Synthesis and Structure−Activity Relationships of 6-Substituted Androst-4-ene Analogs as Aromatase Inhibitors

Series of 6 alpha- and 6 beta-alkyl-substituted androst-4-en-17-ones (18 and 19) and their 17 beta-reduced derivatives (14 and 15)(alkyl: methyl, ethyl, n-propyl, n-pentyl, n-octyl) were synthesized and evaluated as aromatase inhibitors. Androst-4-en-17-ones having an oxygen function (hydroxy, acetoxy, or methoxy group) at C-6 alpha and C-6 beta (4 and 5) were also tested for their abilities to inhibit aromatase. All of the steroids studied inhibited human placental aromatase in a competitive manner. The inhibitory activities of the 6 alpha- and 6 beta-methyl-17-keto steroids 18a and 19a (Ki = 3.1 and 5.3 nM, respectively) as well as the 6 beta- alcohol 5a (Ki = 6.0 nM) were high, and their apparent Ki values were lower than that of the parent 6-unsubstituted 3-deoxy steroid 1 (Ki = 6.8 nM). Elongation of the methyl group decreased affinity for aromatase in relation to carbon number of the alkyl chain in each series, in which the 6 alpha- alkyl steroids 18 essentially had higher affinity for the enzyme than the corresponding 6 beta- isomers 19. The inhibitory activities of the 17 beta-hydroxy analogs 14 and 15 were less potent than those of the corresponding 17-keto steroids. The 6 alpha-ethyl compound 18b, the 6 alpha-oxygenated derivatives 4, and the 6 beta-acetoxy and 6 beta-methoxy analogs 5b and 5c were powerful inhibitors (Ki = 12-24 nM). The methyl steroids (18a and 19a) produced "type I" difference spectra upon interaction with aromatase. These results along with molecular modeling with the PM3 method suggest that compounds 18a and 19a may produce a thermodynamically stable enzyme-inhibitor complex in the hydrophobic binding pocket with a limited accessible volume. A carbonyl group at C-17 of the 6-alkylandrost-4-enes is essential for the tight binding. Moreover, the binding pocket also tolerates a polar hydroxy group at the 6 beta-position rather than at the 6 alpha-position.