Abiraterone acetate is a potent inhibitor of human cytochrome P450c17 (CYP17A1, 17 alpha-hydroxylase/17,20-Iyase) and is clinically used in combination with prednisone for the treatment of castration-resistant prostate cancer. Although many studies have documented the potency of abiraterone (Abi) in a variety of in vitro and in vivo systems for several species, the exact potency of Abi for human CYP17A1 enzyme has not yet been determined, and the structural requirements for high-potency steroidal azole inhibitors are not established. We synthesized 4 Abi analogs differing in the A-B ring substitution patterns: 3 alpha-hydroxy-Delta(4)-Abi (13), 3-keto-Delta(4)-Abi (11), 3-keto-5 alpha-Abi (6), and 3 alpha-hydroxy-5 alpha-Abi (5). We measured the spectral binding constants (K-s) using purified and modified human CYP17A1 along with the determination constants (K-1) applying a native human CYP17A1 enzyme in yeast microsomes for these compounds as well as for ketoconazole. For Abi, 3-keto-Delta(4)-Abi, 3-keto-5 alpha-Abi, and 3 alpha-hydroxy-5 alpha-Abi, the type 2 spectral changes gave the best fit for a quadratic equation, since in these experiments K-s values were 0.1-2.6 nM, much lower than that for ketoconazole and 3 alpha-hydroxy-Delta(4-)Abi (K-s values were 140 and 1660 nM, respectively). Inhibition experiments showed mixed inhibition patterns with K-i values of 7-80 nM. Abi dissociation from the CYP17A1-Abi complex was incomplete and slow; the t(1/2) for dissociation was 1.8 h, with 55% of complex remaining after 5 h. We conclude that Abi and the 3 related steroidal azoles (3-keto-Delta(4)-Abi, 3-keto-5 alpha-Abi, and 3 alpha-hydroxy-5 alpha-Abi), which also mimic natural substrates, are extraordinarily potent inhibitors of human CYP17A1, whereas the 3 alpha-hydroxy-Delta(4)-Abi is moderately potent and comparable to ketoconazole. (C) 2014 Elsevier Ltd. All rights reserved.