含吲哚环2, 4-二氨基嘧啶类化合物的合成及抗肿瘤活性

引用本文: 黎秋, 汪雨, 胡孟金, 陈鹏, 游文玮, 赵培亮. 含吲哚环2, 4-二氨基嘧啶类化合物的合成及抗肿瘤活性[J]. 有机化学, 2017, 37(4): 967-974. doi: 10.6023/cjoc201611006 shu

Citation: Li Qiu, Wang Yu, Hu Mengjin, Chen Peng, You Wenwei, Zhao Peiliang. Synthesis and Biological Activities of Novel 2, 4-Diaminopyrimidine Derivatives Bearing Indole Moiety[J]. Chinese Journal of Organic Chemistry, 2017, 37(4): 967-974. doi: 10.6023/cjoc201611006 shu

含吲哚环2, 4-二氨基嘧啶类化合物的合成及抗肿瘤活性

南方医科大学药学院广东省新药筛选重点实验室广州 510515

通讯作者: 赵培亮, E-mail: plzhao@smu.edu.cn

基金项目:
国家自然科学基金 21102069

广州市珠江科技新星专项 2012J2200051

国家自然科学基金 21372113

摘要: 在课题组前期研究的基础上，设计、合成了23个未见文献报道的含吲哚结构单元的2，4-二氨基嘧啶类化合物，其结构经¹H NMR，¹³C NMR及HRMS确认.并采用噻唑蓝（MTT）法测试了目标化合物对人子宫颈癌细胞（HeLa）、乳腺癌细胞（MD-MBA-231）、前列腺癌细胞（PC-3）、人结肠癌细胞（HCT116）的体外抗肿瘤活性.结果表明，大部分化合物具有一定的肿瘤抑制活性.尤其是2-[5-硝基-2-（6-氯-2-苯甲酰基-1H-吲哚-3-胺基）嘧啶-4-胺基]乙酸乙酯（8j）对HCT116、MD-MBA-231的体外肿瘤抑制活性比阳性对照药品5-氟尿嘧啶分别高出2.0、0.5倍，体现出了较好的进一步研究价值.

关键词:

English

Synthesis and Biological Activities of Novel 2, 4-Diaminopyrimidine Derivatives Bearing Indole Moiety

Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutica Science, Southern Medical University, Guangzhou 510515

Corresponding author: Zhao Peiliang, plzhao@smu.edu.cn

Received Date: 03 November 2016
Revised Date: 19 December 2016
Available Online: 25 April 2017
Fund Project:
the National Natural Science Foundation of China

the Project of Science and Technology New Star in Zhujiang Guangzhou City

the National Natural Science Foundation of China

Abstract: Based on our previous work, twenty-three novel 2, 4-diaminopyrimidine derivatives bearing indole moiety were designed and synthesized. Structures of all compounds were elucidated by ¹H NMR, ¹³C NMR and HRMS. Antiproliferative activities for all these compounds were evaluated by the method of methyl thiazolyl tetrazolium (MTT) assay against four cancer cell lines (HeLa, MD-MBA-231, PC-3 and HCT116), and the results demonstrated that some compounds possessed significant antitumor activities in vitro. Particularly, the most promising ethyl (2-((2-benzoyl-5-chloro-1H-indol-3-yl) amino)-5-nitropyrimidin-4-yl) glycinate (8j) displayed 2.0-and 0.5-fold improvement compared to fluorouracil in inhibiting HCT116, and MD-MBA-231 cell proliferation with IC₅₀ values of 23.15 and 36.88 mmol/L, respectively. These findings suggest that compound 8j may have potential to be developed as a promising lead for the design of novel anticancer small-molecule drugs.

Key words:

2, 4-二氨基嘧啶是一类具有广泛生物活性的含氮杂环化合物, 在药物研发和生物学等方面发挥着非常重要的作用^{[1, 2]}.近年来, 2, 4-二氨基嘧啶衍生物的优异抗肿瘤活性更是吸引了许多药物化学研究者的关注^{[3, 4]}, 不断有新型的2, 4-二氨基嘧啶类化合物被研究和报道, 多个化合物已处于临床或临床前研究阶段, 更有一些化合物已被成功开发为商品化抗癌药物, 如诺华公司研发并于2014年成功上市的用于治疗非小细胞肺癌的色瑞替尼, 以及辉瑞研发的于2015年底获得食品药品监督管理局 (FDA) 上市批准的抗癌药物Palbociclib, 其作为全球首个CDK4/6抑制剂, 被FDA授予突破性疗法认定.因此, 有关2, 4-二氨基嘧啶类化合物的分子设计、合成及抗肿瘤活性的研究是当前抗肿瘤新药创制的热点之一^[5~12].

为了寻找具有高效抗肿瘤活性的先导化合物, 我们课题组在前期工作中^[13~15], 分别合成并报道了一系列含哌啶、哌嗪及长链烷酯基取代的2, 4-二氨基-5-硝基嘧啶衍生物, 发现了一批具有良好抗肿瘤活性的化合物.尤其是烷酯基取代的2, 4-二氨基-5-硝基嘧啶类化合物A, 能抑制多种癌细胞, 且对正常细胞没有明显损伤.此外, 吲哚及其衍生物也是一类具有广泛生物活性的药效基团, 如抗肿瘤^[16~18]、抗菌^[19]、抗惊厥^[20]、抗炎^[21]和糖尿病^[22]等, 因而备受关注.基于以上考虑, 为了进一步丰富2, 4-二氨基-5-硝基嘧啶衍生物的结构类型, 深入研究其生物活性及构效关系规律, 本文以前期发现的具有较强肿瘤抑制活性的化合物A为先导结构, 依据生物活性叠加原理, 将具有广泛生物活性的功能团3-氨基-2-芳甲酰基吲哚引入到2, 4-二氨基-5-硝基嘧啶衍生物中, 设计、合成了23个未见文献报道的含吲哚环的2, 4-二氨基嘧啶类化合物 (Eq. 1), 其结构经¹H NMR, ¹³C NMR以及HRMS的分析确认.目标化合物的合成路线如Scheme 1所示.

图图式1 目标化合物的合成 Figure 图式1. Synthetic routes of the title compounds

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1 结果与讨论

1.1 合成

文献报道的3-氨基-1H-吲哚类化合物的合成方法较少.本文采用邻氨基苯腈为起始原料, 在无溶剂条件下与氯甲酸乙酯进行回流反应6 h, 反应完毕后减压蒸馏除去过量的氯甲酸乙酯, 以93%的收率得到关键中间体N-(2-氰基苯基) 氨基甲酸乙酯.在无水碳酸钾存在下, 以无水N, N-二甲基甲酰胺 (DMF) 为溶剂, 该中间体顺利与α-溴代芳基乙酮在室温下进行Thorpe-Ziegler环化反应高收率地制得了3-氨基-1-乙酯基-2-苯甲酰基吲哚, 最后在乙醇溶剂中, 以10% NaOH为碱经酰胺水解反应得到一系列3-氨基-2-取代芳甲酰基-1H-吲哚类化合物4.以新炒碳酸钾为缚酸剂, 2, 4-二氯-5-硝基嘧啶与甘氨酸乙酯盐酸盐在无水乙醇为溶剂中进行亲核取代反应, 以84%的收率顺利得到的中间体2-氯-5-硝基-4-取代氨基嘧啶.而后, 在乙二醇甲醚溶剂中, 中间体3-氨基吲哚4和2-氯-5-硝基-4-取代氨基嘧啶取代缩合, 经丙酮重结晶, 得目标化合物8a~8w.

1.2 抗肿瘤活性

为了研究目标化合物8a~8w的抗肿瘤活性, 我们将人子宫颈癌细胞 (Hela)、乳腺癌细胞 (MD-MBA-231)、前列腺癌细胞 (PC-3) 以及人结肠癌细胞 (HCT 116) 用不同浓度的化合物处理48 h.细胞存活率由MTT实验确定.阳性对照药物为5-氟尿嘧啶 (5-Fu).测试结果如表 1所示.

表 1 化合物8a~8w对Hela、MD-MBA-231、PC-3和HCT116的IC₅₀值 (μmol/L) Table 1. IC₅₀^a (μmol/L) values of target compounds 8a~8w for Hela, MD-MBA-231, PC-3 and HCT116

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Compd.	Hela	MDA-MB-231	PC-3	HCT 116
8a	70.63	106.80	150.36	70.51
8b	＞200	113.80	＞200	＞200
8c	115.32	＞200	＞200	87.25
8d	＞200	155.2	123.69	76.84
8e	＞200	＞200	＞200	186.34
8f	＞200	＞200	＞200	153.69
8g	108.32	＞200	236.15	＞200
8h	＞200	＞200	＞200	＞200
8i	＞200	＞200	＞200	＞200
8j	60.69	36.88	80.69	23.15
8k	＞200	＞200	＞200	＞200
8l	＞200	＞200	＞200	＞200
8m	60.03	＞200	＞200	153.72
8n	＞200	＞200	＞200	＞200
8o	＞200	＞200	＞200	＞200
8p	108.54	103.30	156.32	60.35
8q	116.85	78.06	100.26	53.21
8r	＞200	＞200	＞200	＞200
8s	＞200	135.81	＞200	＞200
8t	＞200	＞200	＞200	＞200
8u	＞200	＞200	＞200	145.98
8v	93.52	145.69	＞200	68.69
8w	＞200	＞200	＞200	＞200
5-Fu	57.17	53.47	57.04	68.71
^a IC₅₀ values mean of three experiments in replicate.

表 1 化合物8a~8w对Hela、MD-MBA-231、PC-3和HCT116的IC₅₀值 (μmol/L)
Table 1. IC₅₀^a (μmol/L) values of target compounds 8a~8w for Hela, MD-MBA-231, PC-3 and HCT116

从表 1可见, 所合成的化合物对4种人肿瘤细胞的体外生长显示出不同的抑制作用.对于人前列腺癌细胞 (PC-3), 大部分化合物没有表现出显著的肿瘤抑制活性.而对于子宫颈癌细胞 (Hela), 部分化合物 (如化合物8a、8j、8m) 表现出了与阳性对照药品5-氟尿嘧啶相当的抑制活性.对于人乳腺癌细胞 (MD-MBA-231), 化合物8j体现出了明显优于对照药品的体外肿瘤抑制活性.更值得关注的是, 所合成的大部分目标化合物对人结肠癌细胞 (HCT 116) 显示了明显的抑制活性, 其中化合物8j、8p、8q、8v体现了优于5-氟尿嘧啶的抗肿瘤活性, 而化合物8a、8c、8d也显示了与5-氟尿嘧啶处于同一水平的抑制活性.此外, 比较化合物8a与8b~8i的对Hela、MD-MBA-231、PC-3和HCT116的IC₅₀值可以发现, 当在吲哚环-2位的苯环 (R²) 上引入取代基时, 其肿瘤抑制活性均明显降低.而通过分别比较化合物8j与8k~8o, 8q与8r~8u也可以得到同样的结论.说明处于吲哚环-2位的苯环上的取代基对该类化合物的抗肿瘤活性有重要影响.

2 结论

本文设计、合成了23个结构新颖的含吲哚结构单元的2, 4-二氨基嘧啶类化合物, 结构均经¹H NMR、¹³C NMR和HRMS确证.采用MTT法测试了目标产物对肿瘤细胞HeLa、MD-MBA-231、PC-3和HCT116的增殖抑制活性, 结果表明, 部分目标化合物显示了较好的抑制肿瘤细胞增殖活性.尤其是化合物8j对HCT116、MD-MBA-231的体外肿瘤抑制活性比阳性对照药品5-氟尿嘧啶分别高出2.0、0.5倍, 体现出进一步深入研究的潜力.

3 实验部分

3.1 仪器与试剂

熔点用Electrothermal数字熔点仪测定, 温度计未经校正; ¹H NMR和¹³C NMR以DMSO-d₆为溶剂, MERCURY-PLUS400型核磁共振仪测定, 内标为TMS; 高分辨质谱由超高解析度四级杆-飞行时间质谱仪 (agilent 6540) 测定; 薄层层析硅胶用青岛海洋化工厂有限公司生产的GF₂₅₄60型.所有试剂均为分析纯或化学纯, 由进口或国产, 使用前经常规处理. α-溴代芳基乙酮按文献[23]制备.

3.2 中间体的合成

3.3 目标化合物8a~8w的合成

向100 mL单颈圆底烧瓶中, 加入0.256 g (1 mmol) 中间体4和0.26 g (1 mmol) 中间体7及30 mL乙二醇甲醚, 加热至60 ℃, 搅拌, TLC监测.反应完成后, 缓慢倾入水中, 用乙酸乙酯萃取, 干燥, 减压蒸除溶剂, 丙酮重结晶, 得目标化合物8a~8w.

2-[5-硝基-2-(2-苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8a):黄色固体, 收率85.3%. m.p. 212.3~214.5 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.93 (s, 3H, OCH₂CH₃), 3.71~3.74 (m, 2H, OCH₂CH₃), 3.88 (s, 2H, NHCH₂CO₂), 7.07 (t, J＝8.0 Hz, 1H, ArH), 7.30~7.34 (m, 3H, ArH), 7.47~7.53 (m, 3H, ArH), 7.66 (d, J＝8.0 Hz, 2H, ArH), 8.76 (s, 1H, Pyrimidine-H), 8.86 (t, J＝6.0 Hz, 1H, NHCH₂CO₂), 10.07 (s, 1H, NH), 11.80 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 188.0, 169.4, 161.0, 157.7, 155.5, 138.3, 136.1, 132.6, 129.1, 128.3, 125.9, 123.6, 121.2, 120.8, 120.3, 118.9, 113.1, 60.7, 42.3, 14.1. HRMS (ESI) calcd for C₂₃H₂₁N₆O₅ (M+H⁺) 461.1495, found 461.1853.

2-[5-硝基-2-(2-对溴苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8b):黄色固体, 收率57.1%. m.p. 198.5~201.6 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.93 (s, 3H, OCH₂CH₃), 3.71~3.74 (m, 2H, OCH₂CH₃), 3.86 (s, 2H, NHCH₂), 7.08 (t, J＝8.0 Hz, 1H, ArH), 7.33 (t, J＝8.0 Hz, 1H, ArH), 7.48 (d, J＝8.0 Hz, 1H, ArH), 7.55 (dd, J＝16.0, 8.0 Hz, 5H, ArH), 8.81 (s, 1H, Pyrimidine-H), 8.89 (t, J＝6.0 Hz, 1H, NHCH₂CO₂), 10.07 (s, 1H, NH), 11.84 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 186.9, 170.7, 169.4, 157.7, 155.5, 137.4, 136.2, 131.4, 131.0, 130.9, 127.3, 126.3, 126.1, 121.0, 121.3, 120.4, 113.1, 60.3, 42.3, 14.1. HRMS (ESI) calcd for C₂₃H₂₀-BrN₆O₅ (M+H⁺) 540.3382, found 541.0727.

2-[5-硝基-2-(2-对甲氧基苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8c):产率83.4%. m.p. 206~208.8 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.93 (s, 3H, OCH₂CH₃), 3.78 (s, 5H, OCH₂CH₃, OCH₃), 3.88 (s, 2H, NHCH₂CO₂), 6.87 (d, J＝8.0 Hz, 2H, ArH), 7.07 (t, J＝8.0 Hz, 1H, ArH), 7.29~7.52 (m, 3H, ArH), 7.69 (d, J＝12.0 Hz, 2H, ArH), 8.82 (s, 1H, Pyrimidine-H), 8.88 (t, J＝6.0 Hz, 1H, NHCH₂CO₂), 10.08 (s, 1H, NH), 11.74 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 186.7, 169.4, 163.0, 160.9 157.7, 155.5, 135.8, 131.6, 130.9, 125.5, 123.6, 121.1, 120.2, 113.7, 113.0, 60.7, 55.9, 42.3, 14.1. HRMS (ESI) calcd for C₂₄H₂₃N₆O₆ (M+H⁺) 491.4681, found 491.1944.

2-[5-硝基-2-(2-对三氟甲基苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8d):黄色固体, 收率49.1%. m.p. 201.7~204.3 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.93 (s, 3H, OCH₂CH₃), 3.74 (s, 2H, OCH₂CH₃), 3.86 (s, 2H, NHCH₂CO₂), 7.09 (t, J＝8.0 Hz, 1H, ArH), 7.35 (t, J＝8.0 Hz, 1H, ArH), 7.52 (dd, J＝8.0, 24.0 Hz, 2H, ArH), 7.67 (d, J＝8.0 Hz, 2H, ArH), 7.79 (d, J＝8.0 Hz, 2H, ArH), 8.72 (s, 1H, Pyrimidine-H), 8.88 (s, 1H, NHCH₂-CO₂), 10.05 (s, 1H, NH), 11.91 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 186.9, 169.4, 161.2, 157.7, 155.4, 142.0, 136.5, 131.6, 129.6, 127.2, 126.5, 125.5, 125.2, 123.7, 122.8, 121.3, 120.5, 113.2, 60.8, 42.3, 14.1. HRMS (ESI) calcd for C₂₄H₂₀F₃N₆O₅ (M+H⁺) 529.1369 found 529.1661.

2-[5-硝基-2-(2-对甲基苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8e):黄色固体, 收率84.0%. m.p. 225.1~226.3 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.89 (s, 3H, OCH₂CH₃), 2.31 (s, 3H, CH₃), 3.74 (s, 2H, OCH₂CH₃), 3.87 (s, 2H, NHCH₂CO₂), 7.07 (t, J＝8.0 Hz, 1H, ArH), 7.15 (d, J＝8.0 Hz, 2H, ArH), 7.31 (t, J＝8.0 Hz, 1H, ArH), 7.50 (dd, J＝8.0, 16.0 Hz, 2H, ArH), 7.59 (d, J＝8.0 Hz, 2H, ArH), 8.80 (s, 1H, Pyrimidine-H), 8.87 (t, J＝6.0 Hz, 1H, NHCH₂CO₂), 10.05 (s, 1H, NH), 11.76 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 187.7, 169.4, 161.0, 157.7, 155.5, 142.9, 136.0, 135.7, 129.4, 128.9, 125.7, 121.1, 120.2, 113.1, 60.7, 42.3, 21.5, 14.1. HRMS (ESI) calcd for C₂₄H₂₃N₆O₅ (M+H⁺) 475.1652 found 475.1923.

2-[5-硝基-2-(2-对氯苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8f):黄色固体, 收率70.1%. m.p. 229.1~230.4 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.90 (s, 3H, OCH₂CH₃), 3.75 (s, 2H, OCH₂CH₃), 3.87 (s, 2H, NHCH₂CO₂), 7.08 (t, J＝8.0 Hz, 1H, ArH), 7.33 (dd, J＝6.0, 10.0 Hz, 1H, ArH), 7.40 (d, J＝8.0 Hz, 2H, ArH), 7.51 (dd, J＝8.0, 16.0 Hz, 2H, ArH), 7.66 (d, J＝8.0 Hz, 2H, ArH), 8.81 (s, 1H, Pyrimidine-H), 8.88 (t, J＝6.0 Hz, NHCH₂CO₂), 10.07 (s, 1H, NH), 11.84 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 186.7, 169.4, 161.1, 157.7, 155.5, 137.3, 137.1, 136.3, 130.9, 128.4, 127.3, 126.1, 123.6, 121.3, 121.1, 120.4, 119.3, 113.1, 60.8, 42.3, 14.2. HRMS (ESI) calcd for C₂₃H₂₀ClN₆O₅ (M+H⁺) 495.1105, found 495.1356.

2-{5-硝基-2-[2-(3, 4-二氟基苯甲酰基)-1H-吲哚-3-胺基]嘧啶-4-胺基}乙酸乙酯 (8g):黄色固体, 收率80.1%. m.p. 212.6~213.9 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.95 (s, 3H, OCH₂CH₃), 3.75 (s, 2H, OCH₂CH₃), 3.86 (s, 2H, NHCH₂), 7.09 (t, J＝8.0, 1H, ArH), 7.32~7.55 (m, 5H, ArH), 7.61~7.66 (m, 1H, ArH), 8.84 (s, 1H, Pyrimidine-H), 8.90 (t, J＝6.0 Hz, 1H, NHCH₂CO₂), 10.10 (s, 1H, NH), 11.88 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 185.4, 169.3, 161.2, 157.8, 155.5, 153.3, 150.9, 148.2, 136.4, 135.8, 127.1, 126.6, 126.3, 123.6, 121.3, 120.5, 118.4, 118.3, 117.7, 117.6, 113.2, 60.8, 42.3, 14.1. HRMS (ESI) calcd for C₂₃H₁₉F₂N₆O₅ (M+H⁺) 497.1307, found 497.1705.

2-[5-硝基-2-(2-对苯基苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8h):黄色固体, 收率70.2%. m.p. 205.5~207.5 ℃; ¹HNMR (400 MHz, DMSO-d₆) δ: 0.90 (s, 3H, OCH₂CH₃), 3.77 (s, 2H, OCH₂CH₃), 3.90 (s, 2H, NHCH₂), 7.09 (t, J＝8.0 Hz, 1H, ArH), 7.34 (t, J＝6.0 Hz, 1H, ArH), 7.42 (t, J＝6.0 Hz, 1H, ArH), 7.41~7.56 (m, 4H, ArH), 7.63 (t, J＝6.0 Hz, 4H, ArH), 7.76 (d, J＝8.0 Hz, 2H, ArH), 8.75 (s, 1H, Pyrimidine-H), 8.88 (s, 1H, NHCH₂CO₂), 10.12 (s, 1H, NH), 11.83 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆)δ: 187.5, 169.4, 161.1, 157.6, 155.5, 144.1, 139.5, 137.2, 136.1, 129.9, 129.5, 128.6, 127.2, 126.6, 125.9, 121.3, 120.3, 119.1, 113.1, 60.8, 42.3, 14.1. HRMS (ESI) calcd for C₂₉H₂₅N₆O₅(M+H⁺) 537.1808, found 537.2066.

2-[5-硝基-2-(2-间溴苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8i):黄色固体, 收率69.6%. m.p. 200.2~202.8 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.93 (s, 3H, OCH₂CH₃), 3.73 (s, 2H, OCH₂CH₃), 3.86 (s, 2H, NHCH₂), 7.08 (t, J＝6.0 Hz, 1H, ArH), 7.29~7.35 (m, 2H, ArH), 7.49 (t, J＝10.1 Hz, 2H, ArH), 7.62~7.70 (m, 3H, ArH), 8.81 (s, 1H, Pyrimidine-H), 8.91 (s, 1H, NH-CH₂CO₂), 10.04 (s, 1H, NH), 11.86 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆)δ: 186.4, 169.3, 161.1, 157.9, 155.6, 140.5, 136.4, 135.0, 131.5, 130.6, 127.9, 127.1, 126.3, 121.5, 121.4, 120.4, 119.7, 113.2, 60.7, 42.3, 14.1. HRMS (ESI) calcd for C₂₃H₂₀BrN₆O₅ (M+H⁺) 540.3382, found 541.0790.

2-[5-硝基-2-(6-氯-2-苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8j):黄色固体, 收率64.1%. m.p. 239.8~242.6 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.93 (s, 3H, OCH₂CH₃), 3.74 (s, 2H, OCH₂CH₃), 3.87 (s, 2H, NHCH₂), 7.09 (d, J＝8.0 Hz, 1H, ArH), 7.33 (t, J＝8.0 Hz, 2H, ArH), 7.53 (dd, J＝10.0, 14.0 Hz, 3H, ArH), 7.65 (d, J＝8.0 Hz, 2H, ArH), 8.78 (s, 1H, Pyrimidine-H), 8.89 (s, 1H, NHCH₂CO₂), 10.07 (s, 1H, NH), 11.95 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆)δ: 187.9, 169.3, 161.0, 157.8, 155.5, 138.0, 136.2, 132.8, 130.4, 129.1, 128.4, 128.3, 123.0, 122.4, 120.9, 119.0, 122.1, 60.7, 42.4, 14.1. HRMS (ESI) calcd for C₂₃H₂₀ClN₆O₅ (M+H⁺) 495.1105, found 495.1355.

2-[5-硝基-2-(6-氯-2-对甲氧基苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8k):黄色固体, 收率75.3%. m.p. 234.5~236.5 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.93 (s, 3H, OCH₂CH₃), 3.77 (s, 5H, OCH₂CH₃, OCH₃), 3.87 (s, 2H, NHCH₂), 6.86 (d, J＝8.0, 2H, ArH), 7.08 (t, J＝4.0 Hz, 1H, ArH), 7.48~7.54 (m, 2H, ArH), 7.67 (d, J＝8.0 Hz, 2H, ArH), 8.82 (s, 1H, Pyrimidine-H), 8.89 (t, J＝6.0 Hz, 1H, NHCH₂CO₂), 10.06 (s, 1H, NH), 11.89 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆)δ: 186.5, 169.4, 163.1, 161.0, 157.8, 155.5, 136.0, 131.6, 130.6, 130.0, 128.5, 122.9, 120.2, 118.3, 113.7, 112.3, 60.7, 58.4, 42.3, 14.1. HRMS (ESI) calcd for C₂₄H₂₂ClN₆O₆ (M+H⁺) 525.1211, found 525.1398.

2-[5-硝基-2-(6-氯-2-对氯苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8l):产率73.1%. m.p. 266.7~268.9 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.93 (d, 3H, OCH₂CH₃), 3.74 (s, 2H, OCH₂CH₃), 3.86 (s, 2H, NHCH₂), 7.10 (dd, J＝4.0, 8.0 Hz, 1H, ArH), 7.40 (d, J＝8.4 Hz, 2H, ArH), 7.49 (s, 1H, ArH), 7.55 (d, J＝8.0 Hz, 1H, ArH), 7.62~7.65 (m, 2H, ArH), 8.82 (s, 1H, Pyrimidine-H), 8.91 (t, J＝6.0 Hz, NHCH₂CO₂), 10.07 (s, 1H, NH), 11.98 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆)δ: 186.6, 169.3, 161.0, 157.8, 155.5, 137.5, 136.8, 136.4, 130.9, 130.7, 128.5, 128.1, 123.1, 121.0, 119.4, 112.5, 60.8, 42.4, 14.1. HRMS (ESI) calcd for C₂₃H₁₉Cl₂N₆O₅ (M+H⁺) 529.0716, found 529.0816.

2-{5-硝基-2-[2-(2-噻吩甲酰基)-1H-吲哚-3-胺基]嘧啶-4-胺基}乙酸乙酯 (8m):黄色固体.收率69.9%. m.p. 222.2~224.8 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.92 (s, 3H, OCH₂CH₃), 3.74 (s, 2H, OCH₂CH₃), 3.89 (s, 2H, NHCH₂), 7.06~7.13 (m, 2H, ArH), 7.32 (t, J＝8.0 Hz, 1H, ArH), 7.47 (d, J＝8.4 Hz, 1H, ArH), 7.56 (d, J＝8.0 Hz, 1H, ArH), 7.72 (d, J＝3.6 Hz, 1H, ArH), 7.95~7.97 (m, 1H, ArH), 8.87 (s, 1H, Pyrimidine-H), 8.89 (d, J＝4.0 Hz, 1H, NHCH₂CO₂), 10.25 (s, 1H, NH), 11.80 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 179.5, 169.4, 160.9, 157.9, 155.6, 143.6, 135.8, 135.0, 133.9, 128.6, 127.3, 125.7, 123.4, 121.3, 120.3, 118.3, 113.1, 60.7, 58.4, 42.4, 14.4. HRMS (ESI) calcd for C₂₁H₁₉N₆O₅S (M+H⁺) 467.1059, found 467.1063.

2-[5-硝基-2-(6-氯-2-对溴苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8n):黄色固体, 收率60.2%. m.p. 201.6~203.7 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.90 (d, 3H, OCH₂CH₃), 3.75 (s, 2H, OCH₂CH₃), 3.86 (s, 2H, NHCH₂), 7.10 (d, J＝8.0 Hz, 1H, ArH), 7.50 (s, 1H, ArH), 7.55 (dd, J＝8.0, 12.0 Hz, 5H, ArH), 8.83 (s, 1H, Pyrimidine-H), 8.91 (t, J＝6.0 Hz, 1H, NHCH₂CO₂), 10.07 (s, 1H, NH), 11.99 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 186.8, 169.3, 161.0, 157.8, 155.5, 137.1, 136.4, 131.4, 131.0, 130.7, 127.9, 127.7, 126.6, 123.1, 121.0, 119.2, 112.5, 60.9, 42.3, 14.1. HRMS (ESI) calcd for C₂₃H₁₉BrClN₆O₅ (M+H⁺) 574.7832, found 575.0325.

2-[5-硝基-2-(6-氯-2-对甲苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8o):黄色固体, 收率50.8%. m.p. 276.8~278.4 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.87~0.89 (m, 3H, OCH₂CH₃), 2.31 (s, 3H, CH₃), 3.74 (s, 2H, OCH₂CH₃), 3.87 (s, 2H, NHCH₂), 7.09 (d, J＝8.0 Hz, 1H, ArH), 7.49~7.59 (m, 4H, ArH), 7.57 (t, J＝8.0 Hz, 2H, ArH), 8.82 (s, 1H, Pyrimidine-H), 8.90 (t, J＝8.0 Hz, 1H, NHCH₂CO₂), 10.05 (s, 1H, NH), 11.95 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 187.5, 169.3, 160.9, 157.7, 155.5, 143.2, 136.1, 135.4, 132.1, 130.2, 129.4, 129.0, 123.0, 120.8, 112.4, 60.7, 42.3, 21.5, 14.1. HRMS (ESI) calcd for C₂₄H₂₂ClN₆O₅(M+H⁺) 509.1262, found 509.1291.

2-{5-硝基-2-[6-氯-2-(2-噻吩甲酰基)-1H-吲哚-3-胺基]嘧啶-4-胺基}乙酸乙酯 (8p):黄色固体, 收率42.6%. m.p. 287.3~289.3 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.89 (s, 3H, OCH₂CH₃), 3.74 (s, 2H, OCH₂CH₃), 3.89 (s, 2H, NHCH₂), 7.09~7.14 (m, 2H, ArH), 7.49 (s, J＝1.6 Hz, 1H, ArH), 7.58 (d, J＝12.0 Hz, 1H, ArH), 7.70 (d, J＝4.0 Hz, 1H, ArH), 7.99 (d, J＝4.0 Hz, 1H, ArH), 8.90 (s, 1H, Pyrimidine-H), 8.92 (d, J＝4.0 Hz, 1H, NHCH₂CO₂), 10.27 (s, 1H, NH), 11.96 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 179.3, 169.4, 160.9, 157.9, 155.7, 143.3, 135.8, 135.0, 133.9, 128.6, 127.3, 125.7, 123.3, 121.6, 120.3, 118.3, 113.1, 60.7, 42.4, 14.1. HRMS (ESI) calcd for C₂₁H₁₈ClN₆O₅S (M+H⁺) 501.0690, found 501.0846.

2-[5-硝基-2-(6-甲基-2-苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8q):黄色固体, 收率76.3%. m.p. 231.9~233.6 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.95 (s, 3H, OCH₂CH₃), 2.44 (s, 3H, CH₃), 3.77 (s, 2H, OCH₂CH₃), 3.90 (s, 2H, NHCH₂CO), 6.91 (d, J＝8.0 Hz, 1H, ArH), 7.26 (s, 1H, ArH), 7.32 (t, J＝16.0 Hz, 2H, ArH), 7.40 (d, J＝8.0 Hz, 1H, ArH), 7.48 (t, J＝8.0 Hz, 1H, ArH), 7.65 (d, J＝8.0 Hz, 2H, ArH), 8.77 (s, 1H, Pyrimidine-H), 8.87 (t, J＝8.0 Hz, 1H, NHCH₂CO₂), 10.02 (s, 1H, NH), 11.65 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 187.9, 169.4, 161.0, 157.8, 155.5, 138.5, 136.7, 135.5, 132.8, 129.1, 128.4, 127.0, 123.0, 122.4, 120.9, 119.0, 122.1, 60.7, 42.4, 22.1, 14.1. HRMS (ESI) calcd for C₂₄H₂₃N₆O₅ (M+H⁺) 475.1652, found 475.1984.

2-[5-硝基-2-(6-甲基-2-对溴苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8r):黄色固体, 收率76.8%. m.p. 243.1~245.4 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.95 (s, 3H, OCH₂CH₃), 2.43 (s, 3H, CH₃), 3.77 (s, 2H, OCH₂CH₃), 3.88 (s, 2H, NHCH₂CO₂), 6.92 (d, J＝8.0, 1H, ArH), 7.26 (s, 1H, ArH), 7.40 (d, J＝8.0, 1H, ArH), 7.54 (dd, J＝8.0, 16.0 Hz, 4H, ArH), 8.81 (s, 1H, Pyrimidine-H), 8.88 (t, J＝12.0 Hz, 1H, NHCH₂CO₂), 10.00 (s, 1H, NH), 11.69 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 186.7, 169.4, 161.0, 157.7, 155.5, 137.6, 136.8, 135.9, 131.3, 130.9, 126.2, 122.6, 121.1, 112.5, 60.8, 42.3, 22.1, 14.1. HRMS (ESI) calcd for C₂₄H₂₁Br-N₆O₅ (M+H⁺) 554.3647, found 555.0932.

2-[5-硝基-2-(6-甲基-2-对甲氧基苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8s):黄色固体, 收率81.2%. m.p. 245.2.5~247.5 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.95 (s, 3H, OCH₂CH₃), 2.43 (s, 3H, CH₃), 3.77 (s, 5H, OCH₂CH₃, OCH₃), 3.90 (s, 2H, NHCH₂CO₂), 6.88 (dd, J＝8.0, 16.0 Hz, 3H, ArH), 7.25 (s, 1H, ArH), 7.39 (d, J＝8.0 Hz, 1H, ArH), 7.67 (d, J＝8.0 Hz, 2H, ArH), 8.82 (s, 1H, Pyrimidine-H), 8.87 (m, 1H, NHCH₂-CO₂), 10.02 (s, 1H, NH), 11.59 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆)δ: 186.6, 169.4, 162.9, 161.0, 157.7, 155.5, 136.4, 135.1, 131.6, 131.0, 127.3, 122.3, 121.6, 120.2, 118.3, 113.7, 112.3, 60.7, 58.4, 42.3, 22.0, 14.1. HRMS (ESI) calcd for C₂₅H₂₅N₆O₆ (M+H⁺) 505.1757, found 505.1961.

2-[5-硝基-2-(6-甲基-2-对氯苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8t):黄色固体, 收率76.8%. m.p. 243.1~245.4 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.96 (m, 3H, OCH₂CH₃), 2.43 (s, 3H, CH₃), 3.78 (s, 2H, OCH₂CH₃), 3.88 (s, 2H, NHCH₂CO₂), 6.92 (d, J＝8.0 Hz, 1H, ArH), 7.26 (s, 1H, ArH), 7.39 (t, J＝8.0 Hz, 3H, ArH), 7.63 (d, J＝8.0 Hz, 2H, ArH), 8.81 (s, 1H, Pyrimidine-H), 8.88 (t, J＝12.0 Hz, 1H, NHCH₂CO₂), 9.99 (s, 1H, NH), 11.68 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 186.5, 169.4, 161.0, 157.7, 155.5, 152.5, 137.6, 136.8, 135.9, 131.3, 130.9, 126.9, 126.2, 122.6, 121.1, 119.6, 112.5, 60.8, 42.3, 22.1, 14.1. HRMS (ESI) calcd for C₂₄H₂₂ClN₆O₅ (M+H⁺) 509.1262, found 509.1465.

2-[5-硝基-2-(6-甲基-2-对甲基苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8u):黄色固体, 收率72.9%. m.p. 250.5~253.3 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.95 (s, 3H, OCH₂CH₃), 2.30 (s, 3H, CH₃), 2.43 (s, 3H, CH₃), 3.77 (s, 2H, OCH₂CH₃), 3.89 (s, 2H, NHCH₂CO₂), 6.91 (d, J＝8.0 Hz, 3H, ArH), 7.14 (d, J＝8.0 Hz, 2H, ArH), 7.26 (s, 1H, ArH), 7.39 (d, J＝8.0 Hz, 1H, ArH), 7.57 (d, J＝8.0 Hz, 2H, ArH), 8.80 (s, 1H, Pyrimidine-H), 8.87 (t, J＝6.0 Hz, 1H, NHCH₂CO₂), 9.99 (s, 1H, NH), 11.60 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 187.5, 169.4, 161.0, 157.7, 155.5, 136.4, 135.1, 131.6, 131.0, 127.3, 122.3, 121.6, 120.2, 118.3, 113.7, 112.3, 60.7, 42.3, 22.1, 21.5, 14.1. HRMS (ESI) calcd for C₂₅H₂₅N₆O₅ (M+H⁺) 489.4595, found 490.2015.

2-{5-硝基-2-[6-甲基-2-(2-噻吩甲酰基)-1H-吲哚-3-胺基]嘧啶-4-胺基}乙酸乙酯 (8v):黄色固体, 收率50.3%. m.p. 228.4~230.3 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.96 (s, 3H, OCH₂CH₃), 2.44 (s, 3H, CH₃), 3.78 (s, 2H, OCH₂CH₃), 3.92 (s, 2H, NHCH₂CO₂), 6.92 (d, J＝8.0 Hz, 1H, ArH), 7.12 (t, J＝8.0 Hz, 1H, ArH), 7.26 (s, 1H, ArH), 7.45 (d, J＝8.0 Hz, 1H, ArH), 7.70 (d, J＝4.0 Hz, 1H, ArH), 7.95 (d, J＝8.0 Hz, 1H, ArH), 8.88 (s, 1H, Pyrimidine-H), 8.90 (d, J＝4.0 Hz, 1H, NHCH₂CO₂), 10.20 (s, 1H, NH), 11.64 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 179.0, 169.4, 161.8, 157.9, 155.6, 143.7, 136.5, 135.4, 134.8, 133.6, 128.6, 127.3, 125.7, 123.3, 121.6, 120.3, 118.3, 113.1, 60.7, 42.4, 22.0, 14.1. HRMS (ESI) calcd for C₂₂H₂₁N₆O₅S (M+H⁺) 481.1216, found 481.1580.

2-[5-硝基-2-(6-甲基-2-对苯基苯甲酰基-1H-吲哚-3-胺基) 嘧啶-4-胺基]乙酸乙酯 (8w):黄色固体, 收率60.2%. m.p. 205.5~207.5 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.95 (s, 3H, OCH₂CH₃), 3.77 (s, 2H, OCH₂-CH₃), 3.90 (s, 2H, NHCH₂CO₂), 7.10 (dd, J＝4.0, 8.0 Hz, 1H, ArH), 7.41~7.61 (m, 5H, ArH), 7.63 (t, J＝12.0, 4H, ArH), 7.75 (d, J＝8.0 Hz, 2H, ArH), 8.77 (s, 1H, Pyrimidine-H), 8.92 (s, 1H, NHCH₂CO₂), 10.13 (s, 1H, NH), 11.98 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆)δ: 187.3, 169.3, 161.0, 157.7, 155.5, 144.3, 139.5, 136.9, 136.3, 130.5, 129.9, 129.5, 128.6, 127.2, 126.6, 123.1, 122.4, 120.9, 119.2, 112.4, 60.8, 42.4, 14.1. HRMS (ESI) calcd for C₂₉H₂₄ClN₆O₅ (M+H⁺) 571.9831, found 571.1446.

3.4 体外抗肿瘤活性测试

用噻唑蓝 (MTT) 比色法测试了化合物的体外抗肿瘤活性.收集对数期细胞, 调整细胞悬液浓度, 以20000~25000个/mL接种于96孔板, 孵育12~24 h.待细胞贴壁后, 加入不同浓度的药物, 设置3.125, 6.25, 12.5, 25.0, 50.0, 100 mg/mL共6个浓度梯度, 每个浓度4个副孔. 37 ℃、5% CO₂培养箱, 开始计时培养.在加药48 h后取出96孔板, 每孔加入20 mL的5 mg/mL MTT溶液, 37 ℃继续培养4 h.然后, 小心吸弃孔内上清液, 每孔加入150 mL DMSO, 震荡10 min, 使结晶物溶解.选择570 nm波长在酶联免疫检测仪上测定各孔吸光度值 (OD值).按下列公式计算抑制率, 抑制率 (%)＝(1－加药组OD值/对照组OD值)×100%.用GraphPad Prism5软件计算半数抑制量IC₅₀.

辅助材料 (Supporting Information)化合物8a~8w的¹H NMR和¹³C NMR谱图.这些材料可以免费从本刊网站 (http://sioc-journal.cn/) 上下载.

3.2.1 6-取代-3-氨基-1-乙酯基-2-芳甲酰基吲哚 (3) 和6-取代-3-氨基-2-苯甲酰基-1H-吲哚 (4) 的合成

中间体6-取代-3-氨基-1-乙酯基-2-芳甲酰基吲哚 (3) 和6-取代-3-氨基-2-苯甲酰基-1H-吲哚 (4) 分别参照我们课题组前期发表的文献[24, 14]合成.

3.2.2 2-氯-5-硝基-4-取代氨基嘧啶 (7) 的合成

向1.92 g (10.0 mmol) 的2, 4-二氨基嘧啶、10.0 mmol的2-氨基乙酸乙酯盐酸盐和3.04 g (22.0 mmol) 的新炒碳酸钾的20 mL无水乙醇溶液中加入2滴冰醋酸, 搅拌, 室温反应5 h, 薄层色谱 (TLC) 检测.反应完成后减压过滤, 干燥, 柱层析得无色固体, 收率84%. ¹H NMR (400 MHz, CDCl₃) δ: 1.36 (t, J＝7.2 Hz, 3H, CH₃), 4.32 (dd, J＝7.0, 14.2 Hz, 2H, CH₂), 4.42 (d, J＝5.2 Hz, 2H, CH₂), 8.78 (br, 1H, NH), 9.11 (s, 1H, ArH); ESI-MS m/z: 261.9 [M+1]⁺.

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图式1 目标化合物的合成

Scheme 1 Synthetic routes of the title compounds

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表 1 化合物8a~8w对Hela、MD-MBA-231、PC-3和HCT116的IC₅₀值 (μmol/L)

Table 1. IC₅₀^a (μmol/L) values of target compounds 8a~8w for Hela, MD-MBA-231, PC-3 and HCT116

Compd.	Hela	MDA-MB-231	PC-3	HCT 116
8a	70.63	106.80	150.36	70.51
8b	＞200	113.80	＞200	＞200
8c	115.32	＞200	＞200	87.25
8d	＞200	155.2	123.69	76.84
8e	＞200	＞200	＞200	186.34
8f	＞200	＞200	＞200	153.69
8g	108.32	＞200	236.15	＞200
8h	＞200	＞200	＞200	＞200
8i	＞200	＞200	＞200	＞200
8j	60.69	36.88	80.69	23.15
8k	＞200	＞200	＞200	＞200
8l	＞200	＞200	＞200	＞200
8m	60.03	＞200	＞200	153.72
8n	＞200	＞200	＞200	＞200
8o	＞200	＞200	＞200	＞200
8p	108.54	103.30	156.32	60.35
8q	116.85	78.06	100.26	53.21
8r	＞200	＞200	＞200	＞200
8s	＞200	135.81	＞200	＞200
8t	＞200	＞200	＞200	＞200
8u	＞200	＞200	＞200	145.98
8v	93.52	145.69	＞200	68.69
8w	＞200	＞200	＞200	＞200
5-Fu	57.17	53.47	57.04	68.71
^a IC₅₀ values mean of three experiments in replicate.

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