Small-Molecule Inhibitors of 25-Hydroxyvitamin D‑24-Hydroxylase (CYP24A1): Synthesis and Biological Evaluation

The synthesis of imidazole styryl­benz­amide, tert-butyl styryl­imid­azole, and tert-butyl styryl­sulfonate derivatives is described. Evaluation of binding affinity and inhibitory activity against CYP24A1 identified the imid­azole styryl­benz­amides as potent inhibitors of CYP24A1, having selectivity with respect to CYP27B1 comparable with or greater than that of the standard keto­con­azole. Further evaluation of the 3,5-dimethoxy and 3,4,5-trimethoxy derivatives in chronic lymphocytic leukemia cells revealed that co-treatment of 1α,25-dihydroxy­vitamin D3 plus inhibitor coordinately upregulated GADD45α and CDKN1A. Docking experiments on the inhibitors in the CYP24A1 enzyme active site suggest the compounds reach the active site through the vitamin D access tunnel and are exposed to multiple hydro­phobic residues. The imid­azole styryl­benz­amides are optimally positioned to allow inter­action of the imid­azole with the heme, and, in the case of the methoxy derivatives, a hydrogen bond between the 3-methoxy group and Gln82 stabilizes the molecule in a favorable active conformation.