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

✍ Scribed by Charles S. Elmore; Dennis C. Dean; Yong Zhang; Carter Gibson; Herb Jenkins; Allen N. Jones; David G. Melillo

• Publisher: John Wiley and Sons
• Year: 2002
• Tongue: French
• Weight: 107 KB
• Volume: 45
• Category: Article
• ISSN: 0022-2135
• DOI: 10.1002/jlcr.537

✦ Synopsis

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 C 2 H 3 I 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 C 2 H 2 O and NaB 2 H 3 CN gave [C 2 H 3 ]MTX in 52% yield. A previously reported method was utilized to convert [C 2 H 3 ]MTX to [C 2 H 3 ]7-OH-MTX. Preparative HPLC purification of [C 2 H 3 ]7-OH-MTX resulted in extremely low recovery from the column; this was resolved by switching to a column with few free silanols.