title: Analogues of methotrexate and aminopterin with gamma-methylene and gamma-cyano substitution of the glutamate side chain: synthesis and in vitro biological activity.
abstract: Analogues of methotrexate (MTX) and aminopterin (AMT) modified at the gamma-position of the glutamate side chain were synthesized and evaluated as dihydrofolate reductase (DHFR) inhibitors and tumor cell growth inhibitors. Condesations 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 (gamma-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 (gamma-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 (gamma-cyanoMTX). The requisite intermediate gamma-tert-butyl alpha-methyl 4-cyanoglutamate, as a DL-threo/DL-erythro mixture, was prepared from methyl N alpha-Boc-O-tosyl-L-serinate by reaction with sodium tert-butyl cyanoacetate followed by mild trifluoroacetic treatment to selectively remove the Boc group. The gamma-methylene derivatives of MTX and AMT are attractive because of their potential to act as Michael acceptors within the DHFR active site. gamma-CyanoMTX may be viewed as a congener of the nonpolyglutamated MTX analogue gamma-fluoroMTX. In vitro bioassay data for the gamma-methylene and gamma-cyano compounds support the idea that the active site of DHFR, already known for its ability to tolerate modification of the gamma-carboxyl group of MTX and AMT, can likewise accommodate substitution on the gamma-carbon itself.
authors.affiliation: Dana-Farber Cancer Institute, Boston, Massachusetts.
authors.forename: A
authors.initials: A
authors.lastname: Rosowsky
authors.forename: H
authors.initials: H
authors.lastname: Bader
authors.forename: J H
authors.initials: JH
authors.lastname: Freisheim
mesh_terms: D000630:Aminopterin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D003486:Cyanides; D004354:Drug Screening Assays, Antitumor; D005493:Folic Acid Antagonists / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D007202:Indicators and Reagents; D007939:Leukemia L1210; D009682:Magnetic Resonance Spectroscopy; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D051379:Mice; D015394:Molecular Structure; D009904:Optical Rotation; D013055:Spectrophotometry, Infrared; D013329:Structure-Activity Relationship
chemical_list: D003486:Cyanides; D005493:Folic Acid Antagonists; D007202:Indicators and Reagents; D000630:Aminopterin; D008727:Methotrexate