Analogues of methotrexate and aminopterin with γ-methylene and γ-cyano substitution of the glutamate side chain: Synthesis and in vitro biological activity

Article abstract of DOI:10.1021/jm00105a031

Analogues of methotrexate (MTX) and aminopterin (AMT) modified at the γ-position of the glutamate side chain were synthesized and evaluated as dihydrofolate reductase (DHFR) inhibitors and tumor cell growth inhibitors. Condensations of 4-amino-4-deoxy-N¹⁰-methylpteroic acid (mAPA) with dimethyl DL-4-methyleneglutamate in the presence of diethyl phosphorocyanidate (DEPC) followed by alkaline hydrolysis yielded N-(4-amino-4-deoxy-N¹⁰-methylpteroyl)-DL-4- methyleneglutamic acid (γ-methyleneMTX). Condensation of 4-amino-4-deoxy-N¹⁰-formylpteroic acid (fAPA) with dimethyl-DL-4-methyleneglutamate by the mixed carboxylic-carbonic anhydride method yielded N-(4-amino-4-deoxypteroyl)-DL-4-methyleneglutamic acid (γ-methyleneAMT). Also prepared via DEPC coupling was a mixture of the four possible diastereomers of N-(4-amino-4-deoxy-N¹⁰-methylpteroyl)-4-cyanoglutamic acid (γ-cyanoMTX). The requisite intermediate γ-tert-butyl α-methyl 4-cyanoglutamate, as a DL-threo/DL-erythro mixture, was prepared from methyl N(α)-Boc-O-tosyl-L-serinate by reaction with sodium tert-butyl cyanoacetate followed by mild trifluoroacetic treatment to selectively remove the Boc group. The γ-methylene derivatives of MTX and AMT are attractive because of their potential to act as Michael acceptors within the DHFR active site. γ-CyanoMTX may be viewed as a congener of the nonpolyglutamated MTX analogue γ-fluoroMTX. In vitro bioassay data for the γ-methylene and γ-cyano compounds support the idea that the active site of DHFR, already known for its ability to tolerate modification of the γ-carboxyl group of MTX and AMT, can likewise accommodate substitution on the γ-carbon itself.