5‐Deaza‐7‐desmethylene analogues of 5,10‐methylene‐5,6,7,8‐tetrahydrofolic acid and related compounds: Synthesis andin vitrobiological activity

Abstract 1‐[4‐(tert‐Butyloxycarbonyl)phenyl]‐3‐pyrrolidinone and 1‐[3‐(tert‐butyloxycarbonyl)phenyl]‐4‐piperidinone were condensed with ethyl cyanoacetate or malononitrile to form ylidene derivatives, which were then subjected sequentially to (i) catalytic or chemical reduction, (ii) condensation with guanidine, and (iii) gentle tri‐fluoroacetic acid treatment to obtain 3‐(2,4‐diamino‐6(5H)‐oxopyrimidin‐5‐yl)‐1‐(4‐carboxyphenyl)pyrrolidine (27), 4‐(2,4‐diamino‐6(5H)‐oxopyrimidin‐5‐yl)‐1‐(carboxyphenyl)piperidine (35), and 3‐(2,4,6‐triaminopyrimidin‐5‐yl)‐1‐(carboxyphenyl)pyrrolidine (40). Condensation of27, 35, and40with diethyl or di‐tert‐butyl L‐glutamate followed by removal of the ester groups yieldedN‐[4‐[3‐(2,4‐diamino‐6(5H)‐oxopyrimidin‐5‐yl)pyr‐rolidino]benzoyl]‐L‐glutamic acid (13),N‐[4‐[4‐(2,4‐diamino‐6‐(5H)‐oxopyrimidin‐5‐yl)piperidino]benzoyl]‐L‐glutamic acid (14), andN‐[4‐[3‐(2,4,6‐triaminopyrimidin‐5‐yl)pyrrolidino]benzoyl]‐L‐glutamic acid (15). Compounds13and14may be viewed as 5‐deaza‐7‐desmethylene analogues of 5,10‐methylene‐5,6,7,8‐tetrahydrofolic and 5,10‐ethylene‐5,6,7,8‐tetrahydrofolic acid, respectively. Compounds13and15were good substrates for mouse liver folylpolyglutamate synthetase, with Kmvalues of 20 and 18 μMand a relative first‐order rate constant Vmax/Kmof 2.2 (aminopterin = 1.0). In contrast,14was a very poor substrate, with a Kmof 490 μMand a relative Vmax/Kmof 0.052. As expected from its structure,15was a dihydrofolate reductase inhibitor. However its potency was unexceptional (IC50= 1.2 μM). Compounds13and14were inactive at concentrations of up to 100 μM, and likewise showed no activity against thymidylate synthase or glycinamide ribotide formyltransferase, two other key enzymes of folate‐mediated one‐carbon metabolism. Compound15was moderately active as an inhibitor of the growth of cultured tumor cells (SCC25 human squamous cell carcinoma), with an IC50of 0.37 μM(72 hour exposure). By comparison the IC50of aminopterin was 0.0069 μM. Thus, even though15is a good folylpolyglutamate synthetase substrate, the deep‐seated skeletal changes embodied in this structure are unfavorable for DHFR binding and may also be unfavorable for transport into cells.