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. 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. 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. 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)     

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-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. 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.     

Synthesis and biological activity of the 2-desamino and 2-desamino-2-methyl analogues of aminopterin and methotrexate

The previously undescribed 2-desamino and 2-desamino-2-methyl analogues of aminopterin (AMT) and methotrexate (MTX) were synthesized from 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile. The AMT analogues were obtained via a three-step sequence consisting of condensation with di-tert-butyl N-(4-aminobenzoyl)-L-glutamate, heating with formamidine or acetamidine acetate, and mild acidolysis with trifluoroacetic acid. The MTX analogues were prepared similarly, except that 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile was condensed with 4-(N-methylamino)benzoic acid and the resulting product was annulated with formamidine or acetamidine acetate to obtain the 2-desamino and 2-desamino-2-methyl analogues, respectively, of 4-amino-4-deoxy-N10-methylpteroic acid. Condensation with di-tert-butyl L-glutamate in the presence of diethyl phosphorocyanidate followed by ester cleavage with trifluoroacetic acid was then carried out. Retention of the L configuration in the glutamate moiety during this synthesis was demonstrated by rapid and essentially complete hydrolysis with carboxypeptidase G1 under conditions that likewise cleaved the L enantiomer of MTX but left the D enantiomer unaffected. The 2-desamino and 2-desamino-2-methyl analogues of AMT and MTX inhibited the growth of tumor cells, but were very poor inhibitors of dihydrofolate reductase (DHFR). These unexpected results suggested that activity in intact cells was due to metabolism of the 2-desamino compounds to polyglutamates.     

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.     

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.     

Inhibition of human liver folylpolyglutamate synthetase by non-gamma-glutamylatable antifolate analogs

Folylpolyglutamate synthetase (FPGS) catalyzes the gamma-glutamylation of both folates and folate antagonists and has been found to be essential for the survival of mammalian cells. Twelve analogs of the antifolates aminopterin (AMT) and methotrexate (MTX) having the -(CH2)2COOH moiety replaced by -(CH2)nX, where X = SO3H,PO3H2 or NH2, were evaluated as inhibitors of FPGS isolated from human liver. The AMT analogs were consistently found to be better inhibitors than their MTX counterparts, following the order of Km values determined for the parent antifolates as FPGS substrates. For the amino and phosphonate (but not for the sulfonate) compounds, inhibitory efficiencies were markedly dependent on the methylene chain length, with the most effective inhibitors having the groups -(CH2)3NH2(Ki = 0.2 microM) and -(CH2)2PO3H2 (Ki = 1.9 microM). Of those compounds exhibiting Ki values less than 200 microM, six were competitive inhibitors whereas three showed mixed inhibition (Ki' = approximately 6 Ki) when analyzed using AMT as the variable substrate. This demonstration of mixed inhibition of FPGS is consistent with the binding of inhibitor to a second site on the enzyme. Very similar Ki values (0.2-0.3 microM) were obtained for the -(CH2)3NH2 analog of AMT when using folic acid, AMT, MTX, and gamma-glutamyl-MTX as variable substrates, suggesting that the same enzymatic site on FPGS is active in the gamma-glutamylation of these four folyl derivatives. These findings serve to identify structural features which are important for inhibition of human liver FPGS and may therefore prove useful for the design of new compounds having potential as chemotherapeutic agents.     

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.     

Methotrexate analogues. 15. A methotrexate analogue designed for active-site-directed irreversible inactivation of dihydrofolate reductase

N alpha-(4-Amino-4-deoxy-N10-methylpteroyl)-N epsilon-(iodoacetyl)-L-lysine (1) was synthesized as a potential active-site-directed irreversible inhibitor of dihydrofolate reductase (DHFR). In an ultraviolet spectrophotometric assay of dihydrofolate reduction of Lactobacillus casei DHFR, 1 and methotrexate (MTX, 4-amino-4-deoxy-N10-methylpteroyl-L-glutamic acid) had ID50 values of 4.5 and 6.2 nM. The corresponding ID50 values in a competitive radioligand binding assay against [3H]MTX were 31 and 16 nM. Thus, as reversible inhibitors of this enzyme over a short exposure time, 1 and MTX had comparable activity. On the other hand, when L. casei DHFR was incubated for up to 6 h with 0.1 or 1.0 microM 1, a progressive decrease in the ability of [3H]MTX to subsequently displace the drug was observed. When MTX itself was used at the same concentrations, the extent of displacement of [3H]MTX did not decrease with time. These results were consistent with rapid reversible binding of 1 to the enzyme, followed more slowly by covalent bond formation near the active site. The pH profile for this effect followed a curve with a sigmoidal shape. The apparent inflection point near pH 7.2 was consistent with alkylation of a histidine residue.     

Methotrexate analogues. 20. Replacement of glutamate by longer-chain amino diacids: effects on dihydrofolate reductase inhibition, cytotoxicity, and in vivo antitumor activity

Chain-extended analogues of methotrexate were synthesized by condensation of 4-amino-4-deoxy-N10-methylpteroic acid with esters of L-alpha-aminoadipic, L-alpha-aminopimelic, and L-alpha-aminosuberic acids, followed by ester hydrolysis with acid or base. Coupling was accomplished in up to 85% yield by the use of the peptide bond forming reagent diethyl phosphorocyanidate at room temperature. The products were found to bind bacterial (Lactobacillus casei) and mammalian (L1210 mouse leukemia) dihydrofolate reductase with an affinity comparable to methotrexate and were also equitoxic to L1210 cells in culture. Cytotoxicity increased up to 3-fold as the number of CH2 groups in the amino acid side chain was extended from two to five. The alpha-aminoadipate and alpha-aminopimelate analogues were poor substrates for carboxypeptidase G1, confirming that this enzyme has a strict requirement for a C-terminal L-glutamic acid residue. The in vivo antitumor activity of the chain-extended analogues against L1210 leukemia in mice was comparable to that of the parent drug on the qd X 9 schedule, but higher doses were required to achieve the same increase in survival. The results were consistent with findings, reported separately, that these compounds are poor substrates for folate polyglutamate synthetase and therefore would not be expected to form gamma-polyglutamates once they enter a cell. This distinctive property has potential therapeutic implications for the treatment of certain MTX-resistant tumors whose resistance may be associated with a lower than normal capacity to form gamma-polyglutamates in comparison with proliferative tissues such as intestinal mucosa or marrow.     

Influence of lipophilicity and carboxyl group content on the rate of hydroxylation of methotrexate derivatives by aldehyde oxidase.

The influence of lipophilicity and carboxyl group content on the ability of methotrexate (MTX) derivatives to undergo 7-hydroxylation in vitro by partly purified rabbit hepatic aldehyde oxidase was examined. Addition of two to four gamma-glutamyl residues to the MTX molecule caused a progressive decrease in the rate of hydroxylation associated mainly with a decrease in Vmax rather than an increase in Km. These results suggest that the number of carboxyl groups in the side chain has a relatively small effect on affinity for the enzyme active site, but hinders the formation of product. The catalytic efficiency of hydroxylation of MTX tetraglutamate, estimated from Vmax/Km ratios, was 36-fold lower than that of the monoglutamate. In contrast, when the number of carboxyl groups was decreased to one, as in 4-amino-4-deoxy-N10-methylpteroic acid, N alpha-(4-amino-4-deoxy-N10-methylpteroyl)-L-lysine, and gamma-t-butyl-3'-chloromethotexate, enhanced catalytic efficiency was observed, involving both a decrease in Km and an increase in Vmax. The catalytic efficiency of hydroxylation of these three substrates was 88-, 360- and 2100-fold higher than that of MTX. gamma-t-Butyl-3'-chloromethotrexate was a better substrate than gamma-t-butyl-MTX, demonstrating the strong contribution of a lipophilic Cl atom on the phenyl ring. N alpha-(4-Amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine, with two carboxyl groups, showed substrate activity similar to that of MTX. The gamma-t-butyl esters of MTX, 3'-chloromethotrexate, and 3',5'-dichloromethotrexate were compared with the parent acids as inhibitors of the growth of cultured human leukemic lymphoblasts (CEM cells) and an MTX-resistant subline (CEM/MTX) defective in MTX transport and polyglutamylation. Although the esters were less effective than the acids against CEM cells except at high concentrations, they were more effective against CEM/MTX cells. This "collateral sensitivity" of CEM/MTX cells to lipophilic MTX esters is consistent with a decreased ability to take up and utilize reduced folates from the culture medium.     

Methotrexate analogues. 29. Effect of gamma-aminobutyric acid spacers between the pteroyl and glutamate moieties on enzyme binding and cell growth inhibition

A series of "stretched" methotrexate (MTX) analogues containing up to five 4-aminobutyryl (Gab) spacers between the 4-amino-4-deoxy-N10-methylpteroyl (MeAPA) moiety and the glutamate (Glu) side chain was prepared. Interest in these compounds stemmed from their relationship to MTX gamma-polyglutamates, from which they differ only in lacking "internal" alpha-carboxyl groups. The ability of the MeAPA-Gabn-Glu derivatives to inhibit dihydrofolate reductase (DHFR) and thymidylate synthase (TS) in vitro and to inhibit the growth of tumor cells in culture was evaluated. The IC50 for DHFR inhibition increased progressively from 0.082 to 0.84 microM as the number of Gab spacers was varied from one to five. At the same time the introduction of Gab spacers was found to produce substantial TS inhibition (Ki 0.1-0.4 microM) similar to that reported for MTX polyglutamates. Despite the activity of the MeAPA-Gabn-Glu derivatives as combined inhibitors of TS and DHFR, there was a steep loss of cell growth inhibitory potency as the number of Gab spacers was increased. This most likely reflects low cell uptake and the fact that when n greater than 1 there is almost total abolition of substrate activity for folylpolyglutamate synthetase, which had previously been observed with n = 1.     

Synthesis and in vitro biological activity of new deaza analogues of folic acid, aminopterin, and methotrexate with an L-ornithine side chain

The 5-deaza and 5,8-dideaza analogues of N alpha-pteroyl-L-ornithine (Pter-Orn), the 5-deaza, 8-deaza, and 5,8-dideaza analogues of N alpha-(4-amino-4-deoxypteroyl)-L-ornithine (APA-Orn), and the N delta-carboxymethyl derivative of N alpha-(4-amino-4-deoxy-N10-methylpteroyl)-L-ornithine (mAPA-Orn) were synthesized and tested as inhibitors of dihydrofolate reductase (DHFR) and as inhibitors of tumor cell growth in culture. Reductive amination of 2-acetamido-6-formylpyrido[2,3-d]pyrimidine-4(3H)-one with methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate followed by removal of the blocking groups afforded the 5-deaza analogue of Pter-Orn, whereas N-alkylation of methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate with 2-amino-6-(bromomethyl)quinazolin-4(3H)-one and deprotection gave the corresponding 5,8-dideaza analogue. Reductive coupling of 2,4-diaminopyrido[2,3-d]pyrimidine-6-carbonitrile and 4-aminobenzoic acid followed by reaction with 95-97% formic acid yielded 4-amino-4-deoxy-5-deaza-N10-formylpteroic acid, which on condensation with methyl N delta-(benzyloxycarbonyl)-L-ornithinate and deprotection gave the 5-deaza analogue of APA-Orn. A similar sequence starting from 2,4-diamino-quinazoline-6-carbonitrile led to the corresponding 5,8-dideaza compound, whereas treatment of 2,4-diamino-pyrido[3,2-d]pyrimidine-6-methanol with phosphorus tribromide followed by condensation with methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate and deprotection afforded the 8-deaza analogue. For the preparation of the N delta-carboxymethyl derivative of mAPA-Orn, N alpha-(benzyloxycarbonyl)-L-ornithine was subjected to N delta-monoalkylation with glyoxylic acid and sodium cyanoborohydride, followed by N delta-acylation with ethyl trifluoroacetate, N alpha-deprotection by hydrogenolysis, condensation with 4-amino-4-deoxy-N10-methylpteroic acid, and N delta-deprotection by gentle treatment with ammonia. The 2,4-diamino derivatives all inhibited the growth of tumor cells in culture, with IC50 values of 0.2-2 microM, and inhibited purified DHFR with IC50 values of 0.02-0.08 microM. Deletion of ring nitrogens and N delta-carboxymethylation both increased potency in the cell growth assay; however, the ornithine derivatives were less potent than aminopterin or methotrexate.     

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 .     

Synthesis of methotrexate-antibody conjugates by regiospecific coupling and assessment of drug and antitumor activities

In order to increase the retention of drug activity, regiospecific coupling has been used to synthesize conjugates of methotrexate (MTX, 1) with normal rabbit IgG (NRG) and a mouse anti-human renal cancer monoclonal IgG (Dal K-20). MTX gamma-methyl ester (4) was produced either by selective esterification of MTX or by coupling of 4-amino-4-deoxy-N10-methylpteroic acid (2) with suitable glutamic acid derivatives. The MTX gamma-methyl ester (4) was then converted to the corresponding hydrazide 6. An amide-linked conjugate was formed when the MTX gamma-hydrazide (6) was converted to reactive acylating species 7 by using tert-butyl nitrite or trifluoroacetaldehyde, which were reacted with nucleophilic centers, presumably epsilon-amino groups, in native IgG. A hydrazone-linked conjugate was formed when MTX gamma-hydrazide (6) was reacted directly with IgG that had first been oxidized with periodate to form polyaldehyde IgG. The regiospecifically synthesized conjugates were somewhat more effective inhibitors in vitro of dihydrofolate reductase and of colony formation by human renal cancer (Caki-1) cells than were control nonregiospecific conjugates.     

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.