Probing the active site of aromatase with 2-methyl-substituted androstenedione analogs

Mitsuteru Numazawa, , Yoko Watari, Keiko Yamada, Nao Umemura and Wakako Handa

Tohoku Pharmaceutical University, 4-1 Komatsushima-4-chome, Aobaku, Sendai 981-8558, Japan

Received 4 March 2003; revised 28 April 2003; accepted 7 May 2003. ; Available online 18 July 2003.

To gain insight into the spatial nature of the androstenedione (AD) binding (active) site of aromatase in relation to the catalytic function of the enzyme, we synthesized 2,2-dimethylAD (4), 2- and 2-methylADs (5 and 6), 19-oxygenated derivatives of compounds 4 and 6, and 2-methyleneAD (17), and we then tested their inhibitory activity as well as their aromatase reaction (aromatization for 2-methyl and 2-methylene analogs or 19-oxygenation for 2,2-dimethyl steroids ) with human placental aromatase. 2-Methyl and 2-methylene steroids 5, 6, and 17 were good competitive inhibitors of aromatase (Ki=22–68 nM), but less effective compared to the 2,2-dimethyl analog 4 (Ki=8.8 nM), indicating that a combination of 2- and 2-methyl moieties is essential for the formation of a thermodynamically stable inhibitor–aromatase complex. A series of 2-methyl steroids were good substrates for aromatase, whereas 2-methyl steroid 5 was an extremely poor substrate, and a series of 2,2-dimethyl steroids did not serve as substrate, suggesting that a 2-methyl moiety of the 2,2-dimethyl and 2-methyl steroids would prevent the aromatase reaction probably due to steric hindrance in each case. The 2-methylene compound 17 was also aromatized to produce 2-methylestrogen with a low conversion rate where the 1,4-diene structure may have been created before the C10–C19 bond cleavage. Kinetic analysis of the aromatization of androgens revealed that a good substrate was not essentially a good inhibitor for aromatase.

Author Keywords: 2-Methylandrostenedione; 2,2-Dimethylandrostenedione; Aromatase inhibition; Aromatization; 19-Oxygenation; Human placental microsomes

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