肟类衍生物的设计、虚拟筛选、合成与体外抗乙肝病毒活性研究

引用本文

CUI Xin-hua, TAN Jie, ZHOU Min, WEI Wan-xing, LIU Xu, WANG Li-sheng. Design, virtual screening, synthesis and anti-hepatitis B virus of oxime derivatives[J]. Acta Pharm Sin, 2016, 51(10): 1578-1583. 复制到剪切板

崔新华, 谭洁, 周敏, 韦万兴, 刘旭, 王立升. 肟类衍生物的设计、虚拟筛选、合成与体外抗乙肝病毒活性研究[J]. 药学学报, 2016, 51(10): 1578-1583. 复制到剪切板

肟类衍生物的设计、虚拟筛选、合成与体外抗乙肝病毒活性研究

崔新华, 谭洁, 周敏, 韦万兴, 刘旭, 王立升

广西大学化学化工学院, 广西南宁 530004

收稿日期: 2016-05-03; 修回日期: 2016-05-16

基金项目: 国家自然科学基金资助项目（81060261）；广西自然科学基金资助项目（2012GXNSFAA053021）；广西壮族自治区教育厅广西高等学校高水平创新团队及卓越学者计划（2015年起）

^*通讯作者: 韦万兴, Tel: 86-771-3272601, E-mail: wxwei@gxu.edu.cn

摘要: 本文设计了一系列新型结构的肟及肟醚化合物，通过分子对接进行虚拟筛选。以苯、甲苯、甲氧基苯、氯苯为初始原料合成12个新化合物，其中肟类4个、肟醚类8个，并对其结构进行了¹H NMR、¹³C NMR和MS确认。体外活性测试结果表明，部分目标化合物具有一定的抗乙肝病毒活性且毒性较小。其中化合物4B-2抑制活性最好，其对表面抗原（HBsAg）与e抗原（HBeAg）的抑制活性IC₅₀和治疗指数分别为IC_{50 HBsAg}=81.15μmol·L^-1、SI_HBsAg=9.20；IC_{50 HBeAg}=90.66 μmol·L^-1、SI_HBeAg8.24。初步的构效关系显示甲基肟醚类化合物活性较好，为新抗乙肝药物的研究提供参考。

关键词: 肟肟醚合成分子对接抗乙肝病毒活性

Design, virtual screening, synthesis and anti-hepatitis B virus of oxime derivatives

CUI Xin-hua, TAN Jie, ZHOU Min, WEI Wan-xing, LIU Xu, WANG Li-sheng

School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China

Abstract: A series of new oxime and oxime ethers compounds were designed and virtually screened with target using the Molecular Operating Environment (MOE) software. Twelve unreported compounds including 4 oximes and 8 oxime ethers were synthesized with benzene, toluene, methoxybenzene and chlorobenzene as initial raw materials. Structures of compounds were elucidated by ¹H NMR, ¹³C NMR and MS. The results of bioactive screening showed that a part of compounds displayed obviously anti-HBV activities. Inhibitory activities of compounds 4B-2 in secretion of HBsAg and HBeAg were IC_{50 HBsAg}=81.15 μmol·L^-1, SI_HBsAg=9.20 and IC_{50 HBeAg}=90.66 μmol·L^-1, SI_HBeAg8.24, respectively. Preliminary structure-activity relationship study shows that methyl oxime ethers displayed better anti-HBV activities than the oximes.

Key words: oxime oxime ether synthesis molecular docking anti-HBV activity

乙型病毒性肝炎(乙肝)是一种由乙型肝炎病毒(hepatitis B virus, HBV)引起的肝脏感染。可造成急性或慢性疾病, 并伴随引发多器官损害, 成为我国乃至世界范围内危害最为严重的传染病之一^{[1, 2]}。我国不仅有约1亿的慢性乙型肝炎病毒携带者, 且每年新发的感染者多达10万。因此, 乙型肝炎感染以及引起的慢性肝病是严重威胁我国国民健康的一大“杀手”。治疗乙型肝炎是要长期抑制HBV复制, 清除HBV, 缓解肝脏炎症和肝损伤, 预防向肝硬化和肝癌恶化^[3]。目前临床应用最广泛的是核苷类的抗病毒药物。核苷类常用药物有拉米夫定(3TC)、阿德福韦酯、恩替卡韦、替比夫定、替诺福韦酯等, 但是它们大部分都具有用药周期长、易发生耐药性、停药容易反复等缺点^[4−8]。因此, 研发新型结构的疗效高、不良反应少、耐药性小的抗HBV药物仍是医药学界世界性的研究热点。

肟类化合物是一类具有多种生物活性的化合物, 肟醚基团是常用的活性基团之一。肟醚化合物在抗菌、杀螨、杀虫、除草、抗癌、抗病毒和免疫调节方面有重要应用。Qu等^[9]合成的[1-(4-吗啉基)羰基亚甲基苯并咪唑-2-基]氰基甲酮肟醚对生菜灰霉菌与番茄菌核菌具有较高抑制活性。Yang等^[10]合成的吡唑肟醚类化合物显示出良好杀蚊虫、豆蚜和杀螨活性。Hamzeh等^[11]合成的一系列肟醚化合物对鳞翅目夜蛾(Spodopteralitura)有较好的灭杀作用。Park等^[12]合成的吡唑肟醚系列衍生物尤其是5-苯氧基吡唑对人结肠癌细胞(HCT15)和人类中枢神经系统肿瘤细胞(xf-498)表现出强有效的抑制活性。Chen等^[13]合成的一系列新的1-芳基-5-杂环1, 4-戊二烯-3-酮肟醚类化合物, 表现出一定的抗烟草病毒活性。Luo等^[14]合成的查耳酮肟具有较强的免疫抑制活性。但是肟类化合物在抗乙肝病毒的活性方面却很少见报道。Liu等^[15]合成的一系列苯丙素类肟酯化合物具有较好的抗HBV活性, 利用分子对接软件Molecular Operating Environment (MOE)与人类白细胞抗原HLA-A*02:03进行反向对接, 发现此类化合物中的肟基-O-N=C与靶蛋白氨基酸残基之间形成氢键, 从而推测-O-N=C是小分子配体与蛋白受体结合的关键药效基团。以此为基础, 根据药物设计的生物活性基团拼接原理, 设计出一系列肟类衍生物。设计的化合物, 利用MOE软件进行虚拟筛选。以苯、甲苯、甲氧基苯、氯苯为原料, 经傅克反应、酯化反应、肟化反应合成了12个未见文献报道的化合物(合成路线1), 并对目标化合物用¹H NMR、¹³C NMR和MS确证化学结构。以HepG2 2.2.15细胞为模型, 测试目标化合物的细胞毒性及对表面抗原(HBsAg)与e抗原(HBeAg)的抑制活性, 并进行初步的构效关系分析。实验结果显示, 大部分化合物具有一定的抗HBV活性且毒性较低。

结果与讨论 1 虚拟筛选

化合物4A-1～4D-3与人类白细胞抗原3OX8靶点对接结果如表 1所示, 多数化合物的对接作用部位集中在肟基-O-N=C, 这与文献[15]结论一致, 由此可以推测这些化合物可能具有潜在抗HBV活性。其中化合物4C-3、4B-3和4A-3的结果最好, 其分别与靶蛋白相互作用如图 1所示。

Schemes1 Reagents and conditions: (a) Succinic anhydride, AlCl₃, CH₂Cl₂, 0.5 h 0 ℃; 6 h rt; (b) Phenylcarbinol, p-CH₃C₆H₄SO₃H(TsOH), CH₂Cl₂, 4 h 70 ℃, reflux; (c) Hydroxylamine hydrochloride (methoxylamine hydrochloride, benzylhydroxylamine hydrochloride), pyridine, CH₂Cl₂, overnight 65 ℃, reflux.

Table 1 Docking results of synthesized compounds with 3OX8

Figure 1 3D representation showing the binding mode of compounds 4C-3, 4B-3 and 4A-3 into the binding site of 3OX8

由表 1可得活性预测顺序为肟类 < 甲氧肟醚类 < 苄氧肟醚类, 且预测活性普遍高于3TC组。当初始原料取代基(R₁)不同时, 活性预测顺序为:甲氧基取代 > 甲基取代 > 无取代 > 氯取代。

2 化学合成

本文以苯、甲苯、甲基苯、氯苯为初始原料按合成路线1进行合成, 得到了12个目标化合物, 经¹H NMR、¹³C NMR和MS确证了其化学结构。部分中间产物的数据如表 2所示。目标化合物的理化常数及波谱数据见表 3。

Table 2 Partial experimental results of compounds 2A−2D and 3A−3D

Table 3 Physicochemical constants and spectral data of compounds 4A-1−4D-3

Compd.	Physical property, Yield, mp, ¹H NMR, ¹³C NMR, MS
4A-1	White solid, yield 86.7%, mp 92.5−93.6 ℃, ¹H NMR (600 MHz, CDCl₃) δ 9.95 (s, 1H, H-22), 7.65 (d, J=9.8 Hz, 2H, H-3, 5), 7.44−7.38 (m, 8H, H-1, 2, 6, 16, 17, 18, 19, 20), 5.14 (s, 2H, H-14), 3.19 (t, J=4.1 Hz, 2H, H-8), 2.73 (t, J=6.2 Hz, 2H, H-9). ¹³C NMR (151 MHz, CDCl₃) δ 172.89 (C-10), 158.09 (C-7), 135.80 (C-15), 135.21 (C-1), 129.48 (C-4), 128.72 (C-17, 19), 128.60 (C-2, 6), 128.32 (C-3, 5), 128.31 (C-18), 126.39 (C-16, 20), 66.62 (C-14), 30.71 (C-9), 22.17 (C-8). ESI-MS: m/z 284 [M+H]⁺
4A-2	Colourless oily liquid, yield 79.8%, ¹H NMR (600 MHz, CDCl₃) δ 7.72 (d, J=3.8 Hz, 2H, H-3, 5), 7.41 (dt, J=8.9, 4.8 Hz, 8H, H-1, 2, 6, 16, 17, 18, 19, 20), 5.17 (s, 2H, H-14), 4.06 (s, 3H, H-22), 3.18−3.15 (m, 2H, H-8), 2.74−2.66 (m, 2H, H-9). ¹³C NMR (151 MHz, CDCl₃) δ 158.14 (C-10), 142.49 (C-7), 121.71 (C-15), 120.97 (C-1), 115.06 (C-4), 115.05 (C-17, 19), 114.38 (C-2, 6), 114.08 (C-3, 5), 114.07 (C-18), 112.11 (C-16, 20), 52.23 (C-14), 47.86 (C-22), 16.67 (C-9), 8.10 (C-8). ESI-MS: m/z 298 [M+H]⁺
4A-3	Pale yellow oily liquid, yield 78.0%, ¹H NMR (600 MHz, CDCl₃)δ 7.72 (d, J=2.7 Hz, 2H, H-3, 5), 7.50−7.47 (m, 2H, H-2, 6), 7.42 (ddd, J =23.9, 7.9, 4.3 Hz, 11H, H-1, 24, 25, 26, 27, 28, 16, 17, 18, 19, 20), 5.33 (s, 2H, H-14), 5.16 (s, 2H, H-22), 3.21 (t, J=7.3 Hz, 2H, H-8), 2.75− 2.68 (m, 2H, H-9). ¹³C NMR (151 MHz, CDCl₃) δ 172.48 (C-10), 157.13 (C-7), 137.99 (C-23), 135.91 (C-15), 135.21 (C-1), 129.37 (C-4), 128.64 (C-25, 27), 128.63 (C-17, 19), 128.47 (C-2, 6), 128.33 (C-3, 5), 128.31 (C-26), 128.21 (C-18), 127.89 (C-24, 28), 126.43 (C-16, 20), 76.46 (C-22), 66.52 (C-14), 30.92 (C-9), 22.52 (C-8). ESI-MS: m/z 374 [M+H]⁺
4B-1	White solid, yield 87.8%, mp 77.8−79.5 ℃, ¹H NMR (600 MHz, CDCl₃) δ 8.64 (s, 1H, H-23), 7.51 (d, J=8.2 Hz, 2H, H-3, 5), 7.40−7.32 (m, 5H, H-17, 18, 19, 20, 21), 7.20 (d, J=8.2 Hz, 2H, H-2, 6), 5.12 (s, 2H, H-15), 3.16−3.13 (m, 2H, H-8), 2.70−2.67 (m, 2H, H-9), 2.38 (s, 3H, H-14). ¹³C NMR (151 MHz, MeOD) δ 172.87 (C-10), 156.81 (C-7), 138.78 (C-1), 136.07 (C-16), 132.86 (C-4), 128.73 (C-2, 6), 128.12 (C-18, 20), 127.83 (C-19), 127.78 (C-17, 21), 125.92 (C-3, 5), 66.09 (C-15), 30.42 (C-9), 21.37 (C-8), 19.84 (C-14). ESI-MS: m/z 298.144 0 [M+H]⁺, 320.125 9 [M+Na]⁺
4B-2	Colourless oily liquid, yield 77.2%, ¹H NMR (600 MHz, CDCl₃) δ 7.59 (d, J=8.2 Hz, 2H, H-3, 5), 7.43−7.36 (m, 5H, H-17, 18, 19, 20, 21), 7.22 (d, J=8.0 Hz, 2H, H-2, 6), 5.16 (s, 2H, H-15), 4.03 (s, 3H, H-23), 3.14−3.11 (m, 2H, H-8), 2.69−2.66 (m, 2H, H-9), 2.41 (s, 3H, H-14). ¹³C NMR (151 MHz, CDCl₃) δ 172.51 (C-10), 156.71 (C-7), 139.30 (C-1), 135.93 (C-16), 132.33 (C-4), 129.32 (C-2, 6), 128.60 (C-18, 20), 128.38 (C-19), 128.30 (C-17, 21), 126.23 (C-3, 5), 66.48 (C-15), 62.01 (C-23), 30.96 (C-9), 22.31 (C-8), 21.31 (C-14). ESI-MS: m/z 312.158 9 [M+H]⁺, 334.140 4 [M+Na]⁺
4B-3	Pale yellow oily liquid, yield 76.9%, ¹H NMR (600 MHz, CDCl₃)δ 7.60 (d, J=8.4 Hz, 2H, H-3, 5), 7.42 (ddd, J=19.1, 15.0, 6.3 Hz, 10H, H-25, 26, 27, 28, 29, 17, 18, 19, 20, 21), 7.22 (d, J=7.8 Hz, 2H, H-2, 6), 5.29 (s, 2H, H-15), 5.14 (s, 2H, H-23), 3.19−3.16 (m, 2H, H-8), 2.71−2.65 (m, 2H, H-9), 2.41 (s, 3H, H-14). ¹³C NMR (151 MHz, CDCl₃) δ 172.52 (C-10), 157.05 (C-7), 139.37(C-1), 138.04 (C-24), 135.91 (C-16), 132.32 (C-4), 129.30 (C-2, 6), 129.29 (C-26, 28), 128.60 (C-18, 20), 128.42 (C-27), 128.27 (C-19), 128.17 (C-25, 29), 127.81 (C-17, 21), 126.30 (C-3, 5), 76.33 (C-23), 66.47 (C-15), 30.95 (C-9), 22.45 (C-8), 21.32 (C-14). ESI-MS: m/z 388.191 8 [M+H]⁺, 410.173 9 [M+Na]⁺
4C-1	White solid, yield 89.7%, mp 90.8−91.6 ℃, ¹H NMR (600 MHz, CDCl₃) δ 9.45 (s, 1H, H-24), 7.59 (d, J=8.9 Hz, 2H, H-3, 5), 7.42−7.33 (m, 5H, H-18, 19, 20, 21, 22), 6.93 (d, J=8.9 Hz, 2H, H-2, 6), 5.14 (s, 2H, H-16), 3.85 (s, 3H, H-15), 3.19−3.14 (m, 2H, H-8), 2.74−2.68 (m, 2H, H-9). ¹³C NMR (151 MHz, CDCl₃) δ 172.63 (C-10), 160.66 (C-1), 157.49 (C-7), 135.84 (C-17), 128.58 (C-19, 21), 128.29 (C-3, 5), 128.28 (C-20), 127.71 (C-18, 22), 127.50 (C-4), 114.11 (C-2, 6), 66.55 (C-16), 55.35 (C-15), 30.74 (C-9), 21.93 (C-8). ESI-MS: m/z 314.139 3 [M+H]⁺, 336.121 2 [M+Na]⁺
4C-2	Colourless oily liquid, yield 78.9%, ¹H NMR (600 MHz, CDCl₃) δ 7.64 (d, J=8.9 Hz, 2H, H-3, 5), 7.42−7.34 (m, 5H, H-18, 19, 20, 21, 22), 6.92 (d, J=9.0 Hz, 2H, H-2, 6), 5.14 (s, 2H, H-16), 4.01 (s, 3H, H-24), 3.82 (s, 3H, H-15), 3.12−3.09 (m, 2H, H-8), 2.69−2.65 (m, 2H, H-9). ¹³C NMR (151 MHz, CDCl₃) δ 172.46 (C-10), 160.59 (C-1), 156.29 (C-7), 135.96 (C-17), 128.59 (C-19, 21), 128.29 (C-3, 5), 128.27 (C-20), 127.67 (C-18, 22), 127.61 (C-4), 113.98 (C-2, 6), 66.44 (C-16), 61.93 (C-24), 55.26 (C-15), 30.99 (C-9), 22.22 (C-8). ESI-MS: m/z 328.154 9 [M+H]⁺, 350.136 2 [M+Na]⁺
4C-3	Pale yellow oily liquid, yield 74.7%, ¹H NMR (600 MHz, CDCl₃)δ 7.49 (d, J=8.8 Hz, 2H, H-3, 5), 7.40−7.30 (m, 10H, H-26, 27, 28, 29, 30, 18, 19, 20, 21, 22), 6.92 (d, J=8.8 Hz, 2H, H-2, 6), 5.08 (d, J=2.4 Hz, 4H, H-16, 24), 3.84 (s, 3H, H-15), 2.90 (t, J=7.3 Hz, 2H, H-8), 2.65 (t, J=7.3 Hz, 2H, H-9). ¹³C NMR (151 MHz, CDCl₃) δ 172.65 (C-10), 159.87 (C-1), 154.56 (C-7), 138.37 (C-25), 136.03 (C-17), 129.70 (C-27, 29), 128.54 (C-19, 21), 128.26 (C-3, 5), 128.24 (C-20), 128.19 (C-18, 22), 127.92 (C-26, 30), 127.52 (C-28), 125.69 (C-4), 113.51 (C-2, 6), 75.96 (C-24), 66.26 (C-16), 55.25 (C-15), 31.15 (C-9), 30.46 (C-8). ESI-MS: m/z 404.184 4 [M+H]⁺, 426.166 4 [M+Na]⁺
4D-1	White solid, yield 84.4%, mp 85.5−86.6 ℃, ¹H NMR (600 MHz, CDCl₃)δ 9.12 (s, 1H, H-22), 7.55 (d, J=8.6 Hz, 2H, H-3, 5), 7.40−7.32 (m, 7H, H-2, 6, 16, 17, 18, 19, 20), 5.12 (s, 2H, H-14), 3.15−3.12 (m, 2H, H-8), 2.73−2.66 (m, 2H, H-9). ¹³C NMR (151 MHz, CDCl₃) δ 172.41 (C-10), 157.20 (C-7), 135.67 (C-1), 135.54 (C-15), 133.52 (C-2, 6), 128.91 (C-17, 19), 128.60 (C-4), 128.35 (C-3, 5), 128.33 (C-18), 127.64 (C-16, 20), 66.66 (C-14), 30.57 (C-9), 21.86 (C-8). ESI-MS: m/z 340.071 8 [M+Na]⁺
4D-2	Pale yellow oily liquid, yield 74.2%, ¹H NMR (600 MHz, CDCl₃)δ 7.59 (d, J=8.7 Hz, 2H, H-3, 5), 7.37 (td, J=12.9, 8.0 Hz, 7H, H-2, 6, 16, 17, 18, 19, 20), 5.12 (s, 2H, H-14), 4.00 (s, 3H, H-22), 3.08−3.05 (m, 2H, H-8), 2.67−2.59 (m, 2H, H-9). ¹³C NMR (151 MHz, CDCl₃) δ 172.32 (C-10), 155.68 (C-7), 135.76 (C-1), 135.26 (C-15), 133.61 (C-4), 128.75 (C-2, 6), 128.60 (C-17, 19), 128.33 (C-3, 5), 128.31 (C-18), 127.58 (C-16, 20), 66.55 (C-14), 62.21 (C-22), 30.78 (C-9), 22.13 (C-8). ESI-MS: m/z 332.106 0 [M+H]⁺, 354.088 3 [M+Na]⁺
4D-3	Pale yellow oily liquid, yield 70.2%, ¹H NMR (600 MHz, CDCl₃)δ 7.61 (d, J=8.7 Hz, 2H, H-3, 5), 7.43−7.35 (m, 12H, H-2, 6, 24, 25, 26, 27, 28, 16, 17, 18, 19, 20), 5.27 (s, 2H, H-14), 5.11 (s, 2H, H-22), 3.13−3.11 (m, 2H, H-8), 2.68−2.64 (m, 2H, H-9). ¹³C NMR (151 MHz, CDCl₃) δ 172.33 (C-10), 156.04 (C-7), 137.74 (C-23), 135.30 (C-1), 133.61 (C-15), 128.75 (C-2, 6), 128.60 (C-25, 27), 128.45 (C-17, 19), 128.33 (C-4), 128.29 (C-3, 5), 128.18 (C-26), 128.17 (C-18), 127.93 (C-24, 28), 127.65 (C-16, 20), 76.56 (C-22), 66.56 (C-14), 30.78 (C-9), 22.28 (C-8). ESI-MS: m/z 408.136 4 [M+H]⁺, 430.118 7 [M+Na]⁺

Table 3 Physicochemical constants and spectral data of compounds 4A-1−4D-3

3 体外毒性及活性测试

以HepG2 2.2.15细胞为模型, 细胞毒性实验(MTT法)检测目标化合物的细胞毒性; 用酶联免疫法(ELISA法)检测HepG2 2.2.15细胞培养液中HBsAg和HBeAg的表达。细胞毒性及活性结果见表 4。

Table 4 Anti-HBV activity and cytotoxicity of the target compounds in vitro. SI > 2: High efficiency; 1 < SI < 2: Low efficiency and toxic; SI < 1: No efficiency and strong toxicity

毒性实验结果表明, 除4A-1、4B-1、4C-1和4D-1四个肟类化合物之外, 其他化合物的毒性均小于3TC组。活性实验结果表明, 除4A-1、4B-1、4C-1和4D-1四个肟类化合物对HepG2 2.2.15细胞的HBsAg和HBeAg抑制活性较小外, 其他目标化合物均具有一定的抑制活性。4A-1、4B-1、4C-1和4D-1四个肟类化合物对HBsAg和HBeAg治疗指数SI均小于2, 且小于3TC组, 属于低效或无效药物。整体来看, 肟醚类化合物都表现出较低的毒性, 且毒性低于3TC组, 同时表现出较强的抑制HepG2 2.2.15细胞的HBsAg和HBeAg的分泌活性, 且抑制作用大于3TC组。其中以化合物4B-2活性最好, 其HBsAg IC₅₀=81.15 μmol·L⁻¹, HBeAg IC₅₀=90.66 μmol·L⁻¹, 治疗指数分别为SI_HBsAg=9.20和SI_HBeAg=8.24。

4 小结

活性结果最好的化合物4B-2与靶点3OX8对接相互作用3D图如图 2。从对接结合能来说, 化合物4B-2为−20.138 9 kcal·mol⁻¹, 残基Tyr27与-O-N=C的N原子间形成氢键, 距离为2.64 。与具有较好对接结果的苄氧肟醚化合物相比, 甲氧肟醚化合物具有更高的HBsAg和HBeAg的抑制活性。

Figure 2 3D representation showing the binding mode of compound 4B-2 into the binding site of 3OX8

利用MOE-dock分子对接结果预测活性顺序为:肟 < 甲氧基肟醚 < 苄氧基肟醚, 体外活性测试结果显示活性顺序为:肟 < 苄氧基肟醚 < 甲氧基肟醚。横向比较得知, 预测结果为: (R₁基团)甲氧基取代 > 甲基取代 > 无取代 > 氯取代, 体外活性测试结果为: R₁为甲基时最好, 无取代时最差。活性测试结果与利用MOE软件计算预测的活性结果有所出入, 说明模拟对接的靶点可能与化合物的实际作用靶点不同。未来可以将活性较好的化合物与HBV有关蛋白进行反向对接, 确定其作用靶点, 为虚拟筛选此类化合物提供更准确的靶点蛋白。

基于目标化合物的抗HBV活性初步测试结果, 甲氧肟醚类化合物抗HBV活性较好。本文从优化化合物结构方面探索抗HBV活性药物具有一定的指导意义。

实验部分 1 分子对接

计算软件使用MOE 2008.10版本, 对接程序应用MOE-dock模块。

选取人类白细胞抗原HLA-A^*02:03^[21]为靶点蛋白, 来源蛋白质数据库protein data bank (PDB http:// www.rcsb.org/pdb/home/home.do。ID:3OX8), 对设计的化合物进行虚拟筛选。

在MOE中对靶点蛋白进行Protonate 3D加氢加电荷操作, 进行能量最小化操作, 删除水分子、离子等操作。之后进行Site Finder操作, 获取活性位点。在相同条件下, 将设计的化合物与靶点蛋白分别对接。

2 主要仪器与试剂

Bruker 600 Avance Ⅲ核磁共振仪(以CDCl₃或CDOD₃为溶剂, Sigma公司, TMS为内标); Thermo Scientific ITQ 1100质谱仪(Thermo Fisher); WRX-4显微熔点仪(未经过校正); 薄层色谱硅胶(青岛海洋化工有限公司); 其他试剂均为分析纯。

生物活性试验中主要仪器与试剂: Elx800型酶联免疫检测仪(美国BIO-TEK公司); 高糖DMEM (Gibco); MTT (Amresco); G418 (Sigma); 谷氨酰胺(Sigma); 胎牛血清(胎牛血清); HBsAg、HBeAg试剂盒(上海科华生物工程股份有限公司); 3TC (葛兰素史克制药有限公司); 其他试剂均为分析纯。

3 化学合成 3.1 2A～2D的制备

将25 mmol (1 equiv)取代苯溶于30 mL二氯甲烷中, 加入25 mmol (1 equiv)丁二酸酐, 冰水浴搅拌条件下, 在30 min内分批加入37.5 mmol (1.5 equiv)无水AlCl₃, 之后常温反应6 h。薄层色谱检测跟踪反应, 反应结束后加入冰盐酸淬灭, 搅拌10 min, 抽滤, 用适量70%乙醇溶液重结晶, 干燥得产物2A～2D。

3.2 3A～3D的制备

取15 mmol (1 equiv)化合物2A～2D和2 g对甲苯磺酸溶于20 mL二氯甲烷中, 加热到70 ℃, 回流, 加入30 mmol (2 equiv)苄醇。恒温磁力搅拌回流4 h, 薄层色谱检测跟踪反应。反应结束后, 减压旋干二氯甲烷, 用去离子水洗涤, 乙酸乙酯(20 mL×3)萃取, 合并有机层, 用无水硫酸钠干燥, 减压蒸除溶剂, 得粗品。粗品进行柱色谱分离(石油醚−乙酸乙酯, 6:1)得到产物3A～3D。

3.3 4A-1～4D-3的制备

将化合物3A～3D (1 equiv, 3.84 mmol)和盐酸羟胺(或甲氧胺盐酸盐、苄氧胺盐酸盐) (2 equiv, 7.7 mmol)溶于20 mL二氯甲烷中, 加热回流65 ℃, 加入2 mL吡啶, 恒温下磁力搅拌回流过夜, 薄层色谱检测基本反应完全。冷却至室温, 减压蒸去剩余的吡啶, 加入20 mL乙酸乙酯, 用饱和食盐水(20 mL×3)洗涤, 有机相经无水硫酸钠干燥, 过滤, 浓缩得粗品。之后进行柱色谱分离(乙酸乙酯−石油醚, 1:5～1:13)得到产物4A-1～4D-3。

4 体外抗HBV活性实验

参考文献^[22]的方法, 检测目标化合物的细胞毒性和对HBsAg、HBeAg的抑制活性。

用GraphPad Prism 5软件计算半数有毒浓度(CC₅₀)和半数抑制浓度(IC₅₀)。治疗指数SI=CC₅₀/ IC₅₀。

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药学学报 2016, Vol. 51 Issue (10): 1578-1583	PDF