Chemical Mechanism of the Covalent Modification of 5α-Reductases by Finasteride As Probed by Secondary Tritium Isotope Effects

摘要

Finasteride is a potent, specific inhibitor of human steroid 5α-reductases and is used for the treatment of benign prostatic hyperplasia, a ubiquitous condition in aging males. The potency of finasteride in inhibiting 5α-reductases is mainly derived from its time-dependent production of an enzyme-inhibitor complex that is apparently irreversible at neutral pH. The enzyme—inhibitor complex formed with 5α-reductase type 1 is stable under a variety of denaturing conditions, including 6 M Gdn-HCl, 1% SDS, boiling, etc., although the complex formed with the type 2 5α-reductase is less stable under these treatments. These data argue for covalent modification of 5α-reductases by finasteride, although the chemistry of this modification has not been established. Since the Δ~1 double bond of finasteride appears to be essential in the time-dependent inhibition of the type 1 isozyme, it has been proposed that the mechanism of modification may proceed by Michael addition of an enzyme-based nucleophile at C-1 of finasteride. To further investigate the role of the Δ~1 double bond in this time-dependent event, we synthesized [1,2-~3H]finasteride and developed methods for determining the secondary tritium isotope effects on the rate of slow inhibition. Since Michael addition causes sp~2 to sp~3 rehybridization at the unsaturated C-1 and C-2 carbon centers, an inverse isotope effect would be anticipated.