BIOLOGICAL CHARACTERIZATION OF A-RING STEROIDS

Hydroxylated 2,19-methylene-bridged androstenediones were designed as potential mimics of enzyme oxidized intermediates of androstenedione. These compounds exhibited competitive inhibition with low micromolar a ffinities for aromatase. These inhibitory constants (K(i) values) were 10 times greater than the 2,19-methylene-bridged androstenedione cons tant (K(i) = 35-70 Nm). However, expansion of the 2,19-carbon bridge t o ethylene increased aromatase affinity by 10-fold (K(i) = 2 nM). Subs titution of a methylene group with oxygen and sulfur in this expanded bridge resulted in K(i) values of 7 and 20 nM, respectively. When the substituent was an NH group, the apparent inhibitory kinetics changed from competitive to uncompetitive. All of these analogs exhibited time -dependent inhibition of aromatase activity following preincubation of the inhibitor with human placental microsomes prior to measuring resi dual enzyme activity. Part of this inhibition was NADPH cofactor-depen dent for the 2,19-methyleneoxy- but not for the 2,19-ethylene-bridged androstenedione. The time-dependent inhibition for these four analogs was very rapid since they exhibited tau50 values, the t1/2 for enzyme inhibition at infinite inhibitor concentration, of 1 to 3 min. These A -ring-bridged androstenedione analogs represent a novel series of pote nt steroidal aromatase inhibitors. The restrained A-ring bridge contai ning CH2, O, S, or NH could effectively coordinate with the heme of th e P450 aromatase to allow the tight-binding affinities reflected by th eir nanomolar K(i) values.