Methotrexate analogues. 34. Replacement of the glutamate moiety in methotrexate and aminopterin by long-chain 2-aminoalkanedioic acids

Eight previously unreported methotrexate (MTX) and aminopterin (AMT) analogues with the L-glutamate moiety replaced by DL-2-aminoalkanedioic acids containing up to 10 CH2 groups were synthesized from 4-amino-4-deoxy-N10-methylpteroic or 4-amino-4-deoxy-N10-formylpteroic acid. All the compounds were potent inhibitors of purified L1210 mouse leukemia dihydrofolate reductase (DHFR), with IC50's of 0.023-0.034 microM for the MTX analogues and 0.054-0.067 microM for the AMT analogues. The compounds were not substrates for, but were inhibitors of, partially purified mouse liver folylpolyglutamate synthetase (FPGS). Activity was correlated with the number of CH2 groups in the side chain. The IC50's for inhibition of cell growth in culture by the chain-extended MTX analogues were 0.016-0.64 microM against CEM human leukemic lymphoblasts and 0.0012-0.026 microM against L1210 mouse leukemia cells. However, the optimal chain length for growth-inhibitory activity was species-dependent. Our results suggested that CEM cells were inhibited most actively by the analogue with nine CH2 groups, while L1210 cells were most sensitive to the analogue with six CH2 groups. Among the AMT analogues, on the other hand, the most active compound against L1210 cells was the one with nine CH2 groups, which had an IC50 of 0.000 65 microM as compared with 0.0046 microM for MTX and 0.002 microM for AMT. A high degree of cross-resistance was observed between MTX and the chain-extended compounds in two MTX-resistant cell lines, CEM/MTX and L1210/R81. All the MTX analogues were active against L1210 leukemia in mice on a qd X 9 schedule, with optimal increases in lifespan (ILS) of 75-140%. Notwithstanding their high in vitro activity, the AMT analogues were more toxic and less therapeutically effective than MTX analogues of the same chain length even though neither series of compounds possessed FPGS substrate activity. These MTX and AMT analogues are an unusual group of compounds in that they retain the dicarboxylic acid structure of classical antifolates yet are more lipophilic than the parent compounds because they have more CH2 groups and are almost equivalent in vivo to MTX on the same schedule even though they do not form polyglutamates.     

Methotrexate analogues. 32. Chain extension, alpha-carboxyl deletion, and gamma-carboxyl replacement by sulfonate and phosphonate: effect on enzyme binding and cell-growth inhibition

Analogues of methotrexate (MTX) and aminopterin (AMT) with aminophosphonoalkanoic, aminoalkanesulfonic, and aminoalkanephosphonic acid side chains in place of glutamate were synthesized and tested as inhibitors of folylpolyglutamate synthetase (FPGS) from mouse liver. The aminophosphonoalkanoic acid analogues were also tested as inhibitors of dihydrofolate reductase (DHFR) from L1210 murine leukemia cells and as inhibitors of the growth of MTX-sensitive (L1210) and MTX-resistant (L1210/R81) cells in culture. The optimal number of CH2 groups in aminophosphonoalkanoic acid analogues of AMT was found to be two for both enzyme inhibition and cell growth inhibition but was especially critical for activity against FPGS. Deletion of the alpha-carboxyl also led to diminished anti-FPGS activity in comparison with previously studied homocysteic acid and 2-amino-4-phosphonobutyric acid analogues. In the aminoalkanesulfonic acid analogues of MTX without an alpha-carboxyl, anti-FPGS activity was low and showed minimal variation as the number of CH2 groups between the carboxamide and sulfonate moieties was changed from one to four. In similar aminoalkanephosphonic acid analogues of MTX, anti-FPGS activity was also low, was comparable for two and three CH2 groups between the carboxamide and phosphonate moieties, and was diminished by monoesterification of the phosphonate group. These effects demonstrate that the alpha-carboxyl group of folate analogues is involved in binding to the active site of FPGS, and that an alpha-carboxyl group should be retained as part of the structure of FPGS inhibitors.     

Inhibition of dihydrofolate reductase, methotrexate transport, and growth of methotrexate-sensitive and -resistant L1210 leukemia cells in vitro by 5-substituted 2,4-diaminoquinazolines

A series of eighteen 2,4-diaminoquinazoline analogues of folic, isofolic, pteroic and isopteroic acids having various substituents at position 5 was studied. Each compound was evaluated as an inhibitor of L1210 dihydrofolate reductase, methotrexate influx into L1210 leukemia cells, and growth of methotrexate-sensitive and -resistant L1210 cells in vitro. Bridge reversal at positions 9 and 10 reduced the effectiveness of the classical analogues only with regard to the inhibition of the drug-sensitive cells as compared to methotrexate (MTX). Absence of the glutamate moiety adversely affected the potency of the compounds, particularly when coupled with reversal of the 9,10-bridge. However, the presence of -Cl at position 5 restored significantly the potency of these compounds. The pteroate and isopteroate analogue ethyl esters were generally more effective inhibitors of cell growth than their non-esterified counterparts. Regarding the effects of substituents at position 5, the data suggest that -Cl greater than -CH3 greater than -H for inhibition of methotrexate transport and growth of methotrexate-sensitive L1210 cells. The 5-Cl pteroate analogue and its corresponding ethyl ester were highly effective as growth inhibitors of methotrexate-resistant, transport-defective, L1210 cells in vitro.     

Methotrexate analogues. 19. Replacement of the glutamate side chain in classical antifolates by L-homocysteic acid and L-cysteic acid: effect on enzyme inhibition and antitumor activity

Methotrexate (MTX) and aminopterin (AMT) analogues containing L-homocysteic acid or L-cysteic acid in place of L-glutamic acid were synthesized and tested as inhibitors of dihydrofolate reductase from L1210 cells and folyl polyglutamate synthetase from mouse liver. The ID50 against dihydrofolate reductase was comparable for the MTX and AMT analogues (0.04-0.07 microM), whereas the ID50 against folyl polyglutamate synthetase was 3- to 4-fold lower for the AMT analogues (40-60 microM) than for the MTX analogues (100-200 microM). Thus, N10-substitution has a greater effect on binding to folyl polyglutamate synthetase than dihydrofolate reductase. The cytotoxicity of these compounds was assayed in vitro against L1210 cells, and the AMT analogues again proved more potent (ID50 = 0.03-0.05 microM) than the MTX analogues (ID50 = 0.1-0.4 microM). A similarly increased potency was observed for the AMT analogues against L1210 leukemia in vivo. Though differential cell uptake cannot be ruled out as the basis of increased potency, it is possible that part of the activity of the AMT analogues involves interference with the intracellular polyglutamation of reduced folate cofactors, i.e., that they are "self-potentiating antifolates". Of the four compounds reported, the most active was N-(4-amino-4- deoxypteroyl )-L-homocysteic acid, which produced a 138% increase in life span (ILS) in L1210 leukemic mice when given on a modified bid X 10 schedule at a dose of 2 mg/kg. A comparable ILS was obtained with AMT itself at 0.24 mg/kg. Thus, replacement of gamma-CO2H by gamma-SO3H in the side chain does not decrease therapeutic effect. However, a higher dose is required, presumably to offset pharmacological differences reflecting the inability of the sulfonate group to be polyglutamated .     

Methotrexate analogues. 30. Dihydrofolate reductase inhibition and in vitro tumor cell growth inhibition by N epsilon-(haloacetyl)-L-lysine and N delta-(haloacetyl)-L-ornithine analogues and an acivicin analogue of methotrexate

Analogues of methotrexate (MTX) with strong alkylating activity were prepared by replacing the L-glutamate side chain with N omega-haloacetyl derivatives of L-lysine and L-ornithine. Haloacetylation was accomplished in 30-40% yield by reaction of the preformed L-lysine and L-ornithine analogues of MTX with p-nitrophenyl bromoacetate or chloroacetate in aqueous sodium bicarbonate at room temperature. All four haloacetamides were potent inhibitors in spectrophotometric assays measuring noncovalent binding to purified dihydrofolate reductase (DHFR) from L1210 cells. In experiments designed to measure time-dependent inactivation of DHFR from L1210 cells and Candida albicans, the N epsilon-(bromoacetyl)-L-lysine and N delta-(bromoacetyl)-L-ornithine analogues gave results consistent with covalent binding, whereas N epsilon- and N delta-chloroacetyl analogues did not. The N delta-(bromoacetyl)-L-ornithine analogue appeared to be the more reactive one toward both enzymes. Amino acid analysis of acid hydrolysates of the L1210 enzyme following incubation with the bromoacetamides failed to demonstrate the presence of a carboxymethylated residue, suggesting that alkylation had perhaps formed an acid-labile bond. In growth inhibition assays with L1210 cultured murine leukemia cells, the four haloacetamides were all more potent than their nonacylated precursors but less potent than MTX. The greater than 40,000-fold MTX-resistant mutant cell line L1210/R81 was only partly cross-resistant to the haloacetamides. An analogue of MTX with acivicin replacing glutamate was a potent inhibitor of DHFR from chicken liver and L1210 cells but was 200 times less potent than MTX against L1210 cells in culture.     

Synthesis of aza homologues of folic acid.

Folic acid analogues containing an additional nitrogen atom between the phenyl ring and the carbonyl group of the side chain were synthesized. None of the compounds showed significant inhibitory activity against human lymphoblastic leukemia cells (CCRF-CEM) in culture or against Lactobacillus casei (ATCC 7469) growth. Against L1210 leukemia in mice, the aza homologue of folic acid, 4, and the aspartic acid analogue, 14, showed no increase in life span over control animals. These compounds were more toxic in vivo than the corresponding methotrexate analogues. Compound 4 supported the growth of Streptococcus faecium (ATCC 8043), and its tetrahydro derivative supported the growth of Pediococcus cerevisiae (ATCC 8081). These results strongly suggest that 4 can substitute for folate derivatives as cofactors for serine transhydroxymethylase, thymidylate synthetase, and dihydrofolate reductase.     

Chemistry and antitumor evaluation of selected classical 2,4-diaminoquinazoline analogues of folic acid

A series of six 2,4-diaminoquinazoline analogues of folic acid which bear close structural resemblance to methotrexate, 1a, were synthesized by unequivocal routes. Three of these have not been described previously, while complete structural characterization of the remaining compounds is presented for the first time. Each of the compounds was a potent inhibitor of dihydrofolate reductase (DHFR) from rat liver or L1210 leukemia cells having I50 values in a range similar to that of 1a. However, a wide divergence in inhibitory activity toward the growth of human gastrointestinal adenocarcinoma or L1210 leukemia cells in vitro was observed. Compounds having a normal folate configuration at positions 9 and 10 were more inhibitory than their isomeric reversed-bridge counterparts. The N-formyl modifications were the least active of the compounds studied. Unsubstituted or N-methyl modifications competed effectively with tritiated 1a for uptake into L1210 leukemia cells, while N-formyl modifications did not. Against an L1210 cell line resistant to 1a by virtue of altered transport and overproduction of DHFR, partial but not complete cross-resistance was observed for certain analogues. Of the three compounds selected for in vivo evaluation against L1210 leukemia in mice, two had a similar level of antitumor activity to that of 1a. The compound 5,8-dideazamethopterin, 2b, however, was slightly more active than 1a but at substantially reduced dose levels.     

Methotrexate analogues. 31. Meta and ortho isomers of aminopterin, compounds with a double bond in the side chain, and a novel analogue modified at the alpha-carbon: chemical and in vitro biological studies

Five heretofore undescribed analogues of methotrexate (MTX) and aminopterin (AMT) were synthesized and tested as dihydrofolate reductase (DHFR) inhibitors and tumor cell growth inhibitors. The meta isomer of AMT was obtained from 2,4-diamino-6-(bromomethyl)pteridine and m-(aminobenzoyl)-L-glutamic acid, while the ortho isomer was obtained via the same route by using alpha-methyl gamma-tert-butyl o-(aminobenzoyl)-L-glutamate instead of the free acid. Analogues of MTX and AMT containing a double bond in the side chain were prepared from dimethyl D,L-2-amino-4-hexenedioate and 4-amino-4-deoxy-N10-methylpteroic acid and 4-amino-4-deoxy-N10-formylpteroic acid, respectively. Finally, a positional isomer of MTX with the CH2CH2COOH moiety moved from the alpha-carbon to the adjacent carboxamide nitrogen was synthesized from 3-[N-(carboxymethyl)amino]propanoic acid diethyl ester and 4-amino-4-deoxy-N10-methylpteroic acid. The positional isomers of AMT were weak DHFR inhibitors and showed very little growth-inhibitory activity against L1210 murine leukemia cells or the MTX-resistant L1210/R81 mutant line in culture. The MTX and AMT analogues with the CH2CH2COOH moiety replaced by a CH2CH = CHCOOH side chain showed anti-DHFR activity similar to that of the previously described saturated compound N-(4-amino-4-deoxy-N10-methylpteroyl)-L-2-aminoadipic acid, but were less potent than the parent drugs. The MTX analogue with the CH2CH2COOH side chain displaced from C to N was weakly bound to DHFR, confirming the importance of an intact CONH moiety, and showed greatly diminished cell growth inhibitory potency relative to MTX. None of the compounds was a substrate for folylpolyglutamate synthetase (FPGS) from mouse liver. Furthermore, inhibition of folic acid polyglutamylation in vitro at equimolar 500 microM concentrations of drug and substrate was negligible. The structural changes embodied in these five novel compounds are therefore too great for binding to the FPGS active site.     

PTT.119, p-F-Phe-m-bis-(2-chloroethyl)amino-L-Phe-Met ethoxy HCl, a new chemotherapeutic agent active against drug-resistant tumor cell lines

PTT.119 [p-F-Phe-m-bis-(2-chloroethyl)amino-L-Phe-Met ethoxy HCl], a new synthetic tripeptide, was highly effective against the L-phenylalanine mustard (L-PAM) resistant (L1210/L-PAM and P388/L-PAM) tumor lines, as well as the sensitive L1210 leukemia. Cytolytic activity of PTT.119 against all three leukemias was significantly greater than equimolar doses of L-PAM. These in vitro results paralleled the significant increases in mean survival times of hosts and, in some cases, abrogations of tumor formation observed in the in vivo bioassays of PTT.119-treated L1210 and L1210/L-PAM cells. Dose-response studies failed to demonstrate cross-resistance to the tripeptide by L-PAM resistant cells. Doses of PTT.119 required to reduce the viable fraction by 50% (tissue culture dose 50, TCD50) or 100% (TCD100) were 1.3- to 3-fold lower for the L-PAM resistant cells than for the L1210 leukemia. In comparison, L-PAM was unable to completely eliminate cell survival; 0.2 to 3% of the cells in all three leukemias remained viable even at doses of 75 and 163 microM. In similar studies, L1210 leukemia cells made resistant to methotrexate (L1210 MTX) and cisplatin (L1210DDP) were also completely susceptible to PTT.119; TCD50 values of the two resistant lines were 1.94 microM for L1210 MTX and 0.525 microM for L1210DDP compared to 2.38 microM for the susceptible parent L1210S leukemia. Continuous low-dose PTT.119 treatment of MJY-alpha mammary tumor cells for 8 months and exposure of L1210 leukemia to escalating levels of tripeptide for over 100 passages failed to select or induce drug-resistant phenotypes in either cell line. PTT.119 appears to be a poor mutagen and is unlikely to readily increase the probability of drug-resistant mutants in the tumor cell populations.     

Methotrexate analogues-27. Dual inhibition of dihydrofolate reductase and folylpolyglutamate synthetase by methotrexate and aminopterin analogues with a gamma-phosphonate group in the side chain

gamma-Phosphonate analogues of methotrexate (MTX) and aminopterin (AMT) were synthesized from 4-amino-4-deoxy-N10-methylpteroic acid and 4-amino-4-deoxy-N10-formylpteroic acid, respectively, by reaction with methyl D,L-2-amino-4-phosphonobutyrate followed by gentle alkaline hydrolysis. The products were compared with the corresponding D,L-homocysteic acid derivatives as inhibitors of dihydrofolate reductase and folylpolyglutamate synthetase, and as inhibitors of cell growth in culture. The gamma-phosphonates were somewhat less active than either the gamma-sulfonates or the parent drugs as inhibitors of murine dihydrofolate reductase. The MTX gamma-sulfonate and gamma-phosphonate analogues were equally inhibitory toward mouse liver folylpolyglutamate synthetase (Ki = 190 microM), but in the AMT series the gamma-phosphonate (Ki = 8.4 microM) was more potent than the gamma-sulfonate (Ki = 45 microM). The AMT analogues were consistently more inhibitory than the MTX analogues against cultured L1210 murine leukemia cells, but neither the gamma-phosphonates nor the gamma-sulfonates were as potent as their respective parent drugs. The gamma-phosphonate analogue of MTX was three times more potent than MTX against the MTX-resistant mutant line L1210/R81, but the AMT gamma-phosphonate was less potent than AMT; however, these differences were small in comparison with the level of resistance to all these compounds in the L1210/R81 line. The results suggest that N10-methyl and N10-unsubstituted compounds altered at the gamma-position do not necessarily follow identical structure-activity patterns in every test system.     

Methotrexate analogues--XVII. Antitumor activity of 4-amino-4-deoxy-N10-methylpteroyl-D,L-homocysteic acid and its dual inhibition of dihydrofolate reductase and folyl polyglutamate synthetase

A new analogue of methotrexate was synthesized from 4-amino-4-deoxy-N10-methylpteroic acid and D,L-homocysteic acid. The product (mAPA-HCysA) was bound tightly to L1210 mouse leukemia dihydrofolate reductase (IC50 = 1 nM), inhibited L1210 cell proliferation in culture (IC50 = 0.3 microM), and prolonged the survival of L1210 leukemic mice (98% increase in lifespan at 120 mg/kg, qdx9). Studies on the interaction of mAPA-HCysA with partially purified mouse liver folyl polyglutamate synthetase revealed that mAPA-HCysA was not a substrate. Hence, the increased dose of mAPA-HCysA required to inhibit tumor growth in vitro and in vivo relative to methotrexate may reflect, in part, the inability of this compound to form non-effluxing polyglutamates. Folyl polyglutamate synthetase was competitively inhibited by mAPA-HCysA (K1 = 190 +/- 70 microM) when folate was the variable substrate. Thus, mAPA-HCysA is the first known compound to inhibit both mammalian dihydrofolate reductase and mammalian folyl polyglutamate synthetase.     

Methotrexate analogues. 26. Inhibition of dihydrofolate reductase and folylpolyglutamate synthetase activity and in vitro tumor cell growth by methotrexate and aminopterin analogues containing a basic amino acid side chain

Analogues of the antitumor antifolate methotrexate (MTX) were synthesized in which the glutamate (Glu) moiety was replaced by ornithine (Orn), 2,4-diaminobutyric acid (Dab), or 2,3-diaminopropionic acid (Dap). An aminopterin (AMT) analogue with Orn in place of Glu was also synthesized. The MTX analogues were obtained by reaction of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) and N omega-Boc-alpha,omega-diaminoalkanoic acids in the presence of diethyl phosphorocyanidate, followed by deprotection with trifluoroacetic acid (TFA) or by reaction of p-nitrophenyl-mAPA and N omega-Boc-alpha,omega-diaminoalkanoic acids and subsequent treatment with TFA. The AMT analogue (APA-Orn) was synthesized by reaction of p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate with silylated N delta-Boc-L-ornithine in DMF at 55 degrees C for 3 days (45% yield), saponification (83%), and TFA cleavage (89%). APA-Orn was a potent inhibitor of both dihydrofolate reductase (DHFR) from L1210 mouse leukemia (IC50 = 0.072 microM) and partly purified folylpolyglutamate synthetase (FPGS) from mouse liver (Ki = 0.15 +/- 0.06 microM). The MTX analogue (mAPA-Orn) was likewise active against both enzymes, with an IC50 of 0.160 microM for DHFR and a Ki of 20.4 +/- 7.7 microM for FPGS inhibition. The other MTX analogues and the previously reported lysine derivative (mAPA-Lys) showed DHFR affinity similar to that of mAPA-Orn but lacked activity as FPGS inhibitors. The positively charged amino group appears to be detrimental to cellular uptake, as evidenced by the low cytotoxicity of these compounds (IC50 = 0.40-2.4 microM) in comparison with MTX and AMT (IC50 = 0.002 microM) against wild-type L1210 cells. On the other hand, mAPA-Orn and APA-Orn were both more potent than the corresponding Glu derivatives MTX and AMT against L1210/R81 cells, suggesting that in these MTX-resistant cells there may occur a "self-potentiation" process involving enhanced antifolate activity via interference with the polyglutamylation of reduced folates. APA-Orn is the most potent dual inhibitor of DHFR and FPGS discovered to date, but its effectiveness as a therapeutic agent may require some form of prodrug modification to neutralize the terminal amino group of the side chain.     

Methotrexate analogues. 9. Synthesis and biological properties of some 8-alkyl-7,8-dihydro analogues.

A series of 8-alkyl-7,8,-dihydromethotrexate analogues was prepared by direct alkylation of 7,8-dihydromethotrexate, after pilot studies were performed with simpler pteridines. These compounds are tested for in vitro inhibitory activity against Lactobacillus casei and as enzyme inhibitors against dihydrofolate reductase and thymidylate synthetase derived from this organism. All of the analogues were less inhibitory toward dihydrofolate reductase than was methotrexate but were more inhibitory toward thymidylate synthetase. The analogues were also evaluated for in vitro inhibitory activity against the CCRF-CEM human lymphoblastic leukemia cells. In vivo against the L-1210 leukemia in mice, several of the analogues exhibited some antileukemic activity.     

Syntheses and antifolate activity of 5-methyl-5-deaza analogues of aminopterin, methotrexate, folic acid, and N10-methylfolic acid

Evidence indicating that modifications at the 5- and 10-positions of classical folic acid antimetabolites lead to compounds with favorable differential membrane transport in tumor vs. normal proliferative tissue prompted an investigation of 5-alkyl-5-deaza analogues. 2-Amino-4-methyl-3,5-pyridinedicarbonitrile, prepared by hydrogenolysis of its known 6-chloro precursor, was treated with guanidine to give 2,4-diamino-5-methylpyrido[2,3-d]pyrimidine-6-carbonitrile which was converted via the corresponding aldehyde and hydroxymethyl compound to 6-(bromomethyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine. Reductive condensation of the nitrile 8 with diethyl N-(4-amino-benzoyl)-L-glutamate followed by ester hydrolysis gave 5-methyl-5-deazaaminopterin. Treatment of 12 with formaldehyde and Na(CN)BH3 afforded 5-methyl-5-deazamethotrexate, which was also prepared from 15 and dimethyl N-[(4-methylamino)benzoyl]-L-glutamate followed by ester hydrolysis. 5-Methyl-10-ethyl-5-deazaaminopterin was similarly prepared from 15. Biological evaluation of the 5-methyl-5-deaza analogues together with previously reported 5-deazaaminopterin and 5-deazamethotrexate for inhibition of dihydrofolate reductase (DHFR) isolated from L1210 cells and for their effect on cell growth inhibition, transport characteristics, and net accumulation of polyglutamate forms in L1210 cells revealed the analogues to have essentially the same properties as the appropriate parent compound, aminopterin or methotrexate (MTX), except that 20 and 21 were approximately 10 times more growth inhibitory than MTX. In in vivo tests against P388/0 and P388/MTX leukemia in mice, the analogues showed activity comparable to that of MTX, with the more potent 20 producing the same response in the P388/0 test as MTX but at one-fourth the dose; none showed activity against P388/MTX. Hydrolytic deamination of 12 and 20 produced 5-methyl-5-deazafolic acid and 5,10-dimethyl-5-deazafolic acid, respectively. In bacterial studies on the 2-amino-4-oxo analogues, 5-deazafolic acid proved to be a potent inhibitor of Lactobacillus casei DHFR and also the growth of both L. casei ATCC 7469 and Streptococcus faecium ATCC 8043. Its 5-methyl congener 22 is also inhibitory toward L. casei, but its IC50 for growth inhibition is much lower than its IC50 values for inhibition of DHFR or thymidylate synthase from L. casei, suggesting an alternate site of action.     

Methotrexate analogues. 12. Synthesis and biological properties of some aza homologues.

Methotrexate analogues, in which an additional nitrogen atom is inserted between the phenyl ring and the carbonyl group of the side chain, were prepared by photochemical methods. The compounds were less inhibitory toward dihydrofolate reductase and thymidylate synthetase derived from Lactobacillus casei than was methotrexate. They were also less cytotoxic against human lymphoblastic leukemia cells (CCRF-CEM). In vivo against L-1210 leukemia in mice, the aza homologue of methotrexate showed significant antitumor activity (%ILS = 55%) compared to methotrexate (%ILS = 88%).     

Folate analogues altered in the C9-N10 bridge region. 18. Synthesis and antitumor evaluation of 11-oxahomoaminopterin and related compounds

The chemical synthesis of 11-oxahomoaminopterin (1) has been carried out using procedures which were also found to be applicable to the synthesis of 11-oxahomofolic acid (2). Reaction of 1-bromo-4-[p-(caarbomethoxy)phenoxy]-2-butanone (10) with sodium azide gave 1-azido-4-[p-(carbomethoxy)phenoxy]-2-butanone (11). Protection of the carbonyl group of 11 as the ethylene ketal and subsequent base hydrolysis of the product gave 1-azido-4-(p-carboxyphenoxy)-2-butanone ketal (13). The glutamate conjugate 14 was prepared from 13 by the isobutyl chloroformate method and was hydrogenated to diethyl N-[(alpha-amino-2-oxo-4-butanoyl)-p-anisoyl]-L-glutamate ketal (15). Reaction of 15 with 6-chloro-2,4-diamino-5-nitropyrimidine (16) and 2-amino-6-chloro-4-hydroxy-5-nitropyrimidine (17) and deprotection of the corresponding products gave the intermediates 18 and 19, which were elaborated to 1 and 2 using a series of steps involving deprotection, dithionite reduction, cyclization, oxidation, and hydrolysis. Although 11-oxahomoaminopterin showed antifolate activity against two folate-requiring microorganisms and inhibited Lactobacillus casei DHFR, it was inactive against L-1210 leukemia in mice at a maximum dose of 48 mg/kg. Compound Lactobacillus casei DHFR, it was inactive against L-1210 leukemia in mice at a maximum dose of 48 mg/kg. Compound 1 was also tested for its ability to be transported via the methotrexate transport system using the L-1210 and Ehrlich tumor cell lines, and these results are compared with those of related analogues. The growth inhibitory activity of 1 in the L-1210 cell lines in culture was found to be 15 times weaker than that of methotrexate.     

Methotrexate analogues. 25. Chemical and biological studies on the gamma-tert-butyl esters of methotrexate and aminopterin

gamma-tert-Butylaminopterin (gamma-tBAMT), the first example of an aminopterin (AMT) gamma-monoester, was synthesized, and new routes to the known N10-methyl analogue gamma-tert-butyl methotrexate (gamma-tBMTX) were developed. The inhibitory effects of gamma-tBAMT on the activity of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, the growth of L1210 cells and CEM human leukemic lymphoblasts in suspension culture, and the growth of several lines of human squamous cell carcinoma of the head and neck in monolayer culture were compared with the effects of gamma-tBMTX and the parent acids AMT and methotrexate (MTX). Patterns of cross-resistance to gamma-tBAMT, gamma-tBMTX, and AMT among several MTX-resistant cell lines were examined. In vivo antitumor activities of gamma-tBAMT and gamma-tBMTX were compared in mice with L1210 leukemia. While the activity of gamma-tBAMT was very close to that of gamma-tBMTX in the DHFR inhibition assay, the AMT ester was more potent than the MTX ester against cells in culture and against L1210 leukemia in vivo. Only partial cross-resistance was shown against gamma-tBMTX and gamma-tBAMT in cultured cells that were resistant to MTX by virtue of a transport defect or a combination of defective transport and elevated DHFR activity.     

Inhibition of murine thymidylate synthase and human dihydrofolate reductase by 5,8-dideaza analogues of folic acid and aminopterin

A series of 5,8-dideaza analogues of folic acid, isofolic acid, aminopterin, and isoaminopterin were evaluated for inhibition of thymidylate synthase, TS, from mouse L1210 leukemia cells with 10-propargyl-5,8-dideazafolic acid, CB3717, 4a, as the reference inhibitor. These compounds were also tested as inhibitors of human dihydrofolate reductase, DHFR, obtained from WIL2 cells. None of the analogues studied were as potent as 4a toward TS; however, 9-methyl-5,8-dideazaisoaminopterin, 6d, was only 2.5-fold less effective. Compound 4a was prepared by direct alkylation of the di-tert-butyl ester of 5,8-dideazafolic acid followed by hydrolysis of the resulting diethyl ester, which resulted from concomitant transesterification. It was found to be identical with a sample of 4a prepared by earlier methodology by using a variety of spectroscopic techniques. Its isomer, 9-propargyl-5,8-dideazaisofolic acid, 4b, which was synthesized by an analogous approach, was found to be dramatically less inhibitory toward TS than 4a. Each of the 2,4-diamino derivatives, including those possessing an allyl or propargyl group at N9, was an excellent inhibitor of DHFR, having a level of potency similar to that of methotrexate, MTX. However, many of these 5,8-dideazaaminopterin analogues were far more inhibitory toward TS than MTX.     

Synthesis and antifolate properties of 10-alkyl-5,10-dideaza analogues of methotrexate and tetrahydrofolic acid

Synthesis of the 10-methyl and 10-ethyl analogues of 5,10-dideazatetrahydrofolic acid (DDTHF), a potent inhibitor of glycinamide ribotide (GAR) formyltransferase, is reported. Key intermediates in the process were 10-methyl- and 10-ethyl-4-amino-4-deoxy-5,10-dideazapteroic acid. Condensation of the piperidine enamines of branched 4-(p-carbomethoxyphenyl)butyraldehydes with (acetoxymethylene)malononitrile afforded 1,1-dicyano-4-piperidinobutadiene 5a,b. Subsequent reaction with alcoholic ammonium hydroxide yielded the appropriately substituted 2-amino-3-cyanopyridines 6a,b. Ring closure with guanidine gave 10-methyl- and 10-ethyl-4-amino-4-deoxy-5,10-dideazapteroic acids (7a,b). Coupling with diethyl glutamate followed by ester hydrolysis afforded 10-alkyl-5,10-dideazaminopterin analogues 9a,b. Hydrolysis of the 4-amino group of 7a,b yielded the 10-alkylpteroic acids, which were coupled with diethyl glutamate, hydrogenated over PtO2, and saponified to afford 10-alkyl-5,10-dideazatetrahydrofolic acids 13a,b. Aminopterin analogues 9a,b were effective inhibitors of DHFR derived from L1210, but were less potent than methotrexate for inhibition of growth of L1210 in culture. The 10-ethyl (13b) analogue of 5,10-DDTHF was about twice as potent an inhibitor of L1210 cell growth as 5,10-DDTHF, but was only 1/7 as potent for inhibition of GAR formyltransferase. 10-Methyl analogue 13a was similar in potency to 5,10-DDTHF. All of the compounds showed moderately improved transport into L1210 cells relative to methotrexate.     

Regiospecific gamma-conjugation of methotrexate to poly(L-lysine). Chemical and biological studies

Regiospecific syntheses of gamma- and alpha-conjugates of methotrexate and poly(L-lysine) are described. The alpha- and gamma-t-butyl esters, respectively, of methotrexate were coupled to poly(L-lysine) with diphenylphosphoryl azide in N,N-dimethylformamide, the ester-protecting group was cleaved with 15% hydrogen bromide in acetic acid, and small molecules were removed by dialysis. Poly(L-lysine) of Mr = 1,500-8,000 and 8,000-30,000 was used to prepare six different conjugates, which were characterized by ultraviolet absorbance measurement and quantitative amino acid analysis. The degree of substitution varied from one methotrexate per 4.7 lysines to one methotrexate per 10.2 lysines. Dihydrofolate reductase inhibition in a cell-free assay was observed with alpha- and gamma-conjugates, but the latter had the greater affinity (only 3-fold less than that of methotrexate itself). The binding of the conjugates exhibited a slight pH dependence, with affinity being greater at pH 7.2 than at pH 8.5 for both alpha- and gamma-conjugates. Toxicity to cultured rat hepatoma cells (H35) was also greater for the gamma-conjugates, and showed some dependence on the chain-length and degree of substitution of the poly(L-lysine) carrier. Cells resistant to methotrexate by virtue of a transport defect (H35R0.3 line) retained their sensitivity to the gamma-conjugate, but less so to the alpha-conjugate. There was also some retention of sensitivity in a more highly resistant cell line (H35R10) with impaired methotrexate transport and a concomitant increase in dihydrofolate reductase activity. gamma-Conjugation was likewise more favorable in cytotoxicity assays against L1210 murine leukemia cells, and there was partial retention of activity against highly methotrexate-resistant lines (L1210/R71 and L1210/R81) with a transport defect and/or an elevation of dihydrofolate reductase content. In antitumor assays against intraperitoneal L1210 leukemia in mice, a gamma-conjugate with Mr = 8,000-30,000 and one methotrexate per 5.5 lysines produced a 35-75% increase in lifespan when administered intraperitoneally at single doses equivalent to 10-20 mg/kg of methotrexate. A similar increase in lifespan with methotrexate alone on the single-dose regimen required 50-150 mg/kg. An alpha-conjugate of similar Mr and degree of substitution was inactive at nontoxic doses, as were other gamma-conjugates of lower Mr and/or degree of substitution.(ABSTRACT TRUNCATED AT 400 WORDS)