6β-Propynyl-Substituted Steroids: Mechanism-Based Enzyme-Activated Irreversible Inhibitors of Aromatase

The synthesis and aromatase inhibitory profile of 6α- and 6β-propargyl androstenedione and estrenedione are described. The targeted compounds 1 and 2 were prepared by addition of the propargyl Gringard to the 5α,6α-epoxy bisketal 6 or the 5α,6α-epoxy diacetate 7 followed by dehydration of the 6β-propargyl 5α-hydroxy diones 10 and 11 using thionyl chloride. Treatment of the 6β-propargyl analogs 1 and 2 with hydrochloric acid gave the corresponding 6α-propargyl isomers 3 and 4. Inhibitory activity of the synthesized compounds was assessed using a human placental microsomal preparation as the enzyme source and [1β- 3 H]-4-androstenedione as substrate. Under initial velocity assay conditions of low product formation, the inhibitors demonstrated potent inhibition of aromatase, with apparent K i s ranging from 10 to 66 nM, with the K m for androstenedione being 55 nM. 6α-Propargylandrost-4-ene-3,17-dione and 6α-propargylestr-4-ene-3,17-dione were found to be potent competitive inhibitors of aromatase (K i 37 and 66 nM, respectively). On the other hand the 6β-propargylandrost-4-ene-3,17-dione (6β-PAD) and 6β-propargylestr-4-ene-3,17-dione (6β-PED) were found to bind to aromatase with an apparent K i of 10 and 48 nM, respectively, as well as cause rapid time-dependent, first-order inactivation of aromatase in the presence of NADPH, whereas no inactivation was observed in the absence of NADPH. Substrate protects the enzyme from inactivation, but β-mercaptoethanol does not, suggesting that the 6β-propargyl analogs 6β-PAD and 6β-PED are mechanism-based inactivators of aromatase. Energy-minimization calculations and molecular modeling studies indicate three global minima for each of the 6β-propargyl analogs in which one of the conformers is proposed to be responsible for the inactivation of aromatase