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 [lβ-³H]-4-androgtenedione as substrate. Under initial velocity assay conditions of low product formation, the inhibitors demonstrated potent inhibition of aromarase, 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 aromarase 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 6βPAD 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.