Roy L. Kisliuk

Stereospecificity at carbon 6 of formyltetrahydrofolate as a competitive inhibitor of transport and cytotoxicity of methotrexate in vitro

Abstract The unnatural diastereoisomer of l -5-formyltetrahydrofolate was 20-fold less effective as a competitive inhibitor of [ 3 H] methotrexate influx than the natural diastereoisomer during carrier-mediated membrane transport in L1210, S180 and Ehrlich cells. Values derived for K i , were 1.84 to 2.29 μM for the natural derivative and 35.2 to 53.8 μM for the unnatural derivative. Values for K i derived with a chemically synthesized mixture containing equal amounts of both natural and unnatural diastereoisomers were 2-fold greater than values obtained for the natural diastereoisomer. The unnatural diastereoisomer was 100-fold less effective and the chemically synthesized mixture was 2-fold less effective than the natural diastereoisomer in preventing inhibition by methotrexate of L1210 cell growth in culture. These results indicate that the unnatural diastereoisomer competes relatively ineffectively with the natural diastereoisomer or methotrexate for transport in these murine tumor cells.

Single Agents with Designed Combination Chemotherapy Potential: Synthesis and Evaluation of Substituted Pyrimido[4,5-b]indoles as Receptor Tyrosine Kinase and Thymidylate Synthase Inhibitors and as Antitumor Agents

Combinations of antiangiogenic agents (AAs) with cytotoxic agents have shown significant promise in cancer treatment, and several such clinical trials are currently underway. We have designed, synthesized, and evaluated two compounds that each inhibit vascular endothelial growth factor receptor-2 (VEGFR-2) and platelet-derived growth factor receptor-beta (PDGFR-beta) for antiangiogenic effects and also inhibit human thymidylate synthase (hTS) for cytotoxic effects in single agents. The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-chloro-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate benzenethiols. The inhibitory potency of both these single agents against VEGFR-2, PDGFR-beta, and hTS is better than or close to standards. In a COLO-205 xenograft mouse model, one of the analogs significantly decreased tumor growth (tumor growth inhibition (TGI) = 76% at 35 mg/kg), liver metastases, and tumor blood vessels compared with a standard drug and with control and thus demonstrated potent tumor growth inhibition, inhibition of metastasis, and antiangiogenic effects in vivo. These compounds afford combination chemotherapeutic potential in single agents.

Carrier-mediated transport of folate compounds in L1210 cells. Initial rate kinetics and extent of duality of entry routes for folic acid and diastereomers of 5-methyltetrahydrohomofolate in the presence of physiological anions.

Comparison of the kinetic parameters for influx of highly purified [3H]folic acid versus [3H]methotrexate in L1210 cells under anionic buffer conditions showed a marked discordancy. In addition, the kinetics for influx of [3H]folic acid were unchanged in variant L1210 cells defective in [3H]methotrexate transport. In these variant cells, the Vmax for methotrexate was reduced 17-fold and the Km was increased 3-fold. The results show that [3H]folic acid influx is mediated by a system which has a low affinity, but a 20-fold higher capacity, for folate compounds than the classical high-affinity system mediating [3H]methotrexate influx. Since the latter system also exhibits very low affinity for [3H]folic acid, it would not be expected to contribute significantly to the total influx of [3H]folic acid. The high-capacity system for [3H]folic acid influx is different from that believed to mediate pterin influx in L1210 cells since it was not inhibited by adenine, a potent inhibitor of pterin influx. However, exposure of cells to [3H]folic acid in a nonanionic buffer resulted in marked stimulation of initial influx, and a fraction of influx under these conditions was inhibited by methotrexate. These results suggest that anions modulate the extent of multiplicity of [3H]folic acid influx by their known effects on the high-affinity, reduced folate/methotrexate system. The diastereomers, at carbon 6, of [14C]5-methyltetrahydrohomofolate shared both transport systems. The influx Km for the natural diastereomer was one-half that of the unnatural form for both transport systems. Both diastereomers showed a much greater differential in affinity between the two transport systems than did [3H]folic acid. Our results suggest that an analog which could be effectively transported by the low-affinity/high-capacity route may be useful in the treatment of tumors resistant to methotrexate due to a defective high-affinity/low capacity influx system. We also found that incubation of L1210 cells with [3H]folic acid or the natural diastereomer [14C]5-methyltetrahydrohomofolate for 10 min resulted in the formation of a nonexchangeable fraction of radioactivity amounting to 20-40% of the total accumulation. This non-exchangeable fraction may be explained by the accumulation of metabolites other than polyglutamates. Preloading of cells with methotrexate prior to incubation with [3H]folic acid prevented the accumulation of radioactivity as a nonexchangeable fraction.

Potent Dual Thymidylate Synthase and Dihydrofolate Reductase Inhibitors: Classical and Nonclassical 2-Amino-4-oxo-5-arylthio-substituted-6-methylthieno[2,3-d]pyrimidine Antifolates

N-{4-[(2-Amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3- d]pyrimidin-5-yl)sulfanyl]benzoyl}-L-glutamic acid (4) and nine nonclassical analogues 5-13 were synthesized as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. The key intermediate in the synthesis was 2-amino-6-methylthieno[2,3-d]pyrimidin-4(3 H)-one (16), which was converted to the 5-bromo-substituted compound 17 followed by an Ullmann reaction to afford 5-13. The classical analogue 4 was synthesized by coupling the benzoic acid derivative 19 with diethyl L-glutamate and saponification. Compound 4 is the most potent dual inhibitor of human TS (IC 50 = 40 nM) and human DHFR (IC 50 = 20 nM) known to date. The nonclassical analogues 5- 13 were moderately potent against human TS with IC 50 values ranging from 0.11 to 4.6 microM. The 4-nitrophenyl analogue 7 was the most potent compound in the nonclassical series, demonstrating potent dual inhibitory activities against human TS and DHFR. This study indicated that the 5-substituted 2-amino-4-oxo-6-methylthieno[2,3-d]pyrimidine scaffold is highly conducive to dual human TS-DHFR inhibitory activity.

2,4-Diamino-5-methyl-6-substituted arylthio-furo[2,3-d]pyrimidines as novel classical and nonclassical antifolates as potential dual thymidylate synthase and dihydrofolate reductase inhibitors

A novel classical antifolate N-{4-[(2,4-diamino-5-methyl-furo[2,3-d]pyrimidin-6-yl)thio]-benzoyl}-l-glutamic acid 5 and 11 nonclassical antifolates 6-16 were designed, synthesized, and evaluated as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The nonclassical compounds 6-16 were synthesized from 20 via oxidative addition of substituted thiophenols using iodine. Peptide coupling of the intermediate acid 21 followed by saponification gave the classical analog 5. Compound 5 is the first example, to our knowledge, of a 2,4-diamino furo[2,3-d]pyrimidine classical antifolate that has inhibitory activity against both human DHFR and human TS. The classical analog 5 was a nanomolar inhibitor and remarkably selective inhibitor of Pneumocystis carinii DHFR and Mycobacterium avium DHFR at 263-fold and 2107-fold, respectively, compared to mammalian DHFR. The nonclassical analogs 6-16 were moderately potent against pathogen DHFR or TS. This study shows that the furo[2,3-d]pyrimidine scaffold is conducive to dual human DHFR-TS inhibitory activity and to high potency and selectivity for pathogen DHFR.

The effect of 5-alkyl modification on the biological activity of pyrrolo[2,3-d]pyrimidine containing classical and nonclassical antifolates as inhibitors of dihydrofolate reductase and as antitumor and/or antiopportunistic infection agents.

Novel classical antifolates (3 and 4) and 17 nonclassical antifolates (11-27) were synthesized as antitumor and/or antiopportunistic infection agents. Intermediates for the synthesis of 3, 4, and 11-27 were 2,4-diamino-5-alkylsubstituted-7H-pyrrolo[2,3-d]pyrimidines, 31 and 38, prepared by a ring transformation/ring annulation sequence of 2-amino-3-cyano-4-alkyl furans to which various aryl thiols were attached at the 6-position via an oxidative addition reaction using I2. The condensation of alpha-hydroxy ketones with malonodinitrile afforded the furans. For the classical analogues 3 and 4, the ester precursors were deprotected, coupled with diethyl-L-glutamate, and saponified. Compounds 3 (IC50 = 60 nM) and 4 (IC50 = 90 nM) were potent inhibitors of human DHFR. Compound 3 inhibited tumor cells in culture with GI50 <or= 10(-7) M. Nonclassical 17 (IC50 = 58 nM) was a potent inhibitor of Toxoplasma gondii (T. gondii) DHFR with >500-fold selectivity over human DHFR. Analogue 17 was 50-fold more potent than trimethoprim and about twice as selective against T. gondii DHFR.

Crystalline Thymidylate Synthetase From Dichloromethotrexate Resistant Lactobacillus Case I

Abstract A procedure is described for the isolation and crystallization of thymidylate synthetase from a dichloromethotrexate resistant strain of Lactobacillus casei. The recrystallized enzyme shows one band on polyacrylamide gel electrophoresis. The specific activity of the recrystallized enzyme is 216 μmoles of thymidylate formed per hour per mg. at30°C.

Effects of the diastereoisomers of methylenetetrahydrofolate on the reaction catalyzed by thymidylate synthetase

Abstract Diastereoisomers of methylenetetrahydrofolate were prepared and shown to have equal and opposite CD spectra. In addition to being inactive in the spectrophotometric assay for Lactobacillus casei thymidylate synthetase, the diastereoisomer having the unnatural configuration at carbon 6, does not promote removal of tritium from 5-(3H)-dUMP. It is a competitive inhibitor of the reaction with a K1 of 5 × 10−5 M .

Design, synthesis, and molecular modeling of novel pyrido[2,3-d]pyrimidine analogues as antifolates; application of Buchwald-Hartwig aminations of heterocycles.

Opportunistic infections caused by Pneumocystis jirovecii (P. jirovecii, pj), Toxoplasma gondii (T. gondii, tg), and Mycobacterium avium (M. avium, ma) are the principal causes of morbidity and mortality in patients with acquired immunodeficiency syndrome (AIDS). The absence of any animal models for human Pneumocystis jirovecii pneumonia and the lack of crystal structures of pjDHFR and tgDHFR make the design of inhibitors challenging. A novel series of pyrido[2,3-d]pyrimidines as selective and potent DHFR inhibitors against these opportunistic infections are presented. Buchwald-Hartwig coupling reaction of substituted anilines with pivaloyl protected 2,4-diamino-6-bromo-pyrido[2,3-d]pyrimidine was successfully explored to synthesize these analogues. Compound 26 was the most selective inhibitor with excellent potency against pjDHFR. Molecular modeling studies with a pjDHFR homology model explained the potency and selectivity of 26. Structural data are also reported for 26 with pcDHFR and 16 and 22 with variants of pcDHFR.

Interrelations Between Two Pathways of Methionine Biosynthesis in Aerobacter aerogenes

Two pathways for methionine methyl formation, one cobalamin-dependent and one cobalamin-independent, corresponding to those observed in Escherichia coli PA 15 have been found in Aerobacter aerogenes. An initial difficulty in showing the cobalamin-dependent pathway in cell-free extracts proved to be due to the presence of enzymes which caused the removal of adenosylmethionine, a cofactor required for this system. In contrast to E. coli PA 15, A. aerogenes contains holocobalaminmethyl-transferase even when grown in the absence of cobalamin. When cobalamin is added to the growth medium, the cobalamin-independent pathway is repressed.