發展零流感單磷酯衍生物作為有效的抗流感試劑: 合成、生物活性與藥物動力之研究

Abstract

流行性感冒是影響人類健康的主要疾病之一。流感病毒是一種RNA病毒，流感病毒的生命週期中，新形成的流感病毒在離開宿主細胞的時候，神經胺酸酶必須水解血液凝集酶與宿主細胞上唾液酸之間的聯繫，才可以離開宿主細胞而感染其他細胞。神經胺酸酶水解時形成oxonium中間體，而克流感與瑞樂沙的環己烯結構便模擬此中間體以達到抑制流感病毒的效果，其中結構上的羧酸基會與神經胺酸酶的活性中心的精胺酸形成靜電力(Arg118, Arg292 and Arg371)來結合。所以我們實驗室便開發磷酸基團的零流感化合物來取代羧酸基團的oseltamivir以達到更佳結合來有效抑制流感病毒，由實驗結果發現對於H1N1，H5N1與突變H274Y H1N1都較羧酸機化合物更佳。但是零流感化合物的脂溶性低造成生物獲得率較差，為了改善這缺點，我們合成單磷脂零流感化合物來增加脂溶性之外，以可以增加部分的凡德瓦力來加強結合的能力，以達到抑制感冒病毒的目的。在合成上我們使用市售的克流感膠囊先進行Boc保護得到化合物59，接著使用Barton反應得到碘化物76，我們使用不同亞磷酯化合物如二乙基、二丁基、二己基或是二苯丙基亞磷酸與化合物76進行鈀金屬的催化得到不同二磷酯之零流感化合物，將胺基上的Boc去掉之後再與硫脲化物反應得到胍基零流感化合物。接著在鹼性條件之下反應得到單酯零流感化合物之後，最後在移除Boc保護基之後便可獲得零流感與胍基零流感化合物。在分配係數測驗上，我們發現胍基零流感己基單酯95較胍基零流感乙基單酯83有較好的脂溶性，而且胍基零流感己基單酯95也較胍基零流感乙基單酯83與胍基零流感3-苯丙基單酯110有較好的細胞穿透性。然而在生體可用率實驗裡，胍基零流感己基單酯95 (F = 12.6%)之生體可用率略高於胍基零流感乙基單酯83 (F = 12%)。初期在H1N1流感病毒口服投藥的小鼠保護測試上胍基零流感己基單酯95並沒有較胍基零流感乙基單酯83有較好的保護效果，但是胍基零流感己基單酯95與胍基零流感乙基單酯83在鼻吸入方式投藥上都有明顯的保護效用。

Influenza is a major disease to menace human’s health. Influenza virus is a kind of RNA virus. In the life cycle, the new influenza virus will leave the host cell by cutting off the sialic acid moiety of the glycoprotein receptor on host cell surface. Neuraminidase catalyzes such hydrolysis to form an oxonium intermediate from sialoside. The structure of oseltamivir and zanamivir mimic the (oxa)cyclohexene intermediate to inhibit the activity of neuraminidase. Tamiflu™ is a prodrug, and its ester bond will be hydrolyzed by hepatic esterase to form oseltamivir carboxylate (OC), which binds to the three arginine residues (Arg118, Arg292 and Arg371) in the neuraminidase of influenza virus. Our team has synthesized tamiphosphor (the phosphonate congener of OC) and guanidino-tamiphosphor as potent inhibitors against influenza virus. The phosphonic functional group on C-2 rendered better efficiency against avian H5N1, human H1N1 and oseltamivir-resistant H274Y influenza viruses. However, poor lipophilicity of tamiphosphor and guanidino-tamiphosphor caused low bioavailability. To circumvent this drawback, we synthesized phosphonate monoester compounds to increase the lipophilicity with retention of the inhibitory activity against influenza virus. The N-Boc protected OC 59 was prepared from tamiflu capsule, and then converted to the corresponding iodo compound 76 by halo-decarboxylation reaction using Barton’s procedure. The Pd coupling reactions of iodo compound 76 with various dialkyl phosphite compounds such as diethy, dibuty, dihexyl and di(3-phenylpropyl) phosphites, to give the desired phosphonate products. The N-Boc group was deprotected, and the resulting amino group was treated with N-Boc-thiourea reagent to give the N-Boc-guanidino derivatives. The tamiphosphor and guanidino-tamiphosphor was available by the phosphonate dialkyl esters was treated with alkali to give the phosphonate monoesters. Tamiphosphor monoesters and guanidine-tamiphosphor monoesters were finally synthesized after removal of the Boc group. We found monohexyl phosphonate 95 showed better lipophicility than monoethyl phosphonate 83 in the distribution coefficient measurement. Compound 95 also has better cell permeability than compound 83 and the (3-phenyl)propyl phosphonate analog 110. However, the bioavailability of compound 95 (F = 12.6%) was just slightly higher than that of compound 83 (F = 12%). Our preliminary mice test indicated that compound 95 may not be better than 83 for treatment of H1N1 influenza virus infection by oral gavage. Nontheless, both compounds 83 and 95 showed high efficiency in protection of mice from influenza infection by intranasal administration.