5-Alkyl derivatives of dl-tetrahydromethotrexate
have been prepared by reduction of methotrexate by sodium borohydride followed by reductive condensation with aldehyde and sodium borohydride and formation of the calcium salt (l-3).
Methods
Chemicals were obtained as follows: methotrexate, Lederle Laboratories; sodium borohydride, formaldehyde, acetaldehyde, Tris buffer, Hopkin and Williams Ltd; and DEAE-cellulose (D.E. 52), W. and R. Balston Ltd.
Ultraviolet spectra were determined in aqueous solution at pH 1.0, 7.0, and 13.0. Thin-layer chromatography was performed with 0.25 mm depth cellulose powder MN 300 F254 (Macherey, Nagel and Co., Duren, Germany) and developed at room temperature in the dark.
The following solvent systems were used: (a) 0.1 M phosphate buffer, pH 7.0, containing mercaptoethanol (0.5%, v/v).
(b) n-Propanol/water/O.% sp.gr. aqueous ammonia (200: 100: 10, v/v) containing mercaptoethanol (0.5%, v/v).
(c) The organic phase of I-butanol/acetic acid/water (4: 1:5, v/v) containing mercaptoethanol (0.5%, v/v). Compounds were detected as quenching or fluorescent spots when viewed under ultraviolet light emitting at either 254 or 365 mp.
Proton magnetic resonance (PMR) spect,ra were recorded with a Perkin-Elmer RlO spectrometer using tetramethylsilane (TMS) as internal reference in the case of trifluoroacetic acid (TFA) and as external reference in the case of DzO. DEAE-cellulose was provided preswollen and required no further washing. N-CH, and N-CH,CH, determinations were carried out by the Ziesel method.