N(alpha)-(4-Amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-o rnithine (
PT523) is an unusually tight-binding
dihydrofolate reductase (DHFR) inhibitor and is efficiently taken up into cells via the
reduced folate carrier (RFC). Unlike classical DHFR inhibitors with a
glutamate side chain, such as
methotrexate and
aminopterin,
PT523 cannot form polyglutamates. Thus, it resembles lipophilic
antifolates such as
trimetrexate in not requiring metabolic activation by
folylpolyglutamate synthetase in order to produce its
antifolate effect. However, in contrast to
trimetrexate,
PT523 retains growth inhibitory activity in cells with the multidrug resistance phenotype. As part of the preclinical development of this
drug, we have performed systematic modification 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-activity correlations have emerged from this ongoing investigation, and are discussed: (1) the hemiphthaloylornithine side chain has the optimal length; (2) the preferred location of the aromatic carboxyl group is the ortho position; and (3) replacement of the phenyl ring of the
para-aminobenzoic acid moiety 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 all well tolerated. Several of the second generation analogs of
PT523 are more potent DHFR inhibitors and better RFC substrates than
PT523 itself, and are more potent inhibitors of
tumor cell growth in culture.