AU657348B2

AU657348B2 - Specific inhibition of dihydrofolate reductase and compounds therefor

Info

Publication number: AU657348B2
Application number: AU22027/92A
Authority: AU
Inventors: Jeffrey M Blaney; Ivan Kompis; Charles K Marlow
Original assignee: F Hoffmann La Roche AG; Chiron Corp
Current assignee: F Hoffmann La Roche AG; Novartis Vaccines and Diagnostics Inc
Priority date: 1990-11-14
Filing date: 1992-08-31
Publication date: 1995-03-09
Anticipated expiration: 2012-08-31
Also published as: WO1992008461A1; PT99514B; AU2202792A; EP0639075A4; EP0639075A1; PT99514A; IE913974A1; CA2095518A1; JPH07509215A

Description

AUSTRALIA

Patents Act 1990 PROTOS CORPORATION and IVAN KOIIPTS

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT .:Invention Title: Specific Inhibition of Dihydrofolate Reductase and Compounds Therefor 3 a18 a. a 'S *a a.

'a *a a a a.

ft a aqp..

The following statement is a full description of this invention including the best method of performing it known to us:- SPECIFIC INHIBITION OF DIHYDROFOLATE REDUCTASE AND COMPOUNDS THEREFOR Description Technical Field This invention relates to pharmacology and the inhibition of enzymes specific to pathogens. More particularly, the invention relates to methods for specifically inhibiting the enzyme dihydrofolate reductase in fungal pathogens, and compounds therefor.

Background of the Invention Pneumocvstis carinii pneumonia (PCP) is a significant, ife-threatening infection in immunocompromised subjects, and is a leading cause of morbidity and mortality in patients presenting acquired immunodeficiency syndrome (AIDS).

.Since the onset of the AIDS epidemic, the incidence of PCP has risen from 20 approximately 200 cases per year to more than 25,000 cases per year in the United States.

i Due to the lack of a continuous in vitro culture system, and the cumbersome nature of the rat model of PCP, anti-P. carinii therapy has been developed largely on the assumption that P. carinii was a species of protozoa, and thus that anti-protozoal agents were likely to be effective. The two principle therapeutic modalities, trimethoprim/sulfamethoxazole and pentamidine, were developed empirically. However, P. carinii has recently been suggested to belong instead to the Kingdom Fungi Edman et al., Nature (1988) 334:519-22).

-2- Trimethoprim Pat No. 2,909.522) and pyrimethamine, and other dihydrofolate reductase (DHFR) inhibitors are known to be effective anti-bacterial, and anti-protozoal agents due to the central role played by DHFR in the metabolic synthesis of nucleic acid precursors. Despite their efficacy when used in conjunction with a sulfonanaide, trimelnoprim and pyrimethamine are alone poor inhibitors of P. carinii DHFR. For example, trimethoprim and pyrimethamine exhibit 50% inhibition concentrations (ICo) of 8 and 2,500 nM for E. coli DHFR, while ICes for P. carinii DHFR are 39,600 and 2,400 nM, respectively. Other anti-folates have been shown to be more effective inhibitors of P. carinii DHFR, but require concomitant administration of leucovorin to prevent toxicity to the host. Allegra et a. Pat No. 4,694,007) suggested treatment of P. carinii and Toxoplasmosis gondii with 2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)- •o methyl]quinazoline (trimetrexate), on the theory that the DHFR enzyme in this pathogens is more similar to mammalian DHFR than to prokaryotic DHFR.

Prior to the AIDS epidemic, these types of agents were sufficient for treatment of the rare cases of P. carinii pneumonia. However, in the HIV-positive 9* patient, therapy and prophylaxis with the standard anti-P. carinii agents are complicated by frequent toxic and allergic side effects. New compounds that surpass ,the efficacy of the known antifolates in treating PCP are desirable, especially inhibitors having greater selectivity for P. carinii DHFR relative to the host (particularly human) DHFR than known inhibitors such as trimethoprim.

Commonly-owned copending U.S. Patent Application Serial No. 447,181, filed 7 December 1989 disclosed several DHFR inhibitors exhibiting good selectivity for P. carinii DHFR.

i'l-rBY P~Ll~nT PUT-* rraU ~n IT)*w~ r r U'*l ii .11; 1 I I -3- Disclosure of the Invention We have now found compounds which exhibit superior activity against DHFR from fungal pathogens, such as P. carinii, and which exhibit much higher selectivity for the fungal enzyme as compared with the mammalian (human) enzyme. These compounds are represented generally by Formula I: Formula I: a wherein

S

R, is 3-R 3 -4-R 4 -5-R,-benzyl or (N-R 6 )-8-azabicyclo[3.2.1]oct-3-yl; and R, is H; or R, and R, together form where R, and R, are independently selected from the group consisting of H, lower alkoxy, lower alkylthio, lower alkylsulfinyl, vinyl, carboxy-lower alkyl, carboxy-lower alkoxy, dicarboxy-lower alkyl, dicarboxy-lower alkoxy, aryl-lower alkoxy, arylsulfonyllower alkoxy, arylsulfamido-lower alkoxy, and radicals of formula -O(CH,),-COR9, where n is an integer from 0 to 6 and R, is an amino acid; R 4 is selected from the group consisting of lower alkoxy, aryl-lower alkoxy, lower alkylthio, halo, lower alkenyl, lower alkenyloxy, and pyrrolyl; with the proviso fl,4fl4I~flW *4#4t$S~d4fl U -4that R 3

R

4 and R, are not simultaneously methoxy; R 6 is selected from the group consisting of unsubstituted aryl and aryl substituted with one to three radicals selected from the group consisting of halo, lower alkyl, lower alkoxy, lower alkylthio, carboxy, carbamido, carboxy-lower alkyl, and carbamido-lower alkyl; and R 7 is selected from the group consisting of aryl and aryl-lower alkyl, where aryl may be substituted with one to three radicals selected from the group consisting of halo, lower alkyl, lower alkoxy, lower alkylthio, carboxy, carbamido, carboxylower alkyl, and carbamido-lower alkyl; and lower alkyl esters, amides thereof, and pharmaceutically acceptable addition salts.

One aspect of the invention is a method for treating a fungal infection (such as P. carinii) in a mammal by administering an effective amount of a compound of formula I.

i Another aspect of the invention is a composition for treating a fungal infection (such as P. carinii) in a mammal comprising an effective amount of a compound of formula I in combination with a phannaceutically acceptable excipient.

Another aspect of the invention is the use of a compound of formula I to prepare a composition for treating a fungal infecticn (such as P. carinii) in a mammal comprising an effective amount of a compound of formula I in combination '.20 with a pharmaceutically acceptable excipient.

Modes of Carrying Out The Invention A. Definitions The terms "fungal infection" and "fungal pathogen" refer to the infection of a mammal with an organism of the Kingdom Fungi, for example Pneumocvstis carinii Aspergillus, Candida, Fusarium, and the like. The presently preferred method of the invention is the treatment of Pneumocvstis carinii using the compounds of the invention.

The term "pharmaceutically acceptable" refers to compounds and compositions which may be administered to mammals without undue toxicity. Exemplary pharmaceutically acceptable salts include mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.

The term "effective amount" refers to an amount of compound sufficient to exhibit a detectable therapeutic effect. The therapeutic effect may include, for example, without limitation, inhibiting the growth of pathogens, inhibiting or preventing the release of toxins by pathogens, killing pathogens, and preventing the establishment of infection (prophylaxis). The precise effective amount for a subject will depend upon the subject's size and health, the nature of the pathogen, the severity of the infection, and the like. Thus, it is not possible to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation based on the information provided herein.

The term "lower alkyl" refers to saturated straight or branched-chain rad- S icals consisting of carbon and hydrogen having from 1 to 6 carbon atoms, inclusive, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-hexyl, S and the like. "Lower alkoxy" refers to a radical of the form where is 20 lower alkyl as defined above. Suitable examples include methoxy, ethoxy, propoxy, butoxy, and the like. Similarly, "lower alkylthio" refers to radicals of the form and "lower alkylsulfinyl" refers to groups of the form For example, one may employ methylthio, ethylthio, methylsulfinyl, t-butylsulfinyl, and the like. 'Lower alkenyl" refers to straight or branched-chain radicals consisting of carbon and hydrogen having 2-6 carbon atoms and at least one double bond between a pair of carbon atoms, such as ethenyl (vinyl), 2-propenyl, 1-methylethenyl, 2-butenyl, 3-butenyl, and the like.

i- i ICIFnlCI li~n ;rlaylt R(IV h h)llU n *rl -6- The term "carboxy-lower alkyl" refers to radicals having the form where n is an integer from 1 to 6 inclusive. "Dicarboxy-lower alkyl" indicates lower alkyl chains having two COOH groups attached.

"Aryl" denotes cyclic hydrocarbon radicals of 6-10 carbon atoms which exhibit aromatic character, for example phenyl and napthyl.

The term "halo" refers to fluoro, chloro, bromo, and iodo. The term "amino acid" refers to any of the 20 or so commonly occurring amino acids, for example, glycine, alanine, arginine, phenylalanine, glutamic acid, valine, histidine, proline, ornithine, norleucine, and the like. When attached as R, in a radical of the form -O(CH 2 the amino acid will preferably be attached via a peptide bond, i.e by a bond between the amino group of the amino acid and the acyl carbon of the radical.

S. The term "coadministering" means administration of a compound of the invention in combination with a second therapeutic agent. The second therapeutic agent is a dihydropteroate synthase inhibitor, preferably dapsone or a sulfa drug.

Suitable sulfa drugs include, without limitation, sulfadiazine, sulfamethoxazole, and the like. Coadministration may be simultaneous, for example by administering a mixture of the therapeutic agents, or may be accomplished by administration of the agents separately within a short time period.

B. General Method The compounds of the invention are structurally related to the compound trimethoprim (2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine), the synthesis for which is known in the art. See for example U.S. Pat No. 2,909,522, which describes the synthesis of trimethoprim and related compounds. Compounds of formula I may similarly be synthesized by those of ordinary skill in the art.

Syntheses of such compounds are described in the following U.S. patents: Hitchings et al. (2,658,897); Hitchings et al. (2,909,522); Hoffer (3,341,541); Roth (3,772,289); Roth et al. (3,819,629); Roth (3,822,264); Kompis et al.

.9 *0 -7- (3,931,181); Herrling (3,980,649); Liebenow et al. (3,992,379); Kompis (4,024,145); Rosen (4,033,962); Kompis et al. (4,039,543); Jernow et al.

(4,075,209); Perun et al. (4,087,528); Fritschi etal. (4,180, 578); Kompis et al.

(4,203,980); Poe et al. (4,258,045); Daluge et al. (4,438,267); DaII'Asta (4,485,248); Kompis eta (4,515,948); Swaringen et al. (4,568,744); Roth et al.

(4,587,341); Kompis et al. (4,590,270); Daluge et al. (4,603,136); Kompis et al.

(4,792,557); and Seydel et al. (4,912,112).

Presently preferred compounds of the invention are: 2.4-diamino-5-[3 ,5 -dirnethoxy-4.-(2-hydroxyprop-2-yl)benzyl~pyrimidine; 2,4-diamino-5-(3 ,5-dimethoxy-4--N-pyrrolylbenzyl)pyrimidine; #4u. 2,4-diamino-5-(3 ,5-diethoxy-4-N-pyrrolylbeazyl)py-imidine; *9 *n i o 5 3 5 d p o o y 4 y r l l e zy y i i i e 2,4-diaxnino-5-(3,5-diprooxy-4-N-pyrrolylbenzyl)pyrimidine; 2,4-dian-ino-5-(3,5-diethoxy-4.carboethoxybenzyl)pyrimidine; 2,4-diamino-5-(3 ,5-divinyl-4-vinyloxybenzyl)pyd-midine; 2,4-diamino-5- [3-(4-N-acetaminophenyl)sulfonaxninoethoxy-4,5-dimethoxybenzyljpyrnidine; 2,4-diamino-5- [3-(4-aminophenyl)sulfonaminoethoxy-4-methoxybenzyl~pyrimidine; 2,4-cdiamino-5-[3-(4-N-acetaminophenyl)sulfonaminoethoxy-4-bromo-5-methoxybenzyl)- 20 pyrimidine; 2,4-diamino-5-[3-(4-aminophenyl)sulfonaminoethoxy-4-bromo-5-methoxybenzyllpyrimidine; 2,4-diainino-5-(3-methoxy-4,5-dibenzyloxybenzyl)pyimnidine; 2,4-diarnino-5-(4-benzyloxybenzyl)pyrimidine; 2,A-diamino-5-(3,4-dibenzyloxy-5-methoxybenzyl)pyrimidine; 2,4-dianiino-5-(3 2,4-diamino-5-[3,5-diethoxy-4-(propen-2-yl)benzyl]pyrirnidine; 2,4-diamino-5-[3,5 -dimethoxy-4-(propen-2-yl)benzyl]pyrixnidine; 2,4-diamino-5-(3 ,5-dimethoxy-4-methylthiobenzyl)pyrimidine, 8- 2,A-diamino-5-(3-methylsulfinyl-4mehoxy-5-methylthiobenzyl)pyrimidine; 2, 4-ciamino-5- [3-(4,6-dicarboxyhexyloxy)-4-bromo-5-methoxybenzyl3pyrirnidine; 2,4-diarnino-5-[3-(3-carboxymethylamino-3-oxopropoxy)-4-methoxybenzylpyriinidine; 24,4-diamino-5-(,3-[3-(1 ,3-dicarboxypropyl)amino-3-oxopropoxy)-4-bromo-5-methoxybenzyl)pyrimidine; 2,4-diamrino-5-(3 ,5-dimethoxy-4-((2-phenylsulfinyl)acetyI)benzyl~pyrimidine; 2,4-diamino-5-[3-amino-4-methyl-5-(N-pyrrolyl)benzyl]pyrimidine; 2.4-ciiamino-5-(3 ,5-di-N-pyrrolyl-4-methoxybenzy)pyrrdine; 2,4-diainino-5-[3 ,5-di-methoxy-4-(3-hydrocarboxy-l1-oxopropylamino)benzyl]pyrimidine; *a**2,4-diamino-5- [3,5 -dimethoxy-(4-acetaminophenylsulfonaniino)benzyl]pyrimidine; 2,4-diamino-5-(3 ,5-dimethoxy-4-propylbenzyl)pyriridine; 2,4-diamno-5-(3,5-dichloro-4-N-pyrrolylbenzyl)pyrimidine; :9 :2,4-diamino-5-[3,5-dirnethoxy-4-(2-(2-(2-methoxy)ethoxy)ethoxy)ethoxybenzylp y.-iid in e; 2,4-diamino-5-[3-(3icbnzyloxycarbonylnethylamino-3-oxopropoxy)-4-bromo-5 -methoxybenzyflpyhiniidine; 2,4-clianrtno-5- [3-(3-carboxynethylamino-3-oxopropoxy)-4-bromo-5-methoxybenzyll- 99 4*pyrimidine; 2.-diainino-5-[3-inethoxy-4-bromo-5-(4-methylaminobenzamidoethoxy)benzyl]pyrinidine; azabicyclo[3.2. I] octane; 2H,3H-dihydro-5-(2,4-diarLninopyimidin-5-ylmethyl)-6,7-diimethoxybenzofuran; 5-(2,4-dianiinopyrimidin-5-ylmethyl)-7-methoxy-8-bromo- 1,2-benzopyran; 5-(2,4-diainopyrimidin-5-ymethy>-7,8-dimethoxy-1I,2-benzopyran; 2,4-diarnino-5-[3-phenyl-5-(3-methoxyprOpoxy)beflzyllpyrimidile; 2,4-damino-7-(3 ,5 -dirnethoxybenzyl)pyrrolo[2,3-flquinazoline; 2,4-diami,,io-5-[6-(4-methoxybutoxy)naphth-1I-yllpyrimidine; -9- 2 ,4-diamino-5-(2,7-dimethylbenzpyrazol-5-ylmethyl)pyrimidine; 2,4-diamino-5-(4,5,6-trimethoxy-2,3-dihydroinden-1-yl)pyrimidine; and 2,2-dimethyl-5-(2,4-diaminopyrimidin-5-ylmethyl)-7-methoxybenz[b]dioxolane.

The most preferred compounds at present are 2,4-diamino-5-(3,5-diethoxy-4-Npyrrolylbenzyl)pyrirridine and 2,4-diamino-5-[3,5-dimethoxy-4-(2-hydroxyprop-2yl)benzyl]pyrimidine.

Compositions of the invention for administration will generally include an effective amount of a compound of formula I in addition to a pharmaceutically acceptable excipient. Suitable excipients include most carriers approved for oral or parenteral administration, including water, saline, Ringer's solution, Hank's solution, and solutions of glucose, lactose, dextrose, ethanol, glycerol, albumin, and the like. These compositions may optionally include stabilizers, antioxidants, antimicrobials, preservatives, buffering agents, surfactants, and other accessory additives. A presently preferred vehicle comprises about 1 mg/mL serum albumin in phosphate-buffered saline (PBS). A thorough discussion of suitable vehicles for parenteral administration may be found in E.W. Martin, "Remington's Pharmaceutical Sciences" (Mack Pub. Co., current edition).

The precise dosage necessary will vary with the age, size. and condition of the subject, the nature and severity of the disorder to be treated, and the like: 20 thus, a precise effective amount cannot be specified in advance. However, appropriate amounts may be determined by routine experimentation with animal models.

In general terms, an effective dose of compound of formula I will range from about 10 pg/Kg to about 50 mg/Kg. Suitable animal models include the mouse model illustrated in the Examples below. Rats and other rodents have DHFR very similar to the human enzyme, and thus make suitable animal models.

A group of experimental animals is inoculated with 10-100 LDs of Pneumocvstis carin, followed by treatment with a solution of test compound. A negative control group is left untreated, while a positive control group is treated with a standard therapy, such as trimethoprim. Administration of the compounds is prefert "~iaU Ll Pql** gfM *Wfl hl~*,*A~A44bIt$4.4flI lir4hh $lht,~ *li'fntn ,r ably per os using a gavage), but may be parenteral, for example by subcutaneous or intramuscular injection, or by inhalation of an aerosol. The animals are monitored during treatment, and are sacrificed and examined after 60 days for presence of infection.

C. Examples The examples presented below are provided as a further guide to the practitioner of ordinary skill in the art, and are not to be construed as limiting the invention in any way.

example 1 (Demonstration of Activity) A. Materials Buffers were prepared as follows: 4xDHFR buffer: 200 mM Tes, 300 mM BME, 4 mM EDTA, pH +DHF buffer: 2.5 mg/mL BSA, 0.25 mM NADPH, 62.5 IuM dihydrofolate, 2.5x DHFR S"buffer.

-DHF buffer: 2.5 mg/mL BSA, 0.25 mM NADPH, 2.5x DHFR buffer.

Enzyme buffer: 50 mM Tes, 5 mM DTr, 1 mM EDTA, 20% glycerol, 1 mg/mL BSA, 6. 20 pH Dilution buffer: 50 mM Tes, 5 mM DTT, 1 mM EDTA, 1 mg/mL BSA, pH PcDHFR: 5 pg/mL P. carinii DHFR in enzyme buffer.

crude hDHFR: crude recombinant human DHFL. (obtained from Hoffmann-LaRoche) ii enzyme buffer (9.9 mg/mL total protein).

purified hDHFR: purified recombinant human DHFR (obtained from Hoffmann- LaRoche) in enzyme buffer (3.5 mg/mL total protein).

Test compounds were prepared and provided by Hoffmann-LaRoche.

Stock solutions were prepared by dissolving 2-8 mg in dimethylsulfoxide (DMSO) I 11 to prepare 50 mM solutions. Compounds which did not dissolve at 50 mM were diluted se rially to 25, 16.6, or 12.5 mM.

B. Assay Eight compounds (6.7 pL each) were added to wells in one column of a Falcon® 96-well microtiter plate, and diluted with 160 uL of water. The remaining wells received 126 pL of water. The solutions in the first column were then serially diluted (4x) into columns 2-9, with column 10 containing a control sample, and columns 11-12 containing water blanks. The final compound concentrations were 1 mM to 15 nM (after the remaining reagents were added).

Using an 8-channel pipet, 100 pL. of -DHF buffer were added to each well in columns 11 and 12 (blanks containing only water). Next, 100 pL of +DHF buffer were added to each well in columns 1-10. PcDHFR or crude or purified rhDHFR enzyme was then added (25 pL) to each well, and the plate contents mixed using a Titertek shaker. The plates were read on a Molecular Devices Plate Reader at an absorbance of 340 nm, -0.05 O.D. scale, for 10 minutes reading every 10 seconds. The kinetic data was analyzed using Delta Soft software (Biometallics), and the IC 5 o and Ki calculated for both human and P.

carinii enzymes. Selectivity was calculated as Kion/Kip. cn); thus, higher values for selectivity indicate that the compound inhibits the P. carinii enzyme to 20 a greater degree than the human enzyme. The results are shown in Table 1 below.

For purposes of comparison, trimethoprim in this assay exhibits an IC 50 of 20 pM, with a selectivity of 0.1.

12 TABLE 1: Compounds of Formula I R3 R R5IC 50 Selectivity -OMe -OMe -O(CHi 2 2

NHSO

2 4NHAc 2.4 JIM 350 -OMe CMe 2 OH OYe 25 JIM 350 -QEt pyrroly. -QEt 2.4 JIM 310 -OMe pyrrolyl -OMe 29 JIM vinyl vinyloxy vinyl 3.4 JIM 250 H BzO- H 16 JM -OMe BzO- BzO- 8.3 JIM 100 -OMe -C(Me)=CH 2 -OMe 24 JIM 160 -C(Me)=CH 2 -QEt 2.5 pAM 160 0.to -O~e -SMe -O~e 19 JIM 145 -SMe -O~e -S(O)Me 18 JIM 490 to 0 0* -OMe Br -O(CH 2 2 CH (CH 2 2 COOH 0.04 phA 525

COOH

-O~e Br -O(CH2) 2 CO(N-Asp) 2 JIM 4300 H -OMe -O(CH 2 ),COkN-Gly) 9.3 JIM -OMe Br -O(CH 2 2 C0(N-Gly) 10 PMI 8 Me -CBI, Et -CH2ZM,, Bz -CC 6 N-Gly -NHMH 2 700H, N-Asp -NHCH(COOH)C.ii 2 LOOH, 4,-phcayl, Ac -COCH 3 Example 2 ProceedIng as described in Example 1 above, the compounds listed below were assayed and found to exhibit high activity and selectivity, as set forth in Table 11: A) 2,4-diamino-5-(4-benzyloxyl enzyl)pyrixnidine; 13 B) 2,4-ciamino-5-(3,4-dimethoxy-5-benzyloxybenzyl)pyimidine: C) 2,4-diamino-5-(3,4-dibenzyloxy-5-methoxvbenzyl)pyrinidine; D) 2,4-diamino-5-[3,5-clinethoxy-4-(2-hydroxyprop-2-ylbenzyl~pyimdne; E) 2,4-diamino-5-(3 ,5-dimethoxy-4-N-pyrrolylbenzyl)pyrimidine:, F) 2,4-diamino-5-(3,5-diethoxy-4-N-pyrrolylbenzyl)pyimidine; G) 2,4-diamino-5-(3 ,5-divinyl-4-vinyloxybenzyl)pyrimidine; H) 2,4-diamino-5-[3-(4-N-acetamirnophenyl)sulfonanminoethoxy-4,5-dimethoxybenzyl~pyrimidine; 1) 2,4-diamino-5-[3-(4-aminophenyl'jsulfonarninoezhioxy-4-methoxvbenzyl]pyrimidire; 2,4-diamino-5-[3-(4-N-acetaminophenyl)sulfonaniinoethoxy-4-bromo-5- :methoxybenzyllpyrimidine; K) 2,4-dliamino-5-[3-(4-aminophenyl)sulfonaminoethoxy-4-bi benzyl~pyriaidie; L) 2,4-c3 -lmino-5-[3,5 -diethoxy-4-(propen-2-yl)benzyl]pyrimidine; M) 2,4-cliamino-.5-[3 ,5-dimethoxy-4-(propen-2-yl)benzyl~pyximidine; 2,A-diaminr.-5-(3 ,5-dimethoxy-4-methylthiobenzyl1)pyrinmidine; 2,-imn--3mtyslmy mtoy5mtyti~e7v1)- G pyrimidine; 20 P) 2,4-diamino-5-[3-(4,6-dicarboxyhexyloxy)-4-Jromo-5-methoxybenzyl] pyrirnidine; Q) 2,4-diamino-5-I3.(3-carboxymeiylaniino-3-oxopropoxy)-4-methoxybenzyllpyrimidine; R) 2,4-cliamino-5-(3-[3-(1 ,3-dicarboxypropyl)amino-3-oxopropoxy]-4-bromo- S) 2,4-diamino-5-[3,5-climethoxy-4-((2-plenylsufonyl)acetyl)beflzylPpyrimid:ine; T) 2,4-diamino-5-13-amino--mehy-5-(N-pyrroy)bel]py.tflidfle; U) 2,4-diamino- 5-(3,5-di-N-pyrrolyl-4-methoxybelzylpyrimidLC; 14 V) 2,4-diaxnino-5-[3,5-di-methoxy-4-(3-hydrocarboxy- I -oxopropylamin o) benzyl~pyrixnidiiL V) 2,4-diamino-5-[3,5-dimethoxy-(4-acetamiinophenylsulfonamino)benzyl]pyrimidine; X) 2,4-diamino-5-(3,5-diniethoxy-4-propylbenzyl)pyrindine; Y) 2,4-diainino-5-(3,5-dichloro-4-N-pyrrolylbenzyl)pyrimidine; Z) 2,4-diamino-5-[3,5-dimethoxy-4-(2-(2-(2-methoxy)ethoxy)ethoxy)ethoxybenzyl)pyrimidine; AA) 2,A-ciamino-5-[3-(3-benzyloxycarbonyknethylamino-3-oxopropoxy)- 1 BB) 2,4-diamidno-5-[3-(3-carboxymethylamino-3-oxopropoxy)-4-bromo-5methoxybenzyl~pyrinmidine; CC) 2,4-ciamino-5-[3-methoxy-4-bromo-5 -(4-methylaminobenzaniidoethoxy)benzyl)pyriinidine; DD) 3-(2,4-diarniopyrimnidin-5-ylmethyl)-8-(3,5-dimethoxyphenyl)-8-azabicyclot3.2. 1)octae; .EE) 2H,3H-di hydro-5-(2,4-daminopyrimidin-5-yhnethy1)-6,7-dimethoxybenzo- S Sfuran; FE) 5-(2,4-daminopyrimidin-5-ylmethyl)-7-methoxy-8-bromo- 1 .2-benzopyran; 0(3) 5-(2,4-diaminopyiimidin-5-ylmethyl-7,8-dimethoxy- 1,2-benzopyrar:, HH-) 2,4-diamirrio-5-[3-phenyl-5-(3-rnethloxypropoxy)benzyl)pyridine; II) 2,4-diamino-7-(3,5-dimethoxybenzy!)pyrrolo[2,3-fquinazoline; JJ) 2,4-diamino-5-[6-(4-methoxybutoxy)naphth- 1-yl]pyrimidine; KK)Q 2,4-diainno-5-(4,5 ,6-trimethoxy-2,3-dihydroinden- 1 -ylpyimidine; LI) 2,2-dimethyl-5-(2,4-daninopyrimidin-5-ylnethyl)-7-methoxybelbldioxolane; MM) 2,4-diamino-5-(3,5-diethoxy-4carboethoxybenzyl)pyrinidile; and NN) 2,4-dainino-5-(2,7-dinethylbenzpyazol-5-ylmethyl)pyrimidile- 15 TABLE II: Compound IC 50 Selectiv ity A) 16.0 53.2 B) 13.9 3.3 C) 8.3 90.3 D) 24.8 343.5 E) 2.9 29.4 F) 2.4 312.4 G) 3.4 250.5 H) 2.4 248.5 1) 0.57 7.8 J)3.7 29.5 K)0.97 12.1 1)2.5 190.9 M) 24.0 159.8 N) 19.0 143.5 0) 17.5 486.8 P) 0.043 528.0 Q) 9.3 77.7 R) 2.0 4255.0 S) 14.8 36.8 T) 33.5 42.5 U) 4.5 47.7 V) 100.0 85.2 W) 31.0 14.3 X) 20.0 42.6 Y) 10.91 53.2 Z) 36.0 33.1.

*0 0 0*

S.

Sn.

0*S* 0S S

S

n S SS

*S

5t S o 5*

S.

S S 00 00

OS,.

0

S

16 TABLE II (continued): Compound IC 50 Selectivity A.A) 7.9 14.4 BB) 10.0 13.1 CC) 7.8 14.6 DD) 0.016 31.0 EE) 12.9 26.4 FE') 5.9 6.9 GG) 11.5 266.7 HH) 12.2 11.2 II) 0.039 1.2 JJ) 80.0 106.5 KK) 87.0 97.9 L)13.9 15.0 M)8.3 63.6 N)18.0 10.7 S.

S

S.

S. S.

SS S

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Claims

17- What is Claimed: 1. A method for treating fungal infection in a mammal. which method comprises: administering to a mammal infected with a fungal pathogen an effective amount of a compound of formula I: H, '5 A wherein 00 S em.. 00 0 S S a 0e 0s0* 0 0000 4e 0 00 0S 00 0 00 R, is 3-R 3 -4-R 4 -5-R 5 -benzyl or (N-R)-8-azabicyclo[3.2. ]oct-3-yl; S. S 06 00 0590 0 00 S 0 N-R7 and R, s H; or R, and R 2 together form where R 3 and R, are independently selected from the group consist- ing of H, lower alkoxy, lower allcyltbio, lower alylsulfinyl, vinyl, carboxy-lower alkyl, carboxy-lower alkoxy, dicarboxy-lower alkyl, dicarboxy-lower alkoxy, aryl- lower alkoxy, arylsulfonyl-lower alkoxy, arylsulfamido-lower alkoxy, and radicals of formula -O(CH 2 where n is an integer from 0 to 6 and R, is an a ino acid; -I 18 R 4 is selected from the group consisting of lower alkoxy, aryl-lower alkoxy, lower alkylthio, halo, lower alkenyl, lower alkenyloxy, and pyrrolyl; with the proviso that R 3 R 4 and R, are not simultaneously methoxy; R% is selected from the group consisting of unsubstituted aryl and aryl substituted with one to three radicals selected from the group consisting of halo, lower alkyl, lower alkoxy, lower alkylthio, carboxy, carbamido, carboxy- lower alkyl, and carbamido-lower alkyl; and R 7 is selected from the group consisting of aryl and aryl-lower alkyl, where aryl may be substituted with one to three radicals selected from the group consisting of halo, lower alkyl, lower alkoxy, lower alkylthio, carboxy, carbamido, carboxy-lower alkyl, and carbamido-lower alkyl; and lower alkyl esters, amides thereof, and pharmaceutically acceptable addition salts. 2. The method of claim 1, wherein R 2 is H and R, is 3-R 3 3. The method of claim 2. wherein R 3 is methoxy. 4. The method of claim 3, wherein R, is bromo. 5. The method of claim 4, wherein R, is -O(CH 2 ,)CH(COOH)(CH, 2 COOH, or a pharmaceutically acceptable mono- or diester thereof. 6. The method of claim 4, wherein R, is -O(CH,),-COR. 7. The method of claim 6, wherein n is 2 and R 8 is -NHCH(COOH)CH,CH,COOH or a mono- or di-e.,ter thereof. I 61r -19- 8. T1he method of claim 6, wherein n is 2 and R 8 is -NIIC~HCOOH or an ester thereof. 9. The method of claim 3 wherein is methoxy. The method of claim 9, wherein R, is 2-hydroxyprop-2-yl. 11. The method of claim 9, wherein R, is propen-2-yl. 12. The method of claim 9, wherein R, is N-pyrrolyl. 13. 'The method of claim 9, wherein R, is methylthio. *14. The. method of claim 3, wherein R, is benzyloxy. The method of claim 14, wherein R, is benzyloxy. 16. The method of claim 3, wherein R, is methoxy, and R, is -OC'H 2 CH.,NHSO 2 (C 6 H 4 )NHCOCH 3 17. The method of claim 2 wherein R3 is H.

18. The method of claim 9 wherein R, is mf-zhoxy.

19. The method of claim 10 wherein R, is -O(CH 2 ),,-CORs. where n is 2and R, is -NHCH 2 COOH or an ester thereof. The method of claim 9, wherein R 4 is benzyloxy and P., is H. 20

21. The method of claim 2, where R 3 and R, are each ethoxy.

22. The method of claim 21, wherein R, is pyrrolyl.

23. The method of claim 21, wherein R, is propen-2-yl.

24. The method of claim 2, where R 3 and R, are each vinyl. The method of claim 2, where R, is methylthio. R, is 10 methoxy, and R, is methylsulfinyl.

26. The method of claim 1, wherein R, is H and R, is 8-azabicyclo[3 .2.1 )oct-3-yl.

27. The method of claim 26, wherein is selected from the group consisting of 2-naphthyl, 3,5-dimethoxyphenyl, and 4-carboethoxyphenyl.

28. The method of claim 1, wherein R, and R, together form be.. .e 00 00C 0 N-R7 The method of claim 28, wherein R, is benzyl. The method of claim 28 where R 7 is 21

31. The method of claim 2 where R 3 and are propoxy and R, is N-pyrrolyl.

32. The method of claim 1 wherein said fungal pathogen is Pneumocystis carinn.

33. The method of claim 1, further comprising coadrninstering a dihydropteroate synthase inhibitor.

34. The method of claim 33, wherein said dihydropteroate synthase inhibitor is selected from the group consisting of dapsone and sulfa drugs. formula I is selected~ from the group consisting of '2,4-diamino-5-(4-benzyl benzyl)pyrimidinc.; 2,4-diamino-5-(3,4-dimethoxy-5-benzyloxybenzyl mIdie 2,4-diamino-5-(3,4-dibenzyloxy-5-methoxybenzyl)pyrimidine; 2 amidno-5- dimethoxy-4-(2-hydroxyprop-2-yl)benzyl]pyrimidine- 2, o-5-(3 dimethoxy-4-N-pyrrolylbenzyl)pyrimidine; 2,4-dianiin ,5-diethoxy-4-N- pyrrolylbenzyl)pyrimidine; 2,4-diamino-5-(3,5-di -4-vinyloxybenzyl)- pyrirnidine; 2,4-dianmino-5-[3-(4-N-.acetamin methoxybenzyl]pyrirnidine; 2,4-diamin -[3-(4-aminophenyl)sulfonaminoethoxy- 4-methoxyberlzyl]pyrimidine; 2, acetaminophenyl)sulfonamLin xy-4-bromo-5-mcthoxybenzyl]pyrimicline; 2,4-di- amino-5-[3-(4-aminophe pyrirnidine; 2,4-di o-5-[3,5-dethoxy-4-(propen-2-yl)benzyl]pyrinidine; 2,4- diamino-5-[3 ethoxy-4-(propen-2-yl)benzllpyrimidine; 2,4-diarnino-5-(3 dimethoxy nethylthiobenzyl)pyrimidine; 2,4-.diamino-5-(3-methylsuLlinyl-4- A~dNmeth -5-methylthiobenzyl)pyrimidine; 2,4-diaxnino-5-[3-(4,6- 54 .1 !1 bn R A tM III U 21a

315. A method for treating fungal infection in a mammal, which method comprises administering to a mammal infected with a fungal pathogen an effective amount of a compound selected from the group consisting of 2,4-diarnino-5-(4-benzyloxy- benzyl)pyiimidine; 2,4-diamino-5-(3,4-dinaethoxy-5-benzyloxybenzyl)pyrimidine, 2,4-diamino-5-(3,A-dbenzyloxy-5-nethoxybenzyl)py-imidn; 2.4-diamino-5-[3,5- dimeth oxy-4-(2-hydroxyprop-2 -y)benzyl)pyr,= dine; 2,4-dIarnino-5-(3,5-. dirnethoxy-4-N-pyI-rolylbenzyl)pyrimidine; 2,4-diarnino-5-(3,5-diethoxy-4-N- pynrolylbe-nzyl)pyrimidine,; 2,4-diamino-5-(3,5-divinyl-4-vinyloxybenzy1)- pyrirnidine; 2,4-diamino-5-[3-(4-N-acetaminophenyl)sulfonaminoethoxy-4,5-di- methoxybenzyllpyriniidine; 2,4-cliamino>-3-[3-.(4-aminophenyl)sulfonaminoethoxy- 4-rnethoxybenzyllpyrimidine; 2,4-diamino-5-[3-(4-N- 2,4-di- amino-5-[3-(4-ainophenyl)sulfonamnoethoxy-4-brom-5-mehoxybenzyl- pyrimiddine; 2 ,4-diarnino-5- [3 ,5-diethoxy-4-(propen-2-yl)bnzyl]pyinidine; 2,4- seediaminco-5-[3 ,5 -dimethoxy-4-(propen-2-yl)benzyl)yimdine; 2A4-diaxnino-5(3 dimethoxy-4-methyltbilobenzyl)pyrimidine; 2,4-.daxino-5-(3-rnethylsutfinyl-4- 2,4-diwmino-5-[3-(4,6- -methoxybenzyl~pyriidine; 2,4-diamino-5-[3-(3- *foe* -22 carboxymethylarnino-3-oxopropoxy)-4-metlioxybenzyl]pyrimidine; 2,4-diamino-5- 1,3-dicarboxypropyl)amino-3-oxopropoxy]-4-bromo.5-methoxybenzyl)- pyninidine; 2,4-diamino-5-[3,5-dimethoxy-4-((2-phenylsulfonyl)acetyl)benzyl]- pynimidine; 2,4-diamino-5-[3-amino-4-methyl-5-(N-pyrolyl)benzyl]pyimnidine; 2,A-diaxnino-5-(3 ,5 -di-N-pyrrmlyl-4metoxybenzy)pyrinidine; 2,4-diamino-5 di-methoxy-4-(3-hydrocarboxy- I-oxopropylamnino)benzyl]pyrimidine; 2,A-dianiino- 5-[3,5-dimethoxy-(4-acetanirnophenylsulfonamino)benzyl]pyrmidine; 2,4-diamino- 5-(3 ,5-dimnethoxy-4-propylbenzyl)pyimidine; 2,4-diamino-5-(3 ,5-dichloro-4-N- pyrrolylbenzyl)pyrirniidine; 2,A-diamino-5-[3 1 5-cimethoxy-4-(2-(2-(2-methoxy)- :10 ethoxy)ethoxy)ethoxybenzyl~pyrimidine; 2,4-dianaino-5- [3-(3-benzyloxycarbonyl- methylamino-3-oxopropoxy)-4-bromo-5-methoxybenzyl]pyrirnidine; 0*L'3-(3-carboxymcthylamino-3-oxopropoxy)-4-bromo-5-methoxybenzy1] pyiidine; 69 0 2,4-diamino-5-[3-methoxy-4-bromo-5-(4-methylaminobenzamidoethoxy)benzyillpyr- imicline; 3 -(2,4-diatninopyrimidin-5-ylmethyl)-8-(3 ,5-dimethoxyphenyl)-8-aza- :15 bicyclo[3.2. 1 ]octane; 2H,3H-dihydro-5-(2,4-dianiinopyrimidin-5-ylmethy)-6,7-di- methoxybcnzofuran; 5-(2,4-diaminopyrimidin-5-ylmetkiyl)-7-methoxy-8-bromo- 1,2- benzopyran; 5 -(2,4-diarninopyrimidin-5-ylmethyl)-7,8-dimethoxy- 1,2-benzopyran; 2,4--dianino-5-[3-phenyl-5-(3-methoxypropoxy)benzyl]pyrimidine; 2,4-diamino-7- (3,5-dimethoxybenzyl)pyrrolo[2,3-f~quinazoline; 2,4-diamino-5-[6-(4-methoxy- butoxy)naphth-1I-yIlpyrirnidine; 2,4-daniino-5-(4,5 ,6-trimethoxy-2,3-dihydroinden- 1-yl)pyfinidcfne; 2,2-dimetbyl-5-(2,4-daniinopyrimidin-5-ylmethyl)-7-methoxy- benzllb]dioxolane;, 2,4-diamino-5-(3,5-diethoxy-4-carboethoxyben-zyl)pyrinidine; and 2,A-diamino-5-(2,7-dimethylbenzpyrazol-5-ylmethyl)pyrimidine. (57) Abstract Compounds derived from pyrimidines %avng improved activity against fungi such as Pneumocystis carinii, and improvcd selectivity for P. carinii dihydrofalate reductase over human dihydrofolate reductase, are disclosed. P. carinii pneumonia is ad- vantageously treated with the disclosed compounds. 0S