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

J Med Chem. 1991 Jan;34(1):203-8. doi: 10.1021/jm00105a031.


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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Aminopterin / analogs & derivatives*
  • Aminopterin / pharmacology*
  • Animals
  • Cyanides
  • Drug Screening Assays, Antitumor
  • Folic Acid Antagonists* / chemical synthesis*
  • Folic Acid Antagonists* / chemistry
  • Folic Acid Antagonists* / pharmacology
  • Indicators and Reagents
  • Leukemia L1210
  • Magnetic Resonance Spectroscopy
  • Methotrexate / analogs & derivatives*
  • Methotrexate / pharmacology*
  • Mice
  • Molecular Structure
  • Optical Rotation
  • Spectrophotometry, Infrared
  • Structure-Activity Relationship


  • Cyanides
  • Folic Acid Antagonists
  • Indicators and Reagents
  • Aminopterin
  • Methotrexate