Synthesis of methotrexate-3-15N-2-15NH2. A convenient method for preparation of guanidine-15N3

Condensation of guanidine-15N3 with 4-[N(2-amino-3-cyano-5-pyrazinyl-methyl)-N-methylamino] benzoic acid in refluxing methanol afforded 4-amino-4-deoxy-N10-methylpteroic-3-15N-2-15NH2. The mass spectrum clearly indicated no label introduction at the pterin N1 position. Coupling of the latter compound with diethyl L-gluatmate and subsequent alkaline hydrolysis yielded methotrexate-3-15N-2-15NH2. Triply 15N-labeled guanidine was readily prepared from thiocyanic-15N acid (derived from KSC15N) and ammonia-15N by heating the resultant ammonium thiocyanate-15N2 at 1800C, followed by isolation as the water insoluble picrate salt. (author)