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. 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. 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.     

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.     

Analogues of methotrexate and aminopterin with gamma-methylene and gamma-cyano substitution of the glutamate side chain: synthesis and in vitro biological activity

Analogues of methotrexate (MTX) and aminopterin (AMT) modified at the gamma-position of the glutamate side chain were synthesized and evaluated as dihydrofolate reductase (DHFR) inhibitors and tumor cell growth inhibitors. Condesations of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) with dimethyl DL-4-methyleneglutamate in the presence of diethyl phosphorocyanidate (DEPC) followed by alkaline hydrolysis yielded N-(4-amino-4-deoxy-N10-methylpteroyl)-DL-4-methyleneglutamic acid (gamma-methyleneMTX). Condensation of 4-amino-4-deoxy-N10-formylpteroic acid (fAPA) with dimethyl-DL-4-methyleneglutamate by the mixed carboxylic-carbonic anhydride method yielded N-4-amino-4-deoxypteroyl)-DL-4-methyleneglutamic acid (gamma-methyleneAMT). Also prepared via DEPC coupling was a mixture of the four possible diastereomers of N-(4-amino-4-deoxy-N10-methylpteroyl)-4-cyanoglutamic acid (gamma-cyanoMTX). The requisite intermediate gamma-tert-butyl alpha-methyl 4-cyanoglutamate, as a DL-threo/DL-erythro mixture, was prepared from methyl N alpha-Boc-O-tosyl-L-serinate by reaction with sodium tert-butyl cyanoacetate followed by mild trifluoroacetic treatment to selectively remove the Boc group. The gamma-methylene derivatives of MTX and AMT are attractive because of their potential to act as Michael acceptors within the DHFR active site. gamma-CyanoMTX may be viewed as a congener of the nonpolyglutamated MTX analogue gamma-fluoroMTX. In vitro bioassay data for the gamma-methylene and gamma-cyano compounds support the idea that the active site of DHFR, already known for its ability to tolerate modification of the gamma-carboxyl group of MTX and AMT, can likewise accommodate substitution on the gamma-carbon itself.     

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. 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. 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)     

Studies on the mechanism of antitumor action of 2-desamino-2-methyl-5,8-dideazaisofolic acid

The new folate analogue, 2-desamino-2-methyl-5,8-dideazaisofolic acid, 2c, was synthesized and evaluated using a variety of biochemical and antitumor assays. For purposes of comparison, its 2-desamino, 2b, and 2-amino, 2a, counterparts, as well as N10-propargly-5,8-dideazafolic acid, 1a, and the corresponding 2-desamino, 1b, and 2-desamino-2-methyl, 1c, modifications were included in these studies. Compound 2c was found to be a potent inhibitor of the growth of L1210 and MCF-7 cells in culture, being only 2-fold and 5-fold less effective than 1c, respectively. However, although analogue 2c was 189-fold less inhibitory toward L1210 thymidylate synthase (TS) than 1c, its cytotoxicity was reversed completely by thymidine alone which suggests that the compound behaves as a TS inhibitor in cells. Enzymatically synthesized polyglutamates of 2c were substantially more inhibitory toward human TS than the parent compound. Compound 2c was the most efficient substrate for mammalian folyl-polyglutamate synthetase of the compounds studied having a Vmax/Km nearly 12-fold larger than 1c. Both 1c and 2c were effective inhibitors of the uptake of [3H]methotrexate into MOLT-4 cells, implying that each is efficiently transported into tumor cells. These results suggest that a weak inhibitor of TS in vitro can be a potent cytotoxic agent if it can readily gain entry into target cells and be converted to polyglutamated metabolites.     

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.     

Synthesis and antifolate properties of 9-alkyl-10-deazaminopterins

Reformatski condensation of benzyl 2-bromopropionate with 4-carbomethoxybenzaldehyde, followed by dehydration afforded benzyl 2-methyl-p-carbomethoxycinnamate (4a). Hydrogenation over a Pd catalyst gave the hydrocinnamic acid 5a. Conversion to the chloromethyl (6a) and azidomethyl ketone (7a) was followed by hydrogenation to the aminomethyl ketone (8a). Direct N-alkylation by 2,4-diamino-5-nitro-6-chloropyrimidine followed by reductive ring closure in Zn-HOAc and subsequent saponification of the benzoate ester yielded 4-amino-4-deoxy-9-methyl-10-deazapteroic acid (11a). Coupling with diethyl L-glutamate and saponification afforded 9-methyl-10-deazaminopterin (13a). The 9-ethyl analogue (13b) was similarly prepared from benzyl 2-bromobutyrate. The 9-methyl analogue (13a) was 21 times more potent than MTX as an inhibitor of cell growth in L1210 cells. The reason for this enhanced cytotoxicity in L1210 is unclear, since enzyme inhibition and transport parameters were similar to those of MTX. In human Manca leukemia cells growth inhibition was not dramatic and paralleled MTX.     

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.     

Quinazoline antifolates inhibiting thymidylate synthase: 2-desamino derivatives with enhanced solubility and potency

The poor solubility of the thymidylate synthase (TS) inhibiting antifolate 10-propargyl-5,8-dideazafolic acid has posed problems for its clinical use and is probably responsible for its renal toxicity. The insolubility is caused by the 2-amino-3,4-dihydro-4-oxopyrimidine moiety of the drug which stabilizes the solid state by intermolecular hydrogen bonding. In examining this moiety we have removed the 2-amino group and now report on 2-desamino-10-propargyl-5,8-dideazafolic acid (8e) and four analogues with H, Me, Et, and allyl at N10. 3,4-Dihydro-4-oxo-6-methylquinazoline was solubilized by alkylating the lactam nitrogen with chloromethyl pivalate. Reaction with N-bromosuccinimide gave the corresponding 6-bromomethyl compound, which was coupled with diethyl N-(4-aminobenzoyl)-L-glutamate or the appropriate N-substituted derivative thereof. The quinazoline N3 nitrogen and carboxyl groups in the product were simultaneously deprotected by cold alkali in the final step to give the desired five antifolates. These were tested against L1210 TS and it was found that removal of the 2-amino group caused a slight (3-9-fold) loss of TS inhibition. 8e was only 8-fold a lesser TS inhibitor than the parent drug. Inhibition of rat liver dihydrofolate reductase was reduced by over 1 order of magnitude for three compounds tested. All five analogues were more cytotoxic to L1210 cells in culture than their 2-amino counterparts; 8e was 8.5-fold more active with an ID50 of 0.4 microM. This remarkable result probably owes to increased cellular penetration. 8e was 5-fold more soluble than 1 at pH 5.0 and greater than 340-fold more soluble at pH 7.4.     

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.     

Folate analogues. 34. Synthesis and antitumor activity of non-polyglutamylatable inhibitors of dihydrofolate reductase

Five analogues of methotrextate (MTX), 10-deazaaminopterin (10-DAM), and 10-ethyl-10-deazaaminopterin (10-EDAM) in which the glutamate moiety was replaced by either a gamma-methyleneglutamate or beta-hydroxyglutamate were synthesized and evaluated for their antifolate activity. These analogous are 4-amino-4-deoxy-N10-methylpteroyl-beta-hydroxyglutamic acid (1), 4-amino-4-deoxy-10-deazapteroyl-beta-hydroxyglutamic acid (2), 4-amino-4-deoxy-N10-methylpteroyl-gamma-methyleneglutamic acid (3, MMTX), 4-amino-4-deoxy-10-deazapteroyl-gamma-methyleneglutamic acid (4, MDAM), and 4-amino-4-deoxy-10-ethyl-10-deazapteroyl-gamma-methyleneglutamic acid (5, MEDAM). None of these compounds were metabolized to the respective polyglutamate derivative as judged by their inability to serve as substrates for CCRF-CEM human leukemia cell folylpolyglutamate synthetase (FPGS) in vitro. All compounds inhibited recombinant human-dihydrofolate reductase (DHFR) at nearly equivalent magnitude as MTX. Growth-inhibition studies with H35 hepatoma, Manca human lymphoma, and CCRF-CEM human leukemia cells established greater cytotoxic effects with compounds 3-5 than with compounds 1 and 2. gamma-Methyleneglutamate derivatives 3-5 were transported to H35 hepatoma cells better than MTX or beta-hydroxyglutamate derivatives 1 and 2. Compound 3 was 2.5 times better than MTX in competing with folinic acid transport in H35 hepatoma cells. Compound 1 did not have a significant inhibitory effect on folinic acid transport even at 50 microM under identical conditions. The IC50 for compound 1 against H35-hepatoma cell growth was 8.5-fold higher than MTX. Compounds with the gamma-methyleneglutamate moiety (3-5) exhibited almost equal or lower IC50 values than MTX against the growth of CCRF-CEM human leukemia cells. These studies show that on continuous exposure, the non-polyglutamylatable inhibitors DHFR (3-5) can exhibit superior antifolate activity compared to the polyglutamylatable methotrexate, presumably due to their enhanced transport to these cell lines. Compounds 3-5 appear to be excellent models to study the role of polyglutamylation of antifolates in antitumor activity and host toxicity.     

Syntheses and thymidylate synthase inhibitory activity of the poly-gamma-glutamyl conjugates of N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino ]-2-thenoyl]-L-glutamic acid (ICI D1694) and other quinazoline antifolates.

Thirteen poly-gamma-glutamates derived from several novel antifolates have been synthesized by a convergent route. The syntheses of poly-gamma-glutamyl conjugates of N-[5-[N-(3,4-dihydro-2- methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-theno yl]-L-glutamic acid (8) (ICI D1694), 2-desamino-N10-propargyl-5,8-dideazafolic acid (6), 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (7), 2-desamino-2-methyl-N10-propargyl-2'-fluoro-5,8-dideazafolic acid (9), and 2-desamino-2-methyl-4-chloro-N10-propargyl-2'-fluoro-3,5,8-trideazafo lic acid (11) are described. A key step in the route involves coupling of an alpha-tert-butyl-protected poly-gamma-glutamate of the required chain length to the appropriate 5,8-dideazapteroic acid, obtained by carboxypeptidase G2 cleavage of the parent monoglutamate, if available, or by chemical synthesis. Deprotection with trifluoroacetic acid in the final step gave the desired poly-gamma-glutamyl antifolates as their trifluoroacetate salts. As inhibitors of thymidylate synthase, these polyglutamates were more potent in every case than the corresponding non-polyglutamylated drug.     

Synthesis and biological activity of methotrexate analogues with two acid groups and a hydrophobic aromatic ring in the side chain

The heretofore unknown gamma-(m-carboxyanilide) and gamma-(m-boronoanillide) derivatives of methotrexate (MTX) and the gamma-(m-carboxyanilide) derivatives of aminopterin (AMT) were prepared and tested as inhibitors of dihydrofolate reductase (DHFR) and as inhibitors of cell growth in culture with the aim of comparing their activity with that of N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine, a potent antifolate whose side chain likewise contains a hydrophobic aromatic ring with an acid group on the ring. All three anilides were potent DHFR inhibitors, with activity comparable to MTX and AMT. The gamma-(m-boronoanilide) displayed growth inhibitory potency similar to that of the hemiphthaloylornithine analogue, with an IC50 of only 0.7 nM. This compound, which is the most potent of the gamma-amides of MTX tested to date, is also the first reported example of an antifolate with a B(OH)2 group in the side chain and is especially novel because of its potential to form a stable tetrahedral boronate complex by reaction with electron rich OH or NH2 groups in the active site of DHFR or other folate enzymes. In antitumor assays against L1210 leukemia in mice, N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine gave a T/C of greater than 263% at 20 mg/kg (qdx9) and 300% at 16 mg/kg (bidx10), whereas maximally tolerated doses of MTX of 8 mg/kg (qdx9) and 1 mg/kg (bidx10) gave T/C values of 213 and 188%, respectively. MTX gamma-(m-boronoanilide) was also active, with a T/C of 175% at 32 mg/kg (qdx9), the highest dose tested.     

Synthesis and biological evaluation of 5-deazaisofolic acid, 5-deaza-5,6,7,8-tetrahydroisofolic acid, and their N9-substituted analogues

Prompted by recent disclosures concerning the potent antitumor activities of 5-deaza-5,6,7,8-tetrahydrofolic acid and 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF), we have prepared 5-deazaisofolic acid (3a) and 5-deaza-5,6,7,8-tetrahydroisofolic acid (4a). Reductive condensation of 2,6-diamino-3,4-dihydro-4- oxopyrido[2,3-d]pyrimidine with di-tert-butyl N-(4-formylbenzoyl)-L-glutamate and subsequent deprotection with trifluoroacetic acid yielded 5-deazaisofolic acid in good yield. Catalytic hydrogenation of this analogue then gave 4a. The 9-CH3 and 9-CHO modifications of 3a and the 9-CH3 derivative of 4a were also synthesized. Each of the new analogues was evaluated with a variety of folate-requiring enzymes as well as MCF-7 cells in culture. Compound 4a had an IC50 of ca. 1 microM against MCF-7 cells and was nearly 100-fold less potent than DDATHF in this regard. The three oxidized isofolate analogues were all poor inhibitors of tumor cell growth.     

Efficient utilization of the reduced folate carrier in CCRF-CEM human leukemic lymphoblasts by the potent antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L- ornithine (PT523) and its B-ring analogues

The potent nonpolyglutamatable dihydrofolate reductase inhibitor N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-o rnithine (PT523) and six of its B-ring (5-deaza, 8-deaza, and 5,8-dideaza) analogues were compared in terms of their ability to: (a) inhibit the growth of CCRF-CEM human leukemic lymphoblasts, and (b) utilize the reduced folate carrier (RFC) in these cells as measured in a competition assay of [(3)H]methotrexate ([(3)H]MTX) influx. The IC(50) values of the hemiphthaloylornithine derivatives against CCRF-CEM cells after 72 hr of drug exposure varied from 0.64 to 1.3 nM as compared with 14 nM for MTX and 4.4 nM for aminopterin (AMT). The K(i) values of these compounds in the [(3)H]MTX influx assay were in the 0.3 to 0.7 microM range as compared with a K(i) of 5.4 microM for AMT and a K(t) of 7.1 microM for MTX. As a group, the affinities of these compounds for the RFC were approximately 10-fold greater than those of their respective glutamate analogues. These results indicate that, in addition to their previously reported tight binding to dihydrofolate reductase, a property contributing to the high potency of PT523 and its B-ring analogs as inhibitors of tumor cell growth is their strong affinity for the RFC.     

Quinazoline antifolate thymidylate synthase inhibitors: alkyl, substituted alkyl, and aryl substituents in the C2 position

Modification of the potent thymidylate synthase (TS) inhibitor N-[4-[N-[(2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl]-N-prop-2- ynylamino]benzoyl]-L-glutamic acid (1a) has led to the synthesis of quinazoline antifolates bearing alkyl, substituted alkyl, and aryl substituents at C2. In general the synthetic route involved the coupling of the appropriate diethyl N-[4-(alkylamino)benzoyl]-L-glutamate with a C2-substituted 6-(bromo-methyl)-3,4-dihydro-4-oxoquinazoline followed by deprotection using mild alkali. Good enzyme inhibition and cytotoxicity were found with compounds containing small nonpolar groups in the C2 position with the 2-desamino-2-methyl analogue 3a being the most potent. Larger C2 substituents were tolerated by the enzyme, but cytotoxicity was reduced. Highly potent series were followed up by the synthesis of a number of analogues in which the N10 substituent was varied. In this manner a number of interesting TS inhibitors have been prepared. Although none of these was more potent than 1a against the isolated enzyme, over half of the compounds prepared were more potent as cytotoxic agents against L1210 cells in culture. The potential of such compounds as useful antitumor agents was further enhanced by the finding that the improved aqueous solubilities of compounds such as 3a over 1a were reflected in vivo in that 3a was at least 5 times less toxic to mice than 1a.