The effect of side-chain, para-aminobenzoyl region, and B-ring modifications on dihydrofolate reductase binding, influx via the reduced folate carrier, and cytotoxicity of the potent nonpolyglutamatable antifolate N-alpha-(4-amino-4-deoxypteroyl)-N-delta-hemiphthaloyl-L-ornithine

N-alpha-(4-Amino-4-deoxypteroyl)-N-delta-hemiphthaloyl-L-ornithine (PT523) is an unusually tight-binding dihydrofolate reductase (DHFR) inhibitor and is efficiently taken up into cells via the reduced folate carrier (RFC). Un like classical DHFR inhibitors with a glutamate side chain, such as methotr exate and aminopterin, PT523 cannot form polyglutamates, Thus, it resembles lipophilic antifolates such as trimetrexate in not requiring metabolic act ivation by folylpolyglutamate synthetase in order to produce its antifolate effect. However, in contrast to trimetrexate, PT523 retains growth inhibit ory activity in cells with the multidrug resistance phenotype. As part of t he preclinical development of this drug, we have performed systematic modif ication of several regions of the PT523 molecule, with the aim of defining the optimal structural features for DHFR binding, influx into cells via the RFC, and the ability to inhibit cell growth. The following structure-activ ity correlations have emerged from this ongoing investigation, and are disc ussed: (1) the hemiphthaloylornithine side chain has the optimal length; (2 ) the preferred location of the aromatic carboxyl group is the ortho positi on; and (3) replacement of the phenyl ring of the para-aminobenzoic acid mo iety by naphthalene, of nitrogen at the 10-position of the bridge by carbon , and of nitrogen at the 5- and/or 8-position of the B-ring by carbon are a ll well tolerated, Several of the second generation analogs of PT523 are mo re potent DHFR inhibitors and better RFC substrates than PT523 itself, and are more potent inhibitors of tumor cell growth in culture. (C) 2000 Elsevi er Science Inc, All rights reserved.