49 2.8 1.5 3.8 8.1 8.9 3.7 0.79 0.65 2.0 4.6 1.2 1.6 0.36 5.7 1.5

4)

20 2.7 119 3.6 (n 1.6 1.6 58 65 (n 31 41 45 (n 12 9.7 0.14 5.3

3)

3) 3)

2) 4)

A, synthesized at the Dana-Farber Cancer Institute (see reference 37); B, synthesized at the Medical University of South Carolina (see references 1618). Results are the mean IC50s obtained in replicate assays on different days. Numbers (n) in parentheses are the numbers of assays from which the means were determined when a compound was tested more than once. All assays were done with native P. carinii DHFR according to reference 6. Data on rat liver DHFR inhibition by the compounds synthesized at the Medical University of South Carolina were also reported in reference 17. c IC50 (rat liver)/IC50 (P. carinii) ratio calculated from means of pooled data. Numbers greater than 1.0 indicate selectivity for the P. carinii enzyme. d Standard errors are given for entry 1 as a representative example of those compounds for which there were enough replicate data against both enzymes to allow statistical analysis.

most active against rat liver DHFR were the 6,7-bis(4-chlorobenzyl) (entry 42) and 6,7-bis(3-phenylpropyl) (entry 45) derivatives, with IC50s of 8.3 and 2.7 M, respectively. The 6,7bis(3,4-dichlorobenzyl) derivative (entry 43) was less active than the bis(4-chlorobenzyl), showing that meta substitution by an electron-withdrawing group was unfavorable for binding to the mammalian enzyme. This conclusion was further supported by the low potency of the 6,7-bis(3-triuoromethylbenzyl) analog (entry 41). Against P. carinii DHFR, the IC50s of these pteridines ranged from 1.1 M for the 6,7-bis(3-phenylpropyl) derivative to 26 M for the 6,7-bis(3-triuoromethylbenzyl) derivative. Thus, the best and worst inhibitors of the rat liver enzyme were also the best and worst inhibitors of the P. carinii enzyme. On the other hand, while they were not the

most potent, two members of the series, the 6,7-bis(4-n-butylbenzyl) (entry 40) and 6,7-bis(cyclohexylmethyl) (entry 46) derivatives, with IC50s of 3.9 and 2.6 M, respectively, displayed 10-fold selectivity against the P. carinii enzyme. Interestingly, these compounds contained CH2 groups but no halogens in the side chain. However the most potent P. carinii DHFR inhibitor, the 6,7-bis(3-phenylpropyl) derivative, had a selectivity ratio of 3.0. The IC50s of 9,11-diaminonaphtho[1,2-g]pteridine (entry 48) against P. carinii and T. gondii DHFR were 18.4 and 4.9 M, respectively, whereas the IC50 against the rat enzyme was 1.3 M. Thus, this very lipophilic tetracyclic compound, which may also be viewed as an unsymmetrically 6,7-disubstituted 2,4diaminopteridine, lacked selectivity.

82

ROSOWSKY ET AL.

ANTIMICROB. AGENTS CHEMOTHER.

TABLE 2. Inhibition of rat liver and T. gondii DHFR by 2,4-diaminoquinazolines with small substituents at the 5-, 6-, or 7-position
Entry R1 R2 R3 Sourcea Rat liver IC50 ( M)b T. gondii Selectivity ratioc

5-Substituted 29 30 31 32 6-Substituted 33 34 35 36 37 7-Substituted 38 6,7-Disubstituted 39

a

CH3 OCH3 OC2H5 OC3H7 H H H H H H H

H H H H CF3 F Cl I OCH3 H CH3

H H H H H H H H H Cl CH3

A, B A, B A A B B A, B B A A, B A

7.38

2.82 (n 6)d 1.2 (n 2) 11 0.96

4.98

1.87 (n 3)d 0.48 (n 3) 8.7 0.15

1.2 2.5 1.3 0.80 1.3 4.3 2.1 3.7 2.1 0.52 1.0

4.1 452 29 (n 5.9 46 (n 42 (n 7.9

4) 3) 3)

3.1 104 14 1.6 22 81 7.7

A, synthesized at the Dana-Farber Cancer Institute (see reference 35); B, synthesized at the Medical University of South Carolina (see references 1618). b Results are the mean IC50s in replicate assays on different days. Numbers (n) in parentheses are the number of assays from which the means were determined when a compound was tested more than once. All assays were done with native T. gondii DHFR according to reference 8. c IC50 (rat liver)/IC50 (T. gondii) ratio calculated from means of pooled data. Numbers greater than 1.0 indicate selectivity for the T. gondii enzyme. d Standard errors are given for entry 29 as a representative example of those compounds for which there were enough replicate data against both enzymes to allow statistical analysis.

In the assays against T. gondii DHFR, the most active inhibitors were again the 6,7-bis(3-phenylpropyl) and 6,7-bis(cyclohexylmethyl) derivatives, with IC50s of 1.0 and 0.46 M, respectively. However, because of its relatively low IC50 against the rat liver enzyme, the selectivity ratio of the 6,7-bis(3-phenylpropyl) derivative was 3.0, as with the P. carinii enzyme. On the other hand, the 6,7-bis(cyclohexylmethyl) derivative, with a selectivity ratio of 59, was by far the best compound in this set. The fact that these 2,4-diaminopteridines contain a 7-substituent is noteworthy because this feature is generally not considered to be favorable for binding to mammalian DHFR. Our results suggest that a consequence of decreased

afnity for the rat liver enzyme may be an increase in species selectivity. We have previously observed this tendency in another series of inhibitors (38). Other inhibitors. Five examples of the angular tricyclic 1,3diamino-7,8,9,10-tetrahydropyrimido[4,5-c]isoquinoline system were tested as inhibitors of rat liver and P. carinii DHFR (Table 4). These compounds were of interest because they could be viewed as 5,6,7-trisubstituted derivatives of the 2,4diaminopyrido[2,3-d]pyrimidine (5-deazapteridine) ring system, of which PTX is a member. The best compound against the rat liver enzyme was the 6-methyl derivative (entry 50), which had an IC50 of 0.062 M. For the entire group of substituents at the 6-position, IC50s increased in the order Me Et MeS Ph H. The most active compound against P.

TABLE 3. Inhibition of DHFR by 2,4-diamino-6,7disubstituted pteridines TABLE 4. Inhibition of rat liver and P. carinii DHFR by 1,3diamino-7,8,9,10-tetrahydropyrimido[4,5-c]isoquinolines

Entry

R Rat liver

IC50 ( M)a P. carinii T. gondii

40 41 42 43 44 45 46 47 48

4-n-C4H9C6H4CH2 3-CF3C6H4CH2 4-ClC6H4CH2 3,4-Cl2C6H3CH2 C6H4CH2CH2 C6H4CH2CH2CH2 C6H11CH2 2-C10H7CH2 1,2-Naphtho

44 64 8.3 18 4.5 2.7 27 31 1.3

3.9 (11) 26 (2.5) 5.9 (1.4) 4.4 (4.0) 4.9 (0.92) 1.1 (2.5) 2.6 (10) 4.0 ( 7.8) 18.4 ( 0.1)

14 (3.1) 39 (1.6) 2.5 (3.3) 28 (NE) 4.8 (0.94) 1.0 (2.7) 0.46 (59) 31 (NE) 4.9 (0.3)

Entry

IC50 ( M) Rat liver P. carinii

Selectivity ratioa

a Numbers in parentheses are the ratios of IC50 (rat liver)/IC50 (P. carinii) or IC50 (rat liver)/IC50 (T. gondii). Numbers greater than 1.0 indicate selectivity for the P. carinii or T. gondii enzyme; NE, not evaluable.

49 50 51 52 53
a

H CH3 CH3CH2 CH3S C6H5

2.8 0.062 0.18 0.18 0.45

30 1.0 30 30 6.8

0.1 0.1 0.1 0.1 0.1

IC50 (rat liver)/IC50 (P. carinii) ratio.

VOL. 39, 1995

INHIBITORS OF P. CARINII AND T. GONDII DHFR TABLE 5. Inhibition of rat liver, P. carinii, and T. gondii DHFR by 1-aryl-4,6-diamino-1,2-dihydro-s-triazines

83

Entry

R1

R2

R3

R4

R5 Rat liver

IC50 ( M)a P. carinii T. gondii

Phenyl 54 55 56 57 58 59 60 Naphthyl 61 62 63

CH3 CH3 CH3 CH3 CH3 CH3 H Cl Cl

H OCH3 OCH3 OCH3 H H H Cl Cl H

H H OCH3 H OCH3 OCH3 H H H Cl

Cl H OCH3 OCH3 H OCH3

c

H OCH3 H H OCH3 OCH3 H

0.3 60 6.1 90 11.4 100 0.032 9.1 3.1 1.2

20.4 ( 0.1) 60 (NE) 20.4 (0.3) 90 (NE) 12.9 (0.9) 100 (NE) 2.0 ( 0.1) 4.4 (0.5) 3.0 (1.0) 0.84 (1.4)

0.057 (5.3) 60 (NE) 1.2 (5.1) 90 (NE) 16.4 (0.7) 84 (NE) 0.017 (1.9) 1.3 (7.0) 1.4 (0.5) 0.41 (2.9)

a All compounds were isolated and tested as hydrochloride salts. Numbers in parentheses are the ratios of IC50 (rat liver)/IC50 (P. carinii) or IC50 (rat liver)/IC50 (T. gondii). Numbers greater than 1.0 indicate selectivity for the P. carinii or T. gondii enzyme. NE, not evaluable. b R1 n-C11H23. c R5 (4,6-diamino-1,2-dihydro-2,2-dimethyl-s-triazin-1-yl)CH2.

carinii DHFR was again the 6-methyl derivative, but unfortunately its activity was 16-fold lower than against rat liver DHFR. Thus, neither this nor any of the other analogs had the desired selectivity. Ten previously untested 1-aryl-4,6-diamino-1,2-dihydro-striazines were assayed (Table 5). The most active against rat liver DHFR was the bifunctional analog (entry 60) derived from 4,4 -diaminodibenzyl, which had an IC50 of 0.032 M but showed poor selectivity. Among the compounds with a phenyl ring, the most active was the one derived from n-dodecanal (entry 54). The IC50 of this compound with a long-chain monoalkyl group at the 2-position and a 4-chloro substituent on the phenyl ring was 0.3 M. The more conventional derivatives with 2,2-dimethyl substitution and either two or three OCH3 groups on the phenyl ring were all much less active. Interestingly, the 2,5-dimethoxy (entry 55) and 3,4,5-trimethoxy (entry 59) derivatives, which can be viewed as analogs of TMQ and PTX, respectively, were both very poor inhibitors. On the other hand, the 2,3,4-trimethoxy derivative (entry 56) was more active than expected, considering that it contains an ortho group, and actually showed some selectivity for the T. gondii enzyme. Among the halogenated naphthalene derivatives, activity against rat liver DHFR increased in the order 6-Cl 5,6-Cl2 5,7-Cl2. The 5,7-dichloro-2-naphthyl derivative (entry 63) was also the best inhibitor of the P. carinii and T. gondii DHFR, but its selectivity was not as good as that of the less potent 6-chloro analog (entry 61). As a group, three of the triazines proved to

have selectivity ratios in the 5- to 10-fold range for the T. gondii enzyme, but none was selective for the P. carinii enzyme. DISCUSSION In a series of 24 recently tested pyrimethamine analogs, 1 had 6.8-fold selectivity ratio for P. carinii DHFR (6), and 3 had a selectivity ratio for T. gondii enzyme of 10, with 1 showing 60-fold selectivity (8). Several conformationally rigid tricyclic pyrimethamine analogs (36) were also tested, but none was selective (6, 8). Among a group of 23 dihydrotriazines, none had a selectivity ratio greater than 0.9 for P. carinii DHFR (6); however, 5 showed selectivity for the T. gondii enzyme with ratios in the 10- to 30-fold range (8). Several 2,4-diamino-5benzylpyrimidine derivatives related to trimethoprim but with a pyrrole substituent in the phenyl ring have recently been found to have selectivity ratios of 100 for P. carinii DHFR (31, 45). A number of inhibitors of the 2,4-diaminopteridine (30), 2,4-diaminopyrido[2,3-d]pyridine (13), and 2,4-diaminothieno[2,3-d]pyrimidine (38) type have also recently been identied as being 10-fold selective for P. carinii DHFR in some instances and T. gondii DHFR in others. One of these, 2,4-diamino-6-phenylthiomethylpteridine, displayed a remarkable 320-fold selectivity for the T. gondii enzyme (30). Of the 51 compounds tested in the present study against P. carinii and T. gondii, several were found to have IC50s in the 0.01 to 0.1 M range but low selectivity relative to the rat

84

ROSOWSKY ET AL.

ANTIMICROB. AGENTS CHEMOTHER.

FIG. 1. Summary of molecular features found to favor selective binding to P. carinii or T. gondii DHFR.

enzyme. Better selectivity, in fact, was more common among inhibitors whose IC50s were in the 1 to 10 M range. Several such compounds had selectivity ratios of 10-fold or more, and one had a ratio of nearly 60-fold. These values were of the order determined previously for trimethoprim and pyrimethamine (30). By comparison, TMQ and PTX, the two drugs recently introduced into the clinic for treatment of P. carinii and T. gondii infections, showed ratios that were only in the 0.05- to 0.3-fold range (30). It is partly because of this lack of selectivity, and partly because of slower uptake of these drugs by the intact parasite cells, that effective control of opportunistic infections with TMQ or PTX in AIDS patients necessitates fairly high doses of the DHFR inhibitor and coadministration of leucovorin to prevent dose-limiting host toxicity. In order to highlight the molecular features that we believe may contribute to their selectivity, the structures of the best individual compounds identied in this study are depicted in

Fig. 1. Substitution by electron-withdrawing halogen atoms at the 5- and to a lesser degree the 6-position in 2,4-diaminoquinazoline was clearly favorable where P. carinii DHFR selectivity was concerned and was superior to alkoxy substitution. On the other hand, while 7-substitution in 2,4-diaminoquinazoline was unfavorable, this was not the case in the 2,4-diaminopteridine series, where 6,7-disubstitution with large hydrophobic groups yielded unexpectedly good selectivity for both P. carinii and T. gondii DHFR. To our knowledge, this represents a novel nding and may offer a way to achieve selective inhibition of DHFR from these and perhaps other nonmammalian species. In the dihydrotriazine series, a 2,3,4trimethoxyphenyl or 6-chloro-2-naphthyl group at the 1-position and a long-chain alkyl group at the 2-position also seemed to slightly favor selectivity for T. gondii DHFR; however, in contrast to the 5- and 6-substituted quinazolines and 6,7-disubstituted pteridines, these compounds displayed no selectivity for the P. carinii enzyme.

VOL. 39, 1995

INHIBITORS OF P. CARINII AND T. GONDII DHFR

85

It is important to emphasize that this study was not designed to allow us to predict the ability of DHFR inhibitors to block proliferation of intact P. carinii or T. gondii organisms in tissue culture or in mammalian hosts, as this would obviously be inuenced by the rate of drug uptake. Furthermore, the efcacy of these agents in vivo would depend not only on cellular uptake but also on their pharmacokinetic properties in the mammalian host. Nevertheless, because several of the inhibitors that we examined proved to be superior to TMQ, PTX, and even pyrimethamine and trimethoprim, in terms of selectivity for P. carinii and T. gondii DHFR versus mammalian DHFR, they may be viewed as encouraging leads for further structural optimization. In vitro and in vivo assays of the ability of selected examples of these compounds to inhibit pathogen growth in laboratory models are planned in order to address these issues.
ACKNOWLEDGMENTS This work was supported in part by grant RO1-AI29904 (to A.R.) and contract NO1-AI87240 (to S.F.Q.) from the National Institute of Allergy and Infectious Diseases (DHHS) and by a grant from the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (J.B.H.).
REFERENCES 1. Allegra, C. J., J. A. Kovacs, J. C. Drake, J. C. Swan, B. A. Chabner, and H. Masur. 1987. Potent in vitro and in vivo antitoxoplasma activity of the lipid-soluble antifolate trimetrexate. J. Clin. Invest. 79:478482. 2. Araujo, F. G., R. M. Shepard, and J. S. Remington. 1991. In vivo activity of the macrolide antibiotics azithromycin, roxithromycin, and spiramycin against Toxoplasma gondii. Eur. J. Clin. Microbiol. Infect. Dis. 10:519524. 3. Atzori, C., A. Bruno, G. Chichino, E. Bombardelli, M. Scaglia, and M. Ghione. 1993. Activity of bilobalide, a sesquiterpene from Ginkgo biloba, on Pneumocystis carinii. Antimicrob. Agents Chemother. 37:14921496. 4. Bartlett, M. S., and J. W. Smith. 1991. Pneumocystis carinii, an opportunist in immunocompromised patients. Clin. Microbiol. Rev. 4:137149. 5. Berman, E. M., and L. M. Werbel. 1991. The renewed potential for folate antagonists in contemporary cancer chemotherapy. J. Med. Chem. 34:479485. 6. Broughton, M. C., and S. F. Queener. 1991. Pneumocystis carinii dihydrofolate reductase used to screen potential antipneumocystis drugs. Antimicrob. Agents Chemother. 35:13481355. 7. Burchall, J. J., and G. H. Hitchings. 1965. Inhibitor binding analysis of dihydrofolate reductases from various species. Mol. Pharmacol. 1:126136. 8. Chio, L.-C., and S. F. Queener. 1993. Identication of highly potent and selective inhibitors of Toxoplasma gondii dihydrofolate reductase. Antimicrob. Agents Chemother. 37:19141923. 9. Davey, R. T., Jr., and H. Masur. 1990. Recent advances in the diagnosis, treatment, and prevention of Pneumocystis carinii pneumonia. Antimicrob. Agents Chemother. 34:499504. 10. Felton, D. G. I., and G. M. Timmis. 1954. The reaction between N,N-di-2 chloroalkyl-2-naphthylamines and 4-amino-5-nitrosopyrimidines. J. Chem. Soc. 1954:28812886. 11. Fischer, P.-A., and W. Enzensberger. 1987. Neurological complications in AIDS. J. Neurol. 234:269279. 12. Fischl, M. A., G. M. Dickinson, and L. La Voie. 1988. Safety and efcacy of sulfamethoxazole and trimethoprim chemoprophylaxis for Pneumocystis carinii pneumonia in AIDS. JAMA 259:11851189. 13. Gangjee, A., J. Shi, S. F. Queener, L. R. Barrows, and R. L. Kisliuk. 1993. Synthesis of 5-methyl-5-deaza nonclassical antifolates as inhibitors of dihydrofolate reductases and as potential antipneumocystis, antitoxoplasma, and antitumor agents. J. Med. Chem. 36:34373443. 14. Hughes, W. T., V. L. Gray, W. E. Gutteridge, V. S. Latter, and M. Pudney. 1989. Efcacy of a hydroxynaphthoquinone, 566C80, in experimental Pneumocystis carinii pneumonitis. Antimicrob. Agents Chemother. 34:225228. 15. Hughes, W. T., G. Leoung, F. Kramer, S. A. Bazzetta, S. Safrin, P. Frame, N. Clumeck, H. Masur, D. Lancaster, C. Chan, J. Lavelle, J. Rosenstock, J. Falloon, J. Feinbergh, S. LaFon, M. Rogers, and F. Sattler. 1993. Comparison of atovaquone (566C80) with trimethoprim-sulfamethoxazole to treat Pneumocystis carinii pneumonia in patients with AIDS. N. Engl. J. Med. 328:15211527. 16. Hynes, J. B., A. Pathak, C. H. Panos, and C. C. Okeke. 1988. Direct synthesis of 2,4-diaminoquinazolines from 2-uorobenzonitriles. J. Heterocycl. Chem. 25:11731177. 17. Hynes, J. B., A. Tomazic, A. Kumar, V. Kumar, and J. H. Freisheim. 1991. Inhibition of human dihydrofolate reductase by 2,4-diaminoquinazolines bearing simple substituents on the aromatic ring. J. Heterocycl. Chem.28:19811986.

18. Hynes, J. B., A. Tomazic, C. A. Parrish, and O. S. Fetzer. 1991. Further studies on the synthesis of quinazolines from 2-uorobenzonitriles. J. Heterocycl. Chem. 28:13571363. 19. Katlama, C. 1991. Evaluation of the efcacy and safety of clindamycin plus pyrimethamine for induction and maintenance therapy of toxoplasmic encephalitis in AIDS. Eur. J. Clin. Microbiol. Infect. Dis. 10:189191. 20. Kovacs, J. A., C. J. Allegra, J. Beaver, D. Boarman, M. Lewis, J. E. Parrillo, B. Chabner, and H. Masur. 1989. Characterization of de novo folate synthesis in Pneumocystis carinii and Toxoplasma gondii: potential for screening therapeutic agents. J. Infect. Dis. 160:312320. 21. Kovacs, J. A., C. J. Allegra, J. C. Swan, J. C. Drake, J. E. Parrillo, B. A. Chabner, and H. Masur. 1988. Potent antipneumocystis and antitoxoplasma activities of piritrexim, a lipid-soluble antifolate. Antimicrob. Agents Chemother. 32:430433. 22. Kovacs, J. A., J. W. Hiemenz, A. M. Macher, D. Stover, H. W. Murray, J. Shelhamer, H. C. Lane, C. Urmacher, C. Honig, D. L. Longo, M. M. Parker, G. Natanson, J. E. Parrillo, A. S. Fauci, P. A. Pizzo, and H. Masur. 1984. Pneumocystis carinii pneumonia: a comparison between patients with the acquired immunodeciency syndrome and patients with other immunodeciencies. Ann. Intern. Med. 100:663671. 23. Leoung, G. S., J. Mills, P. C. Hopewell, W. Hughes, and C. Wofsy. 1986. Dapsone-trimethoprim for Pneumocystis carinii pneumonia in the acquired immunodeciency syndrome. Ann. Intern. Med. 105:4548. 24. Leport, C., R. Raf, S. Matheron, C. Katiama, B. Regnier, A. G. Saimot, C. Marche, C. Vedrenne, and J. L. Vilde. 1988. Treatment of central nervous system toxoplasmosis with pyrimethamine/sulfadiazine combination in 35 patients with the acquired immunodeciency syndrome. Efcacy of longterm continuous therapy. Am. J. Med. 84:94100. 25. Masur, H. 1990. Problems in the management of opportunistic infections in patients infected with human immunodeciency virus. J. Infect. Dis. 161: 858864. 26. Masur, H., M. A. Polis, C. U. Tuazon, D. Ogata-Arakaki, J. A. Kovacs, D. Katz, D. Hilt, T. Simmons, I. Feuerstein, B. Lundgren, H. C. Lane, B. A. Chabner, and C. J. Allegra. 1992. Salvage trial of trimetrexate-leucovorin for the treatment of cerebral toxoplasmosis. J. Infect. Dis. 167:14221426. 27. Merali, S., and S. R. Meshnick. 1991. Susceptibility of Pneumocystis carinii to artemisinin in vitro. Antimicrob. Agents Chemother. 35:12251227. 28. Montgomery, A., R. J. Debs, J. M. Luce, K. J. Corkery, J. Turner, E. N. Brunette, E. T. Lin, and P. C. Hopewell. 1987. Aerosolized pentamidine as sole therapy for Pneumocystis carinii pneumonia in patients with acquired immunodeciency syndrome. Lancet ii:480482. 29. Ou-Yang, K., E. C. Krug, J. J. Marr, and R. L. Berens. 1990. Inhibition of growth of Toxoplasma gondii by qinghaosu and derivatives. Antimicrob. Agents Chemother. 34:19611965. 30. Piper, J. R., C. A. Johnson, C. A. Hosmer, R. L. Carter, E. R. Pfefferkorn, S. E. Borotz, and S. F. Queener. 1993. Lipophilic antifolates as candidates against opportunistic infections. Adv. Exp. Med. Biol. 338:429433. 31. Queener, S. F., M. S. Bartlett, M. A. Jay, M. M. Durkin, and J. W. Smith. 1987. Activity of lipid-soluble inhibitors of dihydrofolate reductase against Pneumocystis carinii in culture and in a rat model of infection. Antimicrob. Agents Chemother. 31:13231327. 32. Queener, S. F., H. Fujioka, Y. Nishiyama, H. Furukawa, M. S. Bartlett, and J. W. Smith. 1991. In vitro activities of acridone alkaloids against Pneumocystis carinii. Antimicrob. Agents Chemother. 35:377379. 33. Rolston, K. V. I., and G. P. Bodey. 1993. Infections in patients with cancer, p. 24162441. In J. F. Holland, E. Frei III, R. C. Bast, Jr., D. W. Kufe, D. L. Morton, and R. R. Weichselbaum (ed.), Cancer medicine, 3rd ed. Lea & Febiger, Philadelphia. 34. Rosowsky, A., M. Chaykovsky, M. Lin, and E. J. Modest. 1973. Pteridines. 2. New 6.7-disubstituted pteridines as potential antimalarial and antitumor agents. J. Med. Chem. 16:869875. 35. Rosowsky, A., K. K. N. Chen, R. St. Amand, and E. J. Modest. 1973. Chemical and biological studies on 1,2-dihydro-s-triazines. XVIII. Synthesis of 1-(5,6- and 5,7-dichloro-2-naphthyl) derivatives and related compounds as candidate antimalarial and antitumor agents. J. Pharm. Sci. 62:477479. 36. Rosowsky, A., J. H. Freisheim, J. B. Hynes, S. F. Queener, M. Bartlett, J. W. Smith, H. Lazarus, and E. J. Modest. 1989. Tricyclic 2,4-diaminopyrimidines with broad antifolate activity and the ability to inhibit Pneumocystis carinii growth in cultured human lung broblasts in the presence of leucovorin. Biochem. Pharmacol. 38:26772684. 37. Rosowsky, A., J. L. Marini, M. E. Nadel, and E. J. Modest. 1970. Quinazolines. VI. Synthesis of 2,4-diaminoquinazolines from anthranilonitriles. J. Med. Chem. 13:882886. 38. Rosowsky, A., C. E. Mota, J. E. Wright, J. H. Freisheim, J. J. Heusner, J. J. McCormack, and S. F. Queener. 1993. 2,4-Diaminothieno[2,3-d]pyrimidine analogues of trimetrexate and piritrexim as potential inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase. J. Med. Chem. 36:31033112. 39. Rosowsky, A., M. E. Nadel, and E. J. Modest. 1972. Chemical and biological studies on dihydro-s-triazines. XVII. Modications of the the three-component synthesis favoring the formation of 2-guanidino-4-methylquinazoline

86

ROSOWSKY ET AL.

ANTIMICROB. AGENTS CHEMOTHER.

R. M. Black, K. Nollstadt, and K. Bartizal. 1992. Pneumocandins from Zalerion arboricola. IV. Biological evaluation of natural and semisynthetic pneumocandins for activity against Pneumocystis carinii and Candida species. J. Antibiot. 45:18861891. 44. Then, R. L., P. G. Hartman, I. Kompis, and D. Santi. 1993. Selective inhibition of dihydrofolate reductase from problem human pathogens. Adv. Exp. Med. Biol. 338:533536. 45. Walzer, P. D., J. Foy, P. Steele, and M. White. 1993. Synergistic combinations of Ro 11-8958 and other dihydrofolate reductase inhibitors with sulfamethoxazole and dapsone for therapy of experimental pneumocystosis. Antimicrob. Agents Chemother. 37:14361443. 46. Weinberg, G. A., and M. M. Shaw. 1991. Suppressive effect of deferoxamine on the growth of Pneumocystis carinii in vitro. J. Protozool. 38:223S224S.

byproducts. J. Heterocycl. Chem. 9:645650. 40. Rosowsky, A., and N. Papathanasopoulos. 1974. Pyrimido[4,5-c]isoquinolines. 1. Synthesis and biological evaluation of some 6-alkyl-, 6-aralkyl-, and 6-aryl-1,3-diamino-7,8,9,10-tetrahydropyrimido[4,5-c]isoquinolines as potential folate antagonists. J. Med. Chem. 17:12721276. 41. Ruf, B., and H. D. Pohle. 1989. Clindamycin/primaquine for Pneumocystis carinii pneumonia. Lancet ii:626627. 42. Ruf, B., D. Schurmann, F. Bergmann, W. Schuler-Maue, T. Grunewald, H. J. Gottschalk, H. Witt, and H. D. Pohle. 1993. Efcacy of pyrimethamine/ sulfadoxine in the prevention of toxoplasmic encephalitis relapses and Pneumocystis carinii pneumonia in HIV-infected patients. Eur. J. Clin. Microbiol. Infect. Dis. 12:325329. 43. Schmatz, D. M., G. Abruzzo, M. A. Powles, D. C. McFadden, J. M. Balkovec,