雙重抑制劑樂瓦斯他汀–羥胺之結構修飾：改變鋅結合基團及合成金屬錯合物

Abstract

組蛋白去乙醯酶 (HDACs) 會將組蛋白上氮端 (N-) 的離胺酸基團進行去乙醯化反應，藉此影響多種生物機制，在癌症及神經退化疾病的致病機制上都扮演重要的角色。在膽固醇的生合成路徑中，3-羥基-3-甲基戊二酸輔酶A還原酶 (HMGR) 為合成甲羥戊酸的速率決定酵素。市面上他汀類 (statins) 之藥物為 3-羥基-3-甲基戊二酸輔酶A還原酶的抑制劑，普遍用來治療高血酯。現今研究指出，組蛋白去乙醯酶及3-羥基-3-甲基戊二酸輔酶A還原酶之抑制劑為有發展潛力的新型抗癌藥物。 根據里賓斯基五規則 (Lipinski’s “Rule of Five”) 及設計多重功能配體(designed multiple ligands, DMLs) 概念中的接合 (fusing) 策略，我們首先設計且合成了樂瓦斯他汀–羥胺作為有效且低細胞毒性的組蛋白去乙醯酶和3-羥基-3-甲基戊二酸輔酶A還原酶的雙重抑制劑。羥胺 (hydroxylamine) 在組蛋白去乙醯酶抑制中扮演鋅離子配位官能基團 (zinc-binding group)，且取代他汀類藥物中的羧酸官能基，達到抑制3-羥基-3-甲基戊二酸輔酶A還原酶的效果。然而，羥胺基團的藥物動力學 (pharmacokinetics) 特性差且水解後會對生體產生慢性毒性。因此，我們在本篇研究中首先藉由肽鍵耦合反應或親核反應在樂瓦斯他汀上修飾一系列不同鋅離子配位官能基，期望能改善樂瓦斯他汀–羥胺的藥物動力學特性和提升雙重酵素抑制活性。同時我們也合成了親水性較高的氟伐他汀鈉–羥胺(fluvastatin–hydroxamate) 之衍生物。根據離體檢測結果 (enzymatic and cell-based assay)，化合物 28, 32及 34 表現出比其他衍生物高的組蛋白去乙醯酶之抑制活性，因此我們認為強的鋅離子配位官能基團須有自由的末端羥基，且其大小和剛性都會影響抑制活性。氟伐他汀鈉–羥胺擁有跟樂瓦斯他汀–羥胺相當的雙重抑制活性，但其離體細胞檢測 (cell-based assay) 顯示氟伐他汀鈉的高親水性可能減弱了氟伐他汀鈉–羥胺的細胞穿透度 (cell permeability)。總結以上研究，羥胺仍然是目前設計此雙重抑制劑中最適合的鋅離子配位官能基團。 另一方面，過渡金屬經常和多種抑制劑或細胞毒素形成前驅藥物 (prodrug)，在特定的生理環境中被活化而釋放出活性物質，藉此毒殺特定細胞。因此，我們利用不同過渡金屬合成一系列樂瓦斯他汀–羥胺之過渡金屬錯合物，嘗試改善羥胺之藥物動力學特性也建構樂瓦斯他汀–羥胺的控釋效果，提高針對毒殺腫瘤細胞之選擇性。銅二價的錯合物 42 和 43對組蛋白去乙醯酶 6 有比樂瓦斯他汀–羥胺更高的抑制活性。在缺氧及含氧的離體細胞檢測中，銅二價的錯合物 42 和 43表現跟樂瓦斯他汀–羥胺相當且優於鐵三價的錯合物41的酵素抑制活性。在缺氧環境中，鈷三價及釓三價的錯合物49及51比在含氧環境中對組蛋白去乙醯酶 3的抑制活性高了20–40%。在離體細胞檢測中發現銅二價的錯合物 42 和 43加入維他命C作為溫和還原劑後，在特定條件中能促進配體的釋放而提升抑制活性。加入維他命C的酵素和離體細胞檢測皆會繼續進行。紫外光–可見光吸收光譜 (UV–Vis spectra) 可推測此金屬前驅藥物可在缺氧及酸性環境下被活化而釋放出活性物質樂瓦斯他汀–羥胺。在初步的體內異體移植腫瘤檢測 (xenograft model) 中，鐵三價之錯合物 41 能有效的抑制人結直腸癌細胞的生長。在未來也會增加異體移植腫瘤檢測的樣品數。之後也會進行這些金屬錯合物之細胞毒性檢測。在未來我們也將設計利用釓三價的錯合物 51 做為核磁共振成像 (MRI) 的對比劑以觀測樂瓦斯他汀–羥胺在體內的藥物分佈情況。

Histone deacetylases (HDACs) cause deacetylation of the lysine groups on the histone, and play an important role in several diseases including cancer and neurodegenerative disease. 3-Hydroxy-3-methylgluraryl coenzyme A reductase (HMGR) is the rate-controlling enzyme for conversion of HMG-CoA to mevalonic acid in biosynthetic pathway of cholesterol. Statins are a class of HMGR inhibitors used as anti-hypercholesterolemia agents. Nowadays, HDACs and HMGR inhibitors have emerged as potential anti-cancer agents. Based on Lipinski’s “Rule of Five” and fused strategy, we have previously designed and synthesized lovastatin–hydroxamate (LOVA–HA) as the dual-action inhibitor targeting HDACs and HMGR with IC50 values in nanomolar range, but low cytotoxicity to normal cells. The hydroxamate group acts as a zinc binding group (ZBG) for HDACs inhibition and as a surrogate of carboxylic acid in statins. However, the hydroxamate group may have poor pharmacokinetic properties and chronic toxicity after hydrolysis. Therefore, we performed structural modification on LOVA–HA (22) by introducing different ZBGs via amide bond formation with lovastatin (19, in carboxylate form) or direct nucleophilic attack on activated statins in lactone form including lovastatin (11) and fluvastatin (37) to improve pharmacokinetic properties and inhibitory activities for cancer treatment. Based on the in vitro assays, 28, 32 and 34 having terminal N-methylhydroxamic, picolylamine and 2-hydroxycyclohexylamide moieties showed less inhibition than LOVA–HA, indicating that free terminal hydroxyl group, size and rigidity of ZBGs play vital roles in HDAC inhibitory activities. Fluvastatin–hydroxamate (38) showed comparable inhibition against HDAC enzymes but poorer inhibition in HCT116 cells than 22, presumably due to higher hydrophilicity of fluvastatin. Hydroxamate may be still the most suitable and stronger ZBG in dual-action inhibitors so far. Transition metals are often used as chaperones to inhibitors and cytotoxins to form prodrugs which will be activated in specific physiological environments to selectively kill specific cells. Therefore, we synthesized LOVA–HA metallo-complexes in attempts to improve pharmacokinetic properties and construct the controlled-release function. LOVA–HA Cu(II)-complex (a mixture of 42/43) showed a better inhibitory activity against HDAC6 than LOVA–HA. Under either normoxia or hypoxia conditions, LOVA–HA Cu(II)-complex (42/43) had comparable inhibitory activity to LOVA–HA and superior to 41. LOVA–HA Co(III)- and Gd(III)-complexes 49 and 51 showed 20–40% of enhancement in HDAC3 inhibition in hypoxia conditions. The prodrug is activated in hypoxia and acidic environment according to in vitro assays and UV–vis spectra. Based on the preliminary results of the cell-based assays of Cu(II)-complex 42/43, the presence of (L)-ascorbic acid as a mild reducing agent would facilitate the ligand releasing in specific conditions. The enzymatic and cell-based assays of other LOVA–HA metallo-complexes with ascorbic acid are undergoing. 41 had effect on suppressing the tumor growth in a preliminary xenograft model and will repeat this experiment with more mice in a due course. Whether the metallo-complexes are toxic will be determined in a due course. [Gd(LOVA–HA)3]•3H2O (51) may be utilized as a contrast agent in MRI to study the distribution of LOVA–HA in body.