一种哌啶-4-胺基二芳基嘧啶衍生物及其制备方法和用途
技术领域
本发明属于医药技术领域，具体涉及哌啶-4-胺基二芳基嘧啶衍生物及其制备方法和用途。
背景技术
艾滋病（AIDS）即获得性免疫缺陷综合征（Acquired immune deficiency syndrome）是由人类免疫缺陷病毒（Human immunodeficiency virus, HIV）所导致的流行性传染病。
逆转录酶（Reverse transcriptase, RT）在HIV从mRNA逆转录成DNA的过程中起了决定性作用，因此成为抗艾滋病药物设计的重要靶点之一。
在现有的抗HIV药物研究中，非核苷类逆转录酶抑制剂（NNRTIs）因其高效低毒等优点而成为各国药物化学家关注的热点之一。目前，经美国FDA批准上市的抗HIV逆转录酶抑制剂有五种：奈维拉平（Nevirapine）、德拉韦定（Delavirdine）、依非韦伦（Efavirenz）、依曲韦林（Etravirine）、利匹韦林（Rilpivirine）。另外，RDEA806、IDX899、UK-453061正在进行临床研究。研究显示，经典的NNRTIs只作用于HIV-1，而对HIV-2病毒无效。
发明内容
本发明的目的在于提出一种逆转录酶抑制剂哌啶-4-胺基二芳基嘧啶衍生物及其制备方法和用途。，
本发明提出的逆转录酶抑制剂--哌啶-4-胺基二芳基嘧啶衍生物，具有下式Ⅰ所示结构通式：
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其中，R1和R2分别独立选自氢，氰基，硝基，氨基，羟基，卤素，任选被取代的C1~6烷基，任选被取代的C2~6烯基，任选被取代的C2~6炔基，C1~6烷氧基；
R3选自氢，氰基，硝基，氨基，羟基，卤素，磺酸基，羧基，酯基，C1~6烷基，C1~6烷氧基，C1~6烷巯基，C3~6环烷基，C3~6环烷氧基，C3~6环烷氨基，C2~6烯基，C2~6炔基，取代的C2~6烯基，取代的C2~6炔基；
Ar为单取代或多取代的芳环，芳环上的取代基选自氢，氰基，硝基，氨基，羟基，卤素，磺酸基，羧基，酯基，C1~6烷基，C1~6烷氧基，C1~6烷巯基，C3~6环烷基，C3~6环烷氧基，C3~6环烷氨基，C2~6烯基，C2~6炔基，取代的C2~6烯基，取代的C2~6炔基。
本发明还涉及哌啶-4-胺基二芳基嘧啶衍生物，以及其药用盐，其水合物和溶剂化物，其多晶和共晶，其同样生物功能的前体和衍生物。
所述的药用盐包括盐酸盐、氢溴酸盐、硫酸盐、磷酸盐、醋酸盐、甲磺酸盐、对甲苯磺酸盐、酒石酸盐、柠檬酸盐、富马酸盐、苹果酸盐。
本发明还提出上述哌啶-4-胺基二芳基嘧啶衍生物的制备方法，其反应通式如下：
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具体操作步骤如下：
碱性条件下，以上述结构式Ⅱ和Ⅲ所示的化合物为原料，在有机溶剂中，制得化合物Ⅰ；
所述的碱，为如下碱金属氢氧化物：氢氧化锂、氢氧化钠或者氢氧化钾，或为如下碱金属的碳酸盐：碳酸钠、碳酸钾或碳酸铯，或为如下有机碱：三乙胺、DBU或DIEPA；
所述的有机溶剂为N,N-二甲基甲酰胺、四氢呋喃、二氧六环、二甲基亚砜、C1-C4醇，其中的单一溶剂，或者几种的混合溶剂；
X为Cl或者Br；
所述的原料Ⅱ与Ⅲ的摩尔比为2:1~1:2，优选0.9:1~1:0.9；
反应温度为0~100 oC，优选20~40 oC；
反应时间为1~24h，优选2-6h。
本发明还涉及一种药物组合物，该组合物含有有效剂量的上述化合物和相关的药用载体。
本发明还涉及所述化合物或组合物在制备预防和治疗艾滋病药物中的应用。
本发明基于二芳基嘧啶与逆转录酶结合模式，结合计算机辅助药物设计，在中间嘧啶环与右翼的链接子上引入4-胺基哌啶乙酰胺，可与高度保守氨基酸残基Leu234形成氢键，从而增强目标分子与逆转录酶的结合力，进一步提高目标化合物的抗病毒活性。生物活性测试表明，所有化合物均具有较强抗HIV-1病毒活性和较小的细胞毒性，具有较高的选择性指数。
具体实施方式
通过下述实施实例可以更好地理解本发明内容，但是不能限制本发明的内容。
实施例1：终产物Ⅰ的合成
将碳酸钾加入到仲胺Ⅱ的N,N-二甲基甲酰胺的0 oC溶液中，搅拌0.5h，逐滴加入α-溴乙酰苯胺Ⅲ的N,N-二甲基甲酰胺溶液，滴毕，继续在0 oC下搅拌2h，然后升至室温搅拌9h。TLC显示反应完全。倒入水中，过滤，水洗，抽滤。干燥，甲苯重结晶，得到所需固体。
以不同的α-溴乙酰苯胺Ⅲ用上述方法分别制得目标化合物，部分结果如下：
将碳酸钾（17 mmol）加入到3,5-二甲基-4-{[2-(哌啶-4-胺基)嘧啶-4-基]氧基}苯腈Ⅱ（8.5 mmol）的N,N-二甲基甲酰胺（25 mL）的0 oC溶液中，搅拌0.5h，逐滴加入2-溴-N-乙酰苯胺Ⅲ（8.5 mmol）的N,N-二甲基甲酰胺（10 mL）溶液，滴毕，继续在0 oC下搅拌2h，然后升至室温搅拌9h。TLC显示反应完全。倒入350 mL水中，过滤，水洗三次，抽滤。干燥，甲苯重结晶，得到所需固体。
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白色粉状固体；收率81%; 熔点：202.7–203.5 oC; 1H NMR (400 MHz, CDCl3) δ = 1.25-2.04 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.34-2.82 (m, 4H, piperidine), 3.11 (s, 2H, CH2), 4.98 (br s, 1H, NH), 6.14 (d, J = 5.6 Hz, 1H, pyrimidine), 7.10-7.32 (m, 3H, Ph’), 7.40 (s, 2H, Ph), 7.54 (d, J = 5.6 Hz, 2H, Ph’), 8.16 (d, J = 5.6 Hz, 1H, pyrimidine), 9.08 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.51, 32.32, 47.59, 52.68, 62.15, 109.40 (2C), 118.79, 119.51, 124.32, 129.16, 132.42, 133.04, 137.70, 153.53, 160.07, 161.86, 168.49 (2C); IR (KBr): ν = 3343 (s; ν(N-H)), 3234 (s; ν(N-H)), 2223 (s; ν(C≡N)), 1691 (s; ν(C=O)); MS (ESI+) m/z 457 (M+H)+; HRMS calcd for C26H28N6O2 [M+H]+: 457.2352, found: 457.2356; HPLC: tR = 10.90 min, 98.06%。
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操作同上。白色粉状固体；收率79%; 熔点：207.9–208.6 oC; 1H NMR (400 MHz, CDCl3) δ = 1.47-2.04 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.26 (s, 3H, CH3), 2.39-2.88 (m, 4H, piperidine), 3.15 (s, 2H, CH2), 4.98 (br s, 1H, NH), 6.14 (d, J = 5.6 Hz, 1H, pyrimidine), 7.01-7.24 (m, 3H, Ph’), 7.40 (s, 2H, Ph), 8.13-8.17 (m, 2H, pyrimidine + Ph’), 9.24 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.53, 21.48, 32.33, 47.60, 52.66, 62.18, 109.37 (2C), 117.20, 118.81, 126.04, 127.68, 127.71, 132.42, 133.04, 137.52, 138.90, 153.52, 160.12, 161.86, 168.37 (2C); MS (ESI+) m/z 471 (M+H)+; HRMS calcd for C27H30N6O2 [M+H]+: 471.2508, found: 471.2509; HPLC: tR = 11.33 min, 97.26%。
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操作同上。白色粉状固体；收率77%; 熔点：199.0–199.4 oC; 1H NMR (400 MHz, CDCl3) δ = 1.48-2.04 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.34 (s, 5H, CH3 + piperidine), 2.81 (d, J = 8.4 Hz, 2H, piperidine), 3.10 (s, 2H, CH2), 4.97 (br s, 1H, NH), 6.13 (d, J = 5.6 Hz, 1H, pyrimidine), 6.91-7.40 (m, 6H, Ph + Ph’), 8.15 (d, J = 5.6 Hz, 1H, pyrimidine), 9.02 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.53, 21.60, 32.33, 47.55, 52.70, 62.15, 109.36 (2C), 116.57, 118.81, 120.08, 125.11, 128.99, 132.42, 133.03, 137.58, 139.10, 153.51, 160.10, 161.86, 168.44 (2C); MS (ESI+) m/z 471 (M+H)+; HRMS calcd for C27H30N6O2 [M+H]+: 471.2508, found: 471.2507; HPLC: tR = 11.43 min, 98.75%。
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操作同上。白色粉状固体；收率85%; 熔点：185.5–185.9 oC; 1H NMR (400 MHz, CDCl3) δ = 1.47-2.03 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.30 (s, 5H, CH3 + piperidine), 2.80 (d, J = 8.8 Hz, 2H, piperidine), 3.09 (s, 2H, CH2), 4.97 (br s, 1H, NH), 6.13 (d, J = 5.6 Hz, 1H, pyrimidine), 7.11-7.44 (m, 6H, Ph + Ph’), 8.15 (d, J = 5.6 Hz, 1H, pyrimidine), 9.00 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.52, 20.98, 32.32, 47.58, 52.66, 62.12, 109.36 (2C), 118.81, 119.51, 129.62, 132.41, 133.03, 133.90, 135.14, 153.51, 160.10, 161.85, 168.33 (2C); MS (ESI+) m/z 471 (M+H)+; HRMS calcd for C27H30N6O2 [M+H]+: 471.2508, found: 471.2509; HPLC: tR = 11.29 min, 96.14%。
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操作同上。白色粉状固体；收率87%; 熔点：205.0–205.4 oC; 1H NMR (400 MHz, CDCl3) δ = 1.48-1.98 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.29 (s, 8H, 2CH3 + piperidine), 2.80 (d, J = 10.8 Hz, 2H, piperidine), 3.09 (s, 2H, CH2), 4.96 (br s, 1H, NH), 6.13 (d, J = 5.6 Hz, 1H, pyrimidine), 6.75 (s, 1H, Ph’), 7.18 (s, 2H, Ph’), 7.40 (s, 2H, Ph), 8.15 (d, J = 5.6 Hz, 1H, pyrimidine), 8.97 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.54, 17.82, 32.58, 47.57, 52.79, 62.32, 109.36 (2C), 118.80, 120.88, 126.71, 130.43, 132.42, 133.03, 135.90, 153.49, 160.08, 161.85, 168.23 (2C); MS (ESI+) m/z 485 (M+H)+; HRMS calcd for C28H32N6O2 [M+H]+: 485.2665, found: 485.2660; HPLC: tR = 12.18 min, 97.86%。
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操作同上。白色粉状固体；收率92%; 熔点：225.6–226.4 oC; 1H NMR (400 MHz, CDCl3) δ = 1.48-1.98 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.21 (s, 3H, CH3), 2.24 (s, 3H, CH3), 2.30-2.83 (m, 4H, piperidine), 3.09 (s, 2H, CH2), 4.96 (br s, 1H, NH), 6.13 (d, J = 5.6 Hz, 1H, pyrimidine), 7.06-7.31 (m, 3H, Ph’), 7.40 (s, 2H, Ph), 8.15 (d, J = 5.6 Hz, 1H, pyrimidine), 8.95 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.52, 19.30, 19.99, 32.33, 47.58, 52.66, 62.13, 109.35 (2C), 116.98, 118.81, 120.79, 130.10, 132.41, 132.62, 133.02, 135.40, 137.39, 153.52, 160.09, 161.85, 168.29 (2C); MS (ESI+) m/z 485 (M+H)+; HRMS calcd for C28H32N6O2 [M+H]+: 485.2665, found: 485.2669; HPLC: tR = 12.21 min, 97.80%。
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操作同上。白色粉状固体；收率75%; 熔点：194.2–194.9 oC; 1H NMR (400 MHz, CDCl3) δ = 1.47-2.03 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.30-2.83 (m, 4H, piperidine), 3.09 (s, 2H, CH2), 3.78 (s, 3H, OCH3), 4.98 (br s, 1H, NH), 6.13 (d, J = 5.6 Hz, 1H, pyrimidine), 6.85 (d, J = 8.8 Hz, 2H, Ph’), 7.39 (s, 2H, Ph), 7.44 (d, J = 8.8 Hz, 2H, Ph’), 8.15 (d, J = 5.6 Hz, 1H, pyrimidine), 8.95 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.51, 32.30, 47.61, 52.71, 55.60, 62.04, 109.36 (2C), 114.28, 118.80, 121.19, 130.90, 132.41, 133.03, 153.51, 156.40, 160.09, 161.86, 168.21 (2C); MS (ESI+) m/z 487 (M+H)+; HRMS calcd for C27H30N6O3 [M+H]+: 487.2458, found: 487.2462; HPLC: tR = 10.82 min, 98.23%。
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操作同上。白色粉状固体；收率88%; 熔点：224.7–225.5 oC; 1H NMR (400 MHz, CDCl3) δ = 1.49-2.03 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.30-2.85 (m, 4H, piperidine), 3.14 (s, 2H, CH2), 5.05 (br s, 1H, NH), 6.13 (d, J = 5.6 Hz, 1H, pyrimidine), 7.00-7.14 (m, 3H, Ph’), 7.39 (s, 2H, Ph), 8.15 (d, J = 5.6 Hz, 1H, pyrimidine), 8.34 (t, J = 8.0 Hz, 1H, Ph’), 9.53 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.54, 32.42, 47.64, 52.74, 62.11, 109.35 (2C), 114.97 (d, JC-C-F = 18.8 Hz), 118.82, 121.24 (d, JC-C-F = 22.4 Hz), 124.29 (d, JC3-F = 7.6 Hz), 124.75 (d, JC4-F = 3.6 Hz), 126.33 (d, JC3-F = 10.0 Hz), 132.42, 133.03, 151.28 (d, JC-F = 225.2 Hz), 153.70, 160.07, 161.86, 168.70 (2C); MS (ESI+) m/z 475 (M+H)+; HRMS calcd for C26H27FN6O2 [M+H]+: 475.2258, found: 475.2261; HPLC: tR = 11.43 min, 98.55%。
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操作同上。白色粉状固体；收率75%; 熔点：214.9–215.7 oC; 1H NMR (400 MHz, CDCl3) δ = 1.51-2.07 (m, 5H, piperidine), 2.17 (s, 6H, 2CH3), 2.33-2.86 (m, 4H, piperidine), 3.14 (s, 2H, CH2), 5.00 (br s, 1H, NH), 6.17 (d, J = 5.6 Hz, 1H, pyrimidine), 6.80-7.32 (m, 3H, Ph’), 7.43 (s, 2H, Ph), 7.54 (d, J = 10.8 Hz, 1H, Ph’), 8.19 (d, J = 5.6 Hz, 1H, pyrimidine), 9.18 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.53, 32.32, 47.49, 52.68, 62.05, 107.09 (d, JC-C-F = 26.0 Hz), 109.35 (2C), 111.11 (d, JC-C-F = 21.2 Hz), 114.71 (d, JC4-F = 2.8 Hz), 118.82, 130.26 (d, JC3-F = 9.2 Hz), 132.42, 133.03, 139.21 (d, JC3-F = 10.8 Hz), 153.51, 160.12, 161.93, 164.36 (d, JC-F = 254.0 Hz), 168.67 (2C); MS (ESI+) m/z 475 (M+H)+; HRMS calcd for C26H27FN6O2 [M+H]+: 475.2258, found: 475.2252; HPLC: tR = 11.41 min, 98.46%。
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操作同上。白色粉状固体；收率70%; 熔点：194.4–195.2 oC; 1H NMR (400 MHz, CDCl3) δ = 1.47-2.04 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.30-2.82 (m, 4H, piperidine), 3.10 (s, 2H, CH2), 4.93 (br s, 1H, NH), 6.13 (d, J = 5.6 Hz, 1H, pyrimidine), 6.99 (t, J = 8.8 Hz, 2H, Ph’), 7.40 (s, 2H, Ph), 7.49-7.53 (m, 2H, Ph’), 8.15 (d, J = 5.6 Hz, 1H, pyrimidine), 9.06 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.52, 32.28, 47.57, 52.73, 62.01, 109.37 (2C), 115.88 (d, JC-C-F = 22.4 Hz), 118.80, 121.23 (d, JC3-F = 7.8 Hz), 132.41, 133.04, 133.77 (d, JC4-F = 2.7 Hz), 153.51, 160.13, 160.58 (d, JC-F = 241.8 Hz), 161.85, 168.46 (2C); MS (ESI+) m/z 475 (M+H)+; HRMS calcd for C26H27FN6O2 [M+H]+: 475.2258, found: 475.2252; HPLC: tR = 10.85 min, 98.58%。
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操作同上。白色粉状固体；收率83%; 熔点：215.9–216.7 oC; 1H NMR (400 MHz, CDCl3) δ = 1.54-2.05 (m, 5H, piperidine), 2.16 (s, 6H, 2CH3), 2.31-2.88 (m, 4H, piperidine), 3.17 (s, 2H, CH2), 4.90 (br s, 1H, NH), 6.15 (d, J = 5.6 Hz, 1H, pyrimidine), 7.02-7.41 (m, 5H, Ph + Ph’), 8.17 (d, J = 5.6 Hz, 1H, pyrimidine), 8.46 (d, J = 8.0 Hz, 1H, Ph’), 9.96 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.54, 32.50, 47.67, 52.76, 62.27, 109.37 (2C), 118.81, 120.96, 122.71, 124.56, 127.92, 129.15, 132.43, 133.05, 134.64, 153.54, 160.09, 161.89, 168.81 (2C); MS (ESI+) m/z 491 (M+H)+; HRMS calcd for C26H27ClN6O2 [M+H]+: 491.1962, found: 491.1968; HPLC: tR = 12.14 min, 97.43%。
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操作同上。白色粉状固体；收率84%; 熔点：206.1–206.6 oC; 1H NMR (400 MHz, CDCl3) δ = 1.52-2.08 (m, 5H, piperidine), 2.18 (s, 6H, 2CH3), 2.34-2.86 (m, 4H, piperidine), 3.15 (s, 2H, CH2), 4.98 (br s, 1H, NH), 6.18 (d, J = 5.6 Hz, 1H, pyrimidine), 7.10-7.67 (m, 6H, Ph + Ph’), 8.20 (d, J = 5.6 Hz, 1H, pyrimidine), 9.17 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.53, 32.31, 47.54, 52.72, 62.06, 109.38 (2C), 117.46, 118.81, 119.53, 124.33, 130.18, 132.42, 133.04, 134.79, 138.82, 153.52, 160.11, 161.86, 168.65 (2C); MS (ESI+) m/z 491 (M+H)+; HRMS calcd for C26H27ClN6O2 [M+H]+: 491.1962, found: 491.1959; HPLC: tR = 11.95 min, 97.44%。
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操作同上。白色粉状固体；收率86%; 熔点：204.9–205.5 oC; 1H NMR (400 MHz, CDCl3) δ = 1.47-2.04 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.30-2.82 (m, 4H, piperidine), 3.10 (s, 2H, CH2), 4.93 (br s, 1H, NH), 6.13 (d, J = 5.6 Hz, 1H, pyrimidine), 7.27 (d, J = 8.8 Hz, 2H, Ph’), 7.39 (s, 2H, Ph), 7.49 (d, J = 8.8 Hz, 2H, Ph’), 8.15 (d, J = 5.6 Hz, 1H, pyrimidine), 9.10 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.52, 32.30, 47.55, 52.72, 62.04, 109.37 (2C), 118.80, 120.69, 129.14, 129.20, 132.41, 133.04, 136.27, 153.52, 160.13, 161.84, 168.57 (2C); MS (ESI+) m/z 491 (M+H)+; HRMS calcd for C26H27ClN6O2 [M+H]+: 491.1962, found: 491.1957; HPLC: tR = 11.62 min, 98.85%。
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操作同上。白色粉状固体；收率88%; 熔点：186.9–187.8 oC; 1H NMR (400 MHz, CDCl3) δ = 1.51-2.04 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.30-2.86 (m, 4H, piperidine), 3.15 (s, 2H, CH2), 4.91 (br s, 1H, NH), 6.14 (d, J = 5.6 Hz, 1H, pyrimidine), 7.22-7.40 (m, 4H, Ph + Ph’), 8.15 (d, J = 5.6 Hz, 1H, pyrimidine), 8.43 (d, J = 8.8 Hz, 1H, Ph’), 9.95 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.54, 32.50, 47.67, 52.76, 62.27, 109.37 (2C), 118.81, 121,04, 127.03, 128.91, 130.63, 131.35, 132.43, 132.76, 133.05, 153.54, 160.09, 161.89, 168.81 (2C); MS (ESI+) m/z 525 (M+H)+; HRMS calcd for C26H26Cl2N6O2 [M+H]+: 525.1573, found: 525.1567; HPLC: tR = 13.21 min, 97.46%。
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操作同上。白色粉状固体；收率85%; 熔点：235.5–236.3 oC; 1H NMR (400 MHz, CDCl3) δ = 1.47-2.04 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.30-2.81 (m, 4H, piperidine), 3.11 (s, 2H, CH2), 4.94 (br s, 1H, NH), 6.14 (d, J = 5.6 Hz, 1H, pyrimidine), 7.35-7.75 (m, 5H, Ph + Ph’), 8.15 (d, J = 5.6 Hz, 1H, pyrimidine), 9.15 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.52, 32.25, 47.54, 52.73, 61.98, 109.37 (2C), 118.73, 118.80, 121,10, 127.39, 130.66, 132.41, 132.90, 133.04, 137.16, 153.52, 160.12, 161.84, 168.71 (2C); MS (ESI+) m/z 525 (M+H)+; HRMS calcd for C26H26Cl2N6O2 [M+H]+: 525.1573, found: 525.1567; HPLC: tR = 12.62 min, 98.27%。
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操作同上。白色粉状固体；收率79%; 熔点：237.5–238.0 oC; 1H NMR (400 MHz, CDCl3) δ = 1.48-2.04 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.30-2.81 (m, 4H, piperidine), 3.11 (s, 2H, CH2), 4.93 (br s, 1H, NH), 6.14 (d, J = 5.6 Hz, 1H, pyrimidine), 7.08 (s, 1H, Ph’), 7.40 (s, 2H, Ph), 7.51 (s, 2H, Ph’), 8.15 (d, J = 5.6 Hz, 1H, pyrimidine), 9.17 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.52, 32.26, 47.52, 52.74, 61.97, 109.37 (2C), 117.68, 118.80, 124.21, 132.41, 133.04, 135.39, 139.45, 153.52, 160.11, 161.85, 168.79 (2C); MS (ESI+) m/z 525 (M+H)+; HRMS calcd for C26H26Cl2N6O2 [M+H]+: 525.1573, found: 525.1568; HPLC: tR = 13.08 min, 98.02%。
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操作同上。白色粉状固体；收率85%; 熔点：202.8–203.4 oC; 1H NMR (400 MHz, CDCl3) δ = 1.50-2.04 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.30-2.96 (m, 4H, piperidine), 3.18 (s, 2H, CH2), 4.89 (br s, 1H, NH), 6.12 (d, J = 5.6 Hz, 1H, pyrimidine), 7.39-7.40 (m, 4H, Ph + Ph’), 8.15 (d, J = 5.6 Hz, 1H, pyrimidine), 8.93 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.52, 32.28, 47.59, 52.91, 61.69, 109.36 (2C), 118.78, 128.59, 130.96, 132.41, 133.02, 133.33, 133.88, 153.49, 160.08, 161.88, 168.73 (2C); MS (ESI+) m/z 559 (M+H)+; HRMS calcd for C26H25Cl3N6O2 [M+H]+: 559.1183, found: 559.1187; HPLC: tR = 11.85 min, 96.83%。
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操作同上。白色粉状固体；收率89%; 熔点：203.3–204.1 oC; 1H NMR (400 MHz, CDCl3) δ = 1.47-2.04 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.30-2.82 (m, 4H, piperidine), 3.10 (s, 2H, CH2), 4.92 (br s, 1H, NH), 6.13 (d, J = 5.6 Hz, 1H, pyrimidine), 7.39-7.47 (m, 6H, Ph + Ph’), 8.15 (d, J = 5.6 Hz, 1H, pyrimidine), 9.10 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.52, 32.28, 47.53, 52.71, 62.05, 109.36 (2C), 116.78, 118.80, 121.02, 132.08, 132.40, 133.03, 136.76, 153.50, 160.12, 161.84, 168.58 (2C); MS (ESI+) m/z 535 (M+H)+; HRMS calcd for C26H27BrN6O2 [M+H]+: 535.1457, found: 535.1459; HPLC: tR = 11.85 min, 98.16%。
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操作同上。白色粉状固体；收率73%; 熔点：218.6–219.2 oC; 1H NMR (400 MHz, CDCl3) δ = 1.47-2.03 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.30-2.82 (m, 4H, piperidine), 3.13 (s, 2H, CH2), 4.93 (br s, 1H, NH), 6.14 (d, J = 5.6 Hz, 1H, pyrimidine), 7.39 (s, 2H, Ph), 7.59 (d, J = 8.4 Hz, 2H, Ph’), 7.67 (d, J = 8.4 Hz, 2H, Ph’), 8.15 (d, J = 5.6 Hz, 1H, pyrimidine), 9.35 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.51, 32.25, 47.50, 52.69, 62.04, 107.22, 109.37 (2C), 118.79, 118.93, 119.37, 132.39, 133.04, 133.43, 141.59, 153.52, 160.13, 161.82, 169.06 (2C); MS (ESI+) m/z 504 (M+Na)+; HRMS calcd for C27H27N7O2 [M+H]+: 482.2304, found: 482.2309; HPLC: tR = 10.72 min, 97.64%。
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操作同上。白色粉状固体；收率78%; 熔点：227.1–227.8 oC; 1H NMR (400 MHz, CDCl3) δ = 1.48-2.04 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.30-2.81 (m, 4H, piperidine), 3.17 (s, 2H, CH2), 4.93 (br s, 1H, NH), 6.15 (d, J = 5.6 Hz, 1H, pyrimidine), 7.37-7.46 (m, 4H, Ph + Ph’), 8.15 (d, J = 5.6 Hz, 1H, pyrimidine), 8.57 (t, J = 8.2 Hz, 1H, Ph’), 9.85 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.52, 32.41, 47.52, 52.74, 62.00, 106.78 (d, JC3-F = 9.3 Hz), 109.36 (2C), 118.42 (d, JC-C-F = 54.1 Hz), 118.65, 118.80, 121.09 (d, JC-C-F = 24.5 Hz), 129.71 (d, JC4-F = 3.5 Hz), 131.08 (d, JC3-F = 9.8 Hz), 132.41, 133.04, 150.03 (d, JC-F = 245.1 Hz), 153.54, 160.12, 161.85, 169.24 (2C); MS (ESI+) m/z 522 (M+Na)+; HRMS calcd for C27H26FN7O2 [M+H]+: 500.2210, found: 500.2208; HPLC: tR = 11.27 min, 97.67%。
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操作同上。白色粉状固体；收率78%; 熔点：246.9–247.6 oC; 1H NMR (400 MHz, CDCl3) δ = 1.65-2.04 (m, 5H, piperidine), 2.15 (s, 6H, 2CH3), 2.30-2.86 (m, 4H, piperidine), 3.18 (s, 2H, CH2), 4.83 (m, 1H, NH), 6.14 (d, J = 5.6 Hz, 1H, pyrimidine), 7.16 (t, J = 7.8 Hz, 1H, Ph’), 7.40 (s, 2H, Ph), 7.62 (t, J = 7.8 Hz, 1H, Ph’), 8.17 (d, J = 5.6 Hz, 1H, pyrimidine), 8.21 (d, J = 8.4 Hz, 1H, Ph’), 8.87 (d, J = 8.4 Hz, 1H, Ph’),11.94 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.56, 32.15, 47.71, 53.05, 62.56, 109.34 (2C), 118.88, 121.96, 123.32, 125.92, 132.39, 132.44, 132.47, 133.06, 134.62, 153.54, 160.16, 161.87, 170.66 (2C); MS (ESI+) m/z 524 (M+Na)+; HRMS calcd for C26H27N7O4 [M+H]+: 502.2203, found: 502.2198; HPLC: tR = 11.75 min, 97.88%。
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操作同上。白色粉状固体；收率76%; 熔点：240.1–240.8 oC; 1H NMR (400 MHz, CDCl3) δ = 1.44-1.91 (m, 5H, piperidine), 2.08 (s, 6H, 2CH3), 2.76-3.12 (m, 4H, piperidine), 3.19 (s, 2H, CH2), 4.85 (m, 1H, NH), 6.21 (d, J = 5.6 Hz, 1H, pyrimidine), 7.57-8.19 (m, 6H, Ph + Ph’), 8.67 (d, J = 5.6 Hz, 1H, pyrimidine), 10.22 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.57, 32.14, 47.75, 53.03, 62.55, 109.36 (2C), 114.54, 118.86, 119.56, 127.74, 129.82, 132.44, 133.06, 139.47, 148.13, 153.54, 160.16, 161.87, 170.64 (2C); MS (ESI+) m/z 524 (M+Na)+; HRMS calcd for C26H27N7O4 [M+H]+: 502.2203, found: 502.2197; HPLC: tR = 11.49 min, 97.24%。
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操作同上。白色粉状固体；收率90%; 熔点：234.5–235.1 oC; 1H NMR (400 MHz, CDCl3) δ = 1.49-2.04 (m, 5H, piperidine), 2.14 (s, 6H, 2CH3), 2.30-2.82 (m, 4H, piperidine), 3.16 (s, 2H, CH2), 4.93 (m, 1H, NH), 6.15 (d, J = 5.6 Hz, 1H, pyrimidine), 7.40 (s, 2H, Ph), 7.72 (d, J = 9.2 Hz, 2H, Ph’), 8.16 (d, J = 5.6 Hz, 1H, pyrimidine), 8.20 (d, J = 9.2 Hz, 2H, Ph’), 9.48 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.52, 32.28, 47.48, 52.77, 62.05, 109.38 (2C), 118.80, 118.93, 125.26, 132.41, 133.05, 143.40, 143.62, 153.52, 160.11, 161.82, 169.16 (2C); MS (ESI+) m/z 524 (M+Na)+; HRMS calcd for C26H27N7O4 [M+H]+: 502.2203, found: 502.2200; HPLC: tR = 11.13 min, 97.62%。
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操作同上。白色粉状固体；收率93%; 熔点：242.9–243.6 oC; 1H NMR (400 MHz, CDCl3) δ = 1.42-2.05 (m, 5H, piperidine), 2.15 (s, 6H, 2CH3), 2.24-2.80 (m, 4H, piperidine), 3.09 (s, 2H, CH2), 4.93 (m, 1H, NH), 6.14 (d, J = 5.6 Hz, 1H, pyrimidine), 6.79-7.40 (m, 6H, Ph + Ph’), 8.16 (d, J = 5.6 Hz, 1H, pyrimidine), 8.96 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.53, 32.27, 47.46, 52.75, 62.07, 109.34 (2C), 116.14, 118.83, 123.04, 131.13, 132.40, 133.04, 153.55, 154.15, 160.15, 161.84, 169.17 (2C); MS (ESI+) m/z 595 (M+Na)+; HRMS calcd for C26H28N6O3 [M+H]+: 473.2301, found: 473.2305; HPLC: tR = 10.25 min, 98.61%。
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操作同上。白色粉状固体；收率94%; 熔点：228.8–229.7 oC; 1H NMR (400 MHz, CDCl3) δ = 1.42-1.92 (m, 5H, piperidine), 2.08 (s, 6H, 2CH3), 2.76-3.11 (m, 4H, piperidine), 3.06 (s, 2H, CH2), 4.95 (m, 1H, NH), 6.22 (d, J = 5.6 Hz, 1H, pyrimidine), 7.26 (s, 2H, SO2NH2), 7.67 (s, 2H, Ph), 7.74-7.80 (m, 4H, Ph’), 8.19 (d, J = 5.6 Hz, 1H, pyrimidine), 10.04 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 16.52, 32.28, 47.48, 52.77, 62.05, 109.38 (2C), 188.04, 118.80, 129.43, 132.41, 133.05, 136.52, 141.73, 153.52, 160.11, 161.82, 169.16 (2C); MS (ESI+) m/z 558 (M+Na)+; HRMS calcd for C26H29N7O4S [M+H]+: 536.2080, found: 536.2079; HPLC: tR = 9.11 min, 97.65%。
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操作同上。白色粉状固体；收率86%; 熔点：261.3–261.9 oC; 1H NMR (400 MHz, CDCl3) δ = 1.44-1.98 (m, 5H, piperidine), 2.08 (s, 6H, 2CH3), 2.31 (s, 3H, CH3), 2.85-3.12 (m, 4H, piperidine), 3.08 (s, 2H, CH2), 4.97 (m, 1H, NH), 6.24 (d, J = 5.6 Hz, 1H, pyrimidine), 7.25 (s, 2H, SO2NH2), 7.63-8.12 (m, 5H, Ph + Ph’), 8.20 (d, J = 5.6 Hz, 1H, pyrimidine), 9.66 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ = 15.87, 17.41, 31.57, 47.79, 52.47, 61.55, 108.15 (2C), 111.11, 188.66, 120.11, 124.23, 127.65, 132.19, 132.70, 134.64, 139.05, 153.25, 160.54, 161.63, 168.63 (2C); MS (ESI+) m/z 572 (M+Na)+; HRMS calcd for C27H31N7O4S [M+H]+: 550.2236, found: 550.2240; HPLC: tR = 9.61 min, 97.49%。
实施例2：抗HIV生物活性测试
体外细胞水平的抗HIV病毒活性由比利时Katholleke大学的Rega药物研究所测定，主要包括：对HIV感染的MT-4细胞的抑制活性及细胞毒性两方面。方法如下：使化合物在HIV感染的MT-4细胞中，于感染HIV不同时间，用MTT法测定药物对HIV诱变的细胞病变的保护作用，计算使50%的细胞免于HIV诱导的细胞病变所需的浓度半数有效浓度EC50，毒性测定与抗HIV活性实验平行进行，也是在MT-4细胞培养中，用MTT法测定使50%的未感染细胞发生细胞病变的浓度（CC50），并计算选择性指数SI = CC50/EC50。
材料与方法：
各化合物的抗HIV活性由药物对HIV在细胞中引起的细胞病变的抑制作用效率来监控。采用MT-4细胞进行细胞培养。采用的病毒株有：HIV-1病毒株ⅢB及HIV-2病毒株ROD。
具体操作如下：将化合物用DMSO或水溶解后在磷酸盐缓冲食盐水溶液稀释，将3×105 MT-4细胞用100 μL各化合物不同浓度此溶液在37 oC预培养1h，然后向该化合物中加入100 μL适当的病毒稀释液，将细胞于37 oC培养1h。洗涤三次后，将细胞再次分别悬浮于含有或不含有化合物的培养介质中。接着将细胞在5% CO2氛围中，于37 oC下再培养7天，并于感染后第三天用含有或不含有化合物的培养介质替换补充培养液。每种培养液条件重复操作两次。对病毒的细胞病变作用每天都用反向光学显微镜监控。典型来讲，本实验中所用的病毒稀释液常常会在病毒感染后第五天导致细胞病变。药物抑制浓度以药物对病毒细胞病变作用产生50%抑制作用而同时对细胞无直接毒性的浓度（CC50）表示。需要强调的是，当化合物水溶性较差，需要DMSO才能溶解时，DMSO比浓度相对于水来讲，一般低于10%（DMSO在MT-4细胞培养介质中最终浓度小于2%）。因为DMSO能影响测试化合物的抗病毒活性，对含有相同浓度DMSO溶液抗病毒活性对比空白实验也应该平行进行。另外，DMSO最终浓度（1/1000）远远低于HIV-1在T细胞中复制所需的浓度。
本发明用已上市药物德拉韦定（Delavirdine, DEV）、依非韦伦（Efavirenz, EFV）和依曲韦林（Etravirine, ETV）作对照品，部分目标化合物对HIV的抑制活性结果见表1 (Anti-HIV activity and cytotoxicity of compounds 1-26 in MT-4 cells)。
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表1
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a All data represent mean values for at least three separate experiments.
b EC50: effective concentration required to protect the cell against viral cytopathicity by 50% in MT-4 cells.
c CC50: cytotoxic concentration of compound that reduces the normal uninfected MT-4 cell viability by 50%.
d SI: selectivity index, ratio CC50/EC50 (WT)。
实验结果表明，化学通式中所包含的化合物普遍具有较强的抗HIV-1病毒活性，较小的细胞毒性和较高的选择性指数。
本发明不限于上述实例。