甾体-咪唑盐杂合物的设计、合成及细胞毒活性研究
段银1,2, 赵静峰1, 曾祥慧1, 李萍3, 张洪彬1
1.云南大学 教育部自然资源药物化学重点实验室,云南 昆明 650091
2.昆明龙津药业股份有限公司,云南 昆明 650503
3.云南省微生物研究所,云南 昆明 650091
通信作者:张洪彬(1964-),男,云南人,博士生导师,教授,主要从事有机合成及药物化学方面的研究.E-mail:zhanghb@ynu.edu.cn.

作者简介:段 银(1983-),女,云南人,硕士,主管药师,主要从事有机合成方面的研究工作.E-mail:duanyin632@163.com.

摘要

从胆甾醇、豆甾醇、薯蓣皂素出发合成了一系列新型的甾体咪唑盐化合物,其结构经1H NMR、13C NMR、HRMS以及IR波谱确定.对合成的新化合物进行了体外抗肿瘤细胞活性筛选,结果表明,发现3-苄基-1-(2α-胆甾-4-烯-3-酮)咪唑-3-溴盐(10)和3-胡椒苄基-1-(2α-胆甾-4-烯-3-酮)咪唑-3-溴盐(13)具有较好的体外肿瘤生长抑制活性,对乳腺癌细胞株Bcap-37的活性明显优于顺铂.

关键词: 咪唑杂合物; 甾体; 合成; 细胞毒活性
中图分类号:O626.23 文献标志码:A 文章编号:0258-7971(2018)06-1223-10
Design,synthesis and cytotoxic activity of novel hybrid compounds between steroid and imidazolium salts
DUAN Yin1,2, ZHAO Jing-feng1, ZENG Xiang-hui1, LI Ping3, ZHANG Hong-bin1
1.Key Laboratory of Medicinal Chemistry for Natural Resource Ministry of Education,Yunnan University,Kunming 650091,China
2.Kunming Longjin Pharmaceutical Co.,Ltd,Kunming 650503,China
3.Yunnan Institute of Microbiology,Kunming 650091,China
Abstract

A series of hybrid imidazole compounds have been prepared from cholesterol,stigmasterol and diosgenin.Their structures were confirmed by1H NMR,13C NMR,HRMS and IR.These compounds have been evaluated in vitro against a panel of human tumor cell lines.The results showed that 3-benzly-1-(2 α-cholest-4-en-3-one)imidazol-3-ium bromide (10) and 3-(3’,4’-methylenedioxy- benzyl) -1-(2α-cholest-4-en-3-one)imidazol-3-ium bromide (13) exhibited higher inhibitory activities against human breast cancer Bcap-37 than cisplatin (DDP).

Keyword: imidazole hybrid; steroid; synthesis; cytotoxic activity

天然产物一直是药物和药物先导化合物发现的重要源泉.以活性天然产物分子结构为导向, 设计并合成类天然产物库进行生物活性筛选并从中发现先导化合物进行药物临床前研究, 是新药研发的重要途径之一[1].

甾体化合物是动植物中广泛存在并在生命过程中起重要作用的一类天然产物 [2, 3].甾体调控许多生物代谢过程, 甾体衍生物已经是治疗许多疾病的重要药物, 广泛应用在风湿、心血管、胶原性疾病、淋巴性白血病、人体器官移植、抗肿瘤、细菌性脑炎、皮肤病、内分泌失调等疾病的治疗中[4].除了直接药用的部分天然产物外, 大多数甾体类药物都是通过在甾体母体上连接其他药效团半合成而来[5].

咪唑是五元杂环化合物, 是许多活性天然产物的重要结构单元, 其衍生物具有良好的药理活性, 如抗菌、抗炎、抗结核, 尤其是抗肿瘤活性等[6].在咪唑环系基础上合成的咪唑盐类化合物, 由于具有广泛的生物活性, 尤其是抗肿瘤活性而受到国内外有机合成及药物化学工作者的重视[1].将咪唑结构单元引入到一些具有生物活性的化合物中时, 常常会显示出一些特殊的生物活性[7, 8].从药物设计上来看, 咪唑基团的碱性和亲水性可改变甾体的生物活性, 而且, 它还可以作为药物的一部分与生物体内的一些酶相互作用, 比如细胞色素P450等[9].1997年, Yang-zhi Ling等合成了一系列的17位取代的咪唑甾体化合物作为P45017α 抑制剂用于前列腺癌的药物研究中, 他们发现化合物1的活性最好(IC50=21nmol/L)[10].另外, 甾体咪唑还用于加速核糖核酸(RNA)类似物的裂解.1997年, Thorsten Oost和Markus Kalesse将咪唑基和胍基引入甾体的不同位置以模拟核糖核酸酶(RNase).在合成的一系列杂合物中, 化合物2在催化水解模拟底物时活性最好(k=2.0× 10-6 s-1)[11].结构见图1.

图1 化合物1, 2结构Fig.1 Structure of compound 1 and 2

受此启发, 本文设计并合成了一系列甾体咪唑及其盐类化合物, 旨在为进一步的药物化学研究提供物质基础.具体合成路线见图2.

图2 甾体-咪唑盐化合物的合成Fig.2 Synthesis of steroid-imidazolium salts

1 结果与讨论

本文从商品化的甾醇(3a、3b和3c)(图3)出发, 用异丙醇铝在甲苯及丙酮中进行Oppenauer氧化, 从而高收率地分别制备得到α , β -不饱和甾酮化合物4a~c; 化合物4a~c通过碱性条件下的环氧化反应得到4, 5-环氧-3-甾酮化合物5a~c; 化合物5a~c进行重排反应得到2-乙酰氧基-4-烯基-3-甾酮化合物6a~c, 上述反应的收率都在60%以上; 化合物6a~c通过水解反应得到2位羟基的烯酮化合物7a~c; 化合物7a~c进行磺酰化反应后, 得到磺酰化产物8a~c; 化合物8a~c再与咪唑发生取代反应就得到2-咪唑基-4-烯基-3-甾酮化合物9a~c(图4); 甾体咪唑化合物与溴化物进一步在甲苯中回流反应[1], 制备得到6个新的甾体咪唑盐化合物10~15.该类化合物的结构经1H NMR、13C NMR、HRMS以及IR确定(图5).本文对所合成的新化合物10~15做了初步体外细胞毒活性测试, 通过噻唑蓝(MMT)法, 选用人乳腺癌细胞株Bcap-37进行体外细胞毒活性测试, 以顺铂(DDP)作为阳性对照.实验结果表明, 化合物10和13具有较好的抑制活性, IC50值分别为3.7× 10-4、1.1× 10-4 μ g/mL, 优于顺铂, 见表1.

图3 胆甾醇(3a)、豆甾醇(3b)和薯蓣皂素(3c)Fig.3 Cholesterol(3a), stigmasterol(3b) and diosgenin(3c)

图4 甾体-咪唑化合物9a~c的结构Fig.4 Structures of steroid-imidazole hybrid compouds 9a— c

图5 甾体咪唑盐化合物10~15的结构Fig.5 Structures of imidazolium salt compouds 10— 15

表1 化合物10~15的体外细胞毒活性数据 Tab.1 In vitro cytotoxic activities of compounds 10~15
2 结 论

本文从商品化的甾醇出发, 通过Oppenauer氧化、环氧化反应、环氧开环反应、水解脱乙酰氧基反应、甲烷磺酰化反应、咪唑取代反应和成盐7步反应合成了一些新颖的甾体咪唑盐化合物, 其结构经1H NMR、13C NMR、HRMS以及IR波谱确定.对合成的新化合物进行了体外抗肿瘤活性筛选, 结果表明, 化合物10、12、13和15均具有体外肿瘤生长抑制活性, 其中, 化合物10和13具有较好的体外抗肿瘤活性, 对Bcap-37肿瘤细胞株的活性明显优于顺铂, 可以作进一步的结构修饰和更深入的活性研究.

3 实验部分
3.1 仪器与试剂

核磁共振谱用Bruker Avance300M核磁共振仪测定(TMS作内标), 溶剂绝大多数用CDCl3; 红外用Perkin-Elmer1800型FT-IR红外光谱仪, KBr压片测定; 高分辨质谱用QSTAR Pulsar质谱仪测定; 硅胶板GF254和层析用硅胶(0.077~0.172mm和0.054~0.077mm)为青岛海洋化工厂生产; 洗脱剂一般采用石油醚(60~90℃)、乙酸乙酯和丙酮.所有反应试剂来自Acros、Aldrich、Fluka公司提供的商用试剂, 未做任何纯化.

3.2 实验方法

3.2.1 4-烯-3-甾酮的合成(4a~c) 在装有恒压漏斗和冷凝器的500mL 2颈瓶中加入200mL甲苯, 加热蒸馏, 馏出约20mL甲苯(利用共沸蒸馏法除净反应器中的水分), 然后冷至室温加入相当于底物2倍物质的量的异丙醇铝, 在搅拌下加热至50℃, 然后加入10g甾体化合物(即化合物1、2或3)和50mL丙酮的混合溶液, 缓慢升温至84℃, 此时生成的异丙醇被蒸馏出来, 根据薄层层析检测反应进展, 如原料剩余, 可适当加入丙酮继续反应(9~16h), 得到α , β -不饱和酮产物.

化合物4a:白色固体, 产率80%; C27H44O(M=384.64) ; 1H NMR (300MHz, CDCl3)δ :5.68 (1H, s), 2.39~2.30 (4H, m), 2.02~1.97 (2H, dd, J=3.3, 12.9Hz), 1.89~1.82 (2H, m), 1.75~1.67 (1H, dd), 1.66~1.44 (5H, m), 1.42~1.23 (4H, m), 1.13~1.04 (9H, m), 1.01~0.97 (3H, m), 0.89~0.86 (4H, m), 0.84 (3H, dd, J=0.96Hz), 0.82(3H, s), 0.67(3H, s); 13C NMR (75MHz, CDCl3)δ :199.5(C), 171.6(C), 123.7(CH), 56.0(CH), 55.8(CH), 53.7(CH), 42.3(C), 39.6(CH2), 39.4(CH2), 38.5(CH2), 36.1(CH2), 35.7(CH2), 35.6(CH), 35.5(CH), 33.9(CH2), 32.9(CH2), 32.0(CH2), 28.1(CH2), 27.9(CH), 24.1 (CH2), 23.8(CH2), 22.8(CH), 22.5(CH), 21.0(CH2), 18.6(CH3), 17.3(CH3), 11.9(CH3).

化合物4b:白色固体, 产率89%; C29H46O (M=410.67); 1H NMR (300MHz, CDCl3)δ :5.69 (1H, s), 5.16~5.08 (1H, dd, J=8.3, 15.1Hz), 5.03~4.95 (1H, dd, J=8.3, 15.1Hz), 2.35~2.31 (3H, m), 2.29~2.18 (1H, m), 2.01~1.97 (3H, m), 1.88~1.76 (1H, m), 1.74~1.62 (2H, m), 1.60~1.48 (5H, m), 1.46~1.31 (2H, m), 1.21~1.08 (7H, m), 1.02~0.98 (5H, m), 0.98~0.89 (2H, m), 0.83~0.75 (9H, m), 0.70 (3H, s); 13C NMR (75MHz, CDCl3)δ :199.5(C), 171.5(C), 138.1(CH), 129.5(CH), 123.8(CH), 56.1(CH), 56.0(CH), 53.9(CH), 51.3(CH), 42.3(C), 40.5(CH), 39.6(CH2), 38.7(C), 35.8(CH2), 35.7(CH), 34.0(CH2), 33.0(CH2), 32.1(CH2), 31.9(CH), 28.9(CH2), 25.4(CH2), 24.3(CH2), 21.2(2CH3), 21.1(CH2), 19.1(CH3), 17.4(CH3), 12.3(CH3), 12.2 (CH3).

化合物4c:白色固体, 产率79%; C27H40O3 (M=412.60) ; 1H NMR (300MHz, CDCl3)δ :5.67 (s, 1H), 4.39~4.32 (1H, m), 3.43~3.28 (2H, m), 2.37~2.31 (4H, m), 1.99 (2H, m), 1.83~1.23 (14H, m), 1.15~0.80 (10H, m), 0.77~0.73 (6H, m); 13C NMR (75MHz, CDCl3)δ :199.4(C), 171.0(C), 123.8(CH), 109.2(C), 80.5(CH), 66.8(CH2), 62.0(CH), 55.6(CH), 53.7(CH), 41.6(CH), 40.3(CH2), 39.6(C), 38.6(C), 35.6(CH2), 35.1(CH), 33.9(CH2), 32.7(CH2), 32.0 (CH2), 31.6(CH2), 31.3 (CH2), 30.2(CH), 28.7(CH2), 20.7(CH2), 17.3(CH3), 17.1(CH3), 16.3(CH3), 14.4(CH3).

3.2.2 4, 5-环氧-3-甾酮的合成(5a~c) 将1mmol α , β -不饱和酮溶于适量甲醇(加入少量四氢呋喃助溶), 然后加入1mL 6mol/L的氢氧化钠溶液、1mL 30%过氧化氢, 室温下搅拌, 直至原料消失, 用稀盐酸中和, 减压浓缩, 加水并用二氯甲烷萃取, 合并有机相并用无水硫酸钠干燥, 减压浓缩, 柱层析分离纯化产物.

化合物5a:白色固体, 产率68%; C27H44O2 (M=400.64); 1H NMR (300MHz, CDCl3)δ :2.92~2.93 (1H, d), 2.30~2.14 (3H, m), 2.01~1.95 (1H, m), 1.84~1.79 (3H, m), 1.58~1.52 (4H, m), 1.38~1.30 (6H, m), 1.11~0.98 (14H, m), 0.89~0.85 (3H, d), 0.84 (3H, s), 0.83 (3H, s), 0.66 (3H, d, J=2.9Hz); 13C NMR (75MHz, CDCl3)δ :206.8(C), 70.3(C), 62.6(CH), 56.1(CH), 55.8(CH), 46.4(CH), 42.6(C), 39.4(2CH2), 37.1(CH2), 36.0(CH), 35.7(CH), 35.0(C), 32.5(CH2), 30.4(CH2), 29.8(CH2), 28.0(CH2), 27.9(CH), 26.1(CH2), 24.1(CH2), 23.8 (CH2), 22.8(CH3), 22.5(CH3), 21.5(CH2), 18.9(CH3), 18.6(CH3), 12.0(CH3).

化合物5b:白色固体, 产率61%; C29H46O2 (M=426.67) ; 1H NMR (300MHz, CDCl3)δ :5.16~5.08 (1H, dd, J=8.3, 15.1Hz), 5.03~4.95 (1H, dd, J=8.3, 15.1Hz), 2.93 (1H, s), 2.30~1.98 (5H, m), 1.88~1.64 (3H, m), 1.55~1.48 (5H, m), 1.39~1.36 (3H, m), 1.23~0.97 (15H, m), 0.82~0.75 (9H, m), 0.67 ( 3H, s); 13C NMR (75MHz, CDCl3)δ :206.7(C), 138.1(CH), 129.6(CH), 70.3(C), 62.7(CH), 56.0(2CH), 51.3(CH), 46.5(CH), 42.6(C), 40.5(CH), 39.4(CH2), 37.2(C), 35.1(CH), 32.6(CH2), 31.9(CH), 30.5(CH2), 30.0(CH2), 28.8(CH2), 26.3(CH2), 25.4 (CH2), 24.3(CH2), 21.6(CH2), 21.2(CH3), 21.1(CH3), 19.1(CH3), 19.0(CH3), 12.3 (2CH3).

化合物5c:白色固体, 产率71%, C27H40O4 (M=412.60) ; 1H NMR (300MHz, CDCl3)δ :4.45~4.32(1H, m), 3.45~3.32(2H, m), 3.00(1H, α , s), 2.94(1H, β , s).

3.2.3 2-乙酰氧基-4-烯-3-甾酮的合成(6a~c) 依次将1mmolα , β -环氧酮、4mmol无水醋酸钠加入到10mL醋酸中, 然后油浴加热逐渐升温至90~110℃, 薄层层析监测反应进程, 反应完毕后减压浓缩, 然后加水并用乙酸乙酯萃取(3× 20mL), 合并有机相并用无水硫酸钠干燥, 减压浓缩并用柱层析分离纯化产物.

化合物6a:白色固体, 产率74%; C29H46O3 (M=442.67); 1H NMR (300MHz, CDCl3)δ :5.73 (1H, s), 5.47~5.41 (1H, dd, J=5.2, 14.0Hz), 2.36~2.22 (3H, m), 2.16 (3H, s), 2.01 (1H, m), 1.83~1.78 (3H, m), 1.53~1.33 (9H, m), 1.31 (3H, s), 1.15~1.00 (10H, m), 0.91~0.89 (3H, d, J=6.6Hz), 0.87~0.85 (6H, d, J=6.6Hz), 0.70 (3H, s); 13C NMR (75MHz, CDCl3)δ :193.6(C), 171.1(C), 170.2(C), 121.7(CH), 71.3(CH), 56.0(CH), 55.7(CH), 54.3(CH), 42.3(C), 41.4(CH2), 40.6(C), 39.5(2CH2), 36.1(CH2), 35.7(CH), 35.1(CH), 32.5(CH2), 31.9(CH2), 28.1(CH2), 28.0(CH), 24.1 (CH2), 23.8(CH2), 22.8(CH3), 22.5(CH3), 20.9(CH3), 20.9(CH2), 18.6(CH3), 18.1(CH3), 11.9(CH3).

化合物6b:白色固体, 产率72%; C31H48O3 (M=468.71); 1H NMR (300MHz, CDCl3)δ :5.71 (1H, s), 5.46~5.39 (1H, dd, J=5.3, 14.0Hz), 5.16~5.08 (1H, dd, J=8.3, 15.1Hz), 5.04~4.96 (1H, dd, J=8.3, 15.1Hz); 13C NMR (75MHz, CDCl3)δ :193.6(C), 171.1(C), 170.2(C), 138.0(CH), 129.5(CH), 121.7(CH), 71.3(CH), 55.9(2CH), 54.3(CH), 51.2(CH), 42.2(C), 41.4(CH2), 40.6(C), 40.4(CH), 39.3(CH2), 35.1(CH), 32.5(CH2), 31.9(2CH2), 31.9(CH), 28.8(CH2), 25.4(CH2), 24.2(CH2), 21.2(CH3), 21.1(CH3), 20.9(CH3), 19.0(CH3), 18.1(CH3), 12.1 (CH3), 12.0(CH3).

化合物6c:白色固体, 产率63%.

3.2.4 2-羟基-4-烯-3-甾酮的合成(7a~c) 将100mg 2-乙酰氧基α , β -环氧酮投到由100mg碳酸氢钠、1.0mL水和4.0mL甲醇组成的混合液中, 加热回流, 薄层层析监测反应进程.

化合物7a:白色固体, 收率57%; C27H44O2(M=400.64); 1H NMR (300MHz, CDCl3)δ :5.76 (1H, s), 4.27~4.20 (1H, dd, J=1.6, 5.6, 13.7Hz), 3.58~3.57 (1H, d, J=1.8Hz), 2.36~2.30 (3H, dd, J=5.7, 12.6Hz), 2.04~1.99 (1H, d, J=15Hz), 1.85~1.81 (2H, m), 1.57~1.47 (6H, m), 1.29~1.26 (7H, m), 1.12~1.01 (9H, m), 0.89~0.87 (4H, d, J=6.5Hz), 0.85~0.84 (3H, s), 0.83~0.82 (3H, s), 0.68 (3H, s); 13C NMR (75MHz, CDCl3)δ :199.8(C), 173.4(C), 120.2(CH), 69.6(CH), 56.1(CH), 55.8(CH), 54.5(CH), 44.1(CH2), 42.4(C), 40.6(C), 39.5(2CH2), 36.2(CH2), 35.8(CH), 35.1(CH), 32.9(CH2), 32.1(CH2), 28.2(CH2), 28.1(CH), 24.2(CH2), 23.9 (CH2), 22.9(CH3), 22.6(CH3), 20.8(CH2), 18.7(CH3), 18.1(CH3), 12.0(CH3).

化合物7b:淡黄色固体, 产率64%; C29H46O2 (M=426.67) ; 1H NMR (300MHz, CDCl3)δ :5.77 (1H, s), 5.17~5.09 (1H, dd, J=8.3, 15.1Hz), 5.04~4.96 (1H, dd, J=8.3, 15.1Hz), 4.27~4.21 (1H, dd, J=5.6, 13.7Hz), 3.56 (1H, s), 2.37~2.31 (3H, dd, J=5.7, 14.2Hz), 2.02~2.01 (2H, m), 1.93~178 (1H, m), 1.77~1.63 (1H, m), 1.58~1.34 (8H, m), 1.27 (4H, s), 1.23~1.12 (4H, m), 1.04 (3H, s), 0.99 (3H, s), 0.84~0.77 (9H, m), 0.71 (3H, s); 13C NMR (75MHz, CDCl3)δ :199.7(C), 173.3(C), 138.0(CH), 129.4(CH), 120.0(CH), 69.5(CH), 55.8(2CH), 54.43(CH), 51.2(CH), 44.0(CH2), 42.2(C), 40.5(C), 40.4(CH), 39.3(CH2), 35.0(CH), 32.8(CH2), 32.0(CH2), 31.8(CH), 28.8(CH2), 25.3 (CH2), 24.1(CH2), 21.1(CH3), 21.0(CH3), 20.7(CH2), 18.9(CH3), 18.0(CH3), 12.2(CH3), 12.1(CH3).

化合物7c:淡黄色固体, 产率57%; C27H40O4 (M=412.60); 1H NMR (300MHz, CDCl3)δ :5.76 (1H, s), 4.41~4.33 (1H, q, J=7.5, 14.7Hz), 4.26~4.19 (1H, dd, J=8.0Hz), 3.56~3.55 (1H, s, J=1.6Hz), 3.45~3.42 (1H, m), 3.36~3.29 (1H, t), 2.37~2.31 (3H, m), 2.04~1.93 (1H, m), 1.86~1.82 (2H, m), 1.76~1.72 (2H, m), 1.64~1.56 (5H, m), 1.52~1.42 (4H, m), 1.27 (4H, s), 1.12~1.04 (3H, m), 0.95~0.92 (4H, d, J=6.8Hz), 0.79 (3H, s), 0.77~0.75 (3H, d, J=6.2Hz); 13C NMR (75MHz, CDCl3)δ :199.7(C), 172.9(C), 120.3(CH), 109.3(C), 80.6(CH), 69.6(CH), 66.9(CH2), 62.1(CH), 55.6(CH), 54.4(CH), 44.0(CH2), 41.7(CH), 40.7(C), 40.4(C), 39.6(CH2), 34.7(CH), 32.7(CH2), 32.2(CH2), 31.7(CH2), 31.4(CH2), 30.3(CH), 28.9(CH2), 20.6(CH2), 18.1(CH3), 17.2(CH3), 16.4(CH3), 14.5(CH3).

3.2.5 2-甲烷磺酰-4-烯-3-甾酮的合成(8a~c) 在100mL圆底烧瓶中将2α -羟基α , β -不饱和酮甾体溶于足够量的二氯甲烷中, 加入三乙胺(2.4倍物质的量), 冰浴下缓慢滴加甲烷磺酰氯(1.2倍物质的量), 滴加完毕后, 撤去冰浴室温反应, 薄层层析检测反应进程, 反应约1~3h, 反应完毕后加入水(20mL), 用稀盐酸中和, 分离有机相, 水相用二氯甲烷(3× 10mL)萃取, 合并有机相, 依次用饱和碳酸氢钠溶液, 饱和氯化钠溶液洗涤, 无水硫酸钠干燥, 减压浓缩后得到粗产物(收率90%~100%).

化合物8a:淡黄色固体, 产率95%; C28H46O4S (M=478.73) ; 1H NMR (300MHz, CDCl3)δ :5.72 (1H, s), 5.24~5.18 (1H, dd, J=5.4, 13.9Hz), 3.26 (3H, s), 2.40~2.32 (3H, m), 2.05~2.01 (1H, d, J=12Hz), 1.90~1.83 (3H, m), 1.53~1.43 (5H, m), 1.30 (7H, s), 1.13~0.96 (10H, m), 0.90~0.88 (3H, d, J=6.4Hz), 0.86 (3H, s), 0.83 (3H, s), 0.69 (3H, s); 13C NMR (75MHz, CDCl3)δ :192.7(C), 172.9(C), 121.5(CH), 78.6(CH), 56.1(CH), 55.7(CH), 54.3(CH), 42.8(CH2), 42.4(C), 41.2(C), 39.5(CH3), 39.5(CH2), 39.4(CH2), 36.2(CH2), 35.8(CH), 35.1(CH), 32.6(CH2), 31.9(CH2), 28.2(CH2), 28.1 (CH), 24.1(CH2), 23.9(CH2), 22.9(CH3), 22.6(CH3), 21.0(CH2), 18.7(CH3), 18.3(CH3), 12.0(CH3).

化合物8b:白色固体, 产率93%; C30H48O4S (M=504.67); 1H NMR (300MHz, CDCl3)δ :5.72 (1H, s), 5.24~4.96 (3H, m), 3.25 (3H, s), 2.37~2.31 (3H, m), 2.06~1.98 (2H, m), 1.92~1.85 (2H, m), 1.79~1.62 (1H, m), 1.60~1.44 (7H, m), 1.30 (4H, m), 1.23~1.12 (4H, m), 1.03 (3H, s), 0.98 (3H, s), 0.83~0.76 (9H, m), 0.70 (3H, s); 13C NMR (75MHz, CDCl3)δ :192.6(C), 172.8(C), 138.0(CH), 129.6(CH), 121.4(CH), 78.6(CH), 55.8(2CH), 54.3(CH), 51.3(CH), 42.8(CH2), 42.3(C), 41.2(C), 40.4(CH), 39.4(CH3), 39.3(CH2), 35.0(CH), 32.6(CH2), 31.9(CH2), 31.8(CH), 28.8 (CH2), 25.4(CH2), 24.2(CH2), 21.2(CH3), 21.1(CH3), 20.9(CH2), 19.0(CH3), 18.3(CH3), 12.3(CH3), 12.2(CH3).

化合物8c:白色固体, 产率98%; C28H42O6S (M=506.69); 1H NMR (300MHz, CDCl3)δ :5.72 (1H, s), 5.23~5.16 (1H, dd, J=5.4, 13.9Hz), 4.41~4.33 (1H, q, J=7.4, 14.7Hz), 3.46~3.29 (2H, m), 3.25 (3H, s), 2.38~2.33 (3H, m), 2.04~1.93 (1H, m), 1.88~1.82 (2H, m), 1.80~1.72 (3H, m), 1.64~1.57 (4H, m), 1.46~1.42 (3H, m), 1.31 (4H, s), 1.18~0.98 (4H, m), 0.98~0.92 (4H, d, J=6.8Hz), 0.79 (3H, s), 0.77~0.75 (3H, d, J=6.2Hz); 13C NMR (75MHz, CDCl3)δ :192.5(C), 172.3(C), 121.5(CH), 109.3(C), 80.5(CH), 78.5(CH), 66.9(CH2), 62.0(CH), 55.5(CH), 54.1(CH2), 42.7(CH2), 42.2(CH), 41.2(C), 40.3(C), 39.4(CH3), 39.4(CH2), 34.6(CH), 32.4(CH2), 32.0(CH2), 31.6(CH2), 31.4(CH2), 30.3(CH), 28.8(CH2), 20.7(CH2), 18.3(CH3), 17.2(CH3), 16.3(CH3), 14.5 (CH3).

3.2.6 2-咪唑基-4-烯-3-甾酮的合成(9a~c) 在100mL圆底烧瓶中将甲烷磺酰化产物溶于30mL无水甲苯中, 加入2.5倍物质的量的咪唑, 加热回流反应, 薄层层析检测反应进程, 反应约需3~4d.减压浓缩, 硅胶柱层析分离, 得到甾体咪唑化合物.

化合物9a:淡黄色固体, 产率30%; C30H46N2O (M=450.70); 1H NMR (300MHz, CDCl3)δ :7.48 (1H, s), 7.06 (1H, s), 6.86 (1H, s), 5.81 (1H, s), 4.89~4.82 (1H, m), 2.37~2.35 (3H, d, J=3.9Hz), 2.11~2.05 (2H, q), 1.86 (2H, s), 1.54~1.26 (11H, m), 1.09~1.00 (10H, m), 0.91~0.85 (10H, m), 0.71 (3H, s); 13C NMR (75MHz, CDCl3)δ :192.6(C), 171.8(C), 136.8(CH), 128.8(CH), 121.8(CH), 118.1(CH), 58.3(CH), 55.9(CH), 55.6(CH), 54.0(CH), 43.3(CH2), 42.2(C), 40.4(C), 39.3(CH2), 39.2(CH2), 35.9(CH2), 35.5(CH), 35.0(CH), 32.4(CH2), 31.7(CH2), 28.0(CH2), 27.8(CH), 23.9(CH2), 23.6(CH2), 22.7(CH3), 22.4(CH3), 20.7(CH2), 18.5 (CH3), 18.0(CH3), 11.9(CH3); IR ν max (KBr)/cm-1:3444, 3114, 2941, 2871, 2355(CO2), 1687, 1618, 1500, 1460, 1376, 1237, 1192, 1084, 908, 744, 665; ESI-MS (m/z):451([M+1]+, 23%), 452([M+2]+, 19%).

化合物9b:淡黄色固体, 产率17%; C32H48N2O (M=476.74); 1H NMR (300MHz, CDCl3)δ :7.45 (1H, s), 7.06 (1H, s), 6.84 (1H, s), 5.80 (1H, s), 5.15~4.93 (2H, m), 4.83~4.76 (1H, dd, J=4.4, 14.5Hz), 2.53~2.34 (3H, m), 2.16~1.93 (3H, m), 1.92~1.82 (1H, d), 1.82~1.64 (1H, m), 1.62~1.37 (6H, m), 1.33 (3H, s), 1.27 (1H, s), 1.18~0.97 (10H, m), 0.82~0.72 (10H, m), 0.70 (3H, s); 13C NMR (75MHz, CDCl3)δ :192.8(C), 172.0(C), 138.0(CH), 137.0(CH), 129.5(CH), 129.2(CH), 122.0(CH), 118.2(CH), 58.4(CH), 55.8(2CH), 54.2(CH), 51.2 (CH), 43.5(CH2), 42.2(C), 40.5(C), 40.4(CH), 39.2(CH2), 35.2(CH), 32.6(CH2), 31.8(C), 31.8(CH), 28.8(CH2), 25.4(CH2), 24.2(CH2), 21.1(2CH3), 20.9(CH2), 19.0(CH3), 17.8(CH3), 12.3(CH3), 12.1(CH3); IR ν max (KBr)/cm-1:3428, 3107, 2951, 2865, 1684, 1619, 1499, 1455, 1376, 1228, 1082, 970, 908, 822, 750, 661; ESI-MS (m/z):477([M+1]+, 60%), 422(14%), 400(9%).

化合物9c:淡黄色固体, 产率38%; C30H42N2O3 (M=478.67); 1H NMR (300MHz, CDCl3)δ :7.45(1H, s), 7.06 (1H, s), 6.84 (1H, s), 5.82 (1H, s), 4.82~4.76 (1H, dd, J=4.5, 14.5Hz), 4.40~4.37 (1H, q), 3.43~3.42 (1H, m), 3.36~3.29 (1H, q), 2.37~2.32 (3H, m), 2.10~1.92 (1H, m), 1.99~1.72 (5H, m), 1.71~1.52 (4H, m), 1.51~1.40 (3H, m), 1.35 (3H, s), 1.29~1.10 (2H, m), 1.10~1.04 (4H, m), 0.95~0.93 (3H, d, J=6.8Hz), 0.80 (3H, s), 0.77~0.75 (3H, dd, J=6.2Hz); 13C NMR (75MHz, CDCl3)δ :192.7(C), 171.4(C), 137.0(CH), 129.3(CH), 122.1(CH), 118.2(CH), 109.3(C), 80.5(CH), 66.9(CH2), 62.0(CH), 58.4(CH), 55.4(CH), 54.1(CH), 43.5(CH), 41.6(CH), 40.6(C), 40.3(C), 39.4(CH2), 34.8(CH), 32.5(CH2), 32.0 (CH2), 31.6(CH2), 31.3(CH2), 30.2(CH), 28.8(CH2), 20.7(CH2), 17.8(CH3), 17.1(CH3), 16.3(CH3), 14.5(CH3); IR ν max (KBr)/cm-1:3428, 3107, 2945, 1687, 1624, 1500, 1454, 1376, 1231, 1060, 979, 907, 740, 662; ESI-MS (m/z):479([M+1]+, 44%), 438(31%), 422(100%), 400(86%).

3.2.7 化合物10~15的合成 在25mL圆底烧瓶中加入0.1g甾体咪唑化合物, 再加入溴苄或其衍生物(相当于底物的2倍物质的量), 甲苯(15mL), 回流反应10~24h, 过滤得到固体, 用甲苯洗涤数次, 干燥, 即可得到产物.

化合物10:淡黄色粉末, 产率89%; C37H53BrN2O (M=621.73); 1H NMR (300MHz, CDCl3)δ :10.47 (1H, s), 7.48 (4H, s), 7.35~7.34 (3H, d, J=3.1Hz), 6.05~6.00 (1H, dd, J=3.4, 14.2Hz), 5.76 (1H, s), 5.52 (2H, s), 2.55~2.52 (1H, d, J=8.1Hz), 2.41~2.19 (3H, m), 2.03~1.99 (1H, d, J=11.7Hz), 1.84 (2H, s), 1.55~1.49 (8H, m), 1.32~1.26 (4H, d, J=16.5Hz), 1.11~1.02 (10H, t), 0.87~0.85 (9H, d, J=6.4Hz), 0.69 (3H, s); 13C NMR (75MHz, CDCl3)δ :190.5(C), 173.9(C), 137.1(CH), 133.0(C), 129.3 (3CH), 128.9(2CH), 121.8(CH), 121.5(CH), 121.0(CH), 61.3(CH), 55.9(CH), 55.4(CH), 53.8(CH), 53.1(CH2), 42.2(CH2), 41.6(C), 41.1(C), 39.3(CH2), 39.1(CH2), 36.0(CH2), 35.6(CH), 35.0(CH), 32.7(CH2), 31.8(CH2), 28.0(CH2), 27.9(CH), 24.0(CH2), 23.7 (CH2), 22.7(CH3), 22.5(CH3), 20.8(CH2), 18.5(CH3), 18.2(CH3), 11.8(CH3); IR ν max (KBr)/cm-1:3429, 2942, 1864, 2353(CO2), 1684, 1617, 1584, 1454, 1376, 1161, 713, 650; ESI-MS (m/z):542([M-Br+1]+, 100%), 541([M-Br]+, 100%), 502(33%).

化合物11:淡黄色粉末, 产率80%; C39H55BrN2O (M=647.77); 1H NMR (300MHz, CDCl3)δ :10.49 (1H, s), 7.47~7.44 (4H, t), 7.35 (3H, s), 6.04~5.99 (1H, dd, J=3.3, 12.5Hz), 5.78 (1H, s), 5.52(2H, s), 5.17~4.97 (2H, m), 2.54 (1H, s), 2.33~2.23 (3H, m), 2.01~1.98 (2H, d, J=11.0Hz), 1.85 (1H, s), 1.72 (1H, s), 1.70~1.53 (10H, d), 1.46~0.98 (11H, m), 0.89~0.80 (9H, m), 0.71 (3H, s); 13C NMR (75MHz, CDCl3)δ :190.6(C), 174.0(C), 138.0(CH), 137.2(CH), 132.9(C), 129.3(3CH), 128.9(2CH), 121.8(CH), 121.5(CH), 121.0(2CH), 61.3(CH), 55.6(2CH), 53.9(CH), 53.2(CH2), 51.1(CH), 42.1(C), 41.7(C), 41.1(CH2), 40.4(CH), 39.1(CH2), 35.0(CH), 32.7(CH2), 31.8(CH2), 31.8(CH), 28.7(CH2), 25.3(CH), 24.1 (CH2), 21.1(CH3), 21.0(CH3), 20.8(CH2), 189(CH3), 18.2(CH3), 12.2(CH3), 12.0(CH3); IR ν max (KBr)/cm-1:3427, 3050, 2953, 2866, 2354(CO2), 1685, 1617, 1556, 1454, 1373, 1218, 1161, 1105, 972, 714, 652; ESI-MS (m/z):567([M-Br]+, 100%), 502(95%).

化合物12:淡黄色粉末, 产率80%; C37H49BrN2O3 (M=649.70); 1H NMR (300MHz, CDCl3)δ :10.54 (1H, s), 7.47~7.32 (7H, m), 6.04~5.98 (1H, dd, J=4.2, 14.5Hz), 5.78 (1H, s), 5.51 (2H, s), 4.43~4.35 (1H, m), 3.54~3.35 (2H, m), 2.55~2.24 (4H, m), 1.88~1.84 (1H, m), 1.78~1.73 (2H, m), 1.67~0.78 (27H); 13C NMR (75MHz, CDCl3)δ :190.4(C), 173.5(C), 137.3(CH), 132.9(C), 129.4 (3CH), 128.9(2CH), 122.0(CH), 121.4(CH), 121.1(CH), 109.2(C), 80.4(CH), 66.8(CH2), 61.9(CH), 61.4(CH), 55.3(CH), 53.8(CH), 53.2(CH2), 41.7(C), 41.6(C), 41.2 (CH2), 40.2(CH), 39.2(CH), 34.7(CH), 32.6(CH2), 31.9(CH2), 31.6(CH2), 31.3 (CH2), 30.2 (CH), 28.7(CH2), 20.7(CH2), 18.3(CH3), 17.1(CH3), 16.3(CH3), 14.4(CH3); IR ν max (KBr)/cm-1:3427, 3059, 2942, 2850, 2356(CO2), 1684, 1617, 1556, 1453, 1377, 1228, 1164, 1055, 980, 907, 717, 654; ESI-MS (m/z):569([M-Br]+, 48%), 524(95%), 502(100%).

化合物13:白色粉末; 产率81%; C38H53BrN2O3 (M=665.74); 1H NMR (300MHz, CDCl3)δ :10.33~10.31 (1H, d, J=6.7Hz), 7.50 (1H, s), 7.36 (1H, s), 6.97~6.93 (2H, d, J=11.7Hz), 6.69~6.64 (1H, t), 5.93~5.84 (3H, t), 5.70~5.69 (1H, d, J=4.4Hz), 5.38 (2H, s), 2.45~2.11 (4H, m), 1.94~1.91 (1H, d, J=9.9Hz), 1.77 (2H, s), 1.60~1.30 (8H, t), 1.28~1.12 (4H, d), 1.03 (10H, s), 0.79 (9H, s), 0.61 (3H, s); 13C NMR (75MHz, CDCl3)δ :190.6(C), 174.0(C), 148.3(C), 148.1(C), 136.9 (CH), 126.6(C), 123.2(CH), 121.7(CH), 121.5(CH), 121.0(CH), 109.2(CH), 108.7(CH), 101.4(CH2), 61.2(CH), 55.8(CH), 55.5(CH), 53.8(CH), 52.8(CH2), 42.1(CH2), 41.6(C), 41.0(C), 39.3(CH2), 39.1(CH2), 35.9(CH2), 35.6(CH), 34.9(CH), 32.7(CH2), 31.8(CH2), 28.0(CH2), 27.9(CH), 24.0(CH2), 23.7(CH2), 22.7(CH3), 22.4 (CH3), 22.4(CH2), 18.5 (CH3), 18.1(CH3), 11.8(CH3); IR ν max (KBr)/cm-1:3425, 2942, 2858, 1685, 1617, 1557, 1497, 1450, 1373, 1249, 1161, 1110, 1037, 925, 864, 744; ESI-MS (m/z):585([M-Br]+, 95%), 524(100%), 502(89%).

化合物14:白色粉末; 产率80%; C40H55BrN2O3 (M=691.78); 1H NMR (300MHz, CDCl3)δ :10.28 (1H, s), 7.51 (1H, s), 7.38 (1H, s), 6.96~6.92 (2H, d, J=11.9Hz), 6.66~6.64 (1H, t), 5.93~5.83 (3H, t), 5.68 (1H, s), 5.37 (2H, s), 5.07~4.88 (2H, m), 2.46~2.15 (4H, m), 1.91~1.97 (1H, d, J=11.8Hz), 1.79~1.75 (1H, d, J=9.3Hz), 1.61 (1H, s), 1.43~1.36 (10H, d), 1.17~0.80 (12H, m), 0.73~0.70 (9H, d, J=8.0Hz), 0.61 (3H, s); 13C NMR (75MHz, CDCl3)δ :190.6(C), 173.8(C), 148.3(C), 148.0(C), 137.9 (CH), 136.7(CH), 129.3(CH), 126.6(C), 123.2(CH), 121.7(CH), 121.5(CH), 120.9(CH), 109.2(CH), 108.6(CH), 101.4(CH2), 61.2(CH), 55.5(CH), 53.7(CH), 52.8(CH2), 51.0 (CH), 42.6(CH2), 42.0(C), 41.5(CH), 41.0(C), 40.3(C), 39.0(CH2), 34.9(CH), 32.6(CH2), 31.7(CH2), 31.7(CH), 28.7(CH2), 25.2(CH2), 24.0(CH2), 21.0(2CH3), 20.7(CH2), 18.8 (CH3), 18.1(CH3), 12.1(CH3), 11.9(CH3); IR ν max (KBr)/cm-1:3429, 2953, 2868, 1685, 1618, 1556, 1498, 1451, 1373, 1248, 1161, 1109, 1036, 971, 928, 744; ESI-MS (m/z):611([M-Br]+, 69%), 502(100%).

化合物15:白色粉末; 产率82%; C38H51BrN2O4 (M=679.73); 1H NMR (300MHz, CDCl3)δ :10.48 (1H, s), 7.57~7.55 (2H, d, J=6.2Hz), 7.10 (2H, s), 6.76 (1H, s), 5.93 (2H, s), 5.56~5.45 (3H, d), 4.42~4.21 (2H, d), 3.46~3.37 (2H, t), 2.79~2.55 (3H, m), 2.23~1.32 (17H, m), 1.23~0.0.91 (10H, m), 0.78 (6H, s).

13C NMR (75MHz, CDCl3)δ :148.3(C), 148.1(C), 137.9(C), 135.3(CH), 127.0 (C), 123.8(CH), 123.3(CH), 122.0(CH), 120.3(CH), 109.4(CH), 109.1(C), 108.6(CH2), 101.4(CH2), 80.6(CH), 66.7(CH2), 61.9(CH), 60.5(CH), 56.2(CH), 52.7 (CH2), 49.6 (CH), 41.4(CH), 40.1(C), 39.4(CH2), 39.2(CH2), 37.2(CH2), 36.5(C), 31.8(CH2), 31.7 (CH2), 31.2(CH2), 31.1(CH), 30.1(CH), 29.2(CH2), 28.6(CH2), 20.6 (CH2), 19.3(CH3), 17.0(CH3), 16.2(CH3), 14.4(CH3).

IR ν max (KBr)/cm-1:3424, 3048, 2944, 1632, 1556, 1496, 1445, 1373, 1248, 1161, 1045, 980, 927, 770.

ESI-MS (m/z):599([M-Br]+, 92%), 502(100%).

3.3 生物活性筛选

甾体咪唑盐化合物10~15经纯化及结构鉴定后, 进行了肿瘤细胞毒活性筛选.采用MTT法进行测定:选取乳腺癌(Bcap-37)细胞株, 采用含10%胎牛血清的培养液接种, 加入待测化合物溶液后, 37℃培养48h, 然后选择570nm波长, 用酶联免疫检测(Bio-Rad 680)读取光吸收值, 同一样品重复3次, 最后用两点法(Reed和Muench法)计算获得化合物的IC50值.

The authors have declared that no competing interests exist.

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