Biochemical aromatization of 2-methyleneandrostenedione: Stereochemistry of hydrogen removal at the C-1 position

To explore a stereochemistry of hydrogen removal at C-1 of the powerful aromatase inhibitor 2-methyleneandrostenedione (1), of which the A-ring conformation is markedly different from that of the natural substrate androstenedione (AD), in the course of the aromatase-catalyzed A-ring aromatization producing 2-methylestrone (2), we synthesized [1α-2H]labeled steroid 1 and its [1β-2H]stereoisomer, and the metabolic fate of the C-1 deuterium in aromatization was analyzed by gas chromatography-mass spectrometry (GC-MS) in each. Parallel experiments with the natural substrates [1α-2H] and [1β-2H]ADs were also carried out. The GC-MS analysis indicated that 2-methyl estrogen 2 produced from [1α-2H]labeled substrate 1 retained completely the 1α-deuterium (1β-H elimination), while product 2 obtained from [1β-2H]isomer 1 lost completely the 1β-deuterium. Stereospecific 1β-hydrogen elimination was also observed in the parallel experiments with the labeled ADs as established previously. The results indicate that biochemical aromatization of the 2-methylene steroid 1 proceeds through the 1β-hydrogen removal concomitant with cleavage of the C10-C19 bond, yielding 1(10),4-dienone 9, in a similar manner to that involved in AD aromatization. This would give additional evidence for the stereomechanisms for the last step of aromatization of AD, requiring the stereospecific 1β-hydrogen abstraction and cleavage of the C10-C19 bond, and for the enolization of a carbonyl group at C-3 in the A-ring aromatization.