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.

Further studies on the interaction of nonpolyglutamatable aminopterin analogs with dihydrofolate reductase and the reduced folate carrier as determinants of in vitro antitumor activity.

Thirteen structural analogs of the potent nonpolyglutamatable dihydrofolate reductase inhibitor N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523) with modifications in the side chain, the para-aminobenzoyl moiety, or the 9,10-bridge were evaluated for the ability to inhibit human recombinant dihydrofolate reductase (DHFR), to utilize the reduced folate carrier (RFC) for influx, and to inhibit the growth of CCRF-CEM human leukemia cells in culture. In spectrophotometric assays of the kinetics of the reduction of dihydrofolate by DHFR in the presence of NADPH, these compounds had K(i) values ranging from 0.2 to 1.

Synthesis and in vitro antitumor activity of thiophene analogues of 5-chloro-5,8-dideazafolic acid and 2-methyl-2-desamino-5-chloro-5,8-dideazafolic acid.

N-[5-[N-(2-Amino-5-chloro-3,4-dihydro-4-oxoquinazolin-6-yl)methylamino]-2-thenoyl]-L-glutamic acid (6) and N-[5-[N-(5-chloro-3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl)methylamino]-2-thenoyl]-L-glutamic acid (7), the first reported thiophene analogues of 5-chloro-5,8-dideazafolic acid, were synthesized and tested as inhibitors of tumor cell growth in culture. 4-Chloro-5-methylisatin (10) was converted stepwise to methyl 2-amino-5-methyl-6-chlorobenzoate (22) and 2-amino-5-chloro-3,4-dihydro-6-methyl-4-oxoquinazoline (19). Pivaloylation of the 2-amino group, followed by NBS bromination, condensation with di-tert-butyl N-(5-amino-2-thenoyl)-L-glutamate (28), and stepwise cleavage of the protecting groups with ammonia and TFA yielded.

Synthesis and in vitro antitumor activity of new deaza analogues of the nonpolyglutamatable antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523).

Details are disclosed for the synthesis of N(alpha)-[4-[2-(2,4-diaminoquinazolin-6-yl)ethyl]benzoyl]-N(delta)-hemiphthaloyl-L-ornithine (2) and N(alpha)-[4-[5-(2,4-diaminoteridin-6-yl)pent-1-yn-4-yl]benzoyl]-N(delta)-hemiphthaloyl-L-ornithine (6) as analogues of N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (1, PT523), a nonpolyglutamatable antifolate currently in advanced preclinical development. In a 72 h growth inhibition assay against cultures of CCRF-CEM human leukemic lymphoblasts, the IC(50) of 2 and 6 was 0.69 +/- 0.

Synthesis and enzymatic activation of N-[N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithiny]-L-phenylalanine, a candidate for antibody-directed enzyme prodrug therapy (ADEPT).

N-[N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithinyl]-L-phenylalanine (1), a carboxypeptidase A (CPA) cleavable prodrug was synthesized for use in an antibody directed strategy to improve the therapeutic selectivity of N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (2), an extremely potent nonpoly-glutamatable DHFR inhibitor which is also highly cytotoxic. Compound 1 was shown by HPLC analysis to give a >99% yield of 2 upon incubation with bovine CPA (bCPA) for 20 min at 25 degrees C. In a spectrophotometric kinetic assay with 50 microM dihydrofolate as the competing substrate in the presence of 65 microM NADPH, 1+bCPA stoichiometrically inhibited recombinant human DHFR (rhDHFR) with a K(i) of 0.