設計與合成香豆素骨架的化合物作為類固醇硫酸酯酶抑制劑

Abstract

類固醇硫酸酯酶 (steroid sulfatase, STS)以水解類固醇3號位的硫酸根基團，而調節人體內的雌激素 (estrogen)與雄激素 (androgens)的濃度。在雌激素受體陽性型 (ER+)的乳癌組織中，若類固醇硫酸酯酶過度表達，則會造成體內雌激素濃度上升，導致癌細胞增生及抑制細胞凋亡的結果，因此發展類固醇硫酸酯酶抑制劑有其必要性。 Irosustat (STX64, 50c)是第一個進入臨床二期的類固醇硫酸酯酶抑制劑，我們基於STX64的香豆素-7-胺基磺酸骨架，並根據類固醇硫酸酯酶抑制劑的結構通則，設計且合成一系列的芐基衍生物。在本實驗室的先前研究中，香豆素3號位芐基衍生物如3-(2,3-dimethoxybenzamido)-2-oxo-2H-chromen-7-yl sulfamate (69)在環外形成醯胺鍵仍對STS具有良好活性，於是我們進一步，將香豆素3、4號位擴環以增加平面性，其芐基衍生物在人類胎盤類固醇硫酸酯酶與MCF7乳癌細胞中，普遍具有極佳的抑制活性。因此本研究基於此研究成果，再優化三環香豆素骨架的類固醇硫酸酯酶抑制劑，同時合成單環結構的類固醇硫酸酯酶抑制劑以確認剛性結構是否為合適的發展方向，此外本研究亦將香豆素骨架融入雌激素結構中，並且在2、17號位進行取代基修飾，合成一系列四環香豆素骨架的類固醇硫酸酯酶抑制劑。 其結果顯示，單環結構的類固醇硫酸酯酶抑制劑不具有活性；而在三環香豆素骨架中，若衍生物芐基對位為親脂性基團，則在對位或間位加入氟原子 (70f-h)，能有效提升人類胎盤類固醇硫酸酯酶與MCF7乳癌細胞的抑制活性，更可使kinact/KI的數值大幅提升，而電腦模擬結果表明，三環香豆素衍生物皆能有效避免和類固醇硫酸酯酶活性端內的胺基酸產生碰撞，並在衍生的部位和酵素產生疏水性作用力；在四環香豆素骨架的類固醇型化合物中，則是以2號位為乙基、17號位為酮基取代的76c為最具潛力的類固醇硫酸酯酶抑制劑，生物活性和電腦模擬的結果皆表明，2號位取代基的疏水性質是重要的。這些化合物在人類胎盤類固醇硫酸酯酶與MCF7乳癌細胞的抑制活性效果皆優於STX64，顯示其具有治療雌激素受體陽性型乳癌的潛力，此外這些化合物kinact/KI的數值亦大幅增加，因此下一階段將於乳癌(ER+)動物模型中進行測試，期望最終能為乳癌患者提供新機轉治療藥物。

Steroid sulfatase (STS) regulates the local concentration of estrogen and androgens from systemic precursors through hydrolysis of the sulfate esters of 3-hydroxy steroids. Many evidences have shown that the overexpression of STS in estrogen positive (ER+) breast cancers increased cellular estrogen level and resulted in cancer cells proliferation and anti-apoptosis activities. Thus, the development of STS inhibitor was necessary. Irosustat (STX64, 50c), a coumarin compound, is the first STS inhibitor that enters phase Ⅱ clinical trial. Based on structures of STX64 and followed the structural rules of steroid sulfatase inhibitors, a series of STS inhibitors with coumarin skeleton were designed and synthesized. It was found earlier that 3-benzylcarboxamido coumarin-7-sulfamate (69) was capable to inhibit STS activity. Moreover, ring expansion at 3,4-position of coumarin compounds were synthesized, and most of them displayed excellent inhibitory activity against STS of both human placenta and MCF7 cells. Herein, we reported the comprehensive investigation of substitution for the tricyclic coumarin-based compounds. Furthermore, we also designed and synthesized four-membered rings compounds, as estrogen (E1) mimic, and modified substituents on carbon-2 and carbon-17 position. In this study, monocyclic STS inhibitors had no obvious inhibitory activity, while tricyclic derivatives with hydrophobic group at para-position phenyl ring showed significantly improvement against STS from human placenta and MCF7 cell line and their values of kinact/KI were also elevated, especially for those with fluoride substituent at ortho- or meta-position phenyl ring (70f-h). Besides, the docking model showed that tricyclic derivatives could avoid collision with amino acid in the active site of STS, and the hydrophobic interaction formed between derived part of compounds and enzyme. Among E1S mimic compounds, compound 76c was the most potent STS inhibitor with an IC50 value of 0.31 nM and 0.13 μM in STS from placental microsomes and MCF7 cell line, respectively, and the value of kinact/KI was 43. Both bioassays and docking results showed the hydrophobic property was important on carbon-2 position. The inhibitory activity of these compounds against STS from human placenta and MCF7 cell line were better than STX64, indicating that they are potential agents for breast cancer as adjuvant endocrine therapy (AET). Besides, the values of kinact/KI of these compounds also increased significantly. Hence, they will be tested in animal model bearing ER+ breast cancer in the future.