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.

Structural analysis of a holoenzyme complex of mouse dihydrofolate reductase with NADPH and a ternary complex with the potent and selective inhibitor 2,4-diamino-6-(2'-hydroxydibenz[b,f]azepin-5-yl)methylpteridine.

It has been shown that 2,4-diamino-6-arylmethylpteridines and 2,4-diamino-5-arylmethylpyrimidines containing an O-carboxylalkyloxy group in the aryl moiety are potent and selective inhibitors of the dihydrofolate reductase (DHFR) from opportunistic pathogens such as Pneumocystis carinii, the causative agent of Pneumocystis pneumonia in HIV/AIDS patients. In order to understand the structure-activity profile observed for a series of substituted dibenz[b,f]azepine antifolates, the crystal structures of mouse DHFR (mDHFR; a mammalian homologue) holo and ternary complexes with NADPH and the inhibitor 2,4-diamino-6-(2'-hydroxydibenz[b,f]azepin-5-yl)methylpteridine were determined to 1.9 and 1.4 A resolution, respectively. Structural data for the ternary complex with the potent O-(3-carboxypropyl) inhibitor PT684 revealed no electron density for the O-carboxylalkyloxy side chain. The side chain was either cleaved or completely disordered. The electron density fitted the less potent hydroxyl compound PT684a. Additionally, cocrystallization of mDHFR with NADPH and the less potent 2'-(4-carboxybenzyl) inhibitor PT682 showed no electron density for the inhibitor and resulted in the first report of a holoenzyme complex despite several attempts at crystallization of a ternary complex. Modeling data of PT682 in the active site of mDHFR and P. carinii DHFR (pcDHFR) indicate that binding would require ligand-induced conformational changes to the enzyme for the inhibitor to fit into the active site or that the inhibitor side chain would have to adopt an alternative binding mode to that observed for other carboxyalkyloxy inhibitors. These data also show that the mDHFR complexes have a decreased active-site volume as reflected in the relative shift of helix C (residues 59-64) by 0.6 A compared with pcDHFR ternary complexes. These data are consistent with the greater inhibitory potency against pcDHFR.

Antifolate screening using yeast expressing Plasmodium vivax dihydrofolate reductase and in vitro drug susceptibility assay for Plasmodium falciparum.

The presence of homologous point mutations in the dhfr gene in Plasmodium vivax and Plasmodium falciparum is associated with resistance to antifolate drugs. The spread of antifolate resistance encouraged research for novel antifolate drugs active against both wild-type and dhfr-mutant strains of malaria parasites. Because P. vivax cannot be easily maintained in culture, we transformed a Saccharomyces cerevisiae DHFR-deleted mutant to express wild-type P. vivax dhfr gene and its mutant forms. Twenty-five dicyclic and tricyclic 2,4-diaminopyrimidine derivatives were screened. Six quinazoline compounds showed selective inhibition of yeast transformants expressing P. vivax dhfr genes. The 50% inhibitory concentration (IC(50)) of these six compounds was determined against field isolates of P. falciparum. Our results suggest that a close relationship between the yeast assay based on expression of P. vivax dhfr genes and the in vitro test using P. falciparum parasites in culture is a promising initial step for drug screening.

New insights into DHFR interactions: analysis of Pneumocystis carinii and mouse DHFR complexes with NADPH and two highly potent 5-(omega-carboxy(alkyloxy) trimethoprim derivatives reveals conformational correlations with activity and novel parallel ring stacking interactions.

Structural data are reported for two highly potent antifolates, 2,4-diamino-5-[3',4'-dimethoxy-5'-(5-carboxy-1-pentynyl)]benzylpyrimidine (PY1011), with 5000-fold selectivity for Pneumocystis carinii dihydrofolate reductase (pcDHFR), relative to rat liver DHFR, and 2,4-diamino-5-[2-methoxy-5-(4-carboxybutyloxy)benzyl]pyrimidine (PY957), that has 80-fold selectivity for pcDHFR. Crystal structures are reported for NADPH ternary complexes with PY957 and pcDHFR, refined to 2.2 A resolution; with PY1011 and pcDHFR, refined to 2.0 A resolution; and with PY1011 and mouse DHFR (mDHFR), refined to 2.2 A resolution. These results reveal that the carboxylate of the omega-carboxyalkyloxy side chain of these inhibitors form ionic interactions with the conserved Arg in the substrate binding pocket of DHFR. These data suggest that the enhanced inhibitory activity of PY1011 compared with PY957 is, in part, due to the favorable contacts with Phe69 of pcDHFR by the methylene carbons of the inhibitor side chain that are oriented by the triple bond of the 1-pentynyl side chain. These contacts are not present in the PY957 pcDHFR complex, or in the PY1011 mDHFR complex. In the structure of mDHFR the site of Phe69 in pcDHFR is occupied by Asn64. These data also revealed a preference for an unusual parallel ring stacking interaction between Tyr35 of the active site helix and Phe199 of the C-terminal beta sheet in pcDHFR and by Tyr33 and Phe179 in mDHFR that is independent of bound ligand. A unique His174-His187 parallel ring stacking interaction was also observed only in the structure of pcDHFR. These ring stacking interactions are rarely found in any other protein families and may serve to enhance protein stability.

Calorimetric studies of ligand binding in R67 dihydrofolate reductase.

R67 dihydrofolate reductase (DHFR) is a novel bacterial protein that possesses 222 symmetry and a single active site pore. Although the 222 symmetry implies that four symmetry-related binding sites must exist for each substrate as well as for each cofactor, various studies indicate only two molecules bind. Three possible combinations include two dihydrofolate molecules, two NADPH molecules, or one substrate plus one cofactor. The latter is the productive ternary complex. To explore the role of various ligand substituents during binding, numerous analogues, inhibitors, and fragments of NADPH and/or folate were used in both isothermal titration calorimetry (ITC) and K(i) studies. Not surprisingly, as the length of the molecule is shortened, affinity is lost, indicating that ligand connectivity is important in binding. The observed enthalpy change in ITC measurements arises from all components involved in the binding process, including proton uptake. As a buffer dependence for binding of folate was observed, this likely correlates with perturbation of the bound N3 pK(a), such that a neutral pteridine ring is preferred for pairwise interaction with the protein. Of interest, there is no enthalpic signal for binding of folate fragments such as dihydrobiopterin where the p-aminobenzoylglutamate tail has been removed, pointing to the tail as providing most of the enthalpic signal. For binding of NADPH and its analogues, the nicotinamide carboxamide is quite important. Differences between binary (binding of two identical ligands) and ternary complex formation are observed, indicating interligand pairing preferences. For example, while aminopterin and methotrexate both form binary complexes, albeit weakly, neither readily forms ternary complexes with the cofactor. These observations suggest a role for the O4 atom of folate in a pairing preference with NADPH, which ultimately facilitates catalysis.

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 of the lipophilic antifolate piritrexim via a palladium(0)-catalyzed cross-coupling reaction.

[reaction: see text] A regiospecific and convergent route the lipophilic antifolate piritrexim (PTX) is described in which a key step is a Pd(0)-catalyzed cross-coupling reaction between 2-amino-3-cyano-4-methyl-5-bromopyridine and 2,5-dimethoxybenzylzinc chloride to form 2-amino-4-methyl-5-(2,5-dimethoxybenzyl)nicotinonitrile. To complete the synthesis, the amino group is replaced by a more reactive bromine atom via nonaqueous diazotization with tert-butyl nitrite, and the resultant bromo nitrile is cyclized with guanidine.

Synthesis and in vitro antifolate activity of rotationally restricted aminopterin and methotrexate analogues.

Heretofore unknown analogues of aminopterin (AMT) and methotrexate (MTX) in which free rotation of the amide bond between the phenyl ring and amino acid side chain is prevented by a CH(2) bridge were synthesized and tested for in vitro antifolate activity. The K(i) of the AMT analogue (9) against human dihydrofolate reductase (DHFR) was 34 pM, whereas that of the MTX analogue (10) was 2100 pM. Both compounds were less potent than the parent drugs. However, although the difference between AMT and MTX was <2-fold, the difference between 9 and 10 was 62-fold, suggesting that the effect of N(10)-methyl substitution is amplified in the bridged compounds. The K(i) values of 9 and 10 as inhibitors of [(3)H]MTX influx into CCRF-CEM human leukemia cells via the reduced folate carrier (RFC) were 0.28 and 1.1 muM, respectively. The corresponding K(i) and K(t) values determined earlier for AMT and MTX were 5.4 and 4.7 muM, respectively. Thus, in contrast to its unfavorable effect on DHFR binding, the CH(2) bridge increased RFC binding. In a 72 h growth assay with CCRF-CEM cells, the IC(50) values of 9 and 10 were 5.1 and 140 nM, respectively, a 27-fold difference that was qualitatively consistent with the observed combination of weaker DHFR binding and stronger RFC binding. Although rotationally restricted inhibitors of other enzymes of folate pathway enzymes have been described previously, 9 and 10 are the first reported examples of DHFR inhibitors of this type.

In vitro activities of 2,4-diaminoquinazoline and 2,4-diaminopteridine derivatives against Plasmodium falciparum.

The activities of 28 6-substituted 2,4-diaminoquinazolines, 2,4-diamino-5,6,7,8-tetrahydroquinazolines, and 2,4-diaminopteridines against Plasmodium falciparum were tested. The 50% inhibitory concentrations (IC(50)s) of six compounds were <50 nM, and the most potent compound was 2,4-diamino-5-chloro-6-[N-(2,5-dimethoxybenzyl)amino]quinazoline (compound 1), with an IC(50) of 9 nM. The activity of compound 1 was potentiated by the dihydropteroate synthase inhibitor dapsone, an indication that these compounds are inhibitors of dihydrofolate reductase. Further studies are warranted to assess the therapeutic potential of this combination in vivo.

Synthesis of 2,4-diamino-6-[2'-O-(omega-carboxyalkyl)oxydibenz[b,f]azepin-5-yl]methylpteridines as potent and selective inhibitors of Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium dihydrofolate reductase.

Six previously undescribed N-(2,4-diaminopteridin-6-yl)methyldibenz[b,f]azepines with water-solubilizing O-carboxyalkyloxy or O-carboxybenzyloxy side chains at the 2'-position were synthesized and compared with trimethoprim (TMP) and piritrexim (PTX) as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), and Mycobacterium avium (Ma), three of the opportunistic organisms known to cause significant morbidity and mortality in patients with AIDS and other disorders of the immune system. The ability of the new analogues to inhibit reduction of dihydrofolate to tetrahydrofolate by Pc, Tg, Ma, and rat DHFR was determined, and the selectivity index (SI) was calculated from the ratio IC(50)(rat DHFR)/IC(50)(Pc, Tg, or Ma DHFR). The IC(50) values of the 2'-O-carboxypropyl analogue (10), with SI values in parentheses, were 1.1 nM (1300) against Pc DHFR, 9.9 nM (120) against Tg DHFR, and 2.0 nM (600) against Ma DHFR. The corresponding values for the 2'-O-(4-carboxybenzyloxy) analogue (12) were 1.0 nM (560), 22 nM (21), and 0.75 nM (630). By comparison, the IC(50) and SI values for TMP were Pc, 13 000 nM (14); Tg, 2800 nM (65); and Ma, 300 nM (610). For the prototypical potent but nonselective inhibitors PTX and TMX, respectively, these values were Pc, 13 nM (0.26) and 47 nM (0.17); Tg, 4.3 nM (0.76) and 16 nM (0.50); Ma, 0.61 nM (5.4) and 1.5 nM (5.3). Thus 10 and 12 met the criterion for DHFR inhibitors that combine the high selectivity of TMP with the high potency of PTX and TMX.

Preliminary in vitro studies on two potent, water-soluble trimethoprim analogues with exceptional species selectivity against dihydrofolate reductase from Pneumocystis carinii and Mycobacterium avium.

2,4-Diamino-5-[3',4'-dimethoxy-5'-(5-carboxy-1-pentynyl)]benzylpyrimidine (6) and 2,4-diamino-5-[3',4'-dimethoxy-5'-(4-carboxyphenylethynyl)benzylpyrimidine (7) were synthesized from 2,4-diamino-5-(5'-iodo-3',4'-dimethoxybenzyl)pyrimidine (9) via a Sonogashira reaction with appropriate acetylenic esters followed by saponification, and were tested as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), Mycobacterium avium (Ma), and rat in comparison with the widely used antibacterial agent 2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine (trimethoprim, TMP). The selectivity index (SI) for each compound was calculated by dividing its 50% inhibitory concentration (IC(50)) against rat DHFR by its IC(50) against Pc, Tg, or Ma DHFR. The IC(50) of 6 against Pc DHFR was 1.0 nM, with an SI of 5000. Compound 7 had an IC(50) of 8.2 nM against Ma DHFR, with an SI of 11000. By comparison, the IC(50) of TMP was 12000 nM against Pc, 300 nM against Ma, and 180000 against rat DHFR. The potency and selectivity values of 6 and 7 were not as high against Tg as they were against Pc or Ma DHFR, but nonetheless exceeded those of TMP. Because of the outstanding selectivity of 6 against Pc and of 7 against Ma DHFR, these novel analogues may be viewed as promising leads for further structure-activity optimization.

New 2,4-diamino-5-(2',5'-substituted benzyl)pyrimidines as potential drugs against opportunistic infections of AIDS and other immune disorders. Synthesis and species-dependent antifolate activity.

In a continuing effort to design small-molecule inhibitors of dihydrofolate reductase (DHFR) that combine the enzyme-binding selectivity of 2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine (trimethoprim, TMP) with the potency of 2,4-diamino-5-methyl-6-(2',5'-dimethoxybenzyl)pyrido[2,3-d]pyrimidine (piritrexim, PTX), seven previously undescribed 2,4-diamino-5-[2'-methoxy-5'-(substituted benzyl)]pyrimidines were synthesized in which the substituent at the 5'-position was a carboxyphenyl group linked to the benzyl moiety by a bridge of two or four atoms in length. The new analogues were all obtained from 2,4-diamino-5-(5'-iodo-2'-methoxybenzyl)pyrimidine via a Sonogashira reaction, followed, where appropriate, by catalytic hydrogenation. The new analogues were tested as inhibitors of DHFR from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), and Mycobacterium avium (Ma), three life-threatening pathogens often found in AIDS patients and individuals whose immune system is impaired as a result of treatment with immunosuppressive chemotherapy or radiation. The selectivity index (SI) of each compound was obtained by dividing its 50% inhibitory concentration (IC(50)) against Pc, Tg, or Ma DHFR by its IC(50) against rat DHFR. 2,4-Diamino-[2'-methoxy-5'-(3-carboxyphenyl)ethynylbenzyl]pyrimidine (28), with an IC(50) of 23 nM and an SI of 28 in the Pc DHFR assay, had about the same potency as PTX and was 520 times more potent than TMP. As an inhibitor of Tg DHFR, 28 had an IC(50) of 5.5 nM (510-fold lower than that of TMP and similar to that of PTX) and an SI value of 120 (2-fold better than TMP and vastly superior to PTX). Against Ma DHFR, 28 had IC(50) and SI values of 1.5 nM and 430, respectively, compared with 300 nM and 610 for TMP. Although it had 2.5-fold lower potency than 28 against Ma DHFR (IC(50) = 3.7 nM) and was substantially weaker against Pc and Tg DHFR, 2,4-diamino-[2'-methoxy-5'-(4-carboxyphenyl)ethynylbenzyl]pyrimidine (29), with the carboxy group at the para rather than the meta position, displayed 2200-fold selectivity against the Ma enzyme and was the most selective 2,4-diamino-5-(5'-substituted benzyl)pyrimidine inhibitor of this enzyme we have encountered to date. Additional bioassay data for these compounds are also reported.

Dihydrofolate reductase mutant with exceptional resistance to methotrexate but not to trimetrexate.

Two double (F31A/F34A, I60A/L67G) and one quadruple (F31A/F34A/I60A/L67G) mutant murine dihydrofolate reductases were constructed and evaluated for their ability to impart antifolate resistance. Both I60A/L67G and F31A/F34A/I60A/L67G were found to be unstable and devoid of catalytic activity. The K(i) values for F31A/F34A, methotrexate (MTX), bis-MTX, and PT-523 were found to be 10100-, 4410-, and 617-fold higher than the wild-type enzyme, respectively, but only 13.5-fold higher for trimetrexate (TMTX). These findings suggest that F31A/F34A could be used for gene therapy to render normal cells resistant to MTX but sensitive to TMTX.

Further studies on the interaction of nonpolyglutamatable aminopterin analogs with dihydrofolate reductase and the reduced folate carrier as determinants of in vitro antitumor activity.

Thirteen structural analogs of the potent nonpolyglutamatable dihydrofolate reductase inhibitor N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523) with modifications in the side chain, the para-aminobenzoyl moiety, or the 9,10-bridge were evaluated for the ability to inhibit human recombinant dihydrofolate reductase (DHFR), to utilize the reduced folate carrier (RFC) for influx, and to inhibit the growth of CCRF-CEM human leukemia cells in culture. In spectrophotometric assays of the kinetics of the reduction of dihydrofolate by DHFR in the presence of NADPH, these compounds had K(i) values ranging from 0.2 to 1.3pM, and thus were not greatly different in potency from the parent drug PT523. By comparison, the K(i) values of aminopterin (AMT), methotrexate (MTX), and 10-ethyl-10-deazaaminopterin (EDX) were 3.7, 4.8, and 11pM. In assays of competitive inhibition of [3H]MTX influx into CCRF-CEM cells, the K(i) values ranged from 0.21 to 7.3 micro M, as compared with 0.71, 5.4, and 1.1 micro M for PT523, AMT, and EDX. The K(t) for MTX was also re-analyzed and found to be 4.7 micro M, in better agreement with the literature than our previously reported value of 7.1 micro M. The IC(50) values of these compounds as inhibitors of the growth of CCRF-CEM cells after 72hr of drug exposure ranged from 0.53 to 55nM, and were qualitatively consistent with the other results.

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.

Synthesis of new 2,4-Diaminopyrido[2,3-d]pyrimidine and 2,4-Diaminopyrrolo[2,3-d]pyrimidine inhibitors of Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium dihydrofolate reductase.

A concise new route allowing easy access to five previously unreported 2,4-diamino-6-(substituted benzyl)pyrido[2,3-d]pyrimidines (2a-e) was developed, involving condensation of 2,4-dipivaloylamino-5-bromopyrido[2,3-d]pyrimidine (6) with an organozinc halide in the presence of a catalytic amount of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II).CH(2)Cl(2), followed by removal of the pivaloyl groups with base. Also prepared via a scheme based on the Taylor ring expansion/ring annulation synthesis were three heretofore undescribed 2,4-diamino-5-(substituted benzyl)-7H-pyrrolo[2,3-d]pyrimidines (3b-c). Standard spectrophotometric assays were used to compare the ability of 2a-e and 3b-c to inhibit dihydrofolate reductase (DHFR) from Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium, three examples of opportunistic pathogens to which AIDS patients are highly vulnerable because of their immunocompromised state. For comparison, 13 previously untested 2,4-diamino-6-(substituted benzyl)quinazolines (17a-m) were also evaluated as inhibitors of these enzymes, as well as the enzyme from rat liver. None of the quinazolines or pyridopyrimidines tested was more potent against the P. carinii enzyme than the structurally related reference compound piritrexim (1), and none showed selectivity for the P. carinii enzyme over the rat enzyme. One of the pyridopyrimidines (2c) showed 10-fold selectivity for T. gondii versus rat DHFR, and two of them (2b, 2c) showed selectivity for the M. avium enzyme. However, this gain in species selectivity was achieved at the cost of decreased in potency, as has been noted with many other lipophilic DHFR inhibitors.

Structure-based enzyme inhibitor design: modeling studies and crystal structure analysis of Pneumocystis carinii dihydrofolate reductase ternary complex with PT653 and NADPH.

Structural data are reported for N-(2,4-diaminopteridin-6-yl)methyldibenz[b,f]azepine (PT653), an example of structure-based inhibitor design with 21-fold selectivity for Pneumocystis carinii dihydrofolate reductase (pcDHFR) relative to rat liver dihydrofolate reductase (rlDHFR). These data test the hypothesis that 2,4-diaminopteridines with a bulky N,N-diarylaminomethyl side chain at the 6-position could fit better into the larger active site of pcDHFR than into that of mammalian DHFR. The crystal structure of the ternary complex of NADPH, PT653 and pcDHFR, refined to 2.4 A resolution, reveals that PT653 binds in a different orientation than predicted from modeling studies reported previously [Rosowsky et al. (1999), J. Med. Chem. 42, 4853-4860]. These crystal data show that the pteridine-ring plane is tilted compared with that observed in the crystal structure of the pcDHFR methotrexate (MTX) NADPH ternary complex used as a template to model PT653 binding. Also, as a result of this tilt, the dibenzoazepine ring is bound deeper into the p-aminobenzoyl folate binding pocket of pcDHFR, thereby relieving close intermolecular contacts predicted from the modeling data. By far the most significant structural change, but more subtle in magnitude, is the ligand-induced conformational shift of 1.2 A away from the inhibitor of residues 61-66 in helix C. The other major effect is the unwinding of the short helical segment involving loop 47 which has a different conformation to that observed in other pcDHFR complexes [Cody et al. (1999), Biochemistry, 38, 4303-4312]. The favorable pcDHFR selectivity of PT653 could be a result of ligand-induced fit of the large hydrophobic dibenzazepine ring which occupies regions of the enzyme active site not probed by other antifolates and which take advantage of sequence and conformational differences between the structures of human and pcDHFR. These data suggest that such hydrophobic analogs could be used as lead compounds in the design of more pcDHFR-selective antifolates. Enzyme inhibition data also show that PT653 is 102-fold selective for Toxoplasma gondii (tg) DHFR relative to rlDHFR. Homology-modeling studies of the tgDHFR structure suggest that differences in ligand-binding orientation and enzyme sequence could influence the enhanced selectivity of PT653 for tgDHFR.

Synthesis and in vitro antitumor activity of new deaza analogues of the nonpolyglutamatable antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523).

Details are disclosed for the synthesis of N(alpha)-[4-[2-(2,4-diaminoquinazolin-6-yl)ethyl]benzoyl]-N(delta)-hemiphthaloyl-L-ornithine (2) and N(alpha)-[4-[5-(2,4-diaminoteridin-6-yl)pent-1-yn-4-yl]benzoyl]-N(delta)-hemiphthaloyl-L-ornithine (6) as analogues of N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (1, PT523), a nonpolyglutamatable antifolate currently in advanced preclinical development. In a 72 h growth inhibition assay against cultures of CCRF-CEM human leukemic lymphoblasts, the IC(50) of 2 and 6 was 0.69 +/- 0.044 nM and 1.3 +/- 0.35 nM, respectively, as compared with previously reported values 4.4 +/- 0.10 nM for aminopterin (AMT) and 1.5 +/- 0.39 nM for PT523. In a spectrophotometric assay of dihydrofolate reductase (DHFR) inhibition using dihydrofolate and NADPH as the cosubstrates, the previously unreported compounds 2 and the mixed 10R and 10S diastereomers of 6 had K(i) values of 0.21 +/- 0.05 pM and 0.60 +/- 0.02 pM, respectively, as compared with previously reported values of 3.70 +/- 0.35 pM for AMT and 0.33 +/- 0.04 pM for PT523. Thus, while they were comparable to 1 and several of its previously studied analogues in their ability to bind to DHFR and inhibit the growth of CCRF-CEM cells, 2 and the mixed diastereomers of 6 were several times more active than AMT despite the fact that they cannot form gamma-polyglutamylated metabolites of the type formed in cells from AMT and other classical antifolates with a glutamate side chain.

Synthesis and in vitro antitumor activity of thiophene analogues of 5-chloro-5,8-dideazafolic acid and 2-methyl-2-desamino-5-chloro-5,8-dideazafolic acid.

N-[5-[N-(2-Amino-5-chloro-3,4-dihydro-4-oxoquinazolin-6-yl)methylamino]-2-thenoyl]-L-glutamic acid (6) and N-[5-[N-(5-chloro-3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl)methylamino]-2-thenoyl]-L-glutamic acid (7), the first reported thiophene analogues of 5-chloro-5,8-dideazafolic acid, were synthesized and tested as inhibitors of tumor cell growth in culture. 4-Chloro-5-methylisatin (10) was converted stepwise to methyl 2-amino-5-methyl-6-chlorobenzoate (22) and 2-amino-5-chloro-3,4-dihydro-6-methyl-4-oxoquinazoline (19). Pivaloylation of the 2-amino group, followed by NBS bromination, condensation with di-tert-butyl N-(5-amino-2-thenoyl)-L-glutamate (28), and stepwise cleavage of the protecting groups with ammonia and TFA yielded. Treatment of 9 with acetic anhydride afforded 2,6-dimethyl-5-chlorobenz[1,3-d]oxazin-4-one (31), which on reaction with ammonia, NaOH was converted to 2,6-dimethyl-5-chloro-3,4-dihydroquinazolin-4-one (33). Bromination of, followed by condensation with and ester cleavage with TFA, yielded. The IC(50) of and against CCRF-CEM human leukemic lymphoblasts was 1.8+/-0.1 and 2.1+/-0.8 microM, respectively.

Multiple mechanisms of resistance to methotrexate and novel antifolates in human CCRF-CEM leukemia cells and their implications for folate homeostasis.

We determined the mechanisms of resistance of human CCRF-CEM leukemia cells to methotrexate (MTX) vs. those to six novel antifolates: the polyglutamatable thymidylate synthase (TS) inhibitors ZD1694, multitargeted antifolate, pemetrexed, ALIMTA (MTA) and GW1843U89, the non-polyglutamatable inhibitors of TS, ZD9331, and dihydrofolate reductase, PT523, as well as DDATHF, a polyglutamatable glycinamide ribonucleotide transformylase inhibitor. CEM cells were made resistant to these drugs by clinically relevant intermittent 24 hr exposures to 5-10 microM of MTX, ZD1694, GW1843U89, MTA and DDATHF, by intermittent 72 hr exposures to 5 microM of ZD9331 and by continuous exposure to stepwise increasing concentrations of ZD9331, GW1843U89 and PT523. Development of resistance required only 3 cycles of intermittent drug exposure to ZD1694 and MTA, but 5 cycles for MTX, DDATHF and GW1843U89 and 8 cycles for ZD9331. The predominant mechanism of resistance to ZD1694, MTA, MTX and DDATHF was impaired polyglutamylation due to approximately 10-fold decreased folylpolyglutamate synthetase activity. Resistance to intermittent exposures to GW1843U89 and ZD9331 was associated with a 2-fold decreased transport via the reduced folate carrier (RFC). The CEM cell lines resistant to intermittent exposures to MTX, ZD1694, MTA, DDATHF, GW1843U89 and ZD9331 displayed a depletion (up to 4-fold) of total intracellular reduced folate pools. Resistance to continuous exposure to ZD9331 was caused by a 14-fold increase in TS activity. CEM/GW70, selected by continuous exposure to GW1843U89 was 50-fold resistant to GW1843U89, whereas continuous exposure to PT523 generated CEM/PT523 cells that were highly resistant (1550-fold) to PT523. Both CEM/GW70 and CEM/PT523 displayed cross-resistance to several antifolates that depend on the RFC for cellular uptake, including MTX (95- and 530-fold). CEM/GW70 cells were characterized by a 12-fold decreased transport of [3H]MTX. Interestingly, however, CEM/GW70 cells displayed an enhanced transport of folic acid, consistent with the expression of a structurally altered RFC resulting in a 2.6-fold increase of intracellular folate pools. CEM/PT523 cells displayed a markedly impaired (100-fold) transport of [3H]MTX along with 12-fold decreased total folate pools. In conclusion, multifunctional mechanisms of resistance in CEM cells have a differential impact on cellular folate homeostasis: decreased polyglutamylation and transport defects lead to folate depletion, whereas a structurally altered RFC protein can provoke expanded intracellular folate pools.