Synthesis of [C2H3]methotrexate and [C2H3]7-hydroxymethotrexate

Stable label analogues of methotrexate (MTX) and 7-hydroxymethotrexate (7-OH-MTX) were required for use as internal standards for LC/MS quantitation. A minimum incorporation of stable isotopes to produce a mass increase of 3 in the non-glutamate derived portion of the molecule was necessary for adequate MS detection. The commercial availability of desmethyl MTX (aminopterin, 1) made methylation with C2H3I an attractive option. Surprisingly, all attempted methylations of 1 and the dimethyl ester of 1 failed to provide a significant amount of the methylated aniline, apparently due to attenuated reactivity of the secondary amine towards alkylation. However, reductive amination of diacid 1 with (CH2O)-H-2 and (NaBH3CN)-H-2 gave [(CH3)-H-2]MTX in 52% yield. A previously reported method was utilized to convert [(CH3)-H-2]MTX to [(CH3)-H-2]7-OH-MTX. Preparative HPLC purification of [(CH3)-H-2]7-OH-MTX resulted in extremely low recovery from the column; this was resolved by switching to a column with few free silanols. Copyright (C) 2002 John Wiley Sons, Ltd.