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.     

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.     

Further studies on the pharmacologic effects of the 7-hydroxy catabolite of methotrexate in the L1210 murine leukemia cell

This paper describes studies that further explore the pharmacologic activity of the 7-hydroxy catabolite of methotrexate (7-OH-MTX). A 3-hr exposure of L1210 leukemia cells to 100 microM 7-OH-MTX produced negligible suppression of cell growth despite the build-up of intracellular polyglutamyl congeners to levels 2.7 times greater than the dihydrofolate reductase (DHFR) binding capacity. There was no evidence for direct inhibition of DHFR under these conditions based upon measurements of cellular tetrahydrofolate cofactor and dihydrofolate levels, nor was there suppression of [3H]deoxyuridine incorporation into DNA or [14C]formate incorporation into purines. When the interval of exposure to 100 microM 7-OH-MTX was increased to 6 hr, cell growth was inhibited by 60% and there was mild (approximately 50%) inhibition of purine and thymidylate biosynthesis associated with a small increase in cellular dihydrofolate and a small decline in cellular tetrahydrofolates. Consistent with weak inhibition of DHFR was the absence of significant binding of 7-OH-MTX polyglutamates to DHFR as assessed by gel filtration of cell extracts. Mild direct inhibition of purine biosynthetics by 7-OH-MTX- or MTX-polyglutamyl congeners was demonstrated based upon inhibition of [14C]formate incorporation into purines in cells pretreated with fluorodeoxyuridine so as to prevent tetrahydrofolate cofactor depletion or dihydrofolate polyglutamate build-up. Effects of a 6-hr exposure of cells to 100 microM 7-OH MTX on cell growth were reversed completely by 10 microM leucovorin; effects on cells containing comparable levels of MTX polyglutamyl congeners were unaffected by leucovorin. These studies demonstrate very weak inhibition of L1210 leukemia cell growth and purine, pyrimidine and tetrahydrofolate synthesis by the polyglutamyl congeners of 7-OH-MTX. The data suggest that effects of 7-OH-MTX polyglutamates on folate-requiring enzymes are not likely to play an important role in moderate-dose MTX regimens. However, pharmacologic activity may be expressed in high-dose MTX protocols when high blood levels of 7-OH-MTX are sustained over long intervals to the extent to which polyglutamate congeners accumulate in tumor cells and add to the much more potent inhibitory effects of MTX polyglutamates already present. Pharmacologic activity, however, would be diminished, if not completely reversed, by the concurrent administration of leucovorin.     

Methotrexate analogues. 33. N delta-acyl-N alpha-(4-amino-4-deoxypteroyl)-L-ornithine derivatives: synthesis and in vitro antitumor activity

N delta-Acyl derivatives of the potent folylpolyglutamate synthetase (FPGS) inhibitor N alpha-(4-amino-4-deoxypteroyl)-L-ornithine (APA-L-Orn) were synthesized from N alpha-(4-amino-4-deoxy-N10-formylpteroyl)-L-ornithine by reaction with an N-(acyloxy)succinimide or acyl anhydride, followed by deformylation with base. The N delta-hemiphthaloyl derivative was also prepared from 4-amino-4-deoxy-N10-formylpteroic acid by reaction with persilylated N delta-phthaloyl-L-ornithine, followed by simultaneous deformylation and ring opening of the N delta-phthaloyl moiety with base. The products were potent inhibitors of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, with IC50's ranging from 0.027 and 0.052 microM as compared with 0.072 microM for APA-L-Orn. Several of the N delta-acyl-N10-formyl intermediates also proved to be good DHFR inhibitors. One of them, N alpha-(4-amino-4-deoxy-N10-formylpteroyl)-N delta-(4-chlorobenzoyl)-L- ornithine, had a 2-fold lower IC50 than its deformylated product, confirming that the N10-formyl group is well tolerated for DHFR binding. While N delta-acylation of APA-L-Orn did not significantly alter anti-DHFR activity, inhibition of FPGS was dramatically diminished, supporting the view that the basic NH2 on the end of the APA-L-Orn side chain is essential for the activity of this compound against FPGS. N delta-Acylation of APA-L-Orn markedly enhanced toxicity to cultured tumor cells. However, N delta-acyl derivatives also containing an N10-formyl substituent were less cytotoxic than the corresponding N10-unsubstituted analogues even though their anti-DHFR activity was the same, suggesting that N10-formylation may be unfavorable for transport. Two compounds, the N delta-benzoyl and N delta-hemiphthaloyl derivatives of APA-L-Orn, with IC50's against L1210 cells of 0.89 and 0.75 nM, respectively, were more potent than either methotrexate (MTX) or aminopterin (AMT) in this system. These compounds were also more potent than MTX against CEM human lymphoblasts and two human head and neck squamous cell carcinoma cell lines (SCC15, SCC25) in culture. Moreover, in assays against SCC15/R1 and SCC25/R1 sublines with 10-20-fold MTX resistance, the N delta-hemiphthaloyl derivative of APA-L-Orn showed potency exceeding that of MTX itself against the parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Methotrexate analogues. 28. Synthesis and biological evaluation of new gamma-monoamides of aminopterin and methotrexate

Lipophilic gamma-monoamide derivatives of aminopterin (AMT) were synthesized in high overall yield from 4-amino-4-deoxy-N10-formylpteroic acid and gamma-N-tert-alkyl-, gamma-N-aralkyl-, or gamma-N-arylamides of alpha-benzyl L-glutamate via a modification of the mixed carboxylic-carbonic anhydride coupling method. Coupling was also accomplished with p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate. Compounds obtained in this manner included the gamma-tert-butylamide, gamma-(1-adamantylamide), gamma-benzylamide, gamma-(3,4-dichlorobenzylamide), gamma-(2,6-dichlorobenzylamide), gamma-anilide, gamma-(3,4-methylenedioxyanilide), and gamma-(3,4-dihydroxanilide) derivatives of AMT. Also prepared, from 4-amino-4-deoxy-N10-methylpteroic acid via diethyl phosphorocyanidate coupling, was the gamma-(3,4-methylenedioxyanilide) of MTX. The methylenedioxyanilides were cleaved smoothly to dihydroxyanilides with boron tris(trifluoroacetate) in trifluoroacetic acid. All the gamma-monoamides were tested as inhibitors of purified dihydrofolate reductase (DHFR) from murine L1210 leukemia cells and as inhibitors of the growth of wild-type L1210 cells and a subline (L1210/R81) with high-level resistance to MTX and AMT based mainly on a defect in drug uptake via active transport. Several compounds were also tested against human leukemic lymphoblasts (CEM cells) and a resistant subline (CEM/MTX) whose resistance is likewise based on uptake. The IC50 of the gamma-monoamides against DHFR was 1.5- to 5-fold higher than that of the parent acids, but the IC50 against cultured cells varied over a much broader range, suggesting that uptake and/or metabolism rather than DHFR binding are principal determinants of in vitro growth inhibitory activity for these compounds. gamma-N-Aryl and gamma-N-aralkyl derivatives appeared to be more potent than gamma-N-tert-alkyl derivatives. Where comparison could be made, AMT gamma-monoamides were more potent than MTX gamma-monoamides. Several of the gamma-monoamides showed potency comparable to that of the parent acid against wild-type L1210 and CEM cells; all of them were more potent than MTX against the L1210/R81 subline; and some of the AMT gamma-monoamides were also more potent than the parent acid against resistant CEM/MTX cells. As a group, however, the gamma-monoamides were considerably more active against the murine cells than against the human cells, suggesting that the former may take up the amides better or may be able to metabolize them more efficiently than the parent acids. All the gamma-monoamides were tested in vivo against L1210 leukemia in mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

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. 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.     

Studies with a 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP)-resistant L1210 leukemia cell line without cross-resistance to methotrexate

An L1210 leukemia cell line resistant to 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP) (L1210/DDMP) was developed in vivo by treatment of tumor-bearing mice. Resistance to DDMP was confirmed by subsequent in vivo survival experiments and by in vitro dose-response curves. The L1210/DDMP line demonstrated little cross-resistance to another folate analog, methotrexate (MTX). This was confirmed both in vivo, with survival experiments, and in vitro, using dose-response curves. A statistical analysis of the in vivo data confirmed DDMP resistance with lack of MTX cross-resistance. Dihydrofolate reductase (DHFR) activity in the L1210/DDMP/R₅ line was no greater than in the parent cell line (L1210/S). and the K_m of DHFR for dihydrofolate was the same in the L1210/DDMP/R₅ and L1210/S lines. The K_i for DHFR of the L1210/DDMP/R₅ cell line versus the L1210/S cell line was increased 3.0-fold for MTX and 3.5-fold for DDMP. Total accumulation of [¹⁴C]DDMP was identical in the two cell lines. The explanation for the lack of MTX cross-resistance in the L1210/DDMP/R₅ line is unknown.     

Quinazoline antifolate thymidylate synthase inhibitors: 2'-fluoro-N10-propargyl-5,8-dideazafolic acid and derivatives with modifications in the C2 position

The synthesis of 2'-fluoro-10-propargyl-5,8-dideazafolic acid and its 2-desamino, 2-desamino-2-hydroxymethyl, and 2-desamino-2-methoxy analogues is described. In general the synthetic route involved the coupling of diethyl N-[2-fluoro-4-(prop-2-ynylamino)benzoyl]-L-glutamate with the appropriate 6-(bromomethyl)quinazoline followed by deprotection with mild alkali. These four compounds together with the 2-desamino-2-methyl analogue were tested for their activity against L1210 thymidylate synthase (TS). They were also examined for their inhibition of the growth of the L1210 cell line and of two mutant L1210 cell lines, the L1210:R7A that overproduces dihydrofolate reductase (DHFR) and the L1210:1565 that has impaired uptake of reduced folates. Compared with their non-fluorinated parent compounds, the 2'-fluoro analogues were all approximately 2-fold more potent as TS inhibitors. Similarly, they also showed improved inhibition of L1210 cell growth (1.5-5-fold), and this activity was prevented by co-incubation with thymidine. All had retained or improved activity against both the L1210:R7A and L1210:1565 cell lines.     

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.     

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 .     

Folate analogues. 31. Synthesis of the reduced derivatives of 11-deazahomofolic acid, 10-methyl-11-deazahomofolic acid, and their evaluation as inhibitors of glycinamide ribonucleotide formyltransferase

The Boon-Leigh procedure, involving condensation of a 6-chloro-5-nitropyrimidine (22) with an alpha-amino ketone (20 or 21) followed by reduction of the nitro group, cyclization, and L-glutamylation, led to the formation of 11-deazahomofolate (29) and its 10-methyl derivative (30). The corresponding (6R,S)-5,6,7,8-tetrahydro (4, 5) and 7,8-dihydro (31, 32) derivatives were prepared by catalytic hydrogenation. (6S)-11-Deazatetrahydrohomofolate was prepared from 29 by enzymatic reduction. Compounds 29 and 30 had little effect (IC50 greater than 2 x 10(-5) M) on Lactobacillus casei glycinamide ribonucleotide (GAR) formyltransferase but (6R,S)-11-deazatetrahydrohomofolate (4) is a potent inhibitor of this enzyme (IC50 = 5 x 10(-8) M). It is at least 100 times more inhibitory than 33, the 6S compound, indicating that the 6R component of the mixture having the unnatural configuration at C6 (34) is responsible for the potent inhibition. Compound 4 is a much weaker inhibitor of murine (L1210) and human (MOLT-4) leukemia cell GAR formyltransferases (IC50 greater than 1 x 10(-5) M). (6R,S)-11-Deaza-10-methyltetrahydrohomofolate (5) (IC50 = 1.1 x 10(-5) is 200 times weaker than 4 against L. casei GAR formyltransferase. However, 11-deaza-10-methyldihydrohomofolate (32) is more inhibitory (IC50 = 5.5 x 10(-7) M) than 5 or 30. None of the compounds showed inhibition of L. casei aminoimidazolecarboxamide ribonucleotide (AICAR) formyltransferase, dihydrofolate reductase, or thymidylate synthase. The dihydro derivatives 31 and 32 are 5% as active as dihydrofolate as substrates for L. casei dihydrofolate reductase. Compound 4 showed moderate inhibition of the growth of L. casei, Streptococcus faecium, MOLT-4 cells, and MCF-7 human breast adenocarcinoma cells.     

Methotrexate analogues. 14. Synthesis of new gamma-substituted derivatives as dihydrofolate reductase inhibitors and potential anticancer agents

The gamma-tert-butyl ester (1), gamma-hydrazide (2), gamma-n-butylamide (3), and gamma-benzylamide (4) derivatives of methotrexate (MTX) were synthesized from 4-amino-4-deoxy-N10-methylpteroic acid (APA) and the appropriate blocked L-glutamic acid precursors with the aid of the peptide bond forming reagent diethyl phosphorocyanidate. The affinity of these side chain modified products for dihydrofolate reductase (DHFR) from Lactobacillus casei and L1210 mouse leukemic cells was determined spectrophotometrically or by competitive radioligand binding assay, and their cytotoxicity was evaluated against L1210 leukemic cells in culture. The results provide continuing support for the view that the "gamma-terminal region" of the MTX side chain is an attractive site for molecular modification of this anticancer agent.     

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.     

Analog specific aberrancies in antifolate inhibition of L1210 cell dihydrofolate reductase

During studies with L1210 cells and a variety of folate analogs, large discrepancies were revealed between data on membrane transport, on inhibition of dihydrofolate reductase in cell-free extracts, and on inhibition of growth in culture for 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin, and for 3-deaza, 10-methyl-aminopterin. While aminopterin, 10-methyl (methotrexate)-, 10-ethyl- and 10-propyl-aminopterin were tight binding inhibitors (K_i: 2–3 × 10^?12M) of dihydrofolate reductase in cell-free extracts from L1210 cells, the other four analogs were only weak competitive inhibitors (K_i = 3–300 × 10^?8M). Similar differences among analogs were observed for inhibition of dihydrofolate reductase in cell-free extracts from Sarcoma 180 and Ehrlich cells, but not for this enzyme in microbial cell-free extracts. There were only small differences in the transport of all of the analogs by L1210 cells. Inhibition of L1210 cell growth in culture by 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin and by 3-deaza, 10-methyl-aminopterin, in contrast to the other analogs, was several orders of magnitude greater than that predicted from the data on dihydrofolate reductase inhibition. The extent of binding of 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin, and of 3-deaza and 10-methyl-aminopterin to dihydrofolate reductase in intact L1210 cells, in contradistinction to that seen for the cell-free enzyme preparations, approached that observed for methotrexate; these estimates of drug-enzyme interaction in situ were more predictive of the extent of inhibition by these analogs of L1210 cell growth in culture.     

New quinazolinone-based derivatives as DHFR/EGFR-TK inhibitors: Synthesis,molecular modeling simulations,and anticancer activity

New 2-mercapto-quinazolin-4-one analogs were synthesized and tested for their in vitro anticancer activity, dihydrofolate reductase (DHFR) inhibition, and epidermal growth factor tyrosine kinase (EGFR-TK) inhibition activities. Compound 24 , which is characterized by a 2-benzyl-thio function, showed broad-spectrum anticancer activity with high safety profile and selectivity index. The concentrations of 24 causing 50% growth inhibition (GI₅₀) and total cell growth inhibition (TGI) and its lethal concentration 50 (LC₅₀) were 15.1, 52.5, and 91.2 µM, respectively, using 5-fluorouracil as a positive control. Also, it showed EGFR-TK inhibitory activity with IC₅₀ = 13.40 nM compared to gefitinib (IC₅₀ = 18.14 nM) and DHFR inhibitory potency with 0.30 μM compared to methotrexate (MTX; IC₅₀ = 0.08 μM). In addition, compound 24 caused cell cycle arrest and apoptosis on COLO-205 colon cancer cells. Compounds 37, 21 , and 54 showed remarkable DHFR inhibitory activity with IC₅₀ values of 0.03, 0.08, and 0.08 μM, respectively. The inhibitory properties of these compounds are due to an electron-withdrawing group on the quinazolinone ring, except for compound 54 . In a molecular modeling study, compound 24 showed the same binding mode as gefitinib as it interacted with the amino acid Lys745 via π–π interaction. Compound 37 showed a similar binding mode as MTX through the binding interaction with Lys68, Asn64 via hydrogen bond acceptor, and Phe31 via arene–arene interaction. The obtained model and substitution pattern could be used for further development.     

Methotrexate analogues. 21. Divergent influence of alkyl chain length on the dihydrofolate reductase affinity and cytotoxicity of methotrexate monoesters

n-Octyl, n-dodecyl, and n-hexadecyl alpha- and gamma-esters of methotrexate (MTX) were compared with the previously described alpha- and gamma-n-butyl esters and with MTX as inhibitors of dihydrofolate reductase (DHFR) and human leukemic lymphoblasts (CEM cells) in culture. The overall order of activity in both test systems was MTX greater than MTX gamma-esters greater than MTX alpha-esters. In the DHFR assay the activity of the alpha-esters followed the order C4 greater than C8 congruent to C12 greater than C16, whereas for the gamma-esters this order was C4 congruent to C8 greater than C12 greater than C16. On the other hand, the order of cytotoxic activity in culture in both series was C16 greater than C12 greater than C8 greater than C4. Increasing the alkyl chain length in the ester moiety therefore decreases DHFR affinity but increases cytotoxicity. The most potent member of the compounds tested was the gamma-n-hexadecyl ester, whose IC50 against CEM cells was 0.11 microM as compared with 0.025 microM for MTX. In a comparison of the effect of treatment with the gamma-n-hexadecyl ester (10(-5) M, 1 h) on DNA synthesis in CEM and CEM/MTX cells, the latter of which are 120-fold resistant to MTX by virtue of a transport defect, the ester produced only 4-fold less inhibition in the resistant line than in the parental line. These results suggest possible use of this compound or related derivatives in the treatment of MTX-resistant tumors with impaired transport.     

Synthesis and antifolate evaluation of 10-ethyl-5-methyl-5,10- dideazaaminopterin and an alternative synthesis of 10-ethyl-10- deazaaminopterin (edatrexate).

Previous findings suggesting that 5,10-dialkyl-substituted derivatives of 5,10-dideazaaminopterin warranted study as potential antifolates prompted synthesis of 10-ethyl-5-methyl-5,10- dideazaaminopterin (12a). The key step in the synthetic route to 12a was Wittig condensation of the tributylphosphorane derived from 6-(bromomethyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (7a) with methyl 4-propionylbenzoate. Reaction conditions for the Wittig condensation were developed using the tributylphosphorane prepared from 6-(bromomethyl)-2,4-pteridinediamine (7b) as a model. Each of the respective Wittig products 8a and 8b was obtained in 75-80% yield. Hydrogenation of 8a and 8b at their 9,10-double bond afforded 4-amino-4-deoxy-10-ethyl-5-methyl-5,10-dideazapteroic acid methyl ester (9a) and 4-amino-4-deoxy-10-ethyl-10-deazapteroic acid methyl ester (9b). This route to 9b intersects reported synthetic approaches leading to 10-ethyl-10-deazaaminopterin (10-EDAM, edatrexate), an agent now in advanced clinical trials. Thus the Wittig approach affords an alternative synthetic route to 10-EDAM. Remaining steps were ester hydrolysis of 9a,b to give carboxylic acids 10a,b followed by standard peptide coupling with diethyl L-glutamate to produce diethyl esters 11a,b, which on hydrolysis gave 12a and 10-EDAM (12b), respectively. The relative influx of 12a was enhanced about 3.2-fold over MTX, but as an inhibitor of dihydrofolate reductase (DHFR) from L1210 cells and in the inhibition of L1210 cell growth in vitro, this compound was approximately 20-fold less effective than MTX (DHFR inhibition, Ki = 4.82 +/- 0.60 pM for MTX, 100 pM for 12a; cell growth, IC50 = 3.4 +/- 1.0 nM for MTX, 65 +/- 18 nM for 12a).