Design and synthesis of histidine analogues of folic acid and methotrexate as potential folylpolyglutamate synthetase inhibitors

Folylpolyglutamate synthetase (FPGS) is responsible for the conversion of naturally occurring folates and antifolates to their poly-γ-glutamyl derivatives, which are the forms required for intracellular retention of folates and are also the preferred substrates (cofactors) for most folatedependent enzymes. Folate and methotrexate analogues 6 and 4, with L- histidine in place of L-glutamate, were designed and synthesized as potential FPGS inhibitors. Target compound 5, the Nτ-(carboxymethyl)-L-histidine derivative of 4, was also prepared. Compounds 4 and 5 inhibited the growth of L1210 cells (IC₅₀ values: 0.091 and 0.15 μM, respectively) and were potent inhibitors of L1210 dihydrofolate reductase. No significant inhibition of FPGS by 4, 5, or 6 was observed at the high pH of the standard enzyme assay. This could be the consequence of a lack of protonation of the basic side chains, which is likely to be required for FPGS inhibitory activity. The observed cytotoxicity indicates that partial protonation of the imidazole ring permits cellular uptake of the analogues.