The synthesis and
aromatase inhibitory profile of 6 alpha and 6 beta-propargyl
androstenedione and estrenedione are described. The targeted compounds 1 and 2 were prepared by addition of the propargyl Gringard to the 5 alpha,6 alpha-epoxy bisketal 6 or the 5 alpha,6 alpha-epoxy diacetate 7 followed by
dehydration of the 6 beta-propargyl 5 alpha-hydroxy diones 10 and 11 using
thionyl chloride. Treatment of the 6 beta-propargyl analogs 1 and 2 with
hydrochloric acid gave the corresponding 6 alpha-propargyl isomers 3 and 4. Inhibitory activity of the synthesized compounds was assessed using a human placental microsomal preparation as the
enzyme source and [1 beta-3H]-4-
androstenedione as substrate. Under initial velocity assay condition of low product formation, the inhibitors demonstrated potent inhibition of
aromatase, with apparent KiS ranging from 10 to 66 nM, with the Km for
androstenedione being 55 nM. 6 alpha-Propargylandrost-4-ene-3,17-dione and 6 alpha-propargylestr-4-ene-3,17-dione were found to be potent competitive inhibitors of
aromatase (Ki 37 and 66 nM, respectively). On the other hand the 6 beta-propargylandrost-4-ene-3,17-dione (6 beta-PAD) and 6 beta-propargylestr-4-ene-3,17-dione (6 beta-PED) were found to bind to
aromatase with an apparent Ki 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 beta-
mercaptoethanol does not, suggesting that the 6 beta-propargyl analogs 6 beta-PAD and 6 beta-PED are mechanism-based inactivators of
aromatase. Energy-minimization calculations and molecular modeling studies indicate three global minima for each of the 6 beta-propargyl analogs in which one of the conformers is proposed to be responsible for the inactivation of
aromatase.