1, 2, 3-三氮唑[4, 5-<i>d</i>]嘧啶衍生物的合成及抗肿瘤活性评价

引用本文: 栗娜, 辛景超, 马启胜, 李二冬, 孟娅琪, 包崇男, 杨鹏, 宋攀攀, 崔飞, 陈鹏举, 顾一飞, 赵培荣, 可钰, 刘宏民, 张秋荣. 1, 2, 3-三氮唑[4, 5-d]嘧啶衍生物的合成及抗肿瘤活性评价[J]. 有机化学, 2018, 38(3): 665-671. doi: 10.6023/cjoc201709030 shu

Citation: Li Na, Xin Jingchao, Ma Qisheng, Li Erdong, Meng Yaqi, Bao Chongnan, Yang Peng, Song Panpan, Cui Fei, Cheng Pengju, Gu Yifei, Zhao Peirong, Ke Yu, Liu Hongmin, Zhang Qiurong. Synthesis and Antitumor Activities of Novel 1, 2, 3-Triazolo[4, 5-d]pyrimidine Derivatives[J]. Chinese Journal of Organic Chemistry, 2018, 38(3): 665-671. doi: 10.6023/cjoc201709030 shu

1, 2, 3-三氮唑[4, 5-d]嘧啶衍生物的合成及抗肿瘤活性评价

栗娜 ^a,b, ,
辛景超 ^a,b, ,
马启胜 ^a,b, ,
李二冬 ^a,b, ,
孟娅琪 ^a,b, ,
包崇男 ^a,b, ,
杨鹏 ^a,b, ,
宋攀攀 ^a,b, ,
崔飞 ^a,b, ,
陈鹏举 ^a, ,
顾一飞 ^a, ,
赵培荣 ^b, ,
可钰 ^{a,b,
,} ,
刘宏民 ^{a,b,
,} ,
张秋荣 ^{a,b,
,}

1.
郑州大学药学院郑州 450001
2.
新药创制与药物安全性评价河南省协同创新中心郑州 450001

通讯作者: 可钰, ky@zzu.edu.cn; 刘宏民, liuhm@zzu.edu.cn; 张秋荣, zqr406@sina.com

基金项目:

国家自然科学基金（No.81430085）和郑州市科技局科研（No.141PQYJS554）资助项目

摘要: 为了寻找高效的抗肿瘤药物，设计并合成了一系列新型的1，2，3-三氮唑[4，5-d]嘧啶类衍生物，对这些化合物在人类五种癌细胞MGC-803（人胃癌细胞）、MCF-7（人乳腺癌细胞）、EC-109（人食管癌细胞）、PC-3（人前列腺癌细胞）、Hela（人宫颈癌细胞）进行抗肿瘤活性评价，结果显示大部分化合物具有较好的抗肿瘤活性，其中5-（（（1H-苯并[d]咪唑-2-基）甲基）硫基）-3-苄基-N-（4-氯苯基）-3H-[1, 2, 3]-三氮唑[4，5-d]嘧啶-7-胺（11b）和2，2'-（（5-（（（1H-苯并[d]咪唑-2-基）甲基）硫基）-3-苄基-3H-[1, 2, 3]-三氮唑[4，5-d]嘧啶-7-基）氮烷二基）双（乙烷-1-醇）（11m）对MGC-803和Hela癌细胞的抗肿瘤活性优于对照品氟尿嘧啶.

关键词:

三氮唑
/ 嘧啶
/ 抗肿瘤活性
/ 衍生物
/ 评价

English

Synthesis and Antitumor Activities of Novel 1, 2, 3-Triazolo[4, 5-d]pyrimidine Derivatives

1.
School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001
2.
Collaborative Innovation Center of New Drug Research and Safety Evaluation, Zhengzhou 450001

Corresponding author: Ke Yu, E-mail:ky@zzu.edu.cn; Liu Hongmin, E-mail:liuhm@zzu.edu.cn; Zhang Qiurong, E-mail:zqr406@sina.com

Received Date: 19 September 2017
Revised Date: 25 October 2017
Available Online: 25 March 2018
Fund Project:

Project supported by the National Natural Science Foundation of China (No. 81430085) and the Research Project of Science and Technology Bureau of Zhengzhou City (No.141PQYJS554)

Abstract: In order to find more efficient and economical antitumor drugs, a series of novel 1, 2, 3-triazolo[4, 5-d]pyrimidine derivatives were synthesized and evaluated for their antitumor activities against five human cancer cell lines (MGC-803, MCF-7, EC-109, PC-3 and Hela) in vitro. The results showed that most compounds had good antitumor activities, especially 5-(((1H-benzo[d]imidazol-2-yl)methyl)thio)-3-bezyl-N-(4-chlorophenyl)-3H-[1, 2, 3]triazolo[4, 5-d]pyrimidin-7-amine (11b) and 2, 2'-((5-(((1H-benzo[d]imidazol-2-yl)methyl)thio)-3-benzyl-3H-[1, 2, 3]triazolo[4, 5-d]pyrimidin-7-yl)azanediyl)bis(ethan-1-ol) (11m) exhibited better antitumor activities than 5-fluorouracil against MGC-803 and Hela.

Key words:

肿瘤是当今世界危及人类生命的一种常见的严重疾病, 已成为导致人类死亡的“第二大杀手”.据统计, 2035年全球癌症死亡人数将增加到1315万人, 但是目前上市的绝大多数化疗药物不良反应较多, 很多患者不能耐受以致死亡.因此, 抗肿瘤药物的研究开发在当今生命科学领域中极富挑战性而且意义重大.在过去的几十年里, 1, 2, 3-三氮唑[4, 5-d]嘧啶类化合物因其广泛的生物活性而受到药物化学研究人员的高度关注^[1~3], 多个研究小组将1, 2, 3-三氮唑[4, 5-d]嘧啶和其它药效团进行拼合, 得到了一些具有潜在抗肿瘤活性的化合物(图 1). Dehnhardt等^[1]报道的化合物4 (PIK-402)可以通过充分抑制Akt活化来抑制癌细胞增殖并诱细胞凋亡, 从而达到抗肿瘤的效果; 李中华等^[2]将1, 2, 3-三氮唑[4, 5-d]嘧啶与苄胺等基团结合得到化合物5, 能够以较低的浓度抑制人胃癌细胞(MGC-803)的转移而达到抗肿瘤的作用.

图 1

图 1. 1, 2, 3-三氮唑[4, 5-d]嘧啶衍生物的结构式

Figure 1. Structures of 1, 2, 3-triazolo[4, 5-d]pyrimidine derivatives

下载: 全尺寸图片幻灯片

另外, 苯并咪唑是一类重要的天然产物, 该类化合物具有广泛的生物活性, 如抗肿瘤^[4~9]、拓扑异构酶抑制剂^{[10, 11]}、抗原虫^[12]、抗病毒^[13]和抗菌活性^[14~16]等.本工作以1, 2, 3-三氮唑[4, 5-d]嘧啶为母核, 根据生物活性亚结构拼接原理, 设计并合成一系列的3, 5, 7-三取代-1, 2, 3-三氮唑[4, 5-d]嘧啶衍生物.此外, 我们利用噻唑蓝(MTT)法评价了目标化合物对人胃癌细胞(MGC-803)、人乳腺癌细胞(MCF-7)、人食管癌细胞(EC-109)、人前列腺细胞(PC-3)、人宫颈癌细胞(Hela)的细胞增殖抑制活性.

1. 结果与讨论

1.1 目标化合物的合成

通过文献调研^{[6, 16, 17]}, 氰基乙酰胺和5-(氯甲基)-1H-苯并咪唑是合成8-氮杂嘌呤-苯并咪唑共轭物的关键结构单元.在该方法中, 将苄基溴溶于丙酮中, 并缓慢滴加至叠氮化钠水溶液中, 反应得到化合物6, 化合物6在乙醇钠条件下与氰基乙酰胺环化得到化合物7, 化合物7与二硫化碳缩合得到化合物8, 化合物8与2-(氯甲基)-H-苯并咪唑在水和丙酮的混合溶剂中缩合得到化合物9, 再用三氯氧磷作氯代试剂, 发生氯代反应得到化合物10, 最后, 化合物10经亲核取代反应合成了15个新型的1, 2, 3-三氮唑[4, 5-d]嘧啶衍生物11a~11o (Scheme 1), 其结构经¹H NMR, ¹³C NMR和HRMS表征.

图式 1

图式 1. 化合物11a~11o的合成路线

Scheme 1. Synthesis of compounds 11a~11o

Reagents and conditions: (ⅰ) sodium azide, H₂O, acetone, r.t., 2 h; (ⅱ) cyanoacetamide, EtONa, 85 ℃, 2 h; (ⅲ) carbon disulfide, KOH, EtOH, 85 ℃, 12 h; (ⅳ) H₂O, acetone, 2-(chloromethyl)-H-benzodimidazole, 60 ℃, 2 h; (ⅴ) POCl₃, 110 ℃, 1 h; (ⅵ) isopropanol, 85 ℃

下载: 全尺寸图片幻灯片

1.2 目标化合物的抗肿瘤活性

为了研究新化合物的抗肿瘤活性, 采用噻唑蓝(MTT)比色法, 以5-氟尿嘧啶(5-Fu)作为阳性对照, 对所合成化合物进行体外抗人胃癌细(MGC-803)、人乳腺癌细胞(MCF-7)、人食管癌细胞(EC-109)、人前列腺细胞(PC-3)、人宫颈癌细胞(Hela)的活性测试^[18~21].

从活性数据来看(表 1), 大部分的化合物对所测5种人类癌细胞均有明显的抑制活性, 且伴随着取代基的不同而改变; 化合物11l(8.21 μmol/L)、11m (5.58 μmol/L)和11b (9.51 μmol/L)对于人宫颈癌细胞的抗肿瘤活性优于5-氟尿嘧啶(9.51 μmol/L); 化合物11a~11b对于所测五种人类癌细胞的抗肿瘤活性优于化合物11h~11i, 说明当苯环上引入相同的吸电子基团时, 对位比邻位更有利于化合物抗肿瘤活性的表达; 当苯环的对位引入不同的取代基时, 化合物11a~11c对于五种人类癌细胞的抗肿瘤活性优于化合物11d~11f, 说明苯胺对位为吸电子取代基比给电子取代基更有利于化合物抗肿瘤活性的表达; 当R为脂肪胺时, 化合物11l对于人宫颈癌细胞的抗肿瘤活性优于化合物11a~11k, 说明R是仲胺时更有利于化合物抗肿瘤活性的表达; 化合物11o对五种人类癌细胞无抗肿瘤活性, 说明当R为不饱和杂环时不利于其抗肿瘤活性的表达.

表 1

表 1 化合物9~10和11a~11o对五种人类癌细胞的体外细胞毒活性^a [IC₅₀/(μmol•L^－1)]

Table 1. In vitro cytotoxic activities of synthetic compounds 9~10 and 11a~11o against five human cancer cell lines [IC₅₀/(μmol•L^－1)]

下载: 导出CSV

Compd.	R	MGC-803	MCF-7	EC-109	PC-3	Hela
9	HO	25.93±0.82	6.00±0.31	＞100	72.76±1.15	35.97±1.03
10	Cl	15.56±0.74	52.36±1.18	69.17±1.30	56.83±1.26	31.34±0.82
11a	4-FC₆H₄NH	19.32±0.47	26.67±0.51	26.54±0.61	20.17±0.66	11.76±0.54
11b	4-ClC₆H₄NH	8.75±0.09	10.63±0.14	16.91±0.26	11.56±0.43	9.51±0.49
11c	4-BrC₆H₄NH	46.63±1.20	23.57±0.98	＞100	36.67±0.47	18.16±0.36
11d	4-CH₃C₆H₄NH	78.45±1.11	60.56±1.52	＞100	＞100	45.63±1.22
11e	4-CH₃OC₆H₄NH	67.13±1.38	＞100	80.42±1.55	57.82±1.22	55.88±1.39
11f	4-C₂H₅O₂CC₆H₄NH	73.11±1.43	47.98±1.20	54.39±1.13	45.21±1.41	44.56±1.17
11g	4-CH₃OC₆H₄CH₂NH	31.46±1.09	32.34±0.87	47.45±1.22	＞100	25.61±0.40
11h	2-FC₆H₄NH	＞100	＞100	56.79±1.25	＞100	67.18±1.06
11i	2-ClC₆H₄NH	34.65±0.88	＞100	79.32±1.17	＞100	53.97±1.21
11j	2-CH₃OC₆H₄NH	37.89±1.51	＞100	71.32±1.02	＞100	33.86±0.98
11k	3-CF₃C₆H₄NH	＞100	＞100	62.18±1.21	＞100	63.29±1.02
11l	HO(CH₂)₂NH	20.64±0.45	19.34±0.64	32.39±0.89	15.22±0.99	8.21±0.21
11m	[HO(CH₂)₂]₂N	8.61±0.06	10.72±0.11	12.38±0.33	12.68±0.28	5.58±0.13
11n	C₆H₅CH₂(CH₃)N	37.93±1.12	＞100	＞100	45.61±1.37	56.73±1.23
11o	Morpholin-4-yl	＞100	＞100	＞100	＞100	71.26±1.20
5-Fu^b		9.84±0.87	8.67±0.56	17.88±0.69	9.35±0.57	11.86±0.73
^a Anticancer activity was assayed by exposure for 72 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC₅₀). Dates are presented as the means±SDs of three independent experiments. ^b Positive control.

2. 结论

为了寻找活性更好、合成方法更简洁的抗肿瘤化合物, 本课题组设计并制备出了一系列3, 5, 7-三取代-1, 2, 3-三氮唑[4, 5-d]嘧啶衍生物, 利用核磁共振氢谱、核磁共振碳谱和高分辨质谱等技术证实了目标化合物的结构.运用MTT比色法测定了目标化合物对人类五种癌细胞: MGC-803人胃癌细胞、MCF-7人乳腺癌细胞、EC-109人食管癌细胞、PC-3人前列腺细胞、Hela人宫颈癌细胞的抗肿瘤活性, 其中化合物11m对MGC-803人胃癌细胞和Hela人宫颈癌细胞的抗肿瘤活性要优于5-氟尿嘧啶, 其IC₅₀值分别为8.61, 5.58 μmol/L.

3. 实验部分

3.1 仪器与试剂

¹H NMR和¹³C NMR光谱测定使用DPX-DPX-400超导核磁共振仪, TMS作为内标; 高分辨质谱使用Waters-Micromass公司Q-Tof质谱仪测定; 硅胶:中国青岛海洋化工集团公司; 柱色谱硅胶:上海五四化学试剂厂; 乙酰氯:天津化学试剂有限公司; 冰醋酸:天津永达化学试剂有限公司; N, N-二甲基甲酰胺:广东光华科技有限公司; 无水乙醇:烟台市化工有限公司; 本实验分离纯化所用有机溶剂均为工业级, 经重新蒸馏后使用, 其他试剂均为市售分析纯, 必要时做常规处理.

3.2 实验方法

3.2.1 苄基叠氮(6)的合成

将苄基溴(4.8 mL, 40 mmol)溶于丙酮(40 mL)中, 缓慢滴加至叠氮化钠(2.6 g, 40 mmol)的水(22 mL)溶液中, 室温下迅速搅拌2 h.反应完成后, 除去溶剂, 残余物用乙酸乙酯(20 mL×3)萃取, 合并萃取液, 减压蒸馏除去乙酸乙酯, 得到无色透明油状物6 (5.253 g, 98.0%).

3.2.2 5-氨基-1-苄基-1H-1, 2, 3-三氮唑-4-甲酰胺(7)的合成

化合物7按照文献[22]的方法制备, 表征数据与文献一致.

3.2.3 3-苄基-5-巯基-3H-[1, 2, 3]-三氮唑[4, 5-d]嘧啶-7-醇(8)的合成

将二硫化碳(1.8 mL, 40 mmol)加入到氢氧化钾(1.680 g, 30 mmol)的乙醇(54 mL)溶液中, 加热搅拌.然后将化合物7 (2.170 g, 10 mmol)滴加到上述溶液中, 85 ℃反应12 h.将反应混合物冷却至室温, 过滤, 滤饼用乙醇洗涤3次(10 mL×3), 真空干燥, 得到白色固体8 3.541 g, 产率79.1%. m.p. 334.1~334.7 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 10.70 (s, 1H), 7.79~7.22 (m, 5H), 5.49 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 179.47, 156.89, 151.72, 137.06, 129.09, 128.15, 127.86, 126.38, 48.73; HRMS (ESI) calcd for C₁₁H₁₀N₅OS [M+H]⁺ 260.0606, found 260.0607.

3.2.4 5-((1H-苯并[d]咪唑-2-基)甲基)硫基)-3-苄基-3H-[1, 2, 3]-三氮唑[4, 5-d]嘧啶-7-醇(9)的合成

将化合物8 (2.970 g, 10 mmol)加入到2-(氯甲基)-H-苯并咪唑(1.666 g, 10 mmol)的水(50 mL)和丙酮(25 mL)的混合溶液中, 加热至60 ℃反应2 h.待反应完成后(通过TLC监测)冷却至室温, 过滤, 滤饼用丙酮洗涤, 真空干燥, 得到白色固体9 4.784 g, 产率92.3%. m.p. 242.1~242.8 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.73 (s, 1H), 7.55~7.47 (m, 2H), 7.31~7.25 (m, 2H), 7.25~7.09 (m, 5H), 5.66 (s, 2H), 4.80 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 161.78, 155.90, 150.59, 148.68, 139.17, 135.70, 129.09, 128.88, 128.57, 127.73, 122.30, 115.37, 66.82, 50.07, 28.58; HRMS (ESI) calcd for C₁₉H₁₆N₇OS[M+H]⁺ 390.1137, found 390.1136.

3.2.5 5-(((1H-苯并[d]咪唑-2-基)甲基)硫基)-3-苄基-7-氯-3H-[1, 2, 3]-三氮唑[4, 5-d]嘧啶(10)的合成

化合物9 (0.778 g, 2 mmol)于50 mL单口圆底烧瓶中, 加入10 mL三氯氧磷并搅拌, 升温至110 ℃, 反应1 h.待反应完成后(通过TLC监测), 将反应液冷却至室温, 逐滴加入到碎冰中, 边滴加边搅拌, 析出白色固体, 抽滤, 水洗涤至中性, 真空干燥, 得到白色固体10 0.690 g, 产率84.6%. m.p. 178.4~179.2 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 13.39 (s, 1H), 7.70 (dt, J＝4.4, 3.4 Hz, 2H), 7.55~7.49 (m, 2H), 7.13~7.04 (m, 3H), 6.99 (t, J＝7.4 Hz, 2H), 5.72 (s, 2H), 5.12 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 160.77, 155.72, 151.11, 148.47, 135.75, 131.25, 128.86, 128.32, 128.27, 127.75, 126.26, 114.39, 49.78, 26.25; HRMS (ESI) calcd for C₁₉H₁₅ClN₇S [M+H]⁺ 408.0798, found 408.0794.

3.2.6 目标化合物11a~11o的合成

将不同取代基的苯胺(1 mmol)加入化合物10 (0.408 g, 1 mmol)的异丙醇(5 mL)溶液中搅拌, 加热至85 ℃, 反应完成后(通过TLC监测), 将反应物冷却至室温, 过滤, 滤饼用水洗涤, 真空干燥, 得到目标化合物11a~11o.

5-(((1H-苯并[d]咪唑-2-基)甲基)硫基)-3-苄基-N-(4-氟苯基)-3H-[1, 2, 3]-三氮唑-[4, 5-d]嘧啶-7-胺(11a):白色固体, 产率86.9%. m.p. 270.3~271.2 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.11 (s, 1H), 7.77 (s, 2H), 7.69 (dd, J＝6.1, 3.1 Hz, 2H), 7.50~7.46 (m, 2H), 7.20~7.16 (m, 5H), 7.15~7.03 (m, 2H), 5.78 (s, 2H), 5.00 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 167.65, 160.51, 158.15, 152.05, 149.60, 135.98, 134.53, 131.57, 129.00, 128.34, 126.02, 124.57, 123.38, 115.84, 115.62, 114.35, 49.74, 27.00; HRMS (ESI) calcd for C₂₅H₂₀FN₈S[M+H]⁺ 483.1516, found 483.1512.

5-(((1H-苯并[d]咪唑-2-基)甲基)硫基)-3-苄基-N-(4-氯苯基)-3H-[1, 2, 3]-三氮唑[4, 5-d]嘧啶-7-胺(11b):白色固体, 产率86.8%. m.p. 216.3~216.9 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.19 (s, 1H), 7.82 (d, J＝7.6 Hz, 2H), 7.69 (dd, J＝6.1, 3.1Hz, 2H), 7.58~7.45 (m, 4H), 7.14 (tt, J＝14.8, 7.3 Hz, 5H), 5.79 (s, 2H), 5.01 (s, 2H).¹³C NMR (101MHz, DMSO-d₆) δ: 167.63, 152.05, 151.79, 150.46, 149.72, 146.51, 137.32, 135.97, 135.39, 131.57, 129.00, 128.89, 128.37, 126.04, 124.11, 124.00, 123.98, 123.48, 114.36, 49.75, 27.01; HRMS (ESI) calcd for C₂₅H₂₀ClN₈S[M+H]⁺ 499.1220.

5-(((1H-苯并[d]咪唑-2-基)甲基)硫基)-3-苄基-N-(4-溴苯基)-3H-[1, 2, 3]-三氮唑[4, 5-d]嘧啶-7-胺(11c):白色固体, 产率80.4 %. m.p. 177.8~178.5 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.19 (s, 1H), 7.77 (d, J＝7.9 Hz, 2H), 7.68 (dt, J＝6.6, 3.3 Hz, 2H), 7.58~7.52 (m, 2H), 7.48 (dq, J＝6.6, 3.5 Hz, 2H), 7.20~7.14 (m, 3H), 7.14~7.07 (m, 2H), 5.79 (s, 2H), 5.00 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆)δ: 167.70, 164.33, 158.44, 153.23, 152.01, 151.76, 149.75, 142.66, 137.76, 135.97, 134.05, 131.90, 129.01, 128.38, 125.88, 124.35, 123.51, 116.92, 114.40, 49.76, 25.95; HRMS (ESI) calcd for C₂₅H₂₀BrN₈S[M+H]⁺ 543.0715, found 543.0710.

5-(((1H-苯并[d]咪唑-2-基)甲基)硫基)-3-苄基-N-(4-甲基苯基)-3H-[1, 2, 3]-三氮唑[4, 5-d]嘧啶-7-胺(11d):白色固体, 产率89.8%. m.p. 189.8~190.4 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.01 (s, 1H), 7.70 (dd, J＝6.1, 3.1 Hz, 2H), 7.62 (s, 2H), 7.50 (dt, J＝6.1, 3.2 Hz, 2H), 7.20~7.08 (m, 7H), 5.77 (s, 2H), 4.99 (s, 2H), 2.27 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 167.66, 158.46, 152.08, 149.74, 136.17, 135.65, 131.84, 129.45, 129.00, 128.36, 128.02, 125.88, 124.16, 123.43, 122.62, 116.98, 111.93, 49.71, 27.05, 21.01; HRMS (ESI) calcd for C₂₆H₂₃N₈S[M+H]⁺ 479.1766, found 479.1761.

5-(((1H-苯并[d]咪唑-2-基)甲基)硫基)-3-苄基-N-(4-甲氧基苯基)-3H-[1, 2, 3]-三氮唑[4, 5-d]嘧啶-7-胺(11e):白色固体, 产率69.7%. m.p. 208.3~209.0 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 10.96 (s, 1H), 7.69 (dd, J＝5.7, 3.0 Hz, 2H), 7.63 (d, J＝6.8 Hz, 2H), 7.50 (dd, J＝6.1, 3.1 Hz, 2H), 7.20~7.10 (m, 5H), 6.90 (d, J＝8.8 Hz, 2H), 5.77 (s, 2H), 4.97 (s, 2H), 3.75 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 167.68, 156.73, 152.14, 151.71, 149.36, 136.05, 131.51, 131.08, 129.01, 128.33, 126.05, 124.13, 123.27, 114.35, 114.20, 55.70, 49.68, 27.05; HRMS (ESI) calcd for C₂₆H₂₃N₈OS [M+H]⁺ 495.1716, found 495.1711.

乙基-4-((5-(((1H-苯并[d]咪唑-2-基)甲基)硫基)-3-苄基-3H-[1, 2, 3]-三氮唑[4, 5-d]嘧啶-7-基)氨基)苯甲酸甲酯(11f):白色固体, 产率83.7%. m.p. 250.3~251.0 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.40 (s, 1H), 7.95 (dd, J＝21.2, 8.8 Hz, 4H), 7.78~7.65 (m, 3H), 7.52~7.47 (m, 2H), 7.19~7.14 (m, 3H), 7.09 (dd, J＝15.0, 7.6 Hz, 3H), 5.81 (s, 2H), 5.04 (s, 2H), 4.31 (q, J＝7.1 Hz, 2H), 1.34 (t, J＝7.1 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆)δ: 167.68, 165.70, 152.00, 151.76, 151.13, 149.86, 142.87, 135.93, 131.38, 130.31, 129.01, 128.87, 128.37, 126.13, 125.63, 123.64, 121.61, 114.33, 61.01, 56.48, 49.80, 27.01; HRMS (ESI) calcd for C₂₈H₂₅N₈O₂S[M+H]⁺ 537.1821, found 537.1823.

5-(((1H-苯并[d]咪唑-2-基)甲基)硫基)-3-苄基-N-(4-甲氧基苄基)-3H-[1, 2, 3]-三氮唑[4, 5-d]嘧啶-7-胺(11g):黄色固体, 产率61.5%. m.p. 211.6~212.6 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.28 (s, 1H), 9.59 (t, J＝6.1 Hz, 1H), 7.55 (s, 1H), 7.42 (s, 1H), 7.28 (ddd, J＝20.6, 10.2, 4.7 Hz, 7H), 7.15 (d, J＝5.4 Hz, 2H), 6.84 (dd, J＝35.5, 8.7 Hz, 2H), 5.68 (d, J＝5.0 Hz, 2H), 4.68 (d, J＝5.1 Hz, 2H), 4.64 (d, J＝6.1 Hz, 2H), 3.69 (d, J＝11.8 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 168.94, 158.95, 158.77, 153.49, 151.82, 149.54, 143.67, 136.15, 131.14, 129.47, 129.15, 128.73, 128.53, 128.46, 123.33, 122.39, 121.63, 118.88, 114.33, 114.13, 111.70, 55.45, 49.79, 43.23, 29.14; HRMS (ESI) calcd for C₂₇H₂₅N₈OS [M+H]⁺ 509.1872, found 509.1870.

5-(((1H-苯并[d]咪唑-2-基)甲基)硫基)-3-苄基-N-(2-氟苯基)-3H-[1, 2, 3]-三氮唑[4, 5-d]嘧啶-7-胺(11h):白色固体, 产率71.3%. m.p. 227.4~227.9 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 10.97 (s, 1H), 7.69 (dd, J＝5.9, 3.1 Hz, 2H), 7.54~7.43 (m, 3H), 7.32 (d, J＝5.7 Hz, 1H), 7.28~7.08 (m, 7H), 5.76 (s, 2H), 4.91 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 167.60, 152.01, 149.96, 146.51, 144.10, 135.96, 131.74, 129.03, 128.72, 128.40, 128.34, 125.94, 124.87, 123.21, 116.55, 116.36, 114.41, 49.74, 27.03. HRMS (ESI) calcd for C₂₅H₂₀FN₈S [M+H]⁺ 483.1516, found 483.1511.

5-(((1H-苯并[d]咪唑-2-基)甲基)硫基)-3-苄基-N-(2-氯苯基)-3H-[1, 2, 3]-三氮唑[4, 5-d]嘧啶-7-胺(11i):白色固体, 产率72.6%. m.p. 263.5~264.3 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 10.98 (s, 1H), 7.69 (dt, J＝6.6, 3.3 Hz, 2H), 7.50 (ddd, J＝11.2, 6.5, 4.0 Hz, 4H), 7.36~7.28 (m, 2H), 7.23~7.09 (m, 6H), 5.75 (s, 2H), 4.89 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 167.53, 153.43, 151.98, 150.07, 135.97, 134.76, 131.46, 131.26, 131.16, 131.05, 130.15, 129.79, 129.02, 128.37, 128.07, 126.03, 123.10, 114.38, 100.00, 49.73, 26.95. HRMS (ESI) calcd for C₂₅H₂₀ClN₈S [M+H]⁺ 499.1220, found 499.1218.

5-(((1H-苯并[d]咪唑-2-基)甲基)硫基)-3-苄基-N-(2-甲氧基苯基)-3H-[1, 2, 3]-三氮唑[4, 5-d]嘧啶-7-胺(11j):白色固体, 产率62.9%. m.p. 189.2~189.7 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 10.30 (s, 1H), 7.70 (dd, J＝6.0, 3.1 Hz, 2H), 7.50 (dq, J＝6.4, 3.4 Hz, 3H), 7.21~7.05 (m, 7H), 6.91 (t, J＝7.5 Hz, 1H), 5.75 (s, 2H), 4.92 (s, 2H), 3.72 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 167.34, 153.85, 152.22, 152.13, 150.47, 147.91, 136.36, 136.05, 131.63, 129.01, 128.35, 128.28, 127.02, 125.98, 123.29, 120.62, 114.41, 112.47, 56.09, 49.64, 26.99. HRMS (ESI) calcd for C₂₆H₂₃N₈OS [M+H]⁺ 495.1716, found 495.1711.

5-(((1H-苯并[d]咪唑-2-基)甲基)硫基)-3-苄基-N-(3-三氟甲基苯基)-3H-[1, 2, 3]-三氮唑[4, 5-d]嘧啶-7-胺(11k):白色固体, 产率81.7%. m.p. 263.3~263.8 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.40 (s, 1H), 8.40 (s, 1H), 8.11 (d, J＝7.7 Hz, 1H), 7.69 (dt, J＝6.7, 3.4 Hz, 2H), 7.63 (t, J＝8.0 Hz, 1H), 7.50 (dt, J＝6.2, 3.3 Hz, 3H), 7.17 (dd, J＝14.7, 7.2 Hz, 3H), 7.12~7.04 (m, 2H), 5.81 (s, 2H), 5.02 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 167.60, 152.00, 151.86, 149.78, 139.38, 135.95, 131.51, 130.30, 129.97, 129.65, 129.33, 128.97, 128.87, 128.59, 128.41, 128.36, 126.06, 125.88, 123.50, 123.17, 121.06, 118.52, 114.37, 49.79, 26.85. HRMS (ESI) calcd for C₂₆H₂₀F₃N₈S[M+H]⁺ 533.1484, found 533.1481.

2-((5-(((1H-苯并[d]咪唑-2-基)甲基)硫基)-3-苄基-3H-[1, 2, 3]-三氮唑[4, 5-d]嘧啶-7-基)氨基)乙-1-醇(11l):黄色固体, 产率75.4%. m.p. 122.3~122.9 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.88 (d, J＝92.5 Hz, 1H), 7.49 (s, 2H), 7.30~7.13 (m, 7H), 5.68 (s, 2H), 4.92 (s, 1H), 4.67 (d, J＝8.2 Hz, 2H), 3.70~3.57 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆)δ: 168.87, 168.20, 155.29, 154.04, 151.98, 151.86, 150.84, 149.44, 136.18, 129.13, 128.60, 128.51, 128.45, 123.41, 122.00, 59.73, 49.77, 43.34, 29.08; HRMS (ESI) calcd for C₂₁H₂₁N₈OS [M+H]⁺ 433.1559, found 433.1555.

2, 2'-((5-(((1H-苯并[d]咪唑-2-基)甲基)硫基)-3-苄基-3H-[1, 2, 3]-三氮唑[4, 5-d]嘧啶-7-基)氮烷二基)双(乙烷-1-醇)(11m):白色固体, 产率86.0%. m.p. 174.9~175.6 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.33 (s, 1H), 7.55 (d, J＝7.7 Hz, 1H), 7.42 (d, J＝6.8 Hz, 1H), 7.27 (dd, J＝8.0, 2.3 Hz, 5H), 7.19~7.13 (m, 2H), 5.69 (s, 2H), 5.10 (t, J＝5.6 Hz, 1H), 4.89 (t, J＝5.3 Hz, 1H), 4.67 (s, 2H), 4.33 (t, J＝5.6 Hz, 2H), 3.90 (t, J＝5.8 Hz, 2H), 3.71 (dq, J＝17.4, 5.6 Hz, 4H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 167.83, 153.66, 152.05, 150.65, 143.45, 136.15, 134.95, 129.14, 128.44, 126.88, 123.72, 122.52, 121.73, 118.80, 111.71, 59.65, 58.60, 53.18, 51.70, 49.67, 29.05; HRMS (ESI) calcd for C₂₃H₂₅N₈O₂S[M+H]⁺ 477.1821, found 477.1812.

5-(((1H-苯并[d]咪唑-2-基)甲基)硫基)-N, 3-二苄基N-甲基-3H-[1, 2, 3]-三氮唑[4, 5-d]嘧啶-7-胺(11n):白色固体, 产率57.3%. m.p. 119.7~120.3 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.30 (s, 1H), 7.53 (s, 1H), 7.41 (s, 1H), 7.31 (dd, J＝8.1, 6.3 Hz, 6H), 7.28~7.25 (m, 4H), 7.15 (s, 2H), 5.71 (d, J＝4.3 Hz, 2H), 5.55 (s, 1H), 5.04 (s, 1H), 4.72 (s, 1H), 4.66 (s, 1H), 3.18 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 168.07, 153.80, 151.85, 150.92, 150.62, 143.64, 137.11, 136.10, 136.01, 129.15, 129.05, 128.65, 128.57, 128.21, 127.71, 122.41, 121.63, 118.88, 111.67, 54.62, 52.32, 35.72, 29.15; HRMS (ESI) calcd for C₂₇H₂₅N₈S[M+H]⁺ 493.1923, found 493.1921.

4-(5-(((1H-苯并[d]咪唑-2-基)甲基)硫基)-3-苄基-3H-[1, 2, 3]-三氮唑[4, 5-d]嘧啶-7-基)吗(11o):白色固体, 产率73.5%. m.p. 267.2~268.0 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.30 (s, 1H), 7.54 (s, 1H), 7.41 (s, 1H), 7.30~7.21 (m, 5H), 7.14 (d, J＝3.7 Hz, 2H), 5.70 (s, 2H), 4.66 (s, 2H), 4.51 (s, 2H), 3.96 (s, 2H), 3.75 (s, 2H), 3.64 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆)δ: 168.18, 152.52, 151.91, 150.80, 143.50, 136.06, 135.01, 129.13, 128.49, 123.54, 122.34, 121.71, 121.63, 118.79, 111.60, 49.74, 47.89, 44.20, 29.24; HRMS (ESI) calcd for C₂₃H₂₃N₈OS [M+H]⁺ 459.1716, found 459.1711.

3.2.7 抗肿瘤细胞毒活性实验

称取3~5 mg样品置于1.5 mL EP管中, 用二甲基亚砜(DMSO)配制成100×10³ μg/mL的溶液, 4 ℃保存, 根据实验所需浓度用培养基稀释.取对数生长期的细胞, 消化计数后, 用培养基调整细胞密度, 以4×10³个/孔接种至96孔板, 每孔150 μL培养基, 培养24 h, 弃去培养基, 加入用培养基稀释好的药物, 即将待测样品以100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 μg/mL浓度加入96孔板中, 每个浓度设6个复孔, 另设空白对照组和阴性对照组.药物作用72 h, 每孔加入20 μL 3-(4, 5-二甲基噻唑-2-基)-2, 5-二苯基四唑溴化物(MTT)溶液(5 mg/mL), 37 ℃孵育4 h, 吸去液体, 加入150 μL的DMSO, 振荡均匀, 酶标仪490 nm处检测吸光度值, 试验结果采用SPSS16.0统计软件计算IC₅₀值.

辅助材料(Supporting Information)化合物6~10, 11a~11o的¹H NMR, ¹³C NMR谱图.这些材料可以免费从本刊网站(http://sioc-journal.cn/)上下载.

1. [1]
  Christoph, D. M.; Venkatesam, A. M.; Santos, E. D. J. Med. Chem. 2010, 53, 798. doi: 10.1021/jm9014982
2. [2]
  Li, Z. H.; Liu, X. Q.; Geng, P. F.; Suo, F. Z.; Ma, J. L. ACS Med. Chem. Lett. 2017, 8, 384. doi: 10.1021/acsmedchemlett.6b00423
3. [3]
  Yu, W. Q.; Goddard, C.; Clearfield, E.; Mills, C.; Xiao, T.; Guo, H. T.; Morrey, J. D.; Motter, N. E.; Zhao, K.; Block, T. M.; Cuconati, A.; Xu, X. D. J. Med. Chem. 2011, 54, 5660. doi: 10.1021/jm200696v
4. [4]
  Alper, S.; Arpaci, O. T.; Esin, A. S.; Yalcin, I. Farmaco 2002, 58, 497.
5. [5]
  Zhou, R.; Skibo, E. B. J. Med. Chem. 1996, 39, 4321. doi: 10.1021/jm960064d
6. [6]
  Zhou, W.; Li, H. B.; Xia, C. N.; Zheng, X. M.; Hu, W. X. Bioorg. Med. Chem. Lett. 2009, 19, 1861. doi: 10.1016/j.bmcl.2009.02.081
7. [7]
  Schulz, W. G.; Islam, I.; Skibo, E. B. J. Med. Chem. 1995, 38, 109. doi: 10.1021/jm00001a016
8. [8]
  Schulz, W. G.; Nieman, R. A.; Skibo, E. B. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 11854. doi: 10.1073/pnas.92.25.11854
9. [9]
  Skibo, E. B.; Islam, I.; Heileman, M. J.; Schulz, W. G. J. Med. Chem. 1994, 37, 78. doi: 10.1021/jm00027a010
10. [10]
  El-Sayed, W. A.; Ali, O. M.; Faheem, M. S.; Zied, I. F.; Ab-del-Rahmanb, A. A. H. J. Heterocycl. Chem. 2012, 49, 607. doi: 10.1002/jhet.832
11. [11]
  Rangarajan, M.; Kim, J. S.; Jin, S.; Sim, S. P.; Liu, A.; Pilch, D. S.; Liu, L. F.; LaVoie, E. J. Bioorg. Med. Chem. 2000, 8, 1371. doi: 10.1016/S0968-0896(00)00054-7
12. [12]
  Andrzejewska, M.; Yepez-Mulia, L.; Tapia, A.; Cedillo-Rivera, R.; Laudy, A. E.; Starosciak, B. J.; Kazimierczuk, Z. Eur. J. Pharm. Sci. 2003, 21, 323.
13. [13]
  Chen, J. Y.; Ren, X. X.; Mao, Z. W.; Li, X. Y. J. Coord. Chem. 2012, 65, 2182. doi: 10.1080/00958972.2012.681380
14. [14]
  Ansari, K. F.; Lal, C. Eur. J. Med. Chem. 2009, 44, 4028. doi: 10.1016/j.ejmech.2009.04.037
15. [15]
  Asthana, S.; Shukla, S.; Vargiu, A. V.; Ceccarelli, M.; Ruggerone, P.; Paglietti, G.; Marongiu, M. E.; Blois, S.; Giliberti, G.; La Colla, P. Biochemistry 2013, 52, 3752. doi: 10.1021/bi400107h
16. [16]
  El-Sherif, A. A. J. Solution Chem. 2010, 39, 1562. doi: 10.1007/s10953-010-9593-y
17. [17]
  Chou, S. Y.; Yin, W. K.; Chung, Y. S.; Chang, L. S.; Liu, C. W.; Chen, S. F.; Shih, K. S. Org. Process Res. Dev. 2002, 6, 273. doi: 10.1021/op0100807
18. [18]
  Zheng, Y. C.; Duan, Y. C.; Ma, J. L.; Xu, R. M.; Zi, X.; Lv, W. L.; Wang, M. M.; Ye, X. W.; Zhu, S.; Mobley, D. J. Med. Chem. 2013, 56, 8543. doi: 10.1021/jm401002r
19. [19]
  Ma, L. Y.; Zheng, Y. C.; Wang, S. Q.; Wang, B. J. Med. Chem. 2015, 58, 1705. doi: 10.1021/acs.jmedchem.5b00037
20. [20]
  王超杰, 曹钦坡, 杨慧, 宋攀攀, 薛登启, 崔飞, 顾一飞, 张孝松, 田亚楠, 张秋荣, 刘宏民, 有机化学, 2016, 36, 1626. http://kns.cnki.net/KCMS/detail/detail.aspx?filename=yjhu201607018&dbname=CJFD&dbcode=CJFQWang, C.; Cao, Q.; Yang, H.; Song, P.; Xue, D.; Cui, F.; Gu, Y.; Zhang, X.; Tian, Y.; Zhang, Q.; Liu, H. Chin. J. Org. Chem. 2016, 36, 1626(in Chinese). http://kns.cnki.net/KCMS/detail/detail.aspx?filename=yjhu201607018&dbname=CJFD&dbcode=CJFQ
21. [21]
  宋攀攀, 栗娜, 崔飞, 辛景超, 张孝松, 曹钦坡, 王超杰, 戴文杰, 孟祥川, 刘梦, 常通航, 柳晴怡, 孙月红, 可钰, 张秋荣, 刘宏民, 有机化学, 2017, 37, 2725. http://kns.cnki.net/KCMS/detail/detail.aspx?filename=yjhu201710024&dbname=CJFD&dbcode=CJFQSong, P.; Li, N.; Cui, F.; Xin, J.; Zhang, X.; Cao, Q.; Wang, C.; Dai, W.; Meng, X.; Liu, M.; Chang, T.; Liu, Q.; Sun, Y.; Ke, J.; Zhang, Q.; Liu, H. Chin. J. Org. Chem. 2017, 37, 2725(in Chinese). http://kns.cnki.net/KCMS/detail/detail.aspx?filename=yjhu201710024&dbname=CJFD&dbcode=CJFQ
22. [22]
  Izsák, D.; Klapötke, T. M.; Pflüger, C. Dalton Trans. 2015, 44, 17054. doi: 10.1039/C5DT03044G

图 1 1, 2, 3-三氮唑[4, 5-d]嘧啶衍生物的结构式

Figure 1 Structures of 1, 2, 3-triazolo[4, 5-d]pyrimidine derivatives

下载: 全尺寸图片幻灯片

图式 1 化合物11a~11o的合成路线

Scheme 1 Synthesis of compounds 11a~11o

下载: 全尺寸图片幻灯片

表 1 化合物9~10和11a~11o对五种人类癌细胞的体外细胞毒活性^a [IC₅₀/(μmol•L^－1)]

Table 1. In vitro cytotoxic activities of synthetic compounds 9~10 and 11a~11o against five human cancer cell lines [IC₅₀/(μmol•L^－1)]

Compd.	R	MGC-803	MCF-7	EC-109	PC-3	Hela
9	HO	25.93±0.82	6.00±0.31	＞100	72.76±1.15	35.97±1.03
10	Cl	15.56±0.74	52.36±1.18	69.17±1.30	56.83±1.26	31.34±0.82
11a	4-FC₆H₄NH	19.32±0.47	26.67±0.51	26.54±0.61	20.17±0.66	11.76±0.54
11b	4-ClC₆H₄NH	8.75±0.09	10.63±0.14	16.91±0.26	11.56±0.43	9.51±0.49
11c	4-BrC₆H₄NH	46.63±1.20	23.57±0.98	＞100	36.67±0.47	18.16±0.36
11d	4-CH₃C₆H₄NH	78.45±1.11	60.56±1.52	＞100	＞100	45.63±1.22
11e	4-CH₃OC₆H₄NH	67.13±1.38	＞100	80.42±1.55	57.82±1.22	55.88±1.39
11f	4-C₂H₅O₂CC₆H₄NH	73.11±1.43	47.98±1.20	54.39±1.13	45.21±1.41	44.56±1.17
11g	4-CH₃OC₆H₄CH₂NH	31.46±1.09	32.34±0.87	47.45±1.22	＞100	25.61±0.40
11h	2-FC₆H₄NH	＞100	＞100	56.79±1.25	＞100	67.18±1.06
11i	2-ClC₆H₄NH	34.65±0.88	＞100	79.32±1.17	＞100	53.97±1.21
11j	2-CH₃OC₆H₄NH	37.89±1.51	＞100	71.32±1.02	＞100	33.86±0.98
11k	3-CF₃C₆H₄NH	＞100	＞100	62.18±1.21	＞100	63.29±1.02
11l	HO(CH₂)₂NH	20.64±0.45	19.34±0.64	32.39±0.89	15.22±0.99	8.21±0.21
11m	[HO(CH₂)₂]₂N	8.61±0.06	10.72±0.11	12.38±0.33	12.68±0.28	5.58±0.13
11n	C₆H₅CH₂(CH₃)N	37.93±1.12	＞100	＞100	45.61±1.37	56.73±1.23
11o	Morpholin-4-yl	＞100	＞100	＞100	＞100	71.26±1.20
5-Fu^b		9.84±0.87	8.67±0.56	17.88±0.69	9.35±0.57	11.86±0.73
^a Anticancer activity was assayed by exposure for 72 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC₅₀). Dates are presented as the means±SDs of three independent experiments. ^b Positive control.

下载: 导出CSV

扫一扫看文章

计量

PDF下载量: 9
文章访问数: 2285
HTML全文浏览量: 803

通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142