Cofactor and representative inhibitors of folate-dependent enzymes.

Inhibitors of GAR Tfase that contain sulfur atoms.

Starting from methyl 4-formylbenzoate, the aldehyde was converted to dithiane 11 upon reaction with 1,3-propanedithiol (Scheme 1). The resulting dithiane was alkylated with 1-chloro-3-iodopropane to give 12, and subsequent removal of the dithiane using bis(trifluoro)acetoxyiodobenzene (PIFA) provided 13. Ketone 13 was reduced using the R-Me-CBS15 catalyst in high yield (97%) and high enantiomeric excess (99% ee) to yield alcohol S-14. The absolute stereochemistry of S-14 and its subsequent derivatives were assigned based on the well-established stereochemical preferences of the reagent,16 and confirmed in X-ray structures of both 10R-3 and 10S-3 bound to recombinant human GAR Tfase. The details of these latter studies will be disclosed elsewhere. Similarly, the S-Me-CBS catalyst was utilized to provide the enantiomer of 14 (not shown) and characterization data for this enantiomeric series is provided in the Supporting Information.

The preformed sodium salt of ethyl cyanoacetate was alkylated with S-14 to give the common intermediate S-15 for all three optically active inhibitors. Treatment of S-15 with thiolacetic acid under modified Mitsunobu conditions provided R-16 with inversion of C10 stereochemistry (Scheme 2). Compound R-16 was subsequently deprotected and the free thiol was methylated to yield R-18. Intermediate R-18 was treated with the free base of guanidine under basic conditions to form the pyrimidine R-19 and this intermediate was subsequently saponified to give R-20. Compound R-20 was coupled with di-tert-butyl l-glutamate and subsequently deprotected using trifluoroacetic acid to provide final inhibitor 10R-3. The C10 diastereomer 10S-3 was prepared using an analogous route employing the enantiomeric R-Me-CBS catalyst to provide R-14 (See Supporting Information).

The inhibitor 10S-7 bearing a C10-hydroxyl substituent, was prepared from the common intermediate S-15 (Scheme 3). Compound S-15 was treated with guanidine hydrochloride to give the corresponding pyrimidone S-22 which subsequently was saponified using lithium hydroxide to afford S-23. Intermediate S-23 was then coupled with dimethyl l-glutamate and deprotected under basic conditions to provide the final inhibitor 10S-7.

A key compound for comparison is the C10-methoxy derivative 8 where an oxygen replaces the sulfur of 3 as a probe for its unique importance. The synthesis of inhibitor 10S-8, bearing the C10-methoxy substituent, was accomplished by methylation of the common intermediate S-15 using TMSCHN₂ under acidic conditions17 to give S-25 (Scheme 4). Intermediate S-25 was carried forward employing an analogous route, starting with formation of pyrimidone S-26 and saponification to give S-27. Compound S-27 was coupled with di-tert-butyl l-glutamate hydrochloride and deprotected using trifluoroacetic acid to provide 10S-8.

Finally, 9, which incorporates two C10 sulfur atoms and, in a sense, simultaneously incorporates both C10-diastereomers of 3, was judged as an attractive additional derivative to examine that avoids the presence of two C10-diastereomers. Synthesis of inhibitor 9 started with alkylation of 12 using ethyl cyanoacetate to give 29 (Scheme 5). Intermediate 29 was converted to 9 using the same route detailed for the previous inhibitors, starting with formation of pyrimidine 30 and saponification to give 31. Compound 31 was coupled with di-tert-butyl l-glutamate and deprotected using trifluoroacetic acid to provide 9.