6-BETA-PROPYNYL-SUBSTITUTED STEROIDS - MECHANISM-BASED ENZYME-ACTIVATED IRREVERSIBLE INHIBITORS OF AROMATASE

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-hy droxy diones 10 and 11 using thionyl chloride. Treatment of the 6 beta -propargyl analogs 1 and 2 with hydrochloric acid gave the correspondi ng 6 alpha-propargyl isomers 3 and 4. Inhibitory activity of the synth esized compounds was assessed using a human placental microsomal prepa ration as the enzyme source and [1 beta-H-3]-4-androstenedione as subs trate. Under initial velocity assay conditions of low product formatio n, the inhibitors demonstrated potent inhibition of aromatase, with ap parent K(i)s ranging from 10 to 66 nM, with the K-m for androstenedion e being 55 nM. 6 alpha-Propargylandrost-4-ene-3,17-dione and 6 alpha-p ropargylestr-4-ene-3,17-dione were found to be potent competitive inhi bitors of aromatase (K-i 37 and 66 nM, respectively). On the other han d 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 aro matase with an apparent K-i of 10 and 48 nM, respectively, as well as cause rapid time-dependent, first-order inactivation of aromatase in t he presence of NADPH, whereas no inactivation was observed in the abse nce of NADPH. Substrate protects the enzyme from inactivation, but bet a-mercaptoethanol does not, suggesting that the 6 beta-propargyl analo gs 6 beta-PAD and 6 beta-PED are mechanism-based inactivators of aroma tase. 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 i nactivation of aromatase.