The pregnene derivatives with modifications at the 17,20-side chain and D-ring were synthesized and evaluated as inhibitors of human testicular 17 alpha-hydroxylase/C17,20-lyase. The results demonstrate that compounds which have 20-substituents with moderate to strong dipole properties, such as 20-oxime (3, 20), 20 beta-ol (24, 30), and 20 beta-carboxaldehyde (27), are potent inhibitors of this enzyme complex. The 20-substituents with hydrophobic property were devoid of inhibitory activity, e.g., the dimethylhydrazones 8 and 9. The 16-ene together with 20-oxime (20) showed the most potent inhibition of this enzyme complex, whereas 17(20)-ene modification as in 17(20)-ene-20-carbonitrile (14) did not increase activity in comparison to the 20 beta-carbonitrile (16). The bioisotere of 27 with 20-aza (19) also reduced the inhibitory activity. The results showed that isomeric configurations at the 20-position of some steroidal compounds are important factors which influence the potency of the inhibition significantly (e.g., 20 beta-ols 24 and 30 were 3-5-fold more potent than 20 alpha-ols 23 and 29). As expected, some compounds based on the pregn-5-en-3 beta-ol skeleton, which is similar to the natural substrate of human testicular 17 alpha-hydroxylase/C17,20-lyase in A- and B-rings, showed more potent inhibition than similar compounds which are based on the pregn-4-en-3-one skeleton (e.g., 23-25 compared to 29-31). These results suggest that A- and B-rings make significant contributions to the binding of these steroidal compounds to the 17 alpha-hydroxylase and C17,20-lyase. In comparison to ketoconazole, a nonsteroidal inhibitor of 17 alpha-hydroxylase and C17,20-lyase which has been used in the treatment of prostatic cancer, the steroidal compounds 20, 24, and 27 demonstrate more potent inhibition for this enzyme complex. These inhibitors warrant further investigation in biological systems. The structural features of these compounds may serve as leads in the design of new inhibitors.

The 22-hydroximino-23,24-bisnor-4-cholen-3-one (22-oxime) was synthesized and evaluated as an inhibitor of 17 alpha-hydroxylase/C17,20-lyase in rat testicular microsomes and the 5 alpha-reducatase of human prostatic microsomes from patients with benign prostatic hypertrophy. The 22-oxime demonstrated moderate inhibition for the 17 alpha-hydroxylase (Ki 74 nM vs. Km 29 nM) with progesterone as substrate and potent inhibition (Ki 18 nM vs. Km 76 nM) for the C17,20-lyase activity with 17 alpha-hydroxyprogesterone as substrate. Further investigation of this enzyme with progesterone as substrate demonstrated the inhibition occurred mainly at the 17 alpha-hydroxylation step of the progesterone substrate. The 22-oxime also demonstrated potent and competitive inhibition of 5 alpha-reductase in human prostatic microsomes (Ki 1.4 nM vs. Km 14 nM). When adult male rats were injected subcutaneously (sc) daily with 22-oxime (50 mg/kg/day) for 21 days, the concentrations of serum and testicular testosterone were significantly reduced by 65% and 59%, respectively, in comparison to vehicle-treated controls. Furthermore, both testosterone and DHT concentrations in rat prostatic tissue were significantly decreased by 60% and 44% compared to control tissue. Serum LH concentrations were unchanged in the 22-oxime-treated group compared to the control group. This indicates that the reduction in androgen concentrations in animals treated with this compound is not due to its influence on pituitary feedback mechanisms which result in reduced LH secretion. These results suggest that 22-oxime is effective in reducing androgen synthesis through the inhibition of 17 alpha-hydroxylase, C17,20-lyase, and 5 alpha-reductase both in vitro and in vivo.

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