NOVEL PYRROLO[2,3-D]PYRIMIDINE ANTIFOLATE TNP-351 - CYTOTOXIC EFFECT ON METHOTREXATE-RESISTANT CCRF-CEM CELLS AND INHIBITION OF TRANSFORMYLASES OF DE-NOVO PURINE BIOSYNTHESIS

N-{4-[3-(2,4-Diamino-7H-pyrrolo[2,3-d]pyri- midin-5-yl)propyl]benzoyl} -L-glutamic acid (TNP-351), characterized by a pyrrolo[2,3-d]pyrimidin e ring, is a novel antifolate that exhibits potent antitumor activitie s against mammalian solid tumors. The mechanism of action of TNP-351 w as evaluated using some methotrexate-resistant CCRF-CEM human lymphobl astic leukemia cell lines as well as partially purified enzymes folylp olyglutamate synthetase (FPGS), aminoimidazolecarboxamide ribonucleoti de transformylase (AICARTFase), and glycinamide ribonucleotide transfo rmylase (GARTFase) from parent CCRF-CEM cells. TNP-351 was found to in hibit the growth of L1210 and CCRF-CEM cells in culture, with the dose s effective against 50% of the cells (ED(50) values) being 0.79 and 2. 7 nM, respectively. The growth inhibition caused by TNP-351 was revers ed by leucovorin or a combination of hypoxanthine and thymidine. The m ethotrexate-resistant CCRF-CEM cell line, which has an impaired methot rexate transport, showed less resistance to TNP-351 than to methotrexa te. TNP-351 was also found to be an excellent substrate for FPGS with a Michaelis constant (K-m) of 1.45 mu M and a maximum of velocity (V-m ax) of 1,925 pmol h(-1) mg(-1). Inhibitory activities of TNP-351-G(n) (n = 1-6) for AICARTFase were found to be significantly enhanced with increasing glutamyl chain length [inhibition constants (K-i): G(1), 52 mu M; G(6), 0.07 mu M]. Neither TNP-351 nor its polyglutamates were v ery strong inhibitors of GARTFase. These findings have significant imp lications regarding the mechanism of action of TNP-351.