ACTIVATION MECHANISM OF SUICIDE SUBSTRATE OF AROMATASE

1）雄甾-4-烯-3，6，17-三酮（AT）和4-羟基雄烯二酮（4-OHA）是典型的芳香化酶自杀底物，利用^3H和^3C标记的区域和立体特异性抑制剂研究了它们对芳香化酶的失活机制<14>。将标记的抑制剂分别与人胎盘微粒体在各种条件下孵育。AT通过19-羟基-和19-氧代-AT芳构化产生6-氧代雌激素，其芳香酶结合的代谢物保留了AT的1 β-质子、19-碳和三个19-甲基质子之一。这表明19-氧代AT在失活中起重要作用。另一方面，由4-OHA产生的结合代谢物保留了19-碳，但失去了1 β-质子，这表明新的活化过程将参与失活。2）本研究中合成的雄烯二酮（A）的6 β-烷基衍生物和3-脱氧A的6 α，7 α-环丙烷衍生物被证明是芳香酶的有效竞争性抑制剂。此外，雄甾-5-烯-7，17-二酮，天然底物A的位置异构体，以时间依赖性方式灭活芳香酶。这些结果证明了芳香化酶抑制剂新的构效关系。另一方面，3-脱氧A或AT的19-乙炔基和19，19-二氟衍生物分别是芳香化酶的自杀底物，表明3-脱氧A和AT在芳香化酶反应中均在C-19位被氧化。3）建立了一种以[1 β-^3H] 16 α-羟基A代替[1 β-^3H]A作为底物的新的芳香化酶测定方法。研究结果表明，这将对开发有效的芳香化酶抑制剂，用于治疗乳腺癌非常有用。

We studied on a suicide substrate of aromatase and the results obtained are as follows :1) Androst-4-ene-3,6,17-trione (AT) and 4-hydroxyandrostenedione (4-OHA) are typical aromatase suicide substrates and the mechanisms of inactivation of aromatase were studied by use of the regio- and stereo-specifically labeled inhibitors with ^3H and ^<14>C. The labeled inhibitors were separately incubated with human placental mictosomes under various conditions. AT was aromatized to yield 6-oxoestrogens via 19-hydroxy-and 19-oxo-AT's, and its aromatase-bound metabolite retained 1beta-proton, 19-carbon, and one of three 19-methy1 protons of AT. These show that 19-oxo AT has an important role in the inactivation. On the other hand. the bound metabolite produced from 4-OHA retained the 19-carbon but losed the 1beta-proton, suggesting that new activation process would be involved in the inactivation.2) 6beta-Alkyl derivatives of androstenedione (A) and 6alpha, 7alpha-cyclopropane derivatives of 3-deoxy A, synthesized in this study, were proven to be potent competitive inhibitors of aromatase. Moreover, androst-5-ene-7, 17-dione, a positional isomer of the natural substrate A, inactivated aromatase in a time-dependent manner. These results demonstrate new structure-activity relationship of aromatase inhibitor. On the other hand, 19-Erhynyl and 19,19-difluoro derivatives of 3-deoxy A or AT were suicide substrates of aromatase, respectively, showing that both 3-deoxy A and AT are oxygenated at C-19 by aromatase reaction.3) A new aromatase assay method using [1beta-^3H]16alpha-hydroxy A as a substrate, instead of [1beta-^3H]A, was established. It was shown that this would be very useful for the development of efficient aromatase inhibitor which can be used for treatment of breast cancer.