某些A-homo甾体内酰胺噻唑衍生物的合成及抗肿瘤活性评估

引用本文: 黄燕敏, 庞春玲, 杨春晖, 展军颜, 甘春芳, 庞丽萍, 刘晓兰, 崔建国. 某些A-homo甾体内酰胺噻唑衍生物的合成及抗肿瘤活性评估[J]. 有机化学, 2018, 38(6): 1484-1492. doi: 10.6023/cjoc201710005 shu

Citation: Huang Yanmin, Pang Chunling, Yang Chunhui, Zhan Junyan, Gan Chunfang, Pang Liping, Liu Xiaolan, Cui Jianguo. Synthesis and Evaluation of Some New Aza-A-homocholesteryl-lactam Thiazole Derivatives as Anticancer Agents[J]. Chinese Journal of Organic Chemistry, 2018, 38(6): 1484-1492. doi: 10.6023/cjoc201710005 shu

某些A-homo甾体内酰胺噻唑衍生物的合成及抗肿瘤活性评估

广西师范学院化学与材料科学学院南宁 530001

通讯作者: 崔建国, cuijg1954@126.com

基金项目:

国家自然科学基金（Nos.21462009，21562007）、南宁市科技开发项目（No.20171125-5）和广西高校北部湾石油天然气资源有效利用重点实验室开放课题（No.2014KLOG09）资助项目

摘要: 从睾酮出发，经过3-羰基肟化、Beckmann重排反应构建A-环甾体内酰胺甾核骨架，并以此为母体对17-羟基进行官能团改造与结构修饰，引入一些杂环基团，合成了一些结构新颖的A-Homo甾体内酰胺噻唑衍生物.通过IR、NMR及HRMS等现代分析方法对化合物进行了结构表征，并对化合物进行体外抑制肿瘤细胞生长增殖活性测试，结果表明其中某些化合物对所测试肿瘤细胞具有明显的抑制活性，但对人正常肾上皮细胞没有明显的抑制作用.

关键词:

English

Synthesis and Evaluation of Some New Aza-A-homocholesteryl-lactam Thiazole Derivatives as Anticancer Agents

College of Chemistry and Material Science, Guangxi Teachers Education University, Nanning 530001

Corresponding author: Cui Jianguo, E-mail:cuijg1954@126.com

Received Date: 08 October 2017
Revised Date: 13 December 2017
Available Online: 01 June 2018
Fund Project:

Project supported by the National Natural Science Foundation of China (Nos. 21462009, 21562007), the Technology and Development Project of Nanning City (No. 20171125-5), the Guangxi Colleges and University Key Laboratory Foundation of Beibu Gulf Oil and Natural Gas Resource Effective Utilization (No. 2014KLOG09)

Abstract: Using a testosterone as a raw material, a steroidal skelecton with A-homo-lactam was constructed. Then, some new aza-A-homocholesteryl-lactam thiazole derivatives were synthesized by modifying of the 17-hydroxyl and introducing of various heterocycles, and their structures were determined by IR, NMR and HRMS. The antiproliferative activity of the compounds against different carcinoma cells was assayed. The results show that some compounds display a distinct antiproliferative activity against tested cancer cells and are almost inactive to HEK293T cells.

Key words:

testosterone
/ heterosteroids
/ aza-A-homocholesteryl-lactam thiazole derivatives
/ antiproliferative activity

甾体化合物表现出不同的生物活性, 它们在生命体中起着非常重要的作用^{[1, 2]}.甾体药物是一类人体内源性药物, 它在维持人体健康方面有着不可替代的作用, 除了作为人体的激素类药物使用外, 甾体化合物还具有很强的抗炎、抗菌、抗过敏、降压、利尿、抗病毒和抗肿瘤等药理作用^[3～7].

为了探索开发具有显著抑制肿瘤细胞生长增殖活性的新型甾体化合物, 我们曾合成了系列具有A-Homo^{[8, 9]}、B-Homo^[10]、C-Homo^[11]及D-Homo^[8]增碳内酰胺甾核结构的甾体衍生物, 同时调查了它们抑制不同肿瘤细胞的生长增殖活性.结果表明具有A-Homo及B-Homo甾核结构的甾体衍生物, 当其17-支链结构为长链烷烃类型支链结构时, 这些化合物具有显著的抑制肿瘤细胞生长增殖作用.

杂环化合物是一类广泛应用于医药和农药领域的具有良好生物活性的化合物.噻唑环是一类重要的的五元芳杂环, 环内含有N、S杂原子, 其特殊的结构特征使得噻唑类化合物在化学、药学、生物学和材料科学等诸多领域已获得广泛应用^[12].在医药领域, 噻唑类化合物已经在抗菌、抗结核、抗癌、抗病毒、降血糖、消炎镇痛、抗癫痫、抗寄生虫等药物中获得应用^[13～15].因此, 噻唑类化合物具有广阔的研究和开发前景.

把芳杂环结构引入甾体母体, 所得到的甾体芳杂环化合物往往会出现与甾体母体不同的生理活性, 表现出抗肿瘤、抗菌或抗病毒活性的特征^[16].在前期研究中, 我们曾在去氢表雄酮^[17]的17-位及B-降胆甾醇^[18]的6-位引入不同的噻唑杂环药效团, 并研究了化合物的体外抑制肿瘤细胞生长增殖活性, 结果表明某些化合物具有明显的抑制肿瘤细胞生长增殖活性.

为了探索筛选更为有效的具有抗肿瘤活性的化合物, 作为前面工作的继续, 我们从睾酮出发, 利用Beckmann重排反应构建A-环甾体内酰胺甾核骨架, 并以此为母体对17-羟基进行官能团改造与修饰, 引入不同的噻唑杂环官能团, 合成系列结构新颖的A-Homo甾体内酰胺噻唑衍生物, 进一步评估化合物抑制肿瘤细胞生长增殖活性.

1. 结果与讨论

1.1 化学部分

Scheme 1列出了A-Homo甾体噻唑衍生物7～12的合成路线.使用睾酮为原料, 通过7步反应, 某些具有增环A-Homo内酰胺甾核结构的5-芳基噻唑衍生物被合成.首先, 通过乙酰化反应对睾酮17-位羟基进行保护, 然后对3-羰基肟化, 形成3-肟基产物2.化合物2是一个由(3E)-2和(3Z)-2组成的混合物, 其不需要分离可直接用于下步反应.化合物2在SOCl₂存在的条件下发生Beckmann重排, 甾核的A-环发生扩环反应形成7元环的增环A-Homo内酰胺甾核结构, 得到化合物3.在这里, 由于生成的化合物3中4-羰基与4a-双键共轭, 此化合物较为稳定.所以无论是(3E)-2或者(3Z)-2, 它们发生Beckmann重排后都形成化合物3, 没有出现另外的异构体.接着, 化合物3在13% K₂CO₃乙醇溶液中脱去17-羟基的乙酰保护基, 得到化合物4.化合物4采用Jones试剂氧化, 将17-羟基氧化成为羰基得到化合物5, 化合物5进一步和氨基硫脲反应生成具有17-缩氨硫脲结构的化合物6.最后, 化合物6和具有不同结构特征的α-溴代甲基芳基酮反应, 得到具有不同结构的5-芳基取代-2-甾体噻唑衍生物7～12.

图式 1

图式 1. 化合物7～12的合成路线

Scheme 1. Synthesis of compounds 7～12

Reagents and conditions: (a) Py/Ac₂O; (b) NH₂OH•HCl/CH₃CH₂OH; (c) SOCl₂/ THF; (d) 13% K₂CO₃/CH₃CH₂OH; (e) Jones reagent; (f) NH₂NHCSNH₂; (g) ArCOCH₂Br

下载: 全尺寸图片幻灯片

所有化合物的结构都已通过它们的IR、NMR及HRMS的结构表征.如所生成的化合物7, 在NMR谱中, 在δ 7.29、7.39、7.77处出现了Ph—H的化学位移, 在δ 128.6、127.6、125.9和103.0处出现了Ph—C的化学位移, 说明了在化合物7中存在一个苯环结构.而在¹H NMR中, 化学位移δ 6.81的单峰表明一个噻唑环上质子的存在.在¹³C NMR中, 在δ 170.2, 151.0, 135.1的化学位移说明了噻唑环已经形成.由此可见, 化合物7的结构得到了其相应NMR数据的结构表征.通过化合物7的IR及HREIMS谱, 化合物7的结构得到进一步确证.与此类似, 化合物8～12的结构同样通过了其波谱及光谱的结构表征.

化合物5进一步与4-甲基氨基硫脲反应, 得到化合物13.化合物13与不同的α-溴代甲基芳基酮反应时, 由于缩氨硫脲基4'-位N上的H原子已经被甲基取代, 生成的中间产物不能发生消除形成1, 3-噻唑环, 而是脱去2'-N上的H原子, 形成了一种二氢噻唑吖嗪类型的结构, 生成化合物14～18 (Scheme 2).然而, 当化合物13与α-溴代乙酸乙酯反应时, 则得到了另一种具有氢化咪唑结构的化合物19.

图式 2

图式 2. 化合物13～19的合成路线

Scheme 2. Synthesis of compounds 13～19

Reagents and conditions: (a) NH₂NHCSNHCH₃; (b) ArCOCH₂Br/CH₃CH₂OH; (c) BrCH₂CO₂CH₂CH₃/CH₃CH₂OH

下载: 全尺寸图片幻灯片

当化合物13与α-溴代乙酸乙酯反应时, 由于化合物的空间位阻较小, 因此溴甲基进攻底物缩氨硫脲基上2'-位上碱性较强的N原子(对于α-溴代甲基芳基酮来说, 由于芳基的体积较大, 空间位阻大, 因此反应发生在空间位阻较小的4'-位上N原子), 然后4'-位N原子进攻酯基中的羰基, 脱去一分子乙醇得到化合物19.相应的两类化合物形成的反应机理见Scheme 3所示.

图式 3

图式 3. 化合物14～19形成的不同反应机理

Scheme 3. Reaction mechanisms of formation for compounds 14～19

下载: 全尺寸图片幻灯片

1.2 生物部分

采用噻唑蓝(MTT)法, 人体宫颈癌细胞株(HeLa)、人肝癌细胞株(HEPG2)、人鼻咽癌细胞株(CNE-2)及人体正常细胞人肾上皮细胞系(HEK293T)作为被测试对象, 分别对所合成目标产物及中间体进行抑制细胞生长增殖活性测试, 结果见表 1所示.

表 1

表 1 化合物1～19体外抑制细胞生长增殖活性(MTT法) (IC₅₀, μmol/L)

Table 1. Antiproliferative activity of compounds 1～19 in vitro

下载: 导出CSV

化合物	人体宫颈癌细胞(HeLa)	人鼻咽癌细胞(CNE-2)	人肝癌细胞(HEPG2)	人肾上皮细胞(HEK293T)
1	＞80	＞80	＞80	＞80
2	＞80	＞80	＞80	＞80
3	＞80	＞80	＞80	＞80
4	＞80	＞80	＞80	＞80
5	＞80	＞80	＞80	＞80
6	＞80	＞80	＞80	＞80
7	＞80	＞80	＞80	＞80
8	＞80	＞80	＞80	＞80
9	＞80	＞80	＞80	＞80
10	＞80	＞80	＞80	＞80
11	＞80	＞80	＞80	＞80
12	＞80	＞80	＞80	＞80
13	55.2±4.9	＞80	＞80	＞80
14	19.5±2.1	19.5±1.9	42.5±4.3	＞80
15	＞80	＞80	＞80	＞80
16	＞80	＞80	＞80	＞80
17	22.5±2.3	42.5±3.8	19.5±1.8	＞80
18	＞80	＞80	＞80	＞80
19	42.1±3.9	33.4±2.8	62.5±5.8	＞80
Cisplatin	15.1±1.3	10.2±1.1	10.5±1.0	11.0±0.9
^a Data represent the mean values of three independent determinations.

从表 1中结果可见, 化合物1～12对所测试细胞的抑制活性IC₅₀值都大于80 μmol/L.对于化合物1～12, 它们的结构或是17-支链是一个极性取代基(化合物1～6), 或17-支链是一根具有极性的长链含芳香环的支链(化合物7～12).与我们前面的工作相对比, 过去的研究结果表明, 对于具有A-Homo或B-Homo类型甾体内酰胺甾核结构的甾体化合物, 当其17-支链为胆甾醇或豆甾醇类型的长链脂肪烃支链结构时, 化合物都具有很好的抑制肿瘤细胞生长增殖活性^{[9, 10]}.本文的研究结果进一步表明, 在此类A-Homo甾体内酰胺化合物的结构中, 在其甾核17-位拥有一根长链烷烃取代基对于化合物的细胞毒性影响是非常重要的, 其原因可能是17-长链烃基的存在, 增加了化合物的脂溶性, 也就增强了化合物对细胞壁的穿透能力.

对于噻唑环的N上的H被甲基取代后所形成的二氢噻唑吖嗪化合物14～18, 当噻唑环的5-位取代基为苯基(14)或连接有一个供电子甲氧基的苯基(17)时, 化合物表现出明显的细胞毒性, 而5-位苯基上连接的是吸电子基团时, 化合物15、16、18没有表现出明显的抑制肿瘤细胞活性.同样地, 由于吸电子基团的存在, 增强了化合物的极性, 降低了化合物的脂溶性, 因此不利于提高化合物的细胞毒性.另外, 具有4-氢化- 1, 3-咪唑环结构的化合物19也表现出明显的对肿瘤细胞的抑制活性.在这里值得注意的是, 所有的合成化合物对人体正常细胞HEK293T都没有明显的抑制活性.

2. 结论

以睾酮作为起始原料, 经过17-羟基乙酰化保护, 3-羰基肟化、Beckmann重排反应构建A-环甾体内酰胺甾核骨架.然后对17-乙酰氧基去保护, Jones试剂氧化17-羟基成为17-羰基, 进一步形成17-缩氨硫脲.最后与具有不同结构的α-溴代甲基芳基酮反应, 共7步反应合成得到某些新的具有不同结构特征的5-芳基取代-2-甾体噻唑衍生物及一个甾体氢化咪唑衍生物.通过IR、NMR及HRMS等现代分析方法完成了对化合物的结构表征, 并对化合物进行体外抑制肿瘤细胞生长增殖活性测试, 结果表明, 具有吖嗪结构的甾体噻唑衍生物14和17及具有氢化咪唑环结构的化合物19对所测试的肿瘤细胞表现出明显的抑制活性, 但是对人正常肾上皮细胞却没有明显的抑制作用.

3. 实验部分

3.1 仪器与试剂

睾酮、氨基硫脲及各种α-溴代甲基芳基酮试剂均购于百灵威科技有限公司; 合成试剂、溶剂均为分析纯, 四氢呋喃经重蒸处理.红外光谱测定采用Thermo Scientific Nicolet IS-10傅立叶红外光谱仪; 熔点测定采用X4数字显示显微熔点测定仪, 温度计未经校正; ¹H NMR和¹³C NMR测定采用德国Bruker AV-600型超导高分辨核磁共振仪和Bruker AV-300型超导核磁共振仪; 质谱测定采用美国热电LC QDECA-1000质谱仪.

3.2 实验方法

3.2.1 17-乙酰氧基-4-雄烯-3-酮(1)的制备

称取睾酮1.442 g (5mmol)溶于10 mL吡啶, 完全溶解后滴加乙酸酐3 mL, 室温下搅拌反应, 反应12 h至TLC检测[V(乙酸乙酯): V(石油醚)＝1: 6]无原料点.加入30 mL乙酸乙酯, 然后采用1 mol/mL的HCl洗涤3次, 饱和NaHCO₃及饱和NaCl溶液各洗一次, 无水硫酸钠干燥.过滤, 减压除去溶剂, 得到的固体重结晶得到白色固体产物1.581 g, 产率91.3%. m.p. 139～141 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.83 (s, 3H, 18-CH₃), 1.19 (s, 3H, 19-CH₃), 2.04 (s, 3H, COCH₃), 4.59 (t, J＝7.8 Hz, 1H, C17-H), 5.72 (s, 1H, C4-H); ¹³C NMR (CDCl₃, 75 MHz) δ: 199.4 (3-C), 171.2 (5-C), 170.9 (COCH₃), 123.9 (4-C), 82.4 (17-C), 53.7 (9-C), 50.2 (14-C), 42.4 (13-C), 38.6 (10-C), 36.6 (12-C), 35.7 (1-C), 35.4 (8-C), 33.9 (2-C), 32.7 (6-C), 31.5 (7-C), 27.5 (16-C), 23.5 (15-C), 21.1 (11-C), 20.5 (CH₃C＝O), 17.4 (19-C), 12.0 (18-C); IR (KBr) ν: 3459.00, 2937.95, 2873.13, 1738.18, 1674.47, 1611.63, 1447.09, 1379.78, 1227.70, 1038.23 cm^－1; HR- EIMS calcd for C₂₁H₃₀NaO₃ [M+Na]⁺ 353.2093, found 353.2086.

3.2.2 17-乙酰氧基-4-雄烯-3-肟(2)的制备

称取化合物1 346 mg (1 mmol)溶于70 mL 95%乙醇, 升温至75 ℃, 加入136 mg盐酸羟胺(1 mmol), 完全溶解后加入70 mg CH₃COONa•H₂O (1.007 mmol), 75 ℃下反应12 h, 薄层色谱(TLC)检测[V(乙酸乙酯): V(石油醚)＝1: 4]至无原料点停止反应.减压除去大部分溶剂, 加入适量乙酸乙酯, 水洗, 碳酸氢钠、氯化钠各洗涤一次, 无水硫酸钠干燥.过滤, 减压除去溶剂, 粗产品柱层析分离得到白色固体产物241 mg, 产物2是一个由(3E)-2和(3Z)-2组成的混合物, 其不需要分离可直接用于下步反应.产率66.7%. m.p. 173～174 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.83 (s, 3H, 18-CH₃), 1.07 (s, 3H, 19-CH₃), 2.05 (s, 3H, COCH₃), 3.05 (d, J＝17.1 Hz, 2H, C2-βH), 4.60 (t, J＝8.4 Hz, 1H, C17-H), 5.79 (s, 1H, C4-H), 6.49 (s, 1H, N-OH); ¹³C NMR (CDCl₃, 75 MHz) δ: 171.1 (COCH₃), 159.3 (3-C, Z), 156.8 (3-C, E), 155.2 (5-C, E), 153.7 (5-C, Z), 117.4 (4-C, E), 110.5 (4-C, Z), 82.7 (17-C), 53.6 (9-C), 50.4 (14-C), 42.5 (13-C), 38.9 (10-C), 38.0 (12-C), 36.7 (8-C), 35.6 (7-C), 34.6 (1-C), 32.3 (6-C), 31.7 (2-C), 27.5 (16-C), 23.5 (15-C), 21.2 (11-C), 20.8 (CH₃C＝O), 18.7 (19-C, Z), 17.8 (19-C, E), 12.0 (18-C); IR (KBr) ν: 3414.40, 2927.98, 2840.72, 1738.78, 1636.57, 1434.66, 1373.30, 1235.18, 1035.73 cm^－1; HREIMS calcd for C₂₁H₃₂NO₃ [M+H]⁺ 346.2382, found 346.2387.

3.2.3 17-乙酰氧基-3-氮杂-A-Homo-4a-雄烯-4-酮(3)的制备

取化合物2 207 mg (0.6 mmol)置于干燥的二口烧瓶中, 加入2 mL重蒸的无水四氢呋喃(THF)使之完全溶解, 通入氩气保护.冰水浴保持反应温度0 ℃, 缓缓加入2 mL二氯亚砜, 反应2 h后加入冰水结束反应, 用适量氨水调节溶液pH约等于7, 蒸去大部分THF, 加二氯甲烷萃取, 合并有机相, 用蒸馏水、饱和NaHCO₃溶液、饱和NaCl溶液各洗涤一次, 无水硫酸钠干燥, 旋干.粗产物柱层析分离[TLC检测, V(乙酸乙酯): V(石油醚)＝1: 1]得到白色固体产物168 mg, 产率48%. m.p. 231～233 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.81 (s, 3H, 18-CH₃), 1.14 (s, 3H, 19-CH₃), 2.03 (s, 3H, COCH₃), 2.48 (td, J＝13.5, 4.5 Hz, 1H, C6-H), 3.26～3.09 (m, 2H, C2-H), 4.59 (dd, J＝9.0, 7.8 Hz, 1H, C17-H), 5.72 (s, 1H, C4-H), 6.93 (s, 1H, NH); ¹³C NMR (CDCl₃, 75 MHz) δ: 171.1 (COCH₃), 170.2 (4-C), 160.7 (5-C), 119.1 (5a-C), 82.4 (17-C), 53.2 (9-C), 50.3 (14-C), 44.5 (13-C), 42.6 (1-C), 42.0 (8-C), 36.8 (12-C), 36.6 (2-C), 36.2 (10-C), 35.4 (6-C), 33.2 (7-C), 27.5 (16-C), 23.5 (15-C), 21.3 (11-C), 21.1 (19-C), 12.1 (18-C); IR (KBr) ν: 3299.45, 2930.47, 2875.62, 1733.80, 1663.99, 1596.68, 1444.67, 1242.66, 1040.72 cm^－1; HREIMS calcd for C₂₁H₃₂NO₃ [M+H]⁺ 346.2382, found 346.2379.

3.2.4 A-Homo-3-氮杂-4-氧代睾-4a-烯-17-醇(4)的制备

取化合物3 203.4 mg (0.59mmol)置于干燥的圆底烧瓶中, 加入乙醇30 mL使之完全溶解, 加入13% K₂CO₃水溶液6 mL, 搅拌, 75 ℃下反应3 h, TLC [V(乙酸乙酯): V(石油醚)＝1: 1]监测反应进度, 至无原料点停止反应.蒸出大部分乙醇, 加入适量水, 采用二氯甲烷萃取, 合并有机相, 用蒸馏水、饱和NaHCO₃溶液、饱和NaCl溶液各洗涤一次, 无水硫酸钠干燥, 旋干.粗产物重结晶得淡黄色粉末166 mg, 产率92.7%. m.p. 208～210 ℃; ¹H NMR (DMSO-d₆, 300 MHz) δ: 0.65 (s, 3H, 18-CH₃), 1.08 (s, 3H, 19-CH₃), 2.99 (br s, 2H, C2-H), 5.48 (s, 1H, C4-H), 7.70 (s, 1H, NH); ¹³C NMR (DMSO, 75MHz) δ: 168.7 (4-C), 159.8 (5-C), 119.9 (4a-C), 80.3 (17-C), 53.5 (9-C), 50.5 (14-C), 44.4 (13-C), 43.0 (1-C), 42.4 (8-C), 36.9(12-C), 36.0 (2-C), 35.9 (10-C), 35.6 (6-C), 33.7 (7-C), 30.3 (16-C), 23.5 (15-C), 21.4 (11-C), 21.4 (19-C), 11.7 (18-C); IR (KBr) ν: 3269.53, 2922.99, 2845.71, 1649.03, 1606.65, 1442.11, 1347.37, 1050.69 cm^－1; HREIMS calcd for C₁₉H₃₀NO₂ [M+H]⁺ 304.2277, found 304.2270.

3.2.5 A-Homo-3-氮杂-4-氧代睾-4a-烯-17-酮(5)的制备

150 mg (0.49 mmol)化合物4置于干燥的反应烧瓶中, 加入20 mL丙酮完全溶解, 然后慢慢滴加Jonse试剂2 mL, 冰水浴下搅拌反应4 h, TLC [V(石油醚): V(乙酸乙酯)＝4: 1]监测反应至无原料点, 停止反应.蒸出大部分丙酮, 加入适量水.采用二氯甲烷萃取, 合并有机相, 有机相用饱和NaHCO₃、饱和NaCl溶液各洗涤一次, 无水硫酸钠干燥, 旋干.粗产物采用柱层析分离[TLC检测, V(乙酸乙酯): V(石油醚)＝4: 1]得白色固体产物103.7 mg, 产率70%. m.p. 221～223 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.90 (s, 3H, 18-CH₃), 1.17 (s, 3H, 19-CH₃), 2.58～2.42 (m, 2H, C6-H), 3.28～3.10 (m, 2H, C2-H), 5.75 (s, 1H, C4-H), 6.91 (s, 1H, NH); ¹³C NMR (CDCl₃, 75 MHz) δ: 220.4 (17-C), 170.1 (4-C), 160.1 (5-C), 119.4 (4a-C), 53.3 (9-C), 50.8 (14-C), 47.6 (13-C), 44.5 (1-C), 42.1 (8-C), 36.6 (16-C), 35.1 (2-C), 35.8 (6-C), 35.1 (7-C), 32.4 (12-C), 31.4 (16-C), 21.8 (15-C), 21.3 (11-C), 21.1 (19-C), 13.8 (18-C); IR (KBr) ν: 3269.53, 2922.99, 2845.71, 1649.03, 1606.65, 1442.11, 1347.37, 1050.69 cm^－1; HREIMS calcd for C₁₉H₂₈NO₂ [M+H]⁺ 302.2120, found 302.2118.

3.2.6 A-Homo-3-氮杂-4-氧代睾-4a-烯-17-缩氨硫脲(6)的制备

取化合物5 300 mg (1 mmol)置于100 mL反应烧瓶中, 加入无水乙醇30 mL, 硫代氨基脲120 mg (1.31 mmol), 滴加冰醋酸调节pH至5～6, 将反应装置置于80 ℃油浴中搅拌反应8 h. TLC [V(石油醚): V(乙酸乙酯)＝1: 1]跟踪反应至无原料点, 停止反应.旋干大部分无水乙醇, 加入适量水.采用乙酸乙酯萃取, 有机相用饱和碳酸氢钠、饱和氯化钠水溶液各洗涤一次, 减压除去溶剂, 粗产物柱层析分离得白色固体280.6 mg, 产率75.0%. m.p. 273 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.90 (s, 3H, 18-CH₃), 1.09 (s, 3H, 19-CH₃), 2.65～2.58 (m, 2H, C2-H), 5.81 (s, 1H, C4-H), 6.51 (s, 1H, NH), 7.25 (s, 1H, NH), 8.82 (s, 1H, NH), 9.06 (s, 1H, NH); ¹³C NMR (CDCl₃, 75 MHz) δ: 178.4 (C＝S), 163.0 (4-C), 157.8 (5-C), 150.2 (17-C), 120.6 (4a-C), 53.6 (14-C), 50.9 (9-C), 47.6 (13-C), 47.5 (1-C), 37.9 (12-C), 35.8 (8-C), 35.3 (2-C), 34.6 (10-C), 32.2 (6-C), 31.3 (7-C), 30.9 (16-C), 21.8 (15-C), 20.6 (11-C), 17.7 (19-C), 13.7 (18-C); IR (KBr) ν: 3434.07, 3259.55, 3154.95, 2940.44, 2870.64, 1733.80, 1601.66, 1501.94, 1242.66, 1078.12, 863.71, 572.02 cm^－1; HREIMS calcd for C₂₀H₃₁N₄OS [M+H]⁺ 375.2219, found 375.2215.

3.2.7 5-芳基取代-2-[(A-Homo-3'-氮杂-4'-氧代)睾-4a'-烯-17'-酮腙]-1, 3-噻二唑(7～12)合成

取1 mmol化合物6置于100 mL蒸馏烧瓶内, 加入40 mL无水乙醇使之完全溶解, 向瓶内加入1.5 mmol不同的芳基-α-溴代甲基酮.反应体系置于80 ℃油浴下反应12 h, TLC跟踪反应至无原料点.旋干大部分无水乙醇, 加入适量水, 然后用乙酸乙酯萃取, 有机相用饱和碳酸氢钠、饱和氯化钠溶液各洗涤一次, 减压除去溶剂.得到的固体粗产物柱层析分离得到目标产物.

5-苯基-2-[(A-Homo-3'-氮杂-4'-氧代)睾-4a'-烯-17'-酮腙]-1, 3-噻二唑(7):淡黄色固体, 产率51.0%. m.p. 212～213 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.94 (s, 3H, 18-CH₃), 1.18 (s, 3H, 19-CH₃), 2.58～2.35 (m, 2H, C6-H), 3.25～3.03 (m, 2H, C2-H), 3.50 (s, 1H, N-NH), 5.77 (s, 1H, C4a-H), 6.81 (s, 1H, 4-thiazole-H), 7.18 (s, 1H, NH), 7.29 (t, J＝7.2 Hz, 1H, 4'-PhH), 7.39 (t, J＝6.9 Hz, 2H, 3', 5'-PhH), 7.77 (d, J＝7.2 Hz, 2H, 2', 6'-PhH); ¹³C NMR (CDCl₃, 75 MHz) δ: 170.3 (17-C), 170.2 (2-thiazole-C), 164.8 (4-C), 160.5 (5-C), 151.0 (5-thiazole-C), 135.1 (4-thiazole-C), 128.6 (2', 6'-PhC), 127.6 (1'-PhC), 125.9 (3', 5'-PhC), 119.2 (4a-C), 103.0 (4'-PhC), 53.7 (14-C), 53.0 (9-C), 44.5 (13-C), 44.5 (1-C), 42.1 (12-C), 35.1 (8-C), 34.0 (2-C), 33.1 (10-C), 29.7 (6-C), 26.2 (7-C), 23.6 (16-C), 21.4 (15-C), 21.3 (11-C), 17.2 (19-C), 14.2 (18-C); IR (KBr) ν: 3055.12, 2935.46, 2850.69, 1731.30, 1661.52, 1569.25, 1439.61, 1043.21, 701.66 cm^－1; HREIMS calcd for C₂₈H₃₅N₄OS [M+H]⁺ 475.2532, found 475.2540.

5-(4'-硝基)苯基-2-[(A-Homo-3'-氮杂-4'-氧代)睾-4a'-烯-17'-酮腙]-1, 3-噻二唑(8):棕黄色固体, 产率60.3%. m.p. 217～219 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.94 (s, 3H, 18-CH₃), 1.18 (s, 3H, 19-CH₃), 3.21～3.10 (m, 2H, C2-H), 5.72 (s, 1H, C4a-H), 6.92 (s, 1H, NH), 7.01 (s, 1H, 4-thiazole-H), 7.88 (d, J＝9.0 Hz, 2H, 2', 6'-PhH), 8.23 (d, J＝9.0 Hz, 2H, 3', 5'-PhH); ¹³C NMR (CDCl₃, 75 MHz) δ: 170.5 (17-C), 170.3 (2-thiazole-C), 165.6 (4-C), 160.7 (5-C), 148.8 (4'-PhC), 146.9 (1'-PhC), 140.8 (5-thiazole- C), 126.3 (4-thiazole-C), 124.1 (2', 6'-PhC), 119.2 (3', 5'- PhC), 107.2 (4a-C), 53.7 (14-C), 53.0 (9-C), 44.6 (13-C), 44.6 (1-C), 42.1 (8-C), 36.7 (12-C), 36.2 (2-C), 35.0 (10-C), 34.0 (6-C), 33.1 (7-C), 26.2 (16-C), 23.6 (15-C), 21.4 (11-C), 21.3 (19-C), 17.2 (18-C); IR (KBr) ν: 2937.95, 2850.69, 154.02, 1596.68, 1474.62, 1339.99, 1105.54, 853.74, 716.62, 579.50 cm^－1; HREIMS calcd for C₂₈H₃₃N₅NaO₃S [M+Na]⁺ 542.2202, found 542.2198.

5-(4'-三氟甲基)苯基-2-[(A-Homo-3'-氮杂-4'-氧代)睾-4a'-烯-17'-酮腙]-1, 3-噻二唑(9):淡黄色固体, 产率55.0%. m.p. 228～230 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.92 (s, 3H, 18-CH₃), 1.16 (s, 3H, 19-CH₃), 3.23～3.04 (m, 2H, C2-H), 5.74 (s, 1H, C4a-H), 6.85 (s, 1H, 4-thiazole- H), 7.62 (d, J＝8.4 Hz, 2H, 3', 5'-PhH), 7.80 (d, J＝9.0 Hz, 2H, 2', 6'-PhH), 7.87 (s, 1H, NH); ¹³C NMR (CDCl₃, 75 MHz) δ: 170.6 (17-C), 170.3 (2-thiazole-C), 165.3 (4-C), 160.6 (5-C), 149.6 (5-thiazole-C), 138.2 (4-thiazole-C), 129.3 (d, ²J_C—F＝32.25 Hz, 4'-PhC), 126.0 (2', 6'-PhC), 125.6 (d, ³J_C-F＝3.8 Hz, 3', 5'-PhC), 122.4 (CF₃), 119.2 (1'-PhC), 105.1 (4a-C), 53.7 (14-C), 53.0 (9-C), 44.5 (13-C, 1-C), 42.1 (8-C), 36.6 (12-C), 36.2 (2-C), 35.0 (10-C), 34.0 (6-C), 33.1 (7-C), 26.3 (16-C), 23.5 (15-C), 21.4 (11-C), 21.3 (19-C), 17.1 (18-C); IR(KBr) ν: 3060.11, 2935.46, 2853.19, 1651.52, 1574.24, 1409.70, 1322.44, 843.77, 659.28, 574.52 cm^－1; HREIMS calcd for C₂₉H₃₄N₄F₃OS [M+H]⁺ 543.2405, found 543.2411.

5-(4'-甲氧基)苯基-2-[(A-Homo-3'-氮杂-4'-氧代)睾-4a'-烯-17'-酮腙]-1, 3-噻二唑(10):淡黄色固体, 产率64.0%. m.p. 198～200 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.91 (s, 3H, 18-CH₃), 1.16 (s, 3H, 19-CH₃), 3.22～3.01 (m, 2H, C2-H), 3.83 (s, 3H, OCH₃), 5.75 (s, 1H, C4a-H), 6.64 (s, 1H, 4-thiazole-H), 6.90 (d, J＝8.7 Hz, 2H, 3', 5'-PhH), 7.49 (s, 1H, NH), 7.67 (d, J＝8.7 Hz, 2H, 2', 6'-PhH); ¹³C NMR (CDCl₃, 75 MHz) δ: 170.4 (17-C), 170.4 (2-thiazole- C), 164.9 (4-C), 160.6 (5-C), 159.2 (4'-PhC), 150.7 (5-thiazole-C), 128.1 (4-thiazole-C), 127.2 (2', 6'-PhC), 119.2 (1'-PhC), 113.9 (3', 5'-PhC), 101.2 (4a-C), 55.3 (CH₃O), 53.7 (14-C), 53.0 (9-C), 44.5 (13-C), 44.5 (1-C), 42.1 (8-C), 36.6(12-C), 36.2 (2-C), 35.0 (10-C), 34.0 (6-C), 33.1 (7-C), 26.3 (16-C), 23.6 (15-C), 21.4 (11-C), 21.3 (19-C), 17.1 (18-C); IR (KBr) ν: 3200.10, 2930.47, 1641.55, 1601.66, 1479.50, 1247.65, 1177.84, 1023.27, 836.29, 674.24, 417.45 cm^－1; HREIMS calcd for C₂₉H₃₇N₄O₂S [M+H]⁺ 505.2637, found 505.2632.

5-(4'-溴)苯基-2-[(A-Homo-3'-氮杂-4'-氧代)睾-4a'-烯-17'-酮腙]-1, 3-噻二唑(11):棕红色固体, 产率60.0%. m.p. 198～200 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.93 (s, 3H, 18-CH₃), 1.17 (s, 3H, 19-CH₃), 3.24～3.04 (m, 2H, C2-H), 5.75 (s, 1H, C4a-H), 6.76 (s, 1H, 4-thiazole-H), 7.50 (d, J＝8.4 Hz, 2H, 2', 6'-PhH), 7.51 (s, 1H, NH), 7.59 (d, J＝8.4 Hz, 2H, 3', 5'-PhH); ¹³C NMR (CDCl₃, 75 MHz) δ: 171.0 (17-C), 170.6 (2-thiazole-C), 165.5 (4-C), 161.0 (5-C), 149.7 (5-thiazole-C), 133.8 (4-thiazole-C), 131.7 (3', 5'-PhC), 127.5 (2', 6'-PhC), 121.5 (1'-PhC), 119.1 (4'-PhC), 103.5 (4a-C), 53.6 (14-C), 53.0 (9-C), 44.6 (13-C), 44.5 (1-C), 42.0 (8-C), 36.6 (12-C), 36.2 (2-C), 35.0 (10-C), 34.0 (6-C), 33.1 (7-C), 26.4 (16-C), 23.6 (15-C), 21.4 (11-C), 21.1 (19-C), 17.1 (18-C); IR (KBr) ν: 2927.99, 2853.19, 1661.52, 1559.28, 1474.52, 1247.65, 1065.65, 876.18, 826.32, 724.10 cm^－1; HREIMS calcd for C₂₈H₃₄BrN₄OS [M+H]⁺ 553.1637, found 553.1619.

5-(2'-萘基)-2-[(A-Homo-3'-氮杂-4'-氧代)睾-4a'-烯-17'-酮腙]-1, 3-噻二唑(12):棕红色固体, 产率65.0%. m.p. 234～236 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.89 (s, 3H, 18-CH₃), 1.13 (s, 3H, 19-CH₃), 3.21～3.03 (m, 2H, C2-H), 5.77 (s, 1H, C4a-H), 6.91 (s, 1H, 4-thiazole-H), 7.46～7.48 (m, 2H, 3', 4'-Naphthyl-H), 7.56 (s, 1H, NH), 7.88～7.81 (m, 4H, 5', 6', 7', 8'-Naphthyl-H), 8.24 (s, 1H, 1'-Naphthyl-H); ¹³C NMR (CDCl₃, 75 MHz) δ: 170.9 (17-C), 170.5 (2-thiazole-C), 165.4 (4-C), 160.8 (5-C), 150.9 (5-thiazole-C), 133.6 (10'-Naphthyl-C), 132.9 (9'- Naphthyl-C), 132.4 (5'-Naphthyl-C), 128.3 (2'-Naphthyl- C), 128.2 (4-thiazole-C), 127.7 (4'-Naphthyl-C), 126.3 (7'-Naphthyl-C), 126.0 (8'-Naphthyl-C), 124.8 (6'-Naph- thyl-C), 124.2 (3'-Naphthyl-C), 119.1 (1'-20-C), 103.6 (4a-C), 53.5 (14-C), 52.8 (9-C), 44.5 (13-C), 44.4 (1-C), 42.1 (8-C), 36.6 (12-C), 36.2 (2-C), 34.9 (10-C), 34.0 (6-C), 33.0 (7-C), 26.4 (16-C), 23.5 (15-C), 21.4 (11-C), 21.3 (19-C), 17.1 (18-C); IR (KBr) ν: 3050.14, 2935.46, 1651.52, 1601.66, 1556.79, 1477.01, 1437.12, 1357.34, 1130.47, 878.67, 671.75 cm^－1; HREIMS calcd for C₃₂H₃₇N₄OS [M+H]⁺ 525.2688, found 525.2723.

3.2.8 A-Homo-3-氮杂-4-氧代睾-4a-烯-17-(N-甲基)缩氨硫脲(13)的制备

取化合物5 300 mg (1 mmol)置于100 mL反应瓶中, 加入无水乙醇30 mL, 4-甲基-3-硫代氨基脲126 mg (1.2 mmol), 滴加冰醋酸调节pH至5～6, 将反应装置置于80 ℃油浴中反应8 h. TLC [V(石油醚): V(乙酸乙酯)＝1: 1]跟踪反应至无原料点, 旋干大部分乙醇, 加入适量水.采用二氯甲烷萃取, 合并有机相, 用饱和NaHCO₃及饱和食盐水洗涤, 无水Na₂SO₄干燥.减压除去溶剂得粗产物, 粗产物经柱层析分离[流动相: V(石油醚): V(乙酸乙酯)＝1: 1]得到白色固体324.6 mg, 产率83.6%. m.p. 188～190 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.90 (s, 3H, 18-CH₃), 1.17 (s, 3H, 19-CH₃), 3.20 (s, 3H, N-CH₃), 5.74 (s, 1H, C4a-H), 6.90 (s, 1H, NH), 7.42 (m, 1H, NHCH₃), 8.35 (s, 1H, CONH); ¹³C NMR (CDCl₃, 75MHz) δ: 178.8 (C＝S), 170.1 (4-C), 165.4 (17-C), 160.2 (5-C), 119.3 (4a-C), 53.5 (14-C), 52.9 (9-C), 44.6 (13-C), 44.5 (1-C), 42.1 (8-C), 36.6 (12-C), 36.1 (N-CH₃), 35.0 (2-C), 33.9 (10-C), 33.0 (6-C), 31.0 (7-C), 26.1 (16-C), 23.4 (15-C), 21.3 (11-C), 21.3 (19-C), 17.1 (18-C); IR (KBr) ν: 3409.14, 3252.08, 3154.88, 2960.39, 2830.75, 1731.30, 1599.17, 1504.43, 1247.65, 1078.12, 866.20, 569.53 cm^－1; HREIMS calcd for C₂₁H₃₂N₄NaOS [M+ Na]⁺ 411.2195, found 411.2186.

3.2.9 化合物14～19合成

化合物13 (0.75 mmol)置于100 mL烧瓶中, 加入无水乙醇30 mL, 化合物溶解后加进1 mmol的α-溴代甲基芳基酮或5 mL溴代乙酸乙酯(化合物19的制备), 将反应装置置于80 ℃油浴中反应8 h. TLC [V(石油醚): V(乙酸乙酯)＝1: 4]跟踪反应至无原料点, 旋干大部分乙醇, 加入适量水.采用二氯甲烷萃取, 合并有机相, 用饱和NaHCO₃及饱和食盐水洗涤, 无水Na₂SO₄干燥.减压除去溶剂得粗产物, 粗产物经柱层析分离[流动相: V(石油醚): V(乙酸乙酯)＝1: 4]得到相应的目标产物.

A-Homo-(3-氮杂-4-氧代)睾-4a-烯-17-酮-N-甲基-5-苯基噻唑-2(3H)-吖嗪(14):白色固体, 产率94.2%. m.p. 196～198 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.95 (s, 3H, 18-CH₃), 1.17 (s, 3H, 19-CH₃), 3.20～3.10 (m, 2H, C16- H), 3.26 (s, 3H, N-CH₃), 5.72 (s, 1H, C4a-H), 5.84 (s, 1H, 4-thiazole-H), 7.34～7.31 (m, 2H, Ph-H), 7.45～7.39 (m, 3H, Ph-H), 7.49 (br s, 1H, NH); ¹³C NMR (CDCl₃, 75 MHz) δ: 174.2 (17-C), 170.6 (4-C), 167.8 (2-thiazole-C), 161.2 (5-C), 140.7 (1'-PhC), 131.4 (4'-PhC), 128.9 (2', 6'-PhC), 128.7 (3', 5'-PhC), 128.6 (4-thiazole-C), 118.9 (5-thiazole-C), 98.7 (4a-C), 53.7 (9-C), 53.1(14-C), 44.6 (13-C), 44.1 (1-C), 42.0 (8-C), 36.6 (N-CH₃), 36.3 (12-C), 35.2 (2-C), 34.3 (10-C), 33.4 (6-C), 33.2 (7-C), 27.6 (16-C), 23.4 (15-C), 21.5 (11-C), 21.4 (19-C), 17.1 (18-C); IR (KBr) ν: 3291.99, 3067.62, 2927.99, 2950.59, 1741.27, 1666.48, 1596.69, 1322.44, 1167.87, 1122.99, 1063.16, 876.18, 691.69 cm^－1; HREIMS calcd for C₂₉H₃₆N₄NaOS [M+Na]⁺ 511.2508, found 511.2484.

A-Homo-(3-氮杂-4-氧代)睾-4a-烯-17-酮-N-甲基- 5-(4'-硝基)苯基噻唑-2(3H)-吖嗪(15):白色固体, 产率88.6%. m.p. 156～158 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.97 (s, 3H, 18-CH₃), 1.19 (s, 3H, 19-CH₃), 3.24～3.16 (m, 2H, C2-H), 3.32 (s, 3H, N-CH₃), 5.74 (s, 1H, C4a-H), 6.06 (s, 1H, 4-thiazole-H), 7.09 (s, 1H, N—H), 7.54 (d, J＝8.7 Hz, 2H, 2', 6'-PhH), 8.29 (d, J＝8.7 Hz, 2H, 3', 5'-PhH); ¹³C NMR (CDCl₃, 75MHz) δ: 175.3 (17-C), 170.4(4-C), 167.5 (5-C), 161.3 (2-thiazole-C), 160.6 (4'-PhC), 138.7 (1'-PhC), 134.8 (4-thiazole-C), 129.0 (2', 6'-PhC), 124.1 (3', 5'-PhC), 118.9 (5-thiazole-C), 102.3 (4a-C), 53.7 (9-C), 53.1 (14-C), 47.6 (13-C), 44.6 (1-C), 44.2 (8-C), 42.1 (N-CH3), 36.6 (12-C), 36.3 (2-C), 35.2 (10-C), 34.3 (6-C), 33.6 (7-C), 27.7 (16-C), 23.3 (15-C), 21.3 (11-C), 21.3 (19-C), 17.1 (18-C); IR (KBr) ν: 2930.47, 2850.69, 1736.29, 1651.52, 1599.17, 1342.33, 1177.84, 1103.05, 966.20, 654.29 cm^－1; HREIMS calcd for C₂₉H₃₆N₅O₃S [M+H]⁺ 534.2539, found 534.2518.

A-Homo-(3-氮杂-4-氧代)睾-4a-烯-17-酮-N-甲基- 5-(4'-三氟甲基)苯基噻唑-2(3H)-吖嗪(16):白色固体, 产率87.5%. m.p. 189～190 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.96 (s, 3H, 18-CH₃), 1.18 (s, 3H, 19-CH₃), 3.22～3.17 (m, 2H, C2-H), 3.29 (s, 3H, N-CH₃), 5.74 (s, 1H, C4a-H), 5.95 (s, 1H, 4-thiazole-H), 7.15 (s, 1H, N—H), 7.48 (d, J＝8.1 Hz, 2H, 2', 6'-PhH), 7.69 (d, J＝8.1 Hz, 2H, 3', 5'- PhH); ¹³C NMR (CDCl₃, 75 MHz) δ: 174.9 (17-C), 170.4 (4-C), 167.5 (2-thiazole-C), 161.0 (5-C), 160.3 (1'-PhC), 139.3 (4'-PhC), 134.8 (4-thiazole-C), 128.8 (2', 6'-PhC), 125.8 (3', 5'-PhC), 119.3 (CF₃), 119.0 (5-thiazole-C), 100.6 (4a-C), 53.7 (9-C), 53.1 (14-C), 47.6 (13-C), 44.6 (1-C), 44.2 (8-C), 42.1 (N-CH₃), 36.6 (12-C), 36.3 (2-C), 35.2 (10-C), 34.3 (6-C), 33.6 (7-C), 27.7 (16-C), 23.3 (15-C), 21.3 (11-C), 21.2 (19-C), 17.1 (18-C); IR (KBr) ν: 3304.43, 2925.45, 2850.69, 1733.78, 1663.99, 1479.50, 1317.45, 1165.37, 1125.48, 1063.16, 853.74, 746.54 cm^－1; HREIMS calcd for C₃₀H₃₆F₃N₄OS [M+H]⁺ 557.2562, found 557.2568.

A-Homo-(3-氮杂-4-氧代)睾-4a-烯-17-酮-N-甲基- 5-(4'-甲氧基)苯基噻唑-2(3H)-吖嗪(17):白色固体, 产率86%. m.p. 179～180 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.94 (s, 3H, 18-CH₃), 1.16 (s, 3H, 19-CH₃), 3.18～3.14 (m, 2H, C2-H), 3.24 (s, 3H, N-CH₃), 3.82 (s, 1H, OCH₃), 5.72 (s, 1H, C4a-H), 5.78 (s, 1H, 4-thiazole-H), 6.92 (d, J＝8.4 Hz, 2H, 3', 5'-PhH), 7.24 (d, J＝8.4 Hz, 2H, 2', 6'-PhH), 7.42 (s, 1H, N—H); ¹³C NMR (CDCl₃, 75 MHz) δ: 174.1 (17-C), 170.4 (4-C), 167.8 (5-C), 161.0 (2-thiazole-C), 160.1 (4'-PhC), 140.4 (1'-PhC), 130.0 (2', 6'-PhC), 123.7 (4-thiazole-C), 119.0 (5-thiazole-C), 114.1 (3', 5'-PhC), 97.9 (4a-C), 55.4 (OCH₃), 53.7 (9-C), 53.1 (14-C), 47.6 (13-C), 44.6 (1-C), 44.1 (8-C), 42.1 (N-CH₃), 36.6 (12-C), 36.3 (2-C), 35.2 (10-C), 34.3 (6-C), 33.3 (7-C), 27.6 (16-C), 23.4 (15-C), 21.5 (11-C), 21.4 (19-C), 17.1 (18-C); IR (KBr) ν: 3429.09, 2927.99, 2850.69, 1736.29, 1649.03, 1417.17, 1247.65, 1175.35, 1025.76, 876.18, 664.27 cm^－1; HREIMS calcd for C₃₀H₃₉N₄O₂S [M+H]⁺ 519.2794, found 519.2762.

A-Homo-(3-氮杂-4-氧代)睾-4a-烯-17-酮-N-甲基- 5-(4'-溴)苯基噻唑-2(3H)-吖嗪(18):白色固体, 产率85.0%. m.p. 201～203 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.91 (s, 3H, 18-CH₃), 1.14 (s, 3H, 19-CH₃), 3.22 (s, 3H, N-CH₃), 5.69 (s, 1H, C4a-H), 5.84 (s, 1H, 4-thiazole-H), 7.18 (d, J＝8.1 Hz, 2H, 2', 6'-PhH), 7.51 (d, J＝8.1 Hz, 2H, 3', 5'-PhH), 8.63 (s, 1H, N—H); ¹³C NMR (CDCl₃, 75 MHz) δ: 178.6 (17-C), 170.5 (4-C), 167.5 (5-C), 161.3 (2-thiazole-C), 160.5 (1'-PhC), 139.5 (4-thiazole-C), 131.9 (3', 5'-PhC), 130.1 (2', 6'-PhC), 123.1 (4'-PhC), 118.9 (5-thiazole-C), 99.4 (4a-C), 53.7 (9-C), 53.1 (14-C), 44.6 (13-C), 44.5 (1-C), 44.1 (8-C), 42.0 (NCH₃), 36.6 (12-C), 36.3 (2-C), 35.1 (10-C), 34.3 (6-C), 33.3 (7-C), 27.6 (16-C), 23.3 (15-C), 21.4 (11-C), 21.3 (19-C), 17.1 (18-C); IR (KBr) ν: 3299.45, 2927.99, 2850.69, 1733.80, 1651.52, 1479.50, 1242.66, 1172.85, 1068.14, 818.14, 651.80 cm^－1; HREIMS calcd for C₂₉H₃₆BrN₄OS [M+H]⁺ 567.1793, found 567.1792.

3N-甲基-2-硫代-4-氧代-[(A-Homo-3'-氮杂-4'-氧代)睾-4a'-烯-17'-酮腙]-(5H)-1, 3-咪唑(19):白色固体, 产率74.5%. m.p. 283 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 0.90 (s, 3H, 18-CH₃), 1.12 (s, 3H, 19-CH₃), 3.17 (s, 3H, N- CH₃), 3.66 (s, 2H, 22-CH₂), 5.67 (s, 1H, C4a-H), 7.47 (s, 1H, N—H); ¹³C NMR (CDCl₃, 75 MHz) δ: 181.0 (23-CO- NH), 172.0 (C＝S), 170.4 (4-C), 160.8 (5-C), 159.9 (17-C), 119.1 (4a-C), 63.8 (22-C), 53.5 (9-C), 52.9 (14-C), 44.5 (13-C), 42.0 (1-C), 36.5 (8-C), 36.2 (12-C), 35.1 (2-C), 33.8 (10-C), 33.1 (6-C), 32.3 (7-C), 29.5 (N-CH₃), 27.9 (16-C), 23.1 (15-C), 21.4 (11-C), 21.3 (19-C), 16.9 (18-C); IR (KBr) ν: 3296.95, 2935.46, 2843.21, 1726.32, 1661.50, 1429.64, 1220.22, 1118.01, 995.84, 883.66, 773.96, 646.91, 494.74 cm^－1; HREIMS calcd for C₂₃H₃₃N₄O₂S [M+H]⁺ 429.2324, found 429.2383.

3.3 生物活性评估

3.3.1 材料

RPMI 1640培养基(HyClone公司)、MTT (Sigma公司)、胎牛血清(天杭生物科技有限公司)、青霉素-链霉素溶液(100X) (Beyotime公司)、胰酶(Sigma公司)、DMSO (Amresco公司)、细胞冻存液(90% PBS+10% DMSO)、PBS (8 g NaCl、0.2 g KCl、1.56 g Na₂HPO₄• 2H₂O、0.2 g KH₂PO₄•2H₂O溶于1000 mL三蒸水中).细胞增殖评估利用MTT方法, 采用Thermo Scientific公司MLLTISKAN MK3酶标仪测定.

肿瘤细胞株:人体宫颈癌细胞株(HeLa), 人鼻咽癌细胞株(CNE-2), 人肝癌细胞株(HEPG2)和人肾上皮细胞系(HEK293T).

3.3.2 抑制肿瘤细胞生长增殖活性测定

将处于生长平台期的被测试细胞以约3×10⁴～4×10⁴ cells/mL的密度接种于96孔板, 每孔接种200 μL, 置于CO₂培养箱中培养24 h, 按预设的浓度梯度加入待测样品, 每一梯度设3个平行孔, 同时设对照孔和空白孔.在二氧化碳培养箱中于37 ℃培养72 h后, 每孔加入20 μL的MTT (5 mg/mL), 然后在二氧化碳培养箱中继续温育4 h.抽取上清液后加入200 μL的DMSO, 震荡10 min溶解沉淀, 随后用酶标仪测定OD值.通过下式求出一定浓度下样品对细胞的抑制率:

$ \begin{array}{l} {\rm{抑制率}}\left( \% \right) = \left[{\left( {{\rm{对照OD-空白OD}}} \right)-\left( {{\rm{给药OD-空白OD}}} \right)} \right]/\\ \left( {{\rm{对照OD -空白OD}}} \right) \times 100\% \end{array} $

然后以抑制率对药物浓度作图, 求出每个样品的IC₅₀值.

辅助材料(Supporting Information)化合物的¹H NMR, ¹³C NMR和高分辨质谱谱图.这些材料可以免费从本刊网站(qhttp://sioc-journal.cn/)上下载.

1. [1]
  Festi, D.; Montagnani, M.; Azzaroli, F.; Lodato, F.; Mazzella, G.; Roda, A.; Di-Biase, A. R; Roda, E.; Simoni, P.; Colecchia, A. Curr.Clin.Pharmacol. 2007, 2, 155. doi: 10.2174/157488407780598171
2. [2]
  Ifere, G.; Barr, E.; Equan, A.; Gordon, K.; Singh, U. P.; Chaudhary, J.; Igietseme, J. U.; Ananaba, G. A. Cancer Detect. Prev. 2009, 32, 319. doi: 10.1016/j.cdp.2008.12.002
3. [3]
  Morzycki, J. W. Steroids 2014, 83, 62. doi: 10.1016/j.steroids.2014.02.001
4. [4]
  João, F. S.; Carvalho, M.; Manuel, C. S.; João, N.; Moreira, S. S.; Melo, M. L. S. J.Med. Chem. 2011, 54, 6375. doi: 10.1021/jm200803d
5. [5]
  Jorge, A. R.; Salvador, J. F. S.; Carvalho, Marco A. C.; Neves, S. M.; Silvestre, A. J.; Leitão, M.; Manuel C. S. Nat. Prod.Rep. 2013, 30, 324. doi: 10.1039/C2NP20082A
6. [6]
  Ranju, B.; Pratap, C. A. Chem. Rev. 2014, 114, 6986. doi: 10.1021/cr4002935
7. [7]
  林启福, 崔建国, 甘春芳, 刘亮, 姚秋翠, 黄燕敏, 有机化学, 2012, 32, 2214.Liun, Q.-F.; Cui, J.-G.; Gan, C.-F. Chin. J.Org.Chem. 2012, 32, 2214(in Chinese).
8. [8]
  Huang, Y. M.; Cui, J. G.; Zhong, Z. G.; Gan, C. F.; Zhang, W. Y.; Song, H. C. Bioorg.Med.Chem.Lett. 2011, 21, 3641. doi: 10.1016/j.bmcl.2011.04.093
9. [9]
  Huang, Y. M.; Cui, J. G.; Chen, S. J.; Gan, C. F.; Zhou, A. M. Steroids 2011, 76, 1346. doi: 10.1016/j.steroids.2011.06.013
10. [10]
  Huang, Y. M.; Cui, J. G.; Chen, S. J.; Lin, Q. F.; Song, H. C.; Gan, C.-F.; Su, B.; Zhou, A. M. Mar.Drugs 2014, 12, 1715. doi: 10.3390/md12041715
11. [11]
  Huang, Y. M.; Cui, J. G.; Chen, S. J.; Gan, C.-F.; Yao, Q. C.; Lin, Q. F. Bioorg.Med.Chem.Lett. 2013, 23, 2265. doi: 10.1016/j.bmcl.2012.08.064
12. [12]
  崔胜峰, 王艳, 吕敬松, Damu Guri L. V., 周成合, 中国科学:化学, 2012, 42, 1105. https://www.researchgate.net/profile/Wenjue_Zhong2/publication/270463802_Toxicity_identification_evaluation_TIE_on_the_whole_sediments_collected_from_Taihu_Lake_China/links/5732a76308ae298602da3a80.pdf?inViewer=0&pdfJsDownload=0&origin=publication_detailCui, S. F.; Wang, Y.; Lv, J. S.; Damu Guri L. V.; Zhou C. H. Sci. China Chem. 2012, 42, 1105(in Chinese). https://www.researchgate.net/profile/Wenjue_Zhong2/publication/270463802_Toxicity_identification_evaluation_TIE_on_the_whole_sediments_collected_from_Taihu_Lake_China/links/5732a76308ae298602da3a80.pdf?inViewer=0&pdfJsDownload=0&origin=publication_detail
13. [13]
  Kashfi, K. Adv. Pharmacol. 2009, 57, 31. doi: 10.1016/S1054-3589(08)57002-5
14. [14]
  Sun, L. P.; Jiang, Z.; Gao, L. X.; Liu, X. F.; Quan, Y. C.; Zheng, G. H.; Li, J.; Piao, H. Chin. J. Org. Chem. 2013, 33, 1496. doi: 10.6023/cjoc201212012
15. [15]
  Mohareb, R. M.; Al-Omran, F. Steroids 2012, 77, 1551. doi: 10.1016/j.steroids.2012.09.007
16. [16]
  崔建国, 刘亮, 甘春芳, 肖琦, 黄燕敏, 化学进展, 2014, 26, 320.Cui, J. G.; Liu, L.; Gan, C. F.; Xiao, Q.; Huang, Y. M. Prog. Chem. 2014, 26, 320(in Chinese).
17. [17]
  崔建国, 赵丹丹, 何冬梅, 黄燕敏, 刘志平, 林啟福, 石海信, 甘春芳, 有机化学, 2016, 36, 630. doi: 10.6023/cjoc201509021?viewType=HTMLCui, J. G.; Zhao, D. D.; He, D. M.; Huang, Y. M.; Liu, Z. P.; Liu, X. F.; Shi, H. X.; Gan, C. F. Chin. J. Org. Chem. 2016, 36, 630(in Chinese). doi: 10.6023/cjoc201509021?viewType=HTML
18. [18]
  黄燕敏, 戚斌斌, 崔建国, 甘春芳, 杨春晖, 刘畅, 陈爽, 石海信, 有机化学, 2016, 36, 1602. http://manu19.magtech.com.cn/Jwk_yjhx/CN/abstract/abstract346300.shtmlHuang, Y. M.; Qi, B. B.; Cui, J. G.; Gan, C. F.; Yang, C. H.; Liu, C.; Chen, S.; Shi, H. X. Chin. J. Org. Chem. 2016, 36, 1602(in Chinese). http://manu19.magtech.com.cn/Jwk_yjhx/CN/abstract/abstract346300.shtml

图式 1 化合物7～12的合成路线

Scheme 1 Synthesis of compounds 7～12

Reagents and conditions: (a) Py/Ac₂O; (b) NH₂OH•HCl/CH₃CH₂OH; (c) SOCl₂/ THF; (d) 13% K₂CO₃/CH₃CH₂OH; (e) Jones reagent; (f) NH₂NHCSNH₂; (g) ArCOCH₂Br

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图式 2 化合物13～19的合成路线

Scheme 2 Synthesis of compounds 13～19

Reagents and conditions: (a) NH₂NHCSNHCH₃; (b) ArCOCH₂Br/CH₃CH₂OH; (c) BrCH₂CO₂CH₂CH₃/CH₃CH₂OH

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图式 3 化合物14～19形成的不同反应机理

Scheme 3 Reaction mechanisms of formation for compounds 14～19

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表 1 化合物1～19体外抑制细胞生长增殖活性(MTT法) (IC₅₀, μmol/L)

Table 1. Antiproliferative activity of compounds 1～19 in vitro

化合物	人体宫颈癌细胞(HeLa)	人鼻咽癌细胞(CNE-2)	人肝癌细胞(HEPG2)	人肾上皮细胞(HEK293T)
1	＞80	＞80	＞80	＞80
2	＞80	＞80	＞80	＞80
3	＞80	＞80	＞80	＞80
4	＞80	＞80	＞80	＞80
5	＞80	＞80	＞80	＞80
6	＞80	＞80	＞80	＞80
7	＞80	＞80	＞80	＞80
8	＞80	＞80	＞80	＞80
9	＞80	＞80	＞80	＞80
10	＞80	＞80	＞80	＞80
11	＞80	＞80	＞80	＞80
12	＞80	＞80	＞80	＞80
13	55.2±4.9	＞80	＞80	＞80
14	19.5±2.1	19.5±1.9	42.5±4.3	＞80
15	＞80	＞80	＞80	＞80
16	＞80	＞80	＞80	＞80
17	22.5±2.3	42.5±3.8	19.5±1.8	＞80
18	＞80	＞80	＞80	＞80
19	42.1±3.9	33.4±2.8	62.5±5.8	＞80
Cisplatin	15.1±1.3	10.2±1.1	10.5±1.0	11.0±0.9
^a Data represent the mean values of three independent determinations.

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