药学学报

Compd.	MolDock Score /kcal·mol^-1
Ⅰ₁	-141.137
Ⅰ₂	-145.932
Ⅰ₃	-155.361
Ⅰ₄	-140.963
Ⅰ₅	-147.076
L64	-140.016
Ⅱ₁	-133.509
Ⅱ₂	-148.026
Ⅱ₃	-157.754
Ⅱ₄	-154.117
Ⅱ₅	-141.312
OA	-108.186

Compd.	MolDock Score /kcal·mol^-1
Ⅰ₁	-141.137
Ⅰ₂	-145.932
Ⅰ₃	-155.361
Ⅰ₄	-140.963
Ⅰ₅	-147.076
L64	-140.016
Ⅱ₁	-133.509
Ⅱ₂	-148.026
Ⅱ₃	-157.754
Ⅱ₄	-154.117
Ⅱ₅	-141.312
OA	-108.186

Compd.	Inhibition rate/%
OA	19.7	15.3	> 50	> 50
Ⅰ₁	38.6	39.1	32.8	35.2
Ⅰ₂	43.3	40.0	30.1	36.6
Ⅰ₃	59.5	50.4	9.4	12.6
Ⅰ₄	38.1	41.2	37.5	30.4
Ⅰ₅	41.6	40.2	35.5	39.1
Ⅱ₁	40.4	38.5	34.9	41.7
Ⅱ₂	31.5	36.9	31.5	45.8
Ⅱ₃	63.5	53.2	5.5	10.6
Ⅱ₄	50.4	45.5	19.3	25.2
Ⅱ₅	42.5	36.9	29.5	45.5
AZD8055	60.3	52.8	10.7	13.5

Compd.	Inhibition rate/%
OA	19.7	15.3	> 50	> 50
Ⅰ₁	38.6	39.1	32.8	35.2
Ⅰ₂	43.3	40.0	30.1	36.6
Ⅰ₃	59.5	50.4	9.4	12.6
Ⅰ₄	38.1	41.2	37.5	30.4
Ⅰ₅	41.6	40.2	35.5	39.1
Ⅱ₁	40.4	38.5	34.9	41.7
Ⅱ₂	31.5	36.9	31.5	45.8
Ⅱ₃	63.5	53.2	5.5	10.6
Ⅱ₄	50.4	45.5	19.3	25.2
Ⅱ₅	42.5	36.9	29.5	45.5
AZD8055	60.3	52.8	10.7	13.5

以mTOR激酶为靶点的齐墩果酸衍生物的设计、合成及抗肿瘤活性研究

PDF下载

宋艳玲 ^* , 李玲 , 刘忠岩 , 李杰 , 张蓬勃

药学学报 | 研究论文 2019,54(2): 335-342

收起

药学学报 | 研究论文 2019, 54(2): 335-342

以mTOR激酶为靶点的齐墩果酸衍生物的设计、合成及抗肿瘤活性研究

全屏

宋艳玲^*, 李玲, 刘忠岩, 李杰, 张蓬勃

作者信息

沈阳化工大学制药与生物工程学院, 辽宁沈阳 110142

通讯作者:

*宋艳玲, Tel:13840387750, E-mail:yanlingsong521@126.com

Design, synthesis and anticancer activity studies of oleanolic acid derivatives targeting mTOR

Yan-ling SONG^*, Ling LI, Zhong-yan LIU, Jie LI, Peng-bo ZHANG

Affiliations

Institute of Pharmaceutical and Biological Engineering, Shenyang University of Chemical Technology, Shenyang 110142, China

出版时间: 2019-02-12 doi: 10.16438/j.0513-4870.2018-0890

文章导航

摘要

收起

本文以齐墩果酸（oleanolic acid，OA）为先导化合物进行结构优化，通过引入mTOR小分子抑制剂的重要药效基团脲基或硫脲基，同时拼合多种生物活性片段，设计并合成了10个新的衍生物，其结构经¹H NMR、¹³C NMR和HR-MS确证。对所合成的目标化合物以人肝癌细胞（HepG2）和人胃癌细胞（SGC7901）进行体外抗肿瘤活性测试，结果表明目标化合物对两种肿瘤细胞的抑制活性均明显强于OA，其中I₃和Ⅱ₃对HepG2细胞显示出较强的活性（IC₅₀=9.4和5.5 μmol·L^-1）。分子模拟对接研究表明，目标化合物和mTOR激酶均具有较好的结合能力。对化合物I₃和Ⅱ₃进行mTOR激酶的抑制活性测试，结果表明化合物I₃和Ⅱ₃对mTOR激酶具有较强的抑制作用（IC₅₀=0.83和0.26 μmol·L^-1）。

关键词

齐墩果酸衍生物 / 合成 / 分子对接 / 抗肿瘤活性

Abstract

收起

Ten novel oleanolic acid (OA) derivatives containing urea or thiourea group were designed and synthesized, the chemical structures were confirmed by ¹H NMR, ¹³C NMR and HR-MS. All of these compounds were evaluated for the inhibitory activity against growth of HepG2 and SGC7901 cells. The results showed that compounds I₃ and Ⅱ₃ exhibited significant antitumor activities with IC₅₀ of 9.4 and 5.5 μmol·L^-1, respectively. Molecular docking studies showed that all these compounds exhibit inhibitory ability against mTOR kinase. Compounds I₃ and Ⅱ₃ were further evaluated for the inhibitory activity against mTOR kinase. The results showed that I₃ and Ⅱ₃ exhibited strong inhibitory effect on mTOR kinase with IC₅₀ values of 0.83 and 0.26 μmol·L^-1.

Key words

oleanolic acid derivative / synthesis / molecular docking / anti-tumor activity

引用本文

宋艳玲, 李玲, 刘忠岩, 李杰, 张蓬勃. 以mTOR激酶为靶点的齐墩果酸衍生物的设计、合成及抗肿瘤活性研究. 药学学报, 2019 , 54 (2) : 335 -342 . DOI: 10.16438/j.0513-4870.2018-0890

Yan-ling SONG, Ling LI, Zhong-yan LIU, Jie LI, Peng-bo ZHANG. Design, synthesis and anticancer activity studies of oleanolic acid derivatives targeting mTOR[J]. Acta Pharmaceutica Sinica, 2019 , 54 (2) : 335 -342 . DOI: 10.16438/j.0513-4870.2018-0890

正文

收起

哺乳动物雷帕霉素靶蛋白(mTOR)是一种丝氨酸/苏氨酸激酶, 其介导的PI3K-Akt-mTOR信号通路在肿瘤细胞增殖、分化、凋亡及蛋白质合成、血管生成过程中发挥重要作用^{[1, 2]}。目前, 通过与ATP竞争mTOR的催化靶位从而抑制mTOR激酶的小分子抑制剂已成为抗肿瘤药物的研究热点^[3-5]。齐墩果酸(oleanolic acid, OA)是一种五环三萜类天然产物, 具有独特的手性刚性骨架及良好的生物活性和生物相容性^[6-9]。近年来的研究发现, OA具有诱导肿瘤细胞分化、诱导肿瘤细胞凋亡、抑制肿瘤血管生成和肿瘤转移等作用^[10-12], 但分子作用机制尚未明确。也有研究表明OA能够抑制人前列腺癌细胞PC-3和乳腺癌细胞MCF-7中激活状态的mTOR^[13], 并且OA对人白血病HL-60细胞的PI3K表达也具有抑制作用^[14]。因此, mTOR信号通路可能是齐墩果酸抗肿瘤作用的重要靶点。

通过对mTOR小分子抑制剂构效关系研究发现, PKI587、PKI179、PF-05139962、L64和NVP-BEZ235等结构中的脲基是重要的药效基团, 结构式见图 1^[15]。PDB数据库中L64与mTOR激酶(PDB: 3IBE)的共晶结构分析表明, L64可插入靶蛋白活性口袋并与周围的氨基酸残基紧密结合, 其结构中脲基上的氧原子和氢原子分别与LYS83、ASP964和ASP841各形成一个牢固的氢键, 进一步说明脲基是其抗肿瘤作用的重要药效基团(图 2)。已有的OA构效关系研究表明C-3位羰基和C-28位酰胺化能显著提高抗肿瘤活性^[16]; C-28位引入哌嗪基团, 其水溶性和生物活性也会明显增强^[17]。本研究以C-3位羰基OA为先导化合物, 通过C-28引入哌嗪基团作为连接臂, 再利用拼合原理引入脲基及疏水性芳环基团, 设计并合成5个衍生物Ⅰ₁~Ⅰ₅, 通过将脲基置换成硫脲基设计并合成5个衍生物Ⅱ₁~Ⅱ₅, 见图 3。同时对目标化合物进行体外抗肿瘤活性测试和分子模拟对接研究。

结果与讨论

收起

1 化学部分

以OA为起始原料, 经过C-3位氧化、C-28位酰氯化反应和取代反应得到重要中间体化合物3, 化合物3进一步和取代的异氰酸酯或异硫氰酸酯反应, 合成了10个齐墩果酸衍生物, 见合成路线1。目标化合物结构经¹H NMR、¹C NMR和HR-MS确证, 数据见实验部分。

2 分子对接

以PDB数据库中mTOR为靶点(PDB编号: 3IBE), 作为分子对接受体模型。将3IBE导入计算机辅助药物设计软件Molegro Virtual Docker (MVD 6.0), 根据cavity的探测选择最佳对接区域(center x: 42.33 y: 15.87 z: 32.79, radius: 15)。将受体蛋白中原有配体替换为目标化合物, 利用Docking wizard向导设定配体的对接运算次数后, 选择打分函数MolDock Score[GRID]与构象搜寻MolDock SE^[18], 分别对化合物(Ⅰ₁~Ⅰ₅和Ⅱ₁~Ⅱ₅)与受体对接的结合能进行评分, 评分结果见表 1。结果表明, 目标化合物的结合能均明显高于OA, 其中化合物Ⅰ₃、Ⅱ₃和Ⅱ₄高于L64。选取MD score绝对值最高的分子构象作为与靶点的模拟对接模型, 利用Discovery studio 4.0分析配体与氨基酸残基作用的图像。目标化合物均能较好的插入到mTOR-ATP催化区域, 并与周围多个氨基酸紧密连接。对比化合物Ⅰ₃、Ⅱ₃和L64与靶蛋白的分子对接图(图 4), 结果表明化合物Ⅰ₃、Ⅱ₃和L64与靶蛋白具有相同的氢键结合位点, 分子结构中的脲基或硫脲基能与靶蛋白氨基酸残基LYS833\ASP841和ASP964形成氢键。

3 生物活性评价

3.1 目标化合物对人癌细胞株的体外增殖抑制实验

选取HepG2和SGC7901细胞, 采用MTT法, 以AZD8055作为阳性对照药物, 测试目标化合物Ⅰ₁~Ⅰ₅和Ⅱ₁~Ⅱ₅的体外抗肿瘤活性, 测试结果见表 2。结果表明目标化合物对两种肿瘤细胞的抑制活性均明显强于OA, 其中Ⅰ₃和Ⅱ₃对HepG2细胞显示出较强的活性, IC₅₀值分别是9.4和5.5 μmol·L^-1。

3.2 mTOR激酶抑制实验

选取化合物Ⅰ₃和Ⅱ₃进行mTOR激酶抑制实验, 实验由Invitrogen公司完成。结果表明化合物Ⅰ₃和Ⅱ₃对mTOR激酶具有较好的抑制作用, IC₅₀值分别为0.83和0.26 μmol·L^-1。

4 小结

本文以OA为起始原料, 通过在OA-28位引入哌嗪基团, 并以此为连接臂, 引入mTOR小分子抑制剂的药效基团脲基, 再用硫脲基替换脲基, 设计并合成了10个新的OA衍生物, 目标化合物结构经¹H NMR、¹C NMR和HR-MS确证。通过计算机辅助药物设计分子对接方法预测目标化合物和mTOR激酶的结合能, 结果表明目标化合物的结合能均明显高于OA, 其中化合物Ⅰ₃、Ⅱ₃和Ⅱ₄的结合能高于化合物L64, 且化合物Ⅰ₃、Ⅱ₃和L64与mTOR激酶具有相同的氢键结合位点。采用MTT法考察目标化合物的体外抗肿瘤活性, 结果表明目标化合物对人肝癌细胞(HepG2)和人胃癌细胞(SGC7901)的抑制活性明显高于OA, 说明以哌嗪作为连接臂, 连入脲基或硫脲基, 其抗肿瘤活性显著增强。mTOR激酶抑制活性测试结果表明化合物Ⅰ₃和Ⅱ₃具有较强的mTOR激酶抑制活性。实测活性与分子模拟对接预测的结果相关性较好, 本研究结果对进一步优化设计齐墩果酸衍生物作为mTOR激酶抑制剂的研究具有参考价值。

实验部分

收起

Büchi B-540熔点测定仪; BrukerARX-600型核磁共振分析仪(DMSO-d₆为溶剂, TMS为内标); Autospec Ultima-TOF质谱测定仪; WZZ-1S (2s)自动旋光仪; 齐墩果酸(质量分数 > 98%)购于陕西慈缘生物技术有限公司; 柱色谱200~300目硅胶、薄层色谱硅胶GF254, 购于青岛海洋化工厂; 显色剂为10%硫酸-乙醇溶液; 体外抗肿瘤活性测试所用的RPMI-1640培养基(含10%胎牛血清、100 μg·mL^-1青霉素、100 μg·mL^-1链霉素)、溴化四氮唑盐(MTT)、胰蛋白酶(Trypsin)和标准胎牛血清(FBS)、活性测试所用HepG2和SGC7901细胞由沈阳药科大学药理教研室提供。所有试剂均为市售分析纯或化学纯。

1 化合物的合成

1.1 3-氧代-齐墩果烷型-12-烯-28-羧酸(1)的制备

将齐墩果酸(0.500 g, 1.095 mmol)溶解于30 mL丙酮中, 冰盐浴下缓慢滴加30滴新配置的Jones试剂, 室温反应2 h, TLC监测反应终点。加入异丙醇15 mL淬灭, 继续反应30 min。乙酸乙酯萃取3次, 合并有机相, 饱和食盐水洗涤3次, 无水硫酸钠干燥, 抽滤, 蒸干溶剂, 得到白色固体0.483 g, 产率为96%, mp 218.7~221.3 ℃ (文献值mp 220.3~222.1 ℃^[19])。

1.2 3-氧代-齐墩果烷型-12-烯-28-酰氯(2)的制备

将化合物1 (0.500 g, 1.020 mmol)溶解于30 mL无水二氯甲烷中, 加热至40 ℃, 搅拌下缓慢滴加草酰氯(0.698 g, 5.499 mmol), 回流反应2 h, TLC监测反应终点。减压蒸干溶剂, 加入环己烷(10 mL×2)洗涤, 蒸干溶剂, 得到白色固体0.488 g, 产率为97%, 密闭备用。

1.3 化合物(3)的制备

在25 mL茄形瓶中加入哌嗪(1.455 g, 16.889 mmol), 冰盐浴冷却。将化合物2 (1.000 g, 2.114 mmol)溶解于10 mL无水二氯甲烷中, 缓慢滴加到降至0 ℃的哌嗪溶液中, 再依次加入无水DMAP (0.013 g, 0.106 mmol)和无水吡啶(0.248 g, 3.135 mmol), 室温反应3 h, TLC监测反应终点。二氯甲烷萃取3次, 合并有机相, 饱和食盐水洗涤3次, 无水硫酸钠干燥, 抽滤, 减压蒸干溶剂, 得到淡黄色固体。经硅胶柱色谱分离纯化[洗脱剂:甲醇-二氯甲烷=1:150 (V/V)]得到白色固体0.864 g, 产率为86%, mp 149.9~151.2 ℃。HR-MS (m/z): 523.402 8 [M+H]⁺; ¹H NMR (600 MHz, DMSO-d₆) δ 5.21 (t, J = 3.7 Hz, 1H, H-12), 3.38~3.30 (m, 4H, piperazinyl-H-2', 6'), 2.86~2.78 (m, 4H, piperazinyl-H-3', 5'), 1.15 (s, 3H, CH₃), 1.13~1.11 (m, 1H, piperazinyl-H-4'), 1.09 (s, 3H, CH₃), 1.04 (s, 6H, 2CH₃), 0.93 (d, J = 3.2 Hz, 3H, CH₃), 0.90 (s, 3H, CH₃), 0.78 (s, 3H, CH₃); ¹³C NMR (101MHz, CDCl₃) δ 127.62, 174.55, 151.61, 144.45, 139.87, 129.34, 129.26, 128.58, 123.49, 121.92, 121.85, 55.26, 51.86, 51.67, 49.71, 49.38, 47.60, 47.45, 46.32, 43.85, 43.33, 41.96, 40.15, 39.42, 37.68, 34.29, 33.97, 33.08, 31.15, 30.73, 28.84, 28.45, 27.32, 27.08, 26.12, 23.94, 22.07, 20.86, 20.75, 17.46, 16.22。

1.4 化合物Ⅰ₁~Ⅰ₅的制备

1.4.1 化合物Ⅰ₁的制备

将苯基异氰酸酯(0.054 g, 0.453 mmol)溶于10 mL无水1, 2-二氯乙烷中, 置于常温下搅拌; 将溶于5 mL无水1, 2-二氯乙烷的化合物3 (0.158 g, 0.302 mmol)加入三乙胺(0.183 g, 1.808 mmol), 混合均匀后, 缓慢滴入苯基异氰酸酯中, 常温, 过夜反应, TLC跟踪监测反应终点。二氯甲烷萃取3次, 合并有机相, 饱和食盐水洗涤3次, 无水硫酸钠干燥, 抽滤, 减压蒸干溶剂, 得到淡黄色固体。粗品经硅胶色谱纯化分离[洗脱剂:甲醇-二氯甲烷=1:80 (V/V)]得到白色固体0.118 g, 产率为75%, mp 159.6~160.8 ℃; [α]_D²⁵+40.5 (c 0.50, CHCl₃); HR-MS (m/z): 642.455 2 [M+H]⁺; ¹H NMR (600 MHz, DMSO-d₆) δ 7.54~7.50 (m, 2H, ph-H-2', 6'), 7.39~7.35 (m, 2H, ph-H-3', 5'), 7.06~7.04 (m, 1H, ph-H-4'), 5.24 (t, J = 3.7 Hz, 1H, H-12), 3.62~3.54 (m, 4H, piperazinyl-H-3', 5'), 3.50~3.42 (m, 4H, piperazinyl-H-2', 6'), 1.08 (s, 3H, CH₃), 0.97 (s, 3H, CH₃), 0.91 (s, 6H, 2CH₃), 0.86 (s, 6H, 2CH₃), 0.70 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 217.62, 174.55, 151.61, 144.45, 139.87, 129.34, 129.26, 128.58, 123.49, 121.92, 121.85, 55.26, 51.86, 51.67, 49.71, 49.38, 47.60, 47.45, 46.32, 43.85, 43.33, 41.96, 40.15, 39.42, 37.68, 34.29, 33.97, 33.08, 31.15, 30.73, 28.84, 28.45, 27.32, 27.08, 26.12, 23.94, 22.07, 20.86, 20.75, 17.46, 16.22。

1.4.2 化合物Ⅰ₂的制备

按照Ⅰ₁的制备方法, 由化合物3 (0.158 g, 0.302 mmol)与邻甲苯异氰酸酯(0.060 g, 0.453 mmol)反应。粗品经硅胶色谱纯化分离[洗脱剂:甲醇-二氯甲烷=1:80 (V/V)]得到白色固体0.114 g, 产率为72%, mp 170.5~171.8 ℃; [α]_D²⁵+48.6 (c 0.55, CHCl₃); HR-MS (m/z): 656.471 4 [M+H]⁺; ¹H NMR (600 MHz, DMSO-d₆) δ 7.30 (d, J = 6.2 Hz, 1H, ph-H-3'), 7.28~7.24 (m, 2H, ph-H-5', 6'), 7.06~7.04 (m, 1H, ph-H-4'), 3.59~3.51 (m, 4H, piperazinyl-H-3', 5'), 3.47~3.39 (m, 4H, piperazinyl-H-2', 6'), 2.23 (s, 3H, ph-CH₃), 1.14 (s, 3H, CH₃), 1.08 (s, 3H, CH₃), 1.03 (s, 3H, CH₃), 1.00 (s, 3H, CH₃), 0.88 (s, 3H, CH₃), 0.84 (s, 3H, CH₃), 0.78 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 217.65, 174.57, 151.66, 144.42, 134.97, 131.85, 130.98, 129.54, 126.13, 123.26, 114.79, 55.24, 51.88, 51.67, 49.73, 49.40, 47.58, 47.47, 46.32, 43.82, 43.33, 41.95, 40.13, 39.40, 37.64, 34.32, 33.97, 33.04, 31.15, 30.78, 28.79, 28.35, 26.88, 26.80, 26.24, 23.93, 21.91, 21.78, 20.65, 17.47, 17.08, 16.17。

1.4.3 化合物Ⅰ₃的制备

按照Ⅰ₁的制备方法, 由化合物3 (0.158 g, 0.302 mmol)与2-氟-4-甲基苯基异氰酸酯(0.068 g, 0.453 mmol)反应。粗品经硅胶色谱纯化分离[洗脱剂:甲醇-二氯甲烷=1:80 (V/V)]得到白色固体0.118 g, 产率为75%, mp 171.3~173.1 ℃; [α]_D²⁵ +58.9 (c 0.90, CHCl₃); HR-MS (m/z): 674.461 9 [M+H]⁺; ¹H NMR (600 MHz, DMSO-d₆) δ 7.46 (d, J = 6.1 Hz, 1H, ph-H-6'), 7.05 (d, J = 7.2 Hz, 1H, ph-H-5'), 6.95 (s, 1H, ph-H-3'), 5.24 (t, J = 3.7 Hz, 1H, H-12), 3.60~3.52 (m, 4H, piperazinyl-H-3', 5'), 3.48~3.40 (m, 4H, piperazinyl-H-2', 6'), 2.29 (s, 3H, ph-CH₃), 1.15 (s, 3H, CH₃), 1.08 (s, 3H, CH₃), 1.03 (s, 3H, CH₃), 1.00 (s, 3H, CH₃), 0.88 (s, 3H, CH₃), 0.84 (s, 3H, CH₃), 0.79 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 217.58, 174.84, 163.16, 155.63, 144.47, 134.54, 125.38, 123.35, 123.18, 116.54, 116.29, 55.26, 51.67, 51.42, 49.79, 49.64, 47.81, 47.65, 46.28, 43.84, 43.35, 41.94, 39.88, 39.27, 37.62, 34.03, 33.93, 32.75, 31.24, 30.81, 28.79, 28.53, 27.08, 26.64, 26.27, 23.99, 21.91, 21.77, 21.54, 20.82, 17.41, 16.15。

1.4.4 化合物Ⅰ₄的制备

按照Ⅰ₁的制备方法, 由化合物3 (0.158 g, 0.302 mmol)与对甲氧基苯异氰酸酯(0.062 g, 0.453 mmol)反应。粗品经硅胶色谱纯化分离[洗脱剂:甲醇-二氯甲烷=1:80 (V/V)]得到白色固体0.120 g, 产率为76%, mp 165.0~166.4 ℃; [α]_D²⁵+46.5 (c 0.55, CHCl₃); HR-MS (m/z): 672.466 2 [M+H]⁺; ¹H NMR (600 MHz, DMSO-d₆) δ 7.32~7.28 (m, 2H, ph-H-2', 6'), 6.95~6.91 (m, 2H, ph-H-3'), 5.25 (t, J = 3.7 Hz, 1H, H-12), 3.78 (s, 3H, ph-OCH₃), 3.61~3.53 (m, 4H, piperazinyl-H-3', 5'), 3.51~3.43 (m, 4H, piperazinyl-H-2', 6'), 1.00 (s, 3H, CH₃), 0.99 (s, 3H, CH₃), 0.96 (s, 3H, CH₃), 0.95 (s, 3H, CH₃), 0.87 (s, 6H, 2CH₃), 0.70 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 217.61, 174.87, 159.34, 155.65, 144.45, 131.98, 123.38, 120.34, 120.17, 114.82, 114.73, 56.03, 55.24, 51.79, 51.68, 49.86, 49.73, 47.51, 47.46, 46.34, 43.76, 43.34, 41.95, 39.81, 39.38, 37.65, 34.42, 33.93, 32.76, 31.28, 30.75, 28.80, 28.39, 26.72, 26.70, 26.29, 23.97, 21.77, 21.65, 20.96, 17.48, 16.13。

1.4.5 化合物Ⅰ₅的制备

按照Ⅰ₁的制备方法, 由化合物3 (0.158 g, 0.302 mmol)与3-氟苯基异氰酸酯(0.062 g, 0.453 mmol)反应。粗品经硅胶色谱纯化分离[洗脱剂:甲醇-二氯甲烷=1:80 (V/V)]得到白色固体0.099 g, 产率为63%, mp 158.6~159.2 ℃; [α]_D²⁵ +47.6 (c 0.55, CHCl₃); HR-MS (m/z): 660.446 2 [M+H]⁺; ¹H NMR (600 MHz, DMSO-d₆) δ 7.79 (s, 1H, ph-H-2'), 7.41~7.39 (m, 1H, ph-H-5'), 7.35 (d, J = 5.0 Hz, 1H, ph-H-6'), 7.03 (d, J = 7.3 Hz, 1H, ph-H-4'), 5.23 (t, J = 3.7 Hz, 1H, H-12), 3.60~3.52 (m, 4H, piperazinyl-H-3', 5'), 3.50~3.42 (m, 4H, piperazinyl-H-2', 6'), 1.05 (s, 3H, CH₃), 0.97 (s, 3H, CH₃), 0.92 (s, 3H, CH₃), 0.90 (s, 3H, CH₃), 0.85 (s, 6H, 2CH₃), 0.70 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 217.60, 174.58, 163.73, 155.67, 144.64, 137.91, 130.84, 123.51, 117.67, 116.83, 116.44, 55.27, 51.73, 51.68, 49.85, 49.62, 47.69, 47.51, 46.28, 43.79, 43.33, 41.94, 40.15, 39.34, 37.57, 34.26, 33.96, 32.86, 30.72, 30.81, 28.63, 28.35, 26.74, 26.79, 26.28, 23.90, 21.96, 21.72, 20.93, 17.39, 16.17。

1.5 化合物Ⅱ₁~Ⅱ₅的制备

1.5.1 化合物Ⅱ₁的制备

将苯基异硫氰酸酯(0.061 g, 0.453 mmol)溶于10 mL无水1, 2-二氯乙烷中, 置于常温下搅拌; 将溶于5 mL无水1, 2-二氯乙烷的化合物3 (0.158 g, 0.302 mmol)加入三乙胺(0.183 g, 1.808 mmol), 混合均匀后, 缓慢滴入苯基异硫氰酸酯中, 常温, 过夜反应, TLC跟踪监测反应终点。二氯甲烷萃取3次, 合并有机相, 饱和食盐水洗涤3次, 无水硫酸钠干燥, 抽滤, 减压蒸干溶剂, 得到淡黄色固体。粗品经硅胶色谱纯化分离[洗脱剂:甲醇-二氯甲烷=1:80 (V/V)]得到白色固体0.118 g, 产率为75%, mp 134.6~136.4 ℃; [α]_D²⁵+42.3 (c 0.55, CHCl₃); HR-MS (m/z): 658.432 8 [M+H]⁺; ¹H NMR (600 MHz, DMSO-d₆) δ 7.42~7.38 (m, 2H, ph-H-3', 5'), 7.33~7.29 (m, 2H, ph-H-2', 6'), 7.10~7.08 (m, 1H, ph-H-4'), 5.24 (t, J = 3.7 Hz, 1H, H-12), 3.90~3.82 (m, 4H, piperazinyl-H-3', 5'), 3.52~3.44 (m, 4H, piperazinyl-H-2', 6'), 1.08 (s, 3H, CH₃), 0.97 (s, 3H, CH₃), 0.91 (s, 6H, 2CH₃), 0.86 (s, 6H, 2CH₃), 0.70 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 217.57, 182.15, 174.84, 144.68, 138.91, 129.68, 129.54, 128.63, 126.84, 126.65, 56.94, 56.58, 55.67, 50.71, 50.52, 47.86, 47.72, 46.43, 43.84, 43.58, 41.93, 40.18, 39.56, 37.64, 34.33, 33.72, 32.97, 31.20, 30.68, 28.97, 28.43, 27.05, 26.87, 26.19, 23.71, 22.07, 21.83, 20.84, 17.27, 16.11。

1.5.2 化合物Ⅱ₂的制备

按照Ⅱ₁的制备方法, 由化合物3 (0.158 g, 0.302 mmol)与邻甲苯异硫氰酸酯(0.068 g, 0.453 mmol)反应。粗品经硅胶色谱纯化分离[洗脱剂:甲醇-二氯甲烷=1:80 (V/V)]得到白色固体0.114 g, 产率为72%, mp 142.5~143.9 ℃; [α]_D²⁵ +49.8 (c 0.58, CHCl₃); HR-MS (m/z): 672.446 8 [M+H]⁺; ¹H NMR (600 MHz, DMSO-d₆) δ 7.23 (d, J = 6.2 Hz, 1H, ph-H-3'), 7.18~7.16 (m, 1H, ph-H-5'), 7.16 (d, J = 6.5 Hz, 1H, ph-H-6'), 7.12~7.10 (m, 1H, ph-H-4'), 5.24 (t, J = 3.7 Hz, 1H, H-12), 3.87~3.79 (m, 4H, piperazinyl-H-3', 5'), 3.48~3.40 (m, 4H, piperazinyl-H-2', 6'), 2.25 (s, 3H, ph-CH₃), 1.15 (s, 3H, CH₃), 1.08 (s, 3H, CH₃), 1.04 (s, 3H, CH₃), 1.03 (s, 3H, CH₃), 0.93 (s, 3H, CH₃), 0.90 (s, 3H, CH₃), 0.75 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 217.58, 182.26, 174.94, 144.48, 137.02, 136.27, 131.33, 130.18, 127.49, 126.56, 56.92, 56.88, 55.56, 50.81, 50.63, 47.79, 47.60, 46.52, 43.93, 43.58, 42.05, 40.21, 39.36, 37.67, 34.34, 33.92, 32.76, 30.83, 30.68, 28.84, 28.51, 27.09, 26.86, 26.23, 23.97, 21.84, 21.65, 20.63, 18.19, 17.51, 16.18。

5.3 化合物Ⅱ₃的制备

按照Ⅱ₁的制备方法, 由化合物3 (0.158 g, 0.302 mmol)与2-氟-4-甲基苯基异硫氰酸酯(0.076 g, 0.453 mmol)反应。粗品经硅胶色谱纯化分离[洗脱剂:甲醇-二氯甲烷=1:80 (V/V)]得到白色固体0.117 g, 产率为75%, mp 144.2~145.4 ℃; [α]_D²⁵+60.5 (c 0.95, CHCl₃); HR-MS (m/z): 690.438 6 [M+H]⁺; ¹H NMR (600 MHz, DMSO-d₆) δ 7.41~7.39 (m, 1H, ph-H-6'), 7.05 (d, J = 7.1 Hz, 1H, ph-H-5'), 6.96 (s, 1H, ph-H-3'), 5.20 (t, J = 3.7 Hz, 1H, H-12), 3.88~3.80 (m, 4H, piperazinyl-H-3', 5'), 3.46~3.38 (m, 4H, piperazinyl-H-2', 6'), 2.31 (s, 3H, ph-CH₃), 1.11 (s, 3H, CH₃), 1.00 (s, 3H, CH₃), 0.97 (s, 3H, CH₃), 0.95 (s, 3H, CH₃), 0.89 (s, 6H, 2CH₃), 0.72 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 217.60, 182.36, 174.95, 167.89, 144.70, 134.88, 128.34, 125.26, 123.35, 117.62, 116.56, 56.93, 56.78, 55.65, 50.69, 50.41, 47.88, 47.64, 46.52, 43.95, 43.35, 41.94, 40.27, 39.33, 37.78, 34.36, 34.17, 32.92, 31.26, 30.76, 28.84, 28.46, 27.18, 26.76, 26.54, 24.25, 21.97, 21.62, 21.44, 20.85, 17.46, 16.14。

1.5.4 化合物Ⅱ₄的制备

按照Ⅱ₁的制备方法, 由化合物3 (0.158 g, 0.302 mmol)与对甲氧基苯异硫氰酸酯(0.075 g, 0.453 mmol)反应。粗品经硅胶色谱纯化分离[洗脱剂:甲醇-二氯甲烷=1:80 (V/V)]得到白色固体0.119 g, 产率为76%, mp 135.8~137.4 ℃; [α]_D²⁵ +48.5 (c 0.55, CHCl₃); HR-MS (m/z): 688.443 6 [M+H]⁺; ¹H NMR (600 MHz, DMSO-d₆) δ 7.44~7.40 (m, 2H, ph-H-2', 6'), 6.92~6.88 (m, 2H, ph-H-3', 5'), 5.25 (t, J = 3.7 Hz, 1H, H-12), 3.88~3.80 (m, 4H, piperazinyl-H-3', 5'), 3.75 (s, 3H, ph-OCH₃), 3.45~3.37 (m, 4H, piperazinyl-H-2', 6'), 1.01 (s, 3H, CH₃), 0.99 (s, 3H, CH₃), 0.96 (s, 3H, CH₃), 0.95 (s, 3H, CH₃), 0.87 (s, 6H, 2CH₃), 0.70 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 217.56, 182.49, 174.68, 159.65, 144.59, 127.84, 127.67, 123.40, 114.91, 114.65, 113.54, 56.97, 56.75, 55.98, 55.64, 50.46, 50.18, 47.86, 47.62, 46.43, 43.98, 43.52, 42.05, 40.26, 39.57, 37.88, 34.64, 33.98, 32.61, 31.16, 30.92, 28.89, 28.67, 27.16, 26.78, 26.42, 23.63, 21.64, 21.58, 20.96, 17.44, 16.10。

1.5.5 化合物Ⅱ₅的制备

按照Ⅱ₁的制备方法, 由化合物3 (0.158 g, 0.302 mmol)与3-氟苯基异硫氰酸酯(0.069 g, 0.453 mmol)反应。粗品经硅胶色谱纯化分离[洗脱剂:甲醇-二氯甲烷=1:80 (V/V)]得到白色固体0.098 g, 产率为63%, mp 136.6~138.2 ℃; [α]_D²⁵ +51.5 (c 0.60, CHCl₃); HR-MS (m/z): 676.423 6 [M+H]⁺; ¹H NMR (600 MHz, DMSO-d₆) δ 7.66 (s, 1H, ph-H-2'), 7.38~7.36 (m, 1H, ph-H-5'), 7.21 (d, J = 6.1 Hz, 1H, ph-H-6'), 6.95 (d, J = 8.1 Hz, 1H, ph-H-4'), 5.23 (t, J = 3.7 Hz, 1H, H-12), 3.87~3.79 (m, 4H, piperazinyl-H-3', 5'), 3.46~3.38 (m, 4H, piperazinyl-H-2', 6'), 1.08 (s, 3H, CH₃), 0.97 (s, 3H, CH₃), 0.92 (s, 3H, CH₃), 0.90 (s, 3H, CH₃), 0.85 (s, 6H, 2CH₃), 0.69 (s, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 217.52, 182.26, 174.85, 163.57, 144.53, 139.02, 130.95, 123.41, 122.56, 121.85, 116.76, 56.97, 56.65, 55.64, 50.68, 50.49, 47.88, 47.63, 46.25, 43.84, 43.67, 41.94, 40.22, 39.54, 37.61, 34.32, 33.98, 32.67, 31.22, 30.74, 28.91, 28.34, 26.75, 26.65, 26.54, 23.60, 21.97, 21.62, 20.95, 17.55, 16.09。

2 生物活性测试

以AZD8055为阳性对照药, 采用MTT法对合成的目标化合物进行体外抗肿瘤活性测试, 所选用的肿瘤细胞为人肝癌细胞HepG2和人胃癌细胞SGC7901。将对数生长期肿瘤细胞培养在96孔培养板中, 各细胞株每孔接种5×10³个, 每组3个复孔, 置于CO₂恒温培养箱中, 培养24 h备用。给药组药品浓度为10 μmol·L⁻¹, CO₂恒温培养箱中继续培养72 h。每孔20 µL (1 mg·mL^-1) MTT溶液加入, 在37 ℃下孵育4 h, 弃上清液, 加入二甲基亚砜200 µL, 振荡溶解。在酶标仪波长570 nm条件下测定各孔光密度(OD)值, 计算所测化合物对细胞的抑制率和IC₅₀。实验重复3次, 结果取平均值, 计算各组细胞的抑制率, 抑制率= (对照组OD值-药物组OD值)/对照组OD值×100%。

基金

收起

辽宁省自然科学基金资助项目(2015020687)

参考文献

收起

文献

收起

参考文献引证文献

排序方式：

[1]

Yang

, Rudge

, Koos

et al . mTOR kinase structure, mechanism and regulation[J]. Nature, 2013, 497: 217-223.

https://doi.org/10.1038/nature12122

[2]

Dobbin

, Landen

. The importance of the PI3K/AKT/mTOR pathway in the progression of ovarian cancer[J]. Int J Mol Sci, 2013, 14: 8213-8227.

https://doi.org/10.3390/ijms14048213

[3]

Karunakar

, Bandi

, Lalitha

. Three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis and molecular docking of ATP-competitive triazine analogs of human mTOR inhibitors[J]. Med Chem Res, 2012, 21: 1207-1217.

https://doi.org/10.1007/s00044-011-9629-x

[4]

Cho

. Targeting the PI3K/AKT/mTOR pathway in malignancy:rational and clinical outlook[J]. BioDrugs, 2014, 28: 373-381.

https://doi.org/10.1007/s40259-014-0090-5

[5]

, Song

, Zhang

et al . Oleanolic acid inhibits cell survival and proliferation of prostate cancer cells in vitro and in vivo through the PI3K/Akt pathway[J]. Tumor Biol, 2016, 37: 7599-7613.

https://doi.org/10.1007/s13277-015-4655-9

[6]

Chen

, Kong

, Lan

et al . Design, synthesis, and antihepatocellular carcinoma activity of nitric oxide releasing derivatives of oleanolic acid[J]. J Med Chem, 2008, 51: 4834-4838.

https://doi.org/10.1021/jm800167u

[7]

Yan

, Zhang

, Li

et al . A series of oleanolic acid derivatives as anti-hepatitis B virus agents:design, synthesis, and in vitro and in vivo biological evaluation[J]. Molecules, 2016, 21: 402-412.

https://doi.org/10.3390/molecules21040402

[8]

Gutiérrez

. Hypolipidemic and hypoglycemic activities of an oleanolic acid derivative from Malva parviflora on streptozotocin-induced diabetic mice[J]. Arch Pharm Res, 2017, 40: 550-562.

https://doi.org/10.1007/s12272-016-0873-y

[9]

Cheng

, Su

, Huang

et al . Synthesis and cytotoxic evaluation of several oleanolic acid uracil/thymine conjugates[J]. MedChemComm, 2016, 7: 972-981.

https://doi.org/10.1039/C6MD00061D

[10]

, Bao

, Shen

et al . Novel heterocyclic ring-fused oleanolic acid derivatives as osteoclast inhibitors for osteoporosis[J]. MedChemCommn, 2016, 7: 371-377.

https://doi.org/10.1039/C5MD00482A

[11]

Zhao

, Liu

, Li

. Oleanolic acid suppresses the proliferation of lung carcinoma cells by miR-122/Cyclin G1/MEF2D axis[J]. Mol Cell Biochem, 2015, 400: 1-7.

https://doi.org/10.1007/s11010-014-2228-7

[12]

Banno

, Kihisa

, Tokuda

et al . Triterpene acids from the leaves of and their anti-inflammatory and antitumor-promoting effects[J]. Biosci Biotechnol Biochem, 2004, 68: 85-90.

https://doi.org/10.1271/bbb.68.85

[13]

Wang

, Zhu

, Zhang

. Effect of oleanolic acid on the expression of PI3k and Akt genes in leukaem HL-60 cells[J]. Antitumor Pharm, 2013, 4: 64-65. http://cn.bing.com/academic/profile?id=6e20c239457eaddf7e39bbc8383b0844&encoded=0&v=paper_preview&mkt=zh-cn

[14]

Liu

, Wu

, Ma

et al . Oleanolic acid suppresses aerobic glycolysis in cancer cells by switching pyruvate kinase type M isoforms[J]. PLoS One, 2014, 9: e91606/1-e91606/9. http://cn.bing.com/academic/profile?id=aebab5bdce668e95087bf6d3236a29c6&encoded=0&v=paper_preview&mkt=zh-cn

[15]

Tang

, Gong

, Xu

et al . A review of research on mTOR inhibitors[J]. Chin J Org Chem (有机化学), 2011, 31: 1144-1154. http://d.old.wanfangdata.com.cn/OAPaper/oai_doaj-articles_adc7c187d7f44f073bbc49d9203027cc

[16]

Meng

, Feng

, Zhang

et al . Synthesis and anti-tumor activity of oleanolic acid derivatives[J]. Acta Pharm Sin (药学学报), 2015, 50: 469-474. http://www.yxxb.com.cn:8081/aps/CN/abstract/abstract15298.shtml

[17]

Nie

, Luo

, Wang

et al . Synthesis of new α-glucosidase inhibitors based on oleanolic acid incorporating cinnamic amides[J]. Chem Pharm Bull, 2011, 59: 1051-1056.

https://doi.org/10.1248/cpb.59.1051

[18]

Weng

, Zhang

, Lu

et al . Molecular design and computer simulation of small molecule thrombin inhibitor[J]. Cent South Pharm (中南药学), 2013, 11: 565-568. http://cn.bing.com/academic/profile?id=855b978737b3f1eb4c942823bbd78a3e&encoded=0&v=paper_preview&mkt=zh-cn

[19]

Meng

, Nie

, Wang

et al . Synthesis and antitumor activity of oleanolic acid derivatives[J]. Acta Pharm Sin (药学学报), 2011, 46: 1215-1220. http://www.yxxb.com.cn:8081/aps/CN/abstract/abstract14059.shtml

2019年第54卷第2期

PDF下载

146

引用本文

BibTeX

文章信息

doi: 10.16438/j.0513-4870.2018-0890

接收时间：2018-09-30
首发时间：2026-01-26
出版时间：2019-02-12

补充材料

相关文章

文章信息

作者

出版历史

收稿日期：2018-09-30
修回日期：2018-10-29

基金

辽宁省自然科学基金资助项目(2015020687)

作者信息

沈阳化工大学制药与生物工程学院, 辽宁沈阳 110142

通讯作者:

*宋艳玲, Tel:13840387750, E-mail:yanlingsong521@126.com

参考文献

分享链接

https://castjournals.cast.org.cn/joweb/yxxb/CN/10.16438/j.0513-4870.2018-0890

分享至

全文二维码

扫描看全文

引用本文

BibTeX

本文的引用情况

2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

关闭全屏

BibTeX
EndNote
RefWorks
TxT