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R L Kisliuk

Publications and source records attributed to R L Kisliuk.

At least 19 recordsLinked to original sources
PubMed1996

2-amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidines as nonclassical antifolate inhibitors of thymidylate synthase.

Six novel 2-amino-4-oxo-5-[(substituted phenyl)sulfanyl]pyrrolo[2,3-d]pyrimidines 7-12 were synthesized as potential inhibitors of thymidylate synthase (TS) and as antitumor and/or antibacterial agents. The analogues contain a 5-thio substituent with a phenyl, 4'-chlorophenyl, 3',4'-dichlorophenyl, 4'-nitrophenyl, 3',4'-dimethoxyphenyl, and 2'-naphthyl on the sulfur, and were synthesized from the key intermediate 2-(pivaloylamino)-4-oxo-6-methylpyrrolo[2,3-d]-pyrimidine, 17. Appropriately substituted aryl thiols were appended to the 5-position of 17 via an oxidative addition reaction using iodine, ethanol, and water under conditions which also resulted in the deprotection of the 2-amino group. The compounds were evaluated against human, Lactobacillus casei, Escherichia coli, Streptococcus faecium, and Pneumocystis carinii (pc) TSs and against human, rat liver (rl), pc, and Toxoplasma gondii (tg) DHFRs. The nonclassical analogues with the 3',4'-dichloro and the 4'-nitro substituents in the side chain (9 and 10) were more potent than N-[4-[N-[(2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl]-N-prop- 2-ynylamino]benzoyl]-L-glutamic acid (PDDF, 1) and N-[5-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N- methylamino]-2-thenoyl]-L-glutamic acid (ZD1694,2) against human TS. Analogues with the 4'-chloro, 3',4'-dimethoxy, and naphthyl side chains (8, 11 and 12) were more potent than the unsubstituted phenyl analogue (7) but less than 2, 9, and 10 by 1 order of magnitude. They were all poor inhibitors of human, rl, and pc DHFRs (IC50 = 10(-5) M) but moderate inhibitors (IC50 = 10(-6) M) of tg DHFR. The 4-nitro analogue, 10 (EC50 1.5 microM), was comparable to PDDF in its potency as an inhibitor of the growth of the FaDu human squamous cell carcinoma cell line.

Animals↗
PubMed1996

2,4-diamino-5-deaza-6-substituted pyrido[2,3-d]pyrimidine antifolates as potent and selective nonclassical inhibitors of dihydrofolate reductases.

Fifteen novel nonclassical and two classical 2,4-diamino-6-(benzylamino)pyrido[2,3-d]pyrimidine antifolates were synthesized as potential inhibitors of Pneumocystis carinii, (pc) Toxoplasma gondii, (tg) rat liver (rl), and human (h) recombinant dihydrofolate reductases (DHFR). These analogues lack a 5-methyl substitution which has been shown to be important for increased hDHFR inhibitory activity. In addition, they contain a reversal of the C9-N10 bridge present in folates and most antifolates. The synthesis of the compounds involved the reaction of 2,4,6-triaminopyrimidine with the sodium salt of nitromalonaldehyde to afford the key intermediate 2,4-diamino-6-nitropyrido[2,3-d]pyrimidine (7), in a single step. Reduction of 7 to the 2,4,6-triaminopyrido[2,3-d]pyrimidine (8), followed by reductive amination with the appropriate benzaldehydes or phenylacetaldehydes afforded the target compounds. N9 methylation of these analogues was carried out using formaldehyde and sodium cyanoborohydride. The analogues demonstrated significant inhibition of pcDHFR and tgDHFR. N9 methylation significantly increased DHFR inhibitory potency. Compound 11, the 3'4'5'-trimethoxy-substituted analogue with a selectivity ratio of 9.4 for tgDHFR (compared to rlDHFR) was the most selective analogue of the nonclassical series. Compound 22, the N9 methyl 2'5'-dimethoxy-substituted analogue was the most potent analogue against tgDHFR (IC 50 = 6.3 nM) and was the second most selective analogue for tgDHFR (compared to rlDHFR) in the nonclassical series. The naphthyl-substituted analogues 23-25 were generally more potent against rlDHFR than against pcDHFR and tgDHFR. Selected analogues were also evaluated against Streptococcus faecium (sf) DHFR, Escherichia coli (ec) DHFR, Lactobacillus casei (lc) DHFR and tgDHFR with hDHFR as the mammalian reference, under slightly different assay conditions than those employed for rlDHFR. Analogues 11 and 22 had selectivity ratios of greater than 100 for tgDHFR (compared to hDHFR). Analogue 22 in particular, was the most selective analogue of the nonclassical series against tgDHFR (selectivity ratio = 303.5) with excellent potency (28 nM). Analogue 11, also displayed significant selectivity for sfDHFR (selectivity ratio = 4902). Compound 22 was evaluated in vivo for the inhibition of the growth of T.gondii trophozoites in mice, where at 50 mg/kg orally, it demonstrated distinct prolongation of survival without toxicity. Compounds 11, 12 and 21-23 were evaluated as antitumor agents in the National Cancer Institutes preclinical in vitro screening program. Compounds 12, 22, and 23 showed GI50s for tumor growth inhibition in the 10 -6 - 10 -7 M range.

Animals↗
PubMed1995

5-Arylthio-substituted 2-amino-4-oxo-6-methylpyrrolo[2,3-d]pyrimidine antifolates as thymidylate synthase inhibitors and antitumor agents.

Classical antifolate inhibitors of thymidylate synthase (TS) often require the reduced folate uptake system in order to exert their antitumor effects. In addition, these analogues are polyglutamylated via the enzyme folylpoly-gamma-glutamate synthetase (FPGS), which prevents analogue efflux from the cell and usually increases their inhibitory potency against TS. Impaired function of the reduced folate uptake system and that of FPGS are potential sources of resistance to such antifolates. We designed and synthesized a classical 6-5 ring-fused analogue N-[4-[(2-amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3- d]pyrimidin-5-yl)thio]-benzoyl]-L-glutamic acid (5) and a nonclassical 6-5 ring-fused analogue 2-amino-6-methyl-5-(pyridin-4-ylthio)-3,4-dihydro-4-oxo-7H-pyrrolo [2,3- d]pyrimidine (6) as TS inhibitors and antitumor agents. The syntheses of analogues 5 and 6 were achieved via the oxidative addition of the sodium salt of ethyl 4-mercaptobenzoate or 4-mercaptopyridine to 2-(pivaloylamino)-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyri midine (17) in the presence of iodine. For the synthesis of 5 the ester obtained from the reaction was deprotected and coupled with diethyl L-glutamate followed by saponification. Compound 5 was a potent inhibitor of human and bacterial TS with IC50 values of 42 and 21 nM, respectively. Compound 6 was 10-fold less potent than 5 against human TS but more than 4700-fold less potent than 5 against Lactobacillus casei TS. The classical analogue 5 was neither a substrate nor an inhibitor of human FPGS derived from CCRF-CEM cells. Compound 5 was cytotoxic to CCRF-CEM and FaDu tumor cell lines as well as to an FPGS-deficient subline of CCRF-CEM. Thymidine protection studies established that TS was the primary target of 5.

Antineoplastic Agents↗
PubMed1995

Effect of bridge region variation on antifolate and antitumor activity of classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidines.

Variation of the bridge linking the heterocyclic ring and p-aminobenzoyl-L-glutamate portions of our previously described classical 2,4-diaminofuro[2,3-d]pyrimidines 1 and 2 are reported as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS) and as antitumor agents. Specifically -CH2CH2- and -CH2NHCH2- bridged analogues, N-[4-[2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl) ethyl]benzoyl]-L-glutamic acid (3) and N-[4-[[N-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl) methyl]amino]methyl]benzoyl]-L-glutamic acid (4), respectively, were synthesized. Compound 3 was obtained via a Wittig reaction of the tributylphosphonium salt of 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine (5) and methyl 4-formylbenzoate (6) followed by reduction and coupling with the diethyl ester of L-glutamic acid. Compound 4 was synthesized by the nucleophilic displacement of 5 with diethyl N-[4-(aminomethyl)benzoyl]-L-glutamate (15) and saponification. Both analogues were evaluated in vitro as inhibitors of DHFRs from (recombinant) human, human CCRF-CEM cells, and Lactobacillus casei. Compound 3 showed moderate activity (IC50 10(-6)-10(-7) M). Compound 4 was essentially inactive (IC50 10(-5) M, CCRF-CEM). The compounds were also evaluated against TS from (recombinant) human and L. casei and were of low activity (IC50 10(-5) M). The three-atom-bridged analogue 4 was somewhat more inhibitory to human TS than methotrexate (MTX). Compound 3 inhibited the growth of tumor cells in culture (IC50 10(-7) M) while 4 showed a low level of growth inhibitory activity. The inhibition of the growth of leukemia CCRF-CEM cells by both compounds parallels their inhibition of CCRF-CEM DHFR. Analogue 3 was a good substrate for human folylpolyglutamate synthetase (FPGS) derived from CCRF-CEM cells (Km 8.5 microM). Further evaluation of the growth inhibitory activity of 3 against the MTX-resistant subline of CCRF-CEM cells (R30dm) with decreased FPGS indicated that poly-gamma-glutamylation was important for its action. Protection studies with 3 in the FaDu squamous cell carcinoma cell line indicated that inhibition was completely reversed by leucovorin [(6R,S-5-formyltetrahydrofolate] or by a combination of thymidine and hypoxanthine, suggesting an antifolate effect directed at DHFR.

Antineoplastic Agents↗
PubMed1995

Studies on the antitumor effects of analogues of 5,8-dideazaisofolic acid and 5,8-dideazaisoaminopterin.

Six new analogues of 5,8-dideazaisofolic acid and 5,8-dideazaisoaminopterin were synthesized in an effort to obtain enhanced antitumor activity. The modifications included the replacement of the 2-amino group by hydrogen or methyl as well as the inclusion of a methyl substituent at position 9. Based upon activity against L1210 leukemia cells in culture, three of the new analogues together with one compound described previously were evaluated for cytotoxicity in vitro using three human tumor cell lines (Colo 320 DM, Hep G2 and HL-60). The most effective compound was 2-desamino-N9-methyl-5,8-dideazaisoaminopterin (2c) with the HL-60 cells being the most sensitive to its cytotoxic effects. These analogues were evaluated in vitro as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS) from human as well as bacterial (Lactobacillus casei) sources. All four of the 4-amino analogues were most effective toward L. casei DHFR compared with human DHFR, with 2-desamino-2-methyl-5,8-dideazaisoaminopterin (2d) and its 9-methyl derivative (2e) having 818- and 430-fold greater selectivity (L. casei/human). Most of the compounds studied were found to be only modest inhibitors of human TS (I50 values = 1.5 to 20 microM) and were therefore at least 40-fold less inhibitory than 10-propargyl-5,8-dideazafolic acid. Nevertheless, reversal of cytotoxicity studies with thymidine, hypoxanthine and folinic acid using the HL-60 cell line suggested that TS is the primary target for these analogues.

Aminopterin↗
PubMed1995

6-substituted 2,4-diamino-5-methylpyrido[2,3-d]pyrimidines as inhibitors of dihydrofolate reductases from Pneumocystis carinii and Toxoplasma gondii and as antitumor agents.

The synthesis and biological activity of 15 6-substituted 2,4-diamino-5-methylpyrido[2,3-d]-pyrimidines are reported. These compounds were synthesized in improved yields by modifications of procedures previously reported by us. Specifically, dimethoxyphenyl-substituted compounds with H and CH3 at the N-10 position and trimethoxyphenyl-substituted compounds with N-10 ethyl, isopropyl, and propargyl moieties were synthesized. These compounds were evaluated as inhibitors of dihydrofolate reductases (DHFR) from Pneumocystis carinii, Toxoplasma gondii, and rat liver, and selected analogues were evaluated as inhibitors of the growth of T. gondii and tumor cells in culture. All the compounds showed increased selectivity (vs rat liver DHFR) for T. gondii DHFR compared to trimetrexate. In general, for the trimethoxy-substituted analogues, increasing the size of the N-10 substituent from a methyl group to larger groups resulted in a decrease in selectivity and potency for both P. carinii and T. gondii DHFR. For the dimethoxy-substituted analogues, N-10 methylation in general decreased potency but increased selectivity for T. gondii DHFR. In an attempt to improve the cell penetration of these analogues, the N-10 naphthyl-substituted analogues were also synthesized. These analogues displayed excellent cell penetration and inhibition of T. gondii cells in culture. Further, these analogues were potent inhibitors of the growth of tumor cells in the preclinical in-vitro screening program of the National Cancer Institute with IC50s in the nanomolar range.

Animals↗
PubMed1994

Classical and nonclassical furo[2,3-d]pyrimidines as novel antifolates: synthesis and biological activities.

Classical antifolate analogues containing a novel furo[2,3-d]pyrimidine ring system which include N-[4-[N-[(2,4-diaminofuro[2,3-d]pyrimidin-5- yl)methyl]amino]benzoyl]-L-glutamic acid (1) and its N-9 methyl analogue 2 were synthesized as potential dual inhibitors of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) and as antitumor agents. Four nonclassical antifolates, 2,4-diamino-5-(anilinomethyl)furo[2,3-d]pyrimidines 3-6 with 3,4,5-trimethoxy, 3,4,5-trichloro, 3,4-dichloro, and 2,5-dimethoxy substituents, respectively, in the phenyl ring, were also synthesized as potential inhibitors of DHFRs including those from Pneumocystis carinii and Toxoplasma gondii, which are organisms responsible for opportunistic infections in AIDS patients. The classical and nonclassical analogues were obtained via nucleophilic displacements of the key intermediate 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine with the appropriate (p-aminobenzoyl)-L-glutamate or substituted aniline. The key intermediate was in turn synthesized from 2,4-diamino-6-hydroxypyrimidine and 1,3-dichloroacetone. The final compounds were tested in vitro against rat liver, (recombinant) human, P. carinii, T. gondii, and Lactobacillus casei DHFRs. The classical analogues showed moderate to good DHFR inhibitory activity (IC50 10(-6)-10(-8) M) with the N-CH3 analogue 2 about twice as potent as 1. The nonclassical analogues were inactive with IC50S > 3 x 10(-5) M. The classical analogues were also evaluated as inhibitors of TS (L. casei, (recombinant) human and human CCRF-CEM), glycinamide ribonucleotide formyltransferase, and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and were found to be inactive against these enzymes. The classical analogues (particularly 2) were significantly cytotoxic toward a variety of tumor cell lines in culture. The nonclassical analogues were marginally active. Both classical compounds were good substrates for human folylpolyglutamate synthetase. Further evaluation of the cytotoxicity of 1 and 2 in CCRF-CEM cells and its sublines, having defined mechanisms of methotrexate (MTX) resistance, demonstrated that the analogues utilize the reduced folate/MTX-transport system and primarily inhibit DHFR and that poly-gamma-glutamylation was crucial to their mechanism of action. Protection studies in the FaDu squamous cell carcinoma cell line indicated that inhibition was completely reversed by leucovorin or the combination of thymidine plus hypoxanthine. Furthermore, for compounds 1 and 2, in contrast to MTX, the FaDu cells were better protected by thymidine alone than hypoxanthine alone, suggesting a predominantly antithymidylate effect.

AIDS-Related Opportunistic Infections↗
PubMed1993

Synthesis of 5-methyl-5-deaza nonclassical antifolates as inhibitors of dihydrofolate reductases and as potential antipneumocystis, antitoxoplasma, and antitumor agents.

A series of 2,4-diamino-5-methyl-6-(anilinomethyl)pyrido[2,3-d]pyrimidines 4-9 were synthesized as 5-deaza nonclassical antifolates containing trimethoxy, dichloro-, or trichlorophenyl substitutions and a N-H, N-CH3, or N-CHO at the 10-position. The compounds were evaluated as inhibitors of dihydrofolate reductases (DHFR) from Pneumocystis carinii (P. carinii), Toxoplasma gondii (T. gondii), rat liver (RL), and Lactobacillus casei (L. casei); as inhibitors of T. gondii and P. carinii cell growth in culture; and as antitumor agents. The compounds were prepared by modifications of procedures for classical 5-deaza folates. 2,4-Diamino-5-methyl-6-[(3',4',5'-trimethoxy-N- methylanilino)methyl]pyrido[2,3-d]pyrimidine (5a) exhibited high potency as well as selectivity (compared to RL DHFR) for P. carinii and T. gondii DHFR. Compound 5a is one of the most potent and selective nonclassical folate inhibitors of T. gondii DHFR known. The N-10 formyl analogue 2,4-diamino-5-methyl-6-[(N-formyl-3',4',5'-trimethoxyanilino) methyl]pyrido-[2,3-d]pyrimidine (6a) had decreased potency, but it maintained high selectivity for T. gondii DHFR. The corresponding chloro-substituted analogues maintained potency or had decreased potency; N-10 substitution did not increase potency or selectivity to the extent observed in the 3',4',5'-trimethoxy series. Partial reduction of the B ring to afford the dihydro analogue 2,4-diamino-5-methyl-6-[(N-formyl-3',4',5'-trimethoxyanilino) methyl]-5,8-dihydropyrido[2,3-d]pyrimidine (7), its 5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine analogue 8, and 2,4-diamino-5-methyl-6-[(3',4',5'-trimethoxyanilino)methyl]-5,6,7, 8- tetrahydropyrido[2,3-d]pyrimidine (9) resulted in a significant decrease in potency. In T. gondii cell culture inhibitory studies, 2,4-diamino-5-methyl-6-[(3',4',5'- trimethoxyanilino)methyl]pyrido[2,3-d]pyrimidine (4a), 5a, and 6a were less potent compared to their DHFR inhibitory potencies. Against P. carinii cells in culture, 4a and 5a at 10 micrograms/mL were as effective as the clinically used combination of trimethoprim/sulfamethoxazole (50/250 micrograms/mL). With the exception of the B ring reduced analogues 7-9, all of the compounds were significantly cytotoxic to leukemia CCRF-CEM cells in culture. The chloro-substituted analogues, in general, were more potent against a variety of other tumor cells in culture than the trimethoxy analogues. These results were corroborated by the preclinical tumor screening program at the National Cancer Institute where the most potent compound 2,4-diamino-5-methyl-6-[(3',4'-dichloroanilino)methyl]pyrido[2,3- d]pyrimidine (4b) was found to inhibit the growth of 26 tumor cell lines at an IG50 < 1.00 x 10(-8) M.

Animals↗
PubMed1992

5,10-Methylenetetrahydro-5-deazafolic acid and analogues: synthesis and biological activities.

The synthesis of 5,10-methylene-5-deazatetrahydrofolic acid (2), a stable, rigid analogue of 5,10-methylenetetrahydrofolate (1), is reported as a potential inhibitor of thymidylate synthase. The target compound was obtained by a Fisher-indole type cyclization of the hydrazone 16 from 2-amino-6-hydrazino-4-oxopyrimidine (10) and diethyl N-[4-(3-formyl-1-pyrrolyl)benzoyl]-L-glutamate (15) followed by catalytic reduction of the product 17. Similarly, modification of the Fisher-indole type cyclization of the appropriate hydrazone precursors 11 and 12 afforded the nonclassical analogues 3-amino-7,8,9-trimethyl-2H-pyrrolo[3',4':4,5]pyrido[2,3-d]pyrimidin-1- one (4) and 3-amino-8-benzyl-7,9-dimethyl-2H-pyrrolo[3',4':4,5]pyrido [2,3-d]pyrimidin-1-one (5), respectively. The target compound 2, its aromatic precursor 18, and the nonclassical analogue 4 were evaluated as inhibitors of the growth of Manca human lymphoma cells and also as inhibitors of human dihydrofolate reductase, human thymidylate synthase, glycinamide ribonucleotide formyltransferase, and aminoimidazole carboxamide ribonucleotide formyltransferase. Compound 18 showed weak inhibition of lymphoma cell growth (IC50 = 42 microM) and of AICAR formylTF (IC50 = 17 microM). Compounds 2 and 4 did not inhibit lymphoma cell growth or thymidylate synthase. The inactivity of 2 was attributed to its lack of flexibility leading to its inability to bind to thymidylate synthase.

Antineoplastic Agents↗
PubMed1992

Quantitation of folic acid enhancement of antifolate synergism.

Trimetrexate (TMTX), 5,10-dideazatetrahydrofolate (DDATHF), and 10-propargyl-5,8-dideazafolate (PDDF, CB3717) are antifolates whose primary intracellular targets are dihydrofolate reductase, glycinamide ribonucleotide formyltransferase, and thymidylate synthase, respectively. Varying the medium folic acid (PteGlu) concentration over the range of 0.5 to 100 microM increasingly blocks the growth inhibitory effects of the individual antifolates in Manca human lymphoma cells, but increasingly enhances the synergistic interaction of both TMTX + DDATHF and TMTX+ PDDF combinations. Drug interactions were quantitated using the universal response surface approach, which consists of fitting a concentration-effect surface to experimental data with weighted nonlinear regression, enabling the estimation of the synergism parameter, alpha. Estimates for alpha are larger (more intense synergism) for the TMTX + DDATHF combination (alpha = 4.68 +/- 0.66 at 2 microM PteGlu; alpha = 53.6 +/- 3.7 at 40 microM PteGlu) than for the TMTX + PDDF combination (alpha = 0.690 +/- 0.25 at 2 microM PteGlu; alpha = 7.20 +/- 1.8 at 40 microM PteGlu). However, the relative increase brought about by increasing the PteGlu concentration from 2 microM to 40 microM is similar in each instance, 11- and 10-fold, respectively. These experiments suggest that the enhanced cytotoxic interaction brought about by increased PteGlu concentration with the TMTX + DDATHF combination and the TMTX + PDDF combination may share a common mechanism. The dramatic intensity of the synergism between DDATHF and TMTX at 40 microM PteGlu, along with the concept of modulation of antifolate synergism by folates, suggests future in vivo and/or clinical applications of combinations of these (or similar) compounds.

Cell Division↗
PubMed1991

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.

Animals↗
PubMed1991

Synthesis and biological activity of 5,11-methylenetetrahydro-5- deazahomofolic acid.

The synthesis of 5,11-methylene-5-deazatetrahydrohomofolate (5), a stable, semirigid mimic of 5,10-methylenetetrahydrofolate (4) is reported as a potential inhibitor of thymidylate synthases (TS). The key intermediate 3-amino-1-oxo-tetrahydropyrimido[4,5-c] [2,6]naphthyridine (6) was obtained by the regiospecific cyclocondensation of 2,4,6-triaminopyrimidine with ethyl 1-benzyl-3-oxo-4-piperidinecarboxylate followed by halogenation (of the resulting lactam 9) and catalytic hydrogenolysis. Selective reduction of 6 followed by arylation with tert-butyl p-fluorobenzoate, saponification, and coupling with diethyl L-glutamate followed by saponification afforded the target compound 5. The title compound was tested as an inhibitor of the growth of Manca human lymphoma cells and also as an inhibitor of TS from Manca cells and Lactobacillus casei and was found to be inactive. In addition, compound 5 also failed to inhibit glycinamide ribonucleotide formyltransferase from L. casei and from Manca cells.

Cell Line↗
PubMed1991

Folate analogues. 35. Synthesis and biological evaluation of 1-deaza, 3-deaza, and bridge-elongated analogues of N10-propargyl-5,8-dideazafolic acid.

Structural modifications at the pyrimidine ring and at the C9,N10-bridge region of the thymidylate synthase (TS) inhibitors N10-propargyl-5,8-dideazafolate (1; PDDF; CB 3717), 2-desamino-N10-propargyl-5,8-dideazafolate (2, DPDDF), and 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolate (3, DMPDDF) have been carried out. Methods for the synthesis of 2-desamino-N10-propargyl-1,5,8-trideazafolate (4), 2-desamino-2-methyl-N10-propargyl-3,5,8-trideazafolate (5a), and 2-desamino-2-methyl-N10-propargyl-5,8-dideaza-1,2-dihydrofolate (6) have been developed. The bridge-extended analogues isohomo-PDDF (7) and isohomo-DMPDDF (8) contain an additional methylene group interposed between N10 and the phenyl ring of 1 and 3, respectively. All new compounds were evaluated as inhibitors of TS and the growth of tumor cells in culture. Selected analogues were tested as substrates of folylpolyglutamate synthetase (FPGS) and striking differences in substrate activity were observed among these compounds, indicating that structural modifications at the pyrimidine ring of classical antifolates profoundly influence their polyglutamylation. Enzyme inhibition data established that both N1 and N3-H of the pyrimidine ring are essential for efficient binding of quinazoline-type antifolates to human TS.

Biological Transport↗
PubMed1990

Tetrahydrohomofolate polyglutamates as inhibitors of thymidylate synthase and glycinamide ribonucleotide formyltransferase in Lactobacillus casei.

In order to determine the mechanism for the effects of homofolates on growth of Lactobacillus casei, polyglutamated derivatives of homofolate (HPteGlu), dihydrohomofolate and tetrahydrohomofolate (H4HPteGlu) were synthesized and tested as inhibitors of folate-requiring enzymes. The following L. casei enzymes were examined: thymidylate synthase (TS), glycinamide ribonucleotide formyltransferase (GARFT), aminoimidazolecarboxamide ribonucleotide formyltransferase, serine hydroxymethyltransferase and dihydrofolate reductase. Polyglutamates of (6R,S)-H4HPteGlu are potent inhibitors of TS and GARFT. For example, the IC50 values of (6R,S)-H4HPteGlu6 are 0.7 microM for TS and 0.3 microM for GARFT. By contrast, the value for HPteGlu6 is greater than 10 microM for both TS and GARFT. Inhibition of TS and GARFT by (6R,S)-H4HPteGlu derivatives increases with polyglutamate chain length. For TS, the Glu5 and Glu6 derivatives of (6R,S)-H4HPteGlu are 20 and 30 times more potent than the monoglutamate, respectively. For GARFT, the Glu2-6 derivatives are 2-3 times more potent than Glu1. Inhibition of TS and GARFT by (6R,S)-H4HPteGlu polyglutamates is almost entirely due to the unnatural (6R) diastereomer at C-6. Homofolate derivatives are only weak inhibitors of aminoimidazolecarboxamide ribonucleotide formyltransferase, serine hydroxymethyltransferase, and dihydrofolate reductase. We conclude that both TS and GARFT are potential targets of (6R)-H4HPteGlu polyglutamates.

Acyltransferases↗
PubMed1990

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.

Aminopterin↗
PubMed1990

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

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

Acyltransferases↗