Targeting species specific amino acid residues: Design, synthesis and biological evaluation of 6-substituted pyrrolo[2,3-d]pyrimidines as dihydrofolate reductase inhibitors and potential anti-opportunistic infection agents.

To combine the potency of trimetrexate (TMQ) or piritrexim (PTX) with the species selectivity of trimethoprim (TMP), target based design was carried out with the X-ray crystal structure of human dihydrofolate reductase (hDHFR) and the homology model of Pneumocystis jirovecii DHFR (pjDHFR). Using variation of amino acids such as Met33/Phe31 (in pjDHFR/hDHFR) that affect the binding of inhibitors due to their distinct positive or negative steric effect at the active binding site of the inhibitor, we designed a series of substituted-pyrrolo[2,3-d]pyrimidines. The best analogs displayed better potency (IC50) than PTX and high selectivity for pjDHFR versus hDHFR, with 4 exhibiting a selectivity for pjDHFR of 24-fold.

Inhibitors of eIF2α dephosphorylation slow replication and stabilize latency in Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that permanently infects warm-blooded vertebrates through its ability to convert into a latent tissue cyst form. The latent form (bradyzoite) can reinitiate a life-threatening acute infection if host immunity wanes, most commonly in AIDS or organ transplant patients. We have previously shown that bradyzoite development is accompanied by phosphorylation of the parasite eukaryotic initiation factor 2 alpha subunit (eIF2α), which dampens global protein synthesis and reprograms gene expression. In this study, we analyzed the activities of two specific inhibitors of eIF2α dephosphorylation, salubrinal (SAL) and guanabenz (GA). We establish that these drugs are able to inhibit the dephosphorylation of Toxoplasma eIF2α. Our results show that SAL and GA reduce tachyzoite replication in vitro and in vivo. Furthermore, both drugs induce bradyzoite formation and inhibit the reactivation of latent bradyzoites in vitro. To address whether the antiparasitic activities of SAL and GA involve host eIF2α phosphorylation, we infected mutant mouse embryonic fibroblast (MEF) cells incapable of phosphorylating eIF2α, which had no impact on the efficacies of SAL and GA against Toxoplasma infection. Our findings suggest that SAL and GA may serve as potential new drugs for the treatment of acute and chronic toxoplasmosis.

Kinetic and structural analysis for potent antifolate inhibition of Pneumocystis jirovecii, Pneumocystis carinii, and human dihydrofolate reductases and their active-site variants.

A major concern of immunocompromised patients, in particular those with AIDS, is susceptibility to infection caused by opportunistic pathogens such as Pneumocystis jirovecii, which is a leading cause of pneumonia in immunocompromised patients. We report the first kinetic and structural data for 2,4-diamino-6-[(2',5'-dichloro anilino)methyl]pyrido[2,3-d]pyrimidine (OAAG324), a potent inhibitor of dihydrofolate reductase (DHFR) from P. jirovecii (pjDHFR), and also for trimethoprim (TMP) and methotrexate (MTX) with pjDHFR, Pneumocystis carinii DHFR (pcDHFR), and human DHFR (hDHFR). OAAG324 shows a 9.0-fold selectivity for pjDHFR (Ki, 2.7 nM) compared to its selectivity for hDHFR (Ki, 24.4 nM), whereas there is only a 2.3-fold selectivity for pcDHFR (Ki, 6.3 nM). In order to understand the determinants of inhibitory potency, active-site mutations of pj-, pc-, and hDHFR were explored to make these enzymes more like each other. The most unexpected observations were that the variant pcDHFR forms with K37Q and K37Q/F69N mutations, which made the enzyme more like the human form, also made these enzymes more sensitive to the inhibitory activity of OAAG324, with Ki values of 0.26 and 0.71 nM, respectively. A similar gain in sensitivity was also observed for the hDHFR N64F variant, which showed a lower Ki value (0.58 nM) than native hDHFR, pcDHFR, or pjDHFR. Structural data are reported for complexes of OAAG324 with hDHFR and its Q35K and Q35S/N64F variants and for the complex of the K37S/F69N variant of pcDHFR with TMP. These results provide useful insight into the role of these residues in the optimization of highly selective inhibitors of DHFR against the opportunistic pathogen P. jirovecii.

Phosphorylation of eukaryotic initiation factor-2{alpha} promotes the extracellular survival of obligate intracellular parasite Toxoplasma gondii.

While seeking a new host cell, obligate intracellular parasites, such as the protozoan Toxoplasma gondii, must be able to endure the stress of an extracellular environment. The mechanisms Toxoplasma use to remain viable while deprived of a host cell are not understood. We have previously shown that phosphorylation of Toxoplasma eukaryotic initiation factor-2α (TgIF2α) is a conserved response to stress. Here we report the characterization of Toxoplasma harboring a point mutation (S71A) in TgIF2α that prevents phosphorylation. Results show that TgIF2α phosphorylation is critical for parasite viability because the TgIF2α-S71A mutants are ill-equipped to cope with life outside the host cell. The TgIF2α-S71A mutants also showed a significant delay in producing acute toxoplasmosis in vivo. We conclude that the phosphorylation of TgIF2α plays a crucial role during the lytic cycle by ameliorating the stress of the extracellular environment while the parasite searches for a new host cell.

Crystallographic analysis reveals a novel second binding site for trimethoprim in active site double mutants of human dihydrofolate reductase.

In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h) DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (K(i) 49 nM) compared to hDHFR (K(i) 4093 nM). Two mutants that contain one substitution from pj--and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show K(i) values of 593 and 617 nM, respectively; these K(i) values are well above both the K(i) for pjDHFR and are similar to pcDHFR (Q35K/N64F and Q35S/N64F) (305nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes.

CoMFA analysis of tgDHFR and rlDHFR based on antifolates with 6-5 fused ring system using the all-orientation search (AOS) routine and a modified cross-validated r(2)-guided region selection (q(2)-GRS) routine and its initial application.

We report the development of CoMFA analysis models that correlate the 3D chemical structures of 80 compounds with 6-5 fused ring system synthesized in our laboratory and their inhibitory potencies against tgDHFR and rlDHFR. In addition to conventional CoMFA analysis, we used two routines available in the literature aimed at the optimization of CoMFA: all-orientation search (AOS) and cross-validated r(2)-guided region selection (q(2)-GRS) to further optimize the models. During this process, we identified a problem associated with q(2)-GRS routine and modified using two strategies. Thus, for the inhibitory activity against each enzyme (tgDHFR and rlDHFR), five CoMFA models were developed using the conventional CoMFA, AOS optimized CoMFA, the original q(2)-GRS optimized CoMFA and the modified q(2)-GRS optimized CoMFA using the first and the second strategy. In this study, we demonstrate that the modified q(2)-GRS routines are superior to the original routine. On the basis of the steric contour maps of the models, we designed four new compounds in the 2,4-diamino-5-methyl-6-phenylsulfanyl-substituted pyrrolo[2,3-d]pyrimidine series. As predicted, the new compounds were potent and selective inhibitors of tgDHFR. One of them, 2,4-diamino-5-methyl-6-(2',6'-dimethylphenylthio)pyrrolo[2,3-d]pyrimidine, is the first 6-5 fused ring system compound with nanomolar tgDHFR inhibitory activity. The HCl salt of this compound was also prepared to increase solubility. Both forms of the drug were tested in vivo in a Toxoplasma gondii infection mouse model. The results indicate that both forms were active with the HCl salt significantly more potent than the free base.

Design, synthesis, biological evaluation and computational investigation of novel inhibitors of dihydrofolate reductase of opportunistic pathogens.

The present work deals with design, synthesis and biological evaluation of novel, diverse compounds as potential inhibitors of dihydrofolate reductase (DHFR) from opportunistic microorganisms; Pneumocystis carinii (pc), Toxoplasma gondii (tg) and Mycobacterium avium (ma). A set of 14 structurally diverse compounds were designed with varying key pharmacophoric features of DHFR inhibitors, bulky distal substitutions and different bridges joining the distal part and 2,4-diaminopyrimidine nucleus. The designed compounds were synthesized and evaluated in enzyme assay against pc, tg and ma DHFR. The rat liver (rl) DHFR was used as mammalian standard. As the next logical step of the project, flexible molecular docking studies were carried out to predict the binding modes of these compounds in pcDHFR active site and the obtained docked poses were post processed using MM-GBSA protocol for prediction of relative binding affinity. The predicted binding modes were able to rationalize the experimental results in most cases. Of particular interest, both the docking scores and MM-GBSA predicted Delta G(bind) were able to distinguish between the active and low active compounds. Furthermore, good correlation coefficient of 0.797 was obtained between the IC(50) values and MM-GBSA predicted Delta G(bind). Taken together, the current work provides not only a novel scaffold for further optimization of DHFR inhibitors but also an understanding of the specific interactions of inhibitors with DHFR and structural modifications that improve selectivity.

2,4-Diamino-5-methyl-6-substituted arylthio-furo[2,3-d]pyrimidines as novel classical and nonclassical antifolates as potential dual thymidylate synthase and dihydrofolate reductase inhibitors.

A novel classical antifolate N-{4-[(2,4-diamino-5-methyl-furo[2,3-d]pyrimidin-6-yl)thio]-benzoyl}-l-glutamic acid 5 and 11 nonclassical antifolates 6-16 were designed, synthesized, and evaluated as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The nonclassical compounds 6-16 were synthesized from 20 via oxidative addition of substituted thiophenols using iodine. Peptide coupling of the intermediate acid 21 followed by saponification gave the classical analog 5. Compound 5 is the first example, to our knowledge, of a 2,4-diamino furo[2,3-d]pyrimidine classical antifolate that has inhibitory activity against both human DHFR and human TS. The classical analog 5 was a nanomolar inhibitor and remarkably selective inhibitor of Pneumocystis carinii DHFR and Mycobacterium avium DHFR at 263-fold and 2107-fold, respectively, compared to mammalian DHFR. The nonclassical analogs 6-16 were moderately potent against pathogen DHFR or TS. This study shows that the furo[2,3-d]pyrimidine scaffold is conducive to dual human DHFR-TS inhibitory activity and to high potency and selectivity for pathogen DHFR.

Synthesis and biological evaluation of biguanide and dihydrotriazine derivatives as potential inhibitors of dihydrofolate reductase of opportunistic microorganisms.

Twenty-one biguanide and dihydrotriazine derivatives were synthesized and evaluated as inhibitors of dihydrofolate reductase (DHFR) from opportunistic microorganisms: Pneumocystis carinii (pc), Toxoplasma gondii (tg), Mycobacterium avium (ma), and rat liver (rl). The most potent compound in the series was B2-07 with 12 nM activity against tgDHFR. The most striking observation was that B2-07 showed similar potency to trimetrexate, approximately 233-fold improved potency over trimethoprim and approximately 7-fold increased selectivity as compared to trimetrexate against tgDHFR. Molecular docking studies in the developed homology model of tgDHFR rationalized the observed potency of B2-07. This molecule can act as a good lead for further design of molecules with better selectivity and improved potency.

Correlations of inhibitor kinetics for Pneumocystis jirovecii and human dihydrofolate reductase with structural data for human active site mutant enzyme complexes.

To understand the role of specific active site residues in conferring selective dihydrofolate reductase (DHFR) inhibition from pathogenic organisms such as Pneumocystis carinii (pc) or Pneumocystis jirovecii (pj), the causative agent in AIDS pneumonia, it is necessary to evaluate the role of these residues in the human enzyme. We report the first kinetic parameters for DHFR from pjDHFR and pcDHFR with methotrexate (MTX), trimethoprim (TMP), and its potent analogue, PY957. We also report the mutagenesis and kinetic analysis of active site mutant proteins at positions 35 and 64 of human (h) DHFR and the crystal structure determinations of hDHFR ternary complexes of NADPH and PY957 with the wild-type DHFR enzyme, the single mutant protein, Gln35Lys, and two double mutant proteins, Gln35Ser/Asn64Ser and Gln35Ser/Asn64Phe. These substitutions place into human DHFR amino acids found at those sites in the opportunistic pathogens pcDHFR (Q35K/N64F) and pjDHFR (Q35S/N64S). The K(i) inhibition constant for PY957 showed greatest potency of the compound for the N64F single mutant protein (5.2 nM), followed by wild-type pcDHFR (K(i) 22 nM) and then wild-type hDHFR enzyme (K(i) 230 nM). Structural data reveal significant conformational changes in the binding interactions of PY957 in the hDHFR Q35S/N64F mutant protein complex compared to the other hDHFR mutant protein complexes and the pcDHFR ternary complex. The conformation of PY957 in the wild-type DHFR is similar to that observed for the single mutant protein. These data support the hypothesis that the enhanced selectivity of PY957 for pcDHFR is in part due to the contributions at positions 37 and 69 (pcDHFR numbering). This insight will help in the design of more selective inhibitors that target these opportunistic pathogens.

Design, synthesis, and X-ray crystal structures of 2,4-diaminofuro[2,3-d]pyrimidines as multireceptor tyrosine kinase and dihydrofolate reductase inhibitors.

To optimize dual receptor tyrosine kinase (RTK) and dihydrofolate reductase (DHFR) inhibition, the E- and Z-isomers of 5-[2-(2-methoxyphenyl)prop-1-en-1-yl]furo[2,3-d]pyrimidine-2,4-diamines (1a and 1b) were separated by HPLC and the X-ray crystal structures (2.0 and 1.4A, respectively) with mouse DHFR and NADPH as well as 1b with human DHFR (1.5A) were determined. The E- and Z-isomers adopt different binding modes when bound to mouse DHFR. A series of 2,4-diaminofuro[2,3-d]pyrimidines 2-13 were designed and synthesized using the X-ray crystal structures of 1a and 1b with DHFR to increase their DHFR inhibitory activity. Wittig reactions of appropriate 2-methoxyphenyl ketones with 2,4-diamino-6-chloromethyl furo[2,3-d]pyrimidine afforded the C8-C9 unsaturated compounds 2-7 and catalytic reduction gave the saturated 8-13. Homologation of the C9-methyl analog maintains DHFR inhibitory activity. In addition, inhibition of EGFR and PDGFR-beta were discovered for saturated C9-homologated analogs 9 and 10 that were absent in the saturated C9-methyl analogs.

Recombinant bovine dihydrofolate reductase produced by mutagenesis and nested PCR of murine dihydrofolate reductase cDNA.

Recent reports of the slow-tight binding inhibition of bovine liver dihydrofolate reductase (bDHFR) in the presence of polyphenols isolated from green tea leaves has spurred renewed interest in the biochemical properties of bDHFR. Earlier studies were done with native bDHFR but in order to validate models of polyphenol binding to bDHFR, larger quantities of bDHFR are necessary to support structural studies. Bovine DHFR differs from its closest sequence homologue, murine DHFR, by 19 amino acids. To obtain the bDHFR cDNA, murineDHFR cDNA was transformed by a series of nested PCRs to reproduce the amino acid coding sequence for bovine DHFR. The bovine liver DHFR cDNA has an open reading frame of 561 base pairs encoding a protein of 187 amino acids that has a high level of conservation at the primary sequence level with other DHFR enzymes, and more so for the amino acid residues in the active site of the mammalian DHFR enzymes. Expression of the bovine DHFR cDNA in bacterial cells produced a stable recombinant protein with high enzymatic activity and kinetic properties similar to those previously reported for the native protein.

Design and synthesis of classical and nonclassical 6-arylthio-2,4-diamino-5-ethylpyrrolo[2,3-d]pyrimidines as antifolates.

The classical antifolate N-{4-[(2,4-diamino-5-ethyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)sulfanyl]benzoyl}-l-glutamic acid (2) and 15 nonclassical analogues (3-17) were synthesized as potential dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. 5-Ethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (20) served as the key intermediate to which various aryl thiols and a heteroaryl thiol were appended at the 6-position via an oxidative addition reaction. The classical analogue 2 was synthesized by coupling the benzoic acid derivative 18 with diethyl l-glutamate followed by saponification. The classical compound 2 was an excellent inhibitor of human DHFR (IC50 = 66 nM) as well as a two digit nanomolar (<100 nM) inhibitor of the growth of several tumor cells in culture. Some of the nonclassical analogues were potent and selective inhibitors of DHFR from two pathogens (Toxoplasma gondii and Mycobacterium avium) that cause opportunistic infections in patients with compromised immune systems.

Novel boron-containing, nonclassical antifolates: synthesis and preliminary biological and structural evaluation.

Two boron-containing, ortho-icosahedral carborane lipophilic antifolates were synthesized, and the crystal structures of their ternary complexes with human dihydrofolate reductase (DHFR) and dihydronicotinamide adenine dinucleotide phosphate were determined. The compounds were screened for activity against DHFR from six sources (human, rat liver, Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium, and Lactobacillus casei) and showed good to modest activity against these enzymes. The compounds were also tested for antibacterial activity against L. casei, M. tuberculosis H37Ra, and three M. avium strains and for cytotoxic activity against seven different human tumor cell lines. Antibacterial and cytotoxic activity was modest, with one sample, the closo-carborane 4, showing about 10-fold greater activity. The less toxic nido-carborane 2 was also tested as a candidate for boron neutron capture therapy, but showed poor tumor retention and low selectivity ratios for boron distribution in tumor tissue versus normal tissue.

Design, synthesis, and antifolate activity of new analogues of piritrexim and other diaminopyrimidine dihydrofolate reductase inhibitors with omega-carboxyalkoxy or omega-carboxy-1-alkynyl substitution in the side chain.

As part of a search for dihydrofolate reductase (DHFR) inhibitors combining the high potency of piritrexim (PTX) with the high antiparasitic vs mammalian selectivity of trimethoprim (TMP), the heretofore undescribed 2,4-diamino-6-(2',5'-disubstituted benzyl)pyrido[2,3-d]pyrimidines 6-14 with O-(omega-carboxyalkyl) or omega-carboxy-1-alkynyl groups on the benzyl moiety were synthesized and tested against Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium DHFR vs rat DHFR. Three N-(2,4-diaminopteridin-6-yl)methyl)-2'-(omega-carboxy-1-alkynyl)dibenz[b,f]azepines (19-21) were also synthesized and tested. The pyridopyrimidine with the best combination of potency and selectivity was 2,4-diamino-5-methyl-6-[2'-(5-carboxy-1-butynyl)-5'-methoxy]benzyl]pyrimidine (13), with an IC(50) value of 0.65 nM against P. carinii DHFR, 0.57 nM against M. avium DHFR, and 55 nM against rat DHFR. The potency of 13 against P. carinii DHFR was 20-fold greater than that of PTX (IC(50) = 13 nM), and its selectivity index (SI) relative to rat DHFR was 85, whereas PTX was nonselective. The activity of 13 against P. carinii DHFR was 20 000 times greater than that of TMP, with an SI of 96, whereas that of TMP was only 14. However 13 was no more potent than PTX against M. avium DHFR, and its SI was no better than that of TMP. Molecular modeling dynamics studies using compounds 10 and 13 indicated a slight binding preference for the latter, in qualitative agreement with the IC(50) data. Among the pteridines, the most potent against P. carinii DHFR and M. avium DHFR was the 2'-(5-carboxy-1-butynyl)dibenz[b,f]azepinyl derivative 20 (IC(50) = 2.9 nM), whereas the most selective was the 2'-(5-carboxy-1-pentynyl) analogue 21, with SI values of >100 against both P. carinii and M. avium DHFR relative to rat DHFR. The final compound, 2,4-diamino-5-[3'-(4-carboxy-1-butynyl)-4'-bromo-5'-methoxybenzyl]pyrimidine (22), was both potent and selective against M. avium DHFR (IC(50) = 0.47 nM, SI = 1300) but was not potent or selective against either P. carinii or T. gondii DHFR.

Synthesis and biological evaluation of 2,4-diamino-6-(arylaminomethyl)pyrido[2,3-d]pyrimidines as inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase and as antiopportunistic infection and antitumor agents.

A series of 2,4-diamino-6-(arylaminomethyl)pyrido[2,3-d]pyrimidines were synthesized and evaluated as inhibitors of Pneumocystis carinii (pc), Toxoplasma gondii (tg), and rat liver (rl) dihydrofolate reductase (DHFR) and as inhibitors of the growth of tumor cell lines in culture. Compounds 4-15 were designed as part of a continuing effort to examine the effects of substitutions at the 5-position, in the two-atom bridge, and in the side chain phenyl ring on structure-activity/selectivity relationships of 2,4-diaminopyrido[2,3-d]pyrimidines against a variety of DHFRs. Reductive amination of the common intermediate 2,4-diaminopyrido[2,3-d]pyrimidine-6-carbonitrile 16 with the appropriate anilines afforded the target compounds 4-12. Nucleophilic substitution or reductive methylation afforded the N10-methyl target compounds 13-15. As predicted, compounds 4-15 were, in general, less potent against all three DHFRs compared to the corresponding 2,4-diamino-5-methyl analogues previously reported; however, the greater decrease in potency against rlDHFR compared to pcDHFR and tgDHFR resulted in appreciable selectivity toward pathogenic DHFRs from different pathogens. The 2',5'-dichloro analogue 8 showed selectivity ratios (IC(50) against rlDHFR/IC(50) against pcDHFR or tgDHFR) of 15.7 and 23 for pcDHFR and tgDHFR, respectively. Thus, the selectivity of 8 for pcDHFR is higher than the first line clinical agent trimethoprim (TMP). In a P. carinii cell culture study, analogue 8 exhibited 88% cell growth inhibition at a concentration of 10 muM and afforded marginal effects in an in vivo study in the T. gondii mouse model. Selected compounds were evaluated in the National Cancer Institute (NCI) in vitro preclinical antitumor screening program and inhibited the growth of tumor cells in culture at micromolar to submicromolar concentrations and were selected for evaluation in a NCI in vivo hollow fiber assay.

Further studies on 2,4-diamino-5-(2',5'-disubstituted benzyl)pyrimidines as potent and selective inhibitors of dihydrofolate reductases from three major opportunistic pathogens of AIDS.

As part of an ongoing effort to discover novel small-molecule antifolates combining the enzyme-binding species selectivity of trimethoprim (TMP) with the potency of piritrexim (PTX), 10 previously unreported 2,4-diamino-5-(2'-methoxy-5'-substituted)benzylpyrimidines (2-11) containing a carboxyl group at the distal end of the 5'-substituent were synthesized and tested as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), and Mycobacterium avium (Ma), three of the opportunistic pathogens frequently responsible for life-threatening illness in people with impaired immune systems as a result of HIV infection or immunosuppressive chemotherapy. The selectivity index of DHFR inhibition was evaluated by comparing the potency of each compound against the parasite enzymes with its potency against rat liver DHFR. 2,4-Diamino-5-[5'-(5-carboxy-1-pentynyl)-2'-methoxybenzyl]pyrimidine (3) inhibited Pc DHFR with a selectivity index of 79 and was 430 times more potent than TMP. 2,4-Diamino-5-[5'-(4-carboxy-1-butynyl)-2'-methoxybenzyl]pyrimidine (2), with one less carbon than 3 in the side chain, had a selectivity index of 910 against Ma DHFR and was 43 times more potent than TMP. 2,4-Diamino-5-[5'-(5-carboxypentyl)-2'-methoxybenzyl]pyrimidine (6) had a selectivity index of 490 against Tg DHFR and was 320 times more potent than TMP. 2,4-Diamino-5-[5'-(6-carboxy-1-hexynyl)-2'-methoxybenzyl]pyrimidine (4), with one more carbon than 3, was less potent against all three of the parasite enzymes than either 3 or 6 and also had a lower selectivity index than 3 against the Pc enzyme. However, 4 was the only member of the series with a selectivity index of >300 against both Tg and Ma DHFR. Given that PTX is at least 10 times more potent against rat DHFR than against P. carinii or T. gondii DHFR and that the selectivity index of several of the compounds matches or exceeds that of TMP as well as PTX, our results suggest that it may be possible to develop clinically useful nonclassical antifolates that are both potent and selective against the major opportunistic pathogens of AIDS.

Design, synthesis, and biological evaluation of 2,4-diamino-5-methyl-6-substituted-pyrrolo[2,3-d]pyrimidines as dihydrofolate reductase inhibitors.

2,4-diamino-5-methyl-6-(substituted-phenyl)thiopyrrolo[2,3-d]pyrimidines 4-11 were synthesized as dihydrofolate reductase (DHFR) inhibitors against opportunistic pathogens that afflict patients with AIDS. Synthesis was achieved from 2,4-diamino-5-methypyrrolo[2,3-d]pyrimidine and substituted phenylthiols under modified conditions reported by Gangjee et al. Some of these compounds were potent and selective against DHFR from both Toxoplasma gondii and Mycobacterium avium compared to mammalian DHFR. Compound 11 with a 1-naphthyl substituent is 16-fold more potent and equally selective against Toxoplasma gondii DHFR as the clinically used trimethoprim.

Inhibition of Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium dihydrofolate reductases by 2,4-diamino-5-[2-methoxy-5-(omega-carboxyalkyloxy)benzyl]pyrimidines: marked improvement in potency relative to trimethoprim and species selectivity relative to piritrexim.

A series of previously undescribed 2,4-diamino-5-[2-methoxy-5-alkoxybenzyl]pyrimidines (3a-e) and 2,4-diamino-5-[2-methoxy-5-(omega-carboxyalkyloxy)benzyl]pyrimidines (3f-k) with up to eight CH2 groups in the alkoxy or omega-carboxyalkyloxy side chain were synthesized and tested for the ability to inhibit partially purified dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), Mycobacterium avium (Ma), and rat liver in comparison with two standard inhibitors, trimethoprim (1) and piritrexim (2). The latter drug is known to be extremely potent but shows a marked preference for binding to mammalian DHFR, whereas the former is very selective for the parasite enzymes but is a much weaker inhibitor. The underlying strategy for the synthesis of compounds 3a-k was that a hybrid structure embodying some features of both 1 and 2 might possess a more favorable combination of potency and selectivity than either parent drug. The choice of analogues 3f-k was based on the idea that the acidic omega-carboxyl group might interact preferentially with a basic center in the active site of DHFR from any of the parasite species relative to the active site of mammalian DHFR. In addition, the omega-carboxyl group was expected to improve water solubility relative to 1 or 2. In standardized spectrophotometric assays with dihydrofolate as the substrate and NADPH as the cofactor, 2,4-diamino-5-[(2-methoxy-4-carboxybutyloxy)benzyl]pyrimidine (3g) inhibited Pc DHFR with an IC(50) of 0.049 microM and rat DHFR with IC(50) of 3.9 microM. Its potency against Pc DHFR was 140-fold greater than that of 1 and close to that of 2, and its selectivity index, defined as the ratio IC(50)(rat liver)/IC(50)(P. carinii), was 8-fold higher than that of 1 and >10(4)-fold higher than that of 2. Although it was less potent and less selective against Tg than Pc DHFR, it was very potent as well as highly selective against Ma DHFR, with an IC(50) of 0.0058 microM and an IC(50)(rat liver)/IC(50)(M. avium) ratio of >600. Because of this favorable combination of potency and selectivity relative to 1 and 2, compound 3g may be viewed as a promising lead in the search for new antifolates with potential clinical activity against P. carinii and other opportunistic pathogens in patients with AIDS.