4-Amino-4-deoxy-5,8,10-trideazapteroyl-d,l-4'-methyleneglutamic
acid (CH-1504) is the prototype of a potentially therapeutically more selective class of
antifolates for
rheumatoid arthritis treatment. This class is characterized by retention of
dihydrofolate reductase (DHFR; EC 1.5.1.3) as their locus of action and transport by the
reduced folate carrier (RFC; SLC19A1), but their lack of metabolism by known pathways of
antifolate (e.g.,
methotrexate (MTX)) metabolism. Five new
CH-1504 analogs (CHL-001-CHL-005) were synthesized and diastereomers of
CH-1504 itself were obtained by preparative chiral HPLC; all were characterized biochemically. The analogs are not metabolized by
aldehyde oxidase (EC 1.2.3.1),
carboxypeptidase G2 (EC 3.4.17.11), or (excepting CHL-003)
folylpolyglutamate synthetase (EC 6.3.2.17) and thus, unlike MTX, are "metabolism-blocked". All analogs are potent DHFR inhibitors; several are nearly as potent as MTX or
CH-1504. Each analog uses the RFC for transport, although with varying apparent affinities. In contrast, each weakly inhibits other
enzymes of
folate metabolism relevant to
rheumatoid arthritis therapy (
thymidylate synthase (EC 2.1.1.45), two
formyltransferases of
purine biosynthesis (EC 2.1.2.2 and EC 2.1.2.3), and 5,10-
methylenetetrahydrofolate reductase (EC 1.5.1.20)). Biochemical characterization showed one 4'-diastereomer of racemic
CH-1504 was significantly more active than the other. Based on literature data concerning the effect of d- and
l-glutamic acid substitution on
antifolate activity, it is likely that the diastereomer containing l-4'-methylene-glutamic
acid is the more active. Because of concern about potential pharmacokinetic and biochemical effects of d-4'-methylene-glutamic
acid-containing species, these data suggest that future analogs should contain only l-4'-methylene-glutamic
acid. Overall, these data provide several interesting new leads for preclinical development.