药学学报  2018, Vol. 53 Issue (10): 1696-1704   PDF    
含芳氧吡啶酮结构片段的噻唑啉酮脲衍生物的设计、合成及抗肿瘤活性研究
祁宝辉, 杨颖, 宫帼唯, 何欢, 岳续朋, 徐昕, 王雅溶     
遵义医学院珠海校区生物工程系, 广东 珠海 519041
摘要: 本文以cabozantinib为先导物,基于已有的c-Met激酶抑制剂的构效关系,设计并合成了13个结构新颖的小分子抑制剂,其结构经1H NMR、13C NMR和HR-MS确证。采用MTT法对所合成的化合物进行了体外抗肿瘤活性测试,采用实时动态活细胞成像法和流式细胞术对体外抗肿瘤作用机制进行了初步研究。结果表明,所设计的大多数化合物对人非小细胞肺癌细胞A549和人结直肠癌细胞HT-29有较好的抑制作用,活性优于cabozantinib;化合物对HT-29细胞除具有明显的杀伤作用外,还可抑制其增殖,促进细胞凋亡。
关键词: 芳氧吡啶酮     噻唑啉酮脲     设计     合成     抗肿瘤活性    
Design, synthesis and study of anti-tumor activity of thiazolinone urea derivatives bearing aryloxypyridinone fragments
QI Bao-hui, YANG Ying, GONG Guo-wei, HE Huan, YUE Xu-peng, XU Xin, WANG Ya-rong     
Department of Bioengineering, Zhuhai Campus of Zunyi Medical University, Zhuhai 519041, China
Abstract: Taking cabozantinib as leading compound, 13 novel small molecular c-Met inhibitors were designed and synthesized based on the obtained structure-activity relationships (SARs) of c-Met inhibitors. The structures of compounds were confirmed by 1H NMR, 13C NMR and HR-MS. In vitro anti-tumor activity was evaluated by MTT method, and the mechanism was preliminarily disclosed by real-time dynamic living cell imaging and flow cytometry analysis. The results indicated that most of compounds showed good inhibition activity against human non-small-cell carcinoma cell A549 and human colorectal cancer cell HT-29 which was superior to cabozantinb. Compounds showed excellent cytotoxity and anti-proliferative activity against HT-29, and promoted cell apoptosis.
Key words: aryloxypyridinone     thiazolinone ureas     design     synthesis     anti-tumor activity    

间叶上皮细胞转变因子(mesenchymal-epithelia transition factor, MET)是受体酪氨酸激酶家族的一个重要成员[1]。它被证实是受体酪氨酸激酶家族中唯一能与肝细胞生长因子(hepatocyte growth factor, HGF)结合的高亲和受体, 故也被称为肝细胞生长因子受体。异常的HGF/c-Met活性与肿瘤的发生、转移和耐药性等有着十分密切的关系, 它在多种肿瘤组织中呈现异常的高表达、突变及活性改变等特征, 如肺癌、结肠癌、前列腺癌、卵巢癌及头颈部癌等[2, 3]

c-Met激酶位于导致肿瘤形成及转移的众多通路的交叉点, 主要包括丝裂原活化蛋白酶通路、磷酸肌醇3激酶-哺乳动物雷帕霉素靶蛋白通路和转录激活因子等通路[4-7]。因此, 以c-Met激酶为靶点可相对容易地实现对多种肿瘤信号通路的同时干扰, 一旦肿瘤细胞中异常活化的HGF/c-Met信号通路被阻断, 肿瘤细胞就会出现增殖减缓、成瘤性下降或侵袭能力减弱等一系列变化。因而, c-Met激酶已成为肿瘤靶向治疗的重要靶点之一[8]

对c-Met激酶的结构及其作用机制的深入研究表明, 小分子c-Met激酶抑制剂是对其催化结构域进行竞争性结合而产生抑制作用的。c-Met激酶催化结构域为一高度保守的催化核心区域, 含有1个起始端——DFG (天冬氨酸-苯丙氨酸-甘氨酸, Asp-Phe- Gly)序列和1个终止端——含APE (丙氨酸-脯氨酸-谷氨酸, Ala-Pro-Glu)序列的环状活化区域。该区域存在着多种构象, 当DFG序列中的Asp和Phe朝向ATP结合位点时, 为激酶活化构象, 该构象被称为“DFG-in”; 当DFG序列中的Phe朝向ATP结合位点时, 为激酶的非活化构象, 其被称为“DFG-out”[9]

目前, 小分子抑制剂按结合方式主要分为Type Ⅰ、Type Ⅱ及Type Ⅲ三种[10-13]。Type Ⅰ抑制剂主要竞争性抑制激酶的铰链区域, 属于ATP竞争性抑制剂, 针对“DFG-in”构象; “DFG-out”非活化构象与“DFG-in”活化构象相比, 多出一个邻近ATP结合位点的疏水口袋, 亦称为变构位点。它的氨基酸序列不如ATP结合口袋的氨基酸序列保守, 起到调节激酶选择性的作用, 以此为基础的抑制剂被定义为Type Ⅱ抑制剂。此外, 所有的Type Ⅱ抑制剂都含有一个疏水部分, 该部分在氢键形成后立即与变构位点产生范德华力。尽管Type Ⅱ抑制剂是以占据变构位点为特征的, 但它们还可伸入至腺嘌呤区域, 并采取与Tpye Ⅰ抑制剂类似的结合方式与激酶铰链部位的氨基酸残基形成氢键。与Type Ⅰ抑制剂相比, Type Ⅱ抑制剂可改动空间较大, 结构类型较丰富, 主要分为噻吩并吡啶类、氨基吡啶类及喹啉类等几大类, 如图 1所示[14, 15]。其中, cabozantinib于2012年12月被美国FDA批准上市, 临床上主要用于治疗甲状腺髓样癌, 它是目前唯一上市的Type Ⅱ抑制剂。

Figure 1 Structures of representative type II c-Met inhibitors

基于以上及课题组已有的小分子c-Met激酶抑制剂的构效关系[16-18], 本文将潜在药理活性较好的噻唑啉酮脲与吡啶酮结构片段引入至先导物cabozantinib中, 再对其进行局部结构修饰, 设计、合成了13个结构新颖的小分子c-Met激酶抑制剂(如图 2所示), 并对其进行了体外生物活性研究, 初步总结了该类抑制剂的构效关系。

Figure 2 The design of novel type II c-Met inhibitors
结果与讨论 1 化学合成

目标化合物的合成方法如路线1所示, 以市售的2-甲氧基-5-羟基吡啶和3, 4-二氟硝基苯为起始原料, 经亲核取代反应制得中间体a, 它再经还原、酰化、肼解、缩合、环合与脱甲基等过程制得目标化合物g1~g11; 采用类似的方法制得目标化合物g12g13[18, 19]。部分中间体及目标化合物的结构经1H NMR、13C NMR和HR-MS确证, 具体数据见后文实验部分。

2 生物活性评价 2.1 体外抗肿瘤活性及初步构效关系

以cabozantinib为阳性对照物, 采用四甲基偶氮唑盐比色法(MTT法)对合成的化合物进行初步的体外抗肿瘤活性测试, 结果表明该系列化合物对人非小细胞肺癌细胞A549和人结直肠癌细胞HT-29两个细胞系表现出优于人乳腺癌细胞MDA-MB-231的抑制活性。其中, 化合物g2g9的体外抗肿瘤活性明显优于cabozantinib。初步构效关系表明: ① C环(如图 2所示)被氟原子取代时, 对抑制活性较为有利, 尤其是氟原子位于间、对位; ② B环(如图 2所示)的3位为氟原子(g2)取代时, 抗肿瘤活性优于氯原子(g13)和溴原子(g12)取代物(表 1)。

Reagents and conditions: ⅰ) Cs2CO3, DMF, 80 ℃, 6 h; ⅱ) Fe, 90% EtOH-H2O, reflux, 3 h; ⅲ) Phenyl chloroformate, pyridine, CH2Cl2, rt, 4 h; ⅳ) 80% hydrazine hydrate, xylene, 60 ℃, 4 h; ⅴ) Aromatic aldehydes, HOAc, i-PrOH, reflux, 2 h; ⅵ) Mercapto acetic acid, SiCl4, CH2Cl2, reflux, 5 h; ⅶ) TMSCl, NaI, MeCN, reflux, 2 h Scheme1 Synthetic routes of target compounds g1-g13.

Table 1 Anti-tumor activity of target compounds against A549, HT-29 and MDA-MB-231 cells. a: The values were an average of three separate determinations and standard deviations were shown; b: ND: Not determined
2.2 抑制增殖和杀伤作用

为初步了解该系列化合物的体外抗肿瘤作用机制, 采用实时动态活细胞成像法对活性较好的化合物g2进行了高内涵的抗肿瘤活性研究。结果表明, 化合物g2对HT-29细胞的增殖具有一定的抑制作用, 较cabozantinib稍弱, 如图 3图 4所示。此外, 化合物g2对HT-29细胞表现出了明显的杀伤作用, 3个实验浓度下的杀伤作用均优于cabozantinib。在10和3.33 μg·mL-1两个浓度下, 化合物g2的杀伤能力分别为阳性药的1.4倍和2.4倍(以杀死的细胞数计), 如图 5图 6所示。

HT-29 cells were incubated with 0.1% DMSO or exposed to compound g2 or cabozantinib at different concentration. Error bars: ± SD from n = 3 and all SD values are below 0.05 Figure 3 Real-time cell confluence study in HT-29 cell line. The cell population was monitored for 72 h using an IncuCyte ZOOM system in an incubator.

Green fluorescent cells were counted as dead cells Figure 4 Phase-contrast images (merged) of HT-29 cells after 0, 36 and 72 h of treatment with compound g2 (3.33 μg·mL-1), cabozantinib (3.33 μg·mL-1) or not.

HT-29 cells were incubated with 0.1% DMSO or exposed to compound g2 or cabozantinib at different concentration. Error bars: ± SD from n = 3 and all SD values are below 0.05 except the results of 1.11 μg·mL-1 cabo­ zantinib Figure 5 Real-time cytotoxicity study in HT-29 cell line. The dead cell population was monitored for 72 h using an IncuCyte ZOOM system in an incubator.

Green fluorescent cells were counted as dead cells Figure 6 Phase-contrast images (green light) of HT-29 cells after 0, 36 and 72 h of treatment with compound g2 (3.33 μg·mL-1), cabozantinib (3.33 μg·mL-1) or not.
2.3 凋亡检测

通过流式细胞仪的检测发现, 与空白组对比, 化合物g2能够促进HT-29细胞凋亡, 尤其是早期凋亡。如图 7所示, 给药浓度为5.0 μg·mL-1时, 化合物g2作用72 h后, 早期凋亡比例为21.9%, 弱于阳性(76.7%)。当浓度达到10.0 μg·mL-1时, 早期凋亡比例增加到49.5%。早期凋亡的细胞数量随着剂量的增加而增加, 呈现浓度依赖性。

The upper right represents late apoptosis as detected by annexin V/PI staining, the lower right represents early apoptosis as detected by only annexin V staining. The image represents the results from one of three independent experiments Figure 7 Quantitative analysis of massive apoptosis caused by compound g2.
2.4 靶点的初步筛选

为初步了解该系列化合物的作用靶点, 本研究选取体外抗肿瘤活性较好的化合物g2, 分别测试其对c-Met、KDR、c-Kit、c-Src、ALK和HER-2六种激酶的抑制活性。结果显示, 化合物g2对c-Met、KDR和c-Kit激酶表现出不同程度的抑制作用, 对其他几种激酶无明显的抑制作用(表 2)。

Table 2 Kinase profile of compound g2. *The values are average of two independent determinations
3 分子对接

应用Molecular Operating Environment (MOE)软件将活性较优的化合物g2与c-Met蛋白晶体进行模拟对接。结果如图 8所示, 化合物g2在结合口袋内与c-Met的作用力主要有: ①吡啶酮环和噻唑啉酮脲结构片段与Met1160、Asp1222和Glu1127形成牢固的氢键; ②吡啶酮环及三氟取代苯环(C环)分别与Asp1222和Ile1084产生H-π作用; ③单氟取代的苯环(B环)与Phe1223产生的π-π相互作用。

The compound was shown by blue sticks, H-bonds were represented by green dotted lines, and arene-H interaction was shown by red dotted lines Figure 8 The structure of compound g2 and its proposed binding mode with c-Met kinase (PBD ID: 3LQ8).
4 小结

本文以cabozantinib为先导物, 设计、合成了13个含芳氧吡啶酮结构片段的噻唑啉酮脲衍生物作为新型c-Met激酶抑制剂。生物活性研究结果表明, 该类化合物的主要作用靶点为c-Met激酶, 对KDR和c-Kit两种激酶表现出较弱的抑制活性; 对A549和HT-29两种肿瘤细胞表现出一定的体外抗肿瘤活性。其中, 化合物g2对两种肿瘤细胞的杀伤和抑制增殖的作用均明显优于cabozantinib。通过本研究, 进一步拓宽了Type Ⅱ类c-Met激酶抑制剂的结构类型, 对新型抗肿瘤药物的研究具有一定的指导意义。

实验部分

3-(4, 5-二甲基噻唑-2)-2, 5-二苯基四氮唑溴盐(MTT, 美国Sigma公司); 胎牛血清(浙江天杭生物科技股份有限公司); Dulbecco's Modified Eagle Medium (DMEM)培养基、胰蛋白酶消化液及青霉素/链霉素双抗溶液及PBS缓冲液(美国HyClone公司); 激酶试剂盒(日本Carna Biosciences公司); EDTA和ATP (美国Sigma公司); 柱色谱硅胶200~300目(青岛海洋化工厂), 其他所有试剂均为市售分析纯或化学纯。

Bruker AM-400型核磁共振仪(Bruker公司); Agilent 6530四极杆-飞行时间串联质谱仪(美国Agilent公司); 多功能酶联免疫检测仪(美国Thermo Fisher公司); 集热式恒温加热磁力搅拌器(DF-101S) (巩义市予华科技有限公司); FACS Caliur流式细胞仪(美国Beckman Coulter公司); 实时动态活细胞成像系统(美国Essen公司)。

1 化合物的合成 1.1 5-(2-氟-4-硝基苯氧基)-2-甲氧基吡啶(a)的合成

将2-甲氧基-5-羟基吡啶(10.0 g, 80.0 mmol)和3, 4-二氟硝基苯(15.3 g, 96.0 mmol)加入至60 mL的N, N-二甲基甲酰胺(DMF)中, 再加入碳酸铯(39.0 g, 120.0 mmol), 加毕, 80 ℃搅拌6 h。将反应液冷却至室温, 倒入400 mL冰水中, 搅拌, 抽滤, 得土黄色固体15.3 g, 收率72.6%。1H NMR (DMSO-d6, 400 MHz) δ: 7.83 (m, 1H), 7.75 (m, 1H), 7.48 (s, 1H), 7.28 (d, J = 6.0 Hz, 1H), 7.16 (m, 1H), 6.60 (d, J = 6.0 Hz, 1H), 3.86 (s, 3H)。HR-MS (ESI) m/z: 265.058 9 [M+ H]+

1.2 3-氟-4-[(6-甲氧基吡啶-3-基)氧基]苯胺(b)的合成

将中间体a (15.0 g, 56.8 mmol)加入至150 mL的90%乙醇-水溶液中, 再加入还原铁粉(9.5 g, 170.5 mmol)和0.5 mL浓盐酸, 回流3 h。趁热抽滤, 滤液浓缩至约30 mL, 0 ℃冷却析晶, 抽滤, 得黄色固体11.1 g, 收率83.5%。1H NMR (DMSO-d6, 400 MHz) δ: 7.47 (s, 1H), 7.26 (d, J = 6.0 Hz, 1H), 7.17 (m, 1H), 6.75 (m, 1H), 6.68 (m, 1H), 6.60 (d, J = 6.0 Hz, 1H), 4.59 (s, 2H), 3.85 (s, 3H); HR-MS (ESI) m/z: 235.086 5 [M+H]+

1.3 3-氟-4-[(6-甲氧基吡啶-3-基)氧基]苯基氨基甲酸苯酯(c)的合成

将干燥的中间体b (10.0 g, 42.7 mmol)加入至100 mL干燥的二氯甲烷中, 再加入吡啶(10.1 g, 128.2 mmol), 搅拌10 min; 0 ℃下, 滴入氯甲酸苯酯(10.0 g, 64.1 mmol), 滴毕, 室温继续搅拌4 h。向反应液中加入50 mL饱和碳酸氢钠水溶液, 分出有机相, 水洗一次, 减压蒸除二氯甲烷, 得黄色油状物(直接用于下一步)。

1.4 N-{3-氟-4-[(6-甲氧基吡啶-3-基)氧基]苯基}氨基脲(d)的合成

将上述所得油状物c加入至50 mL二甲苯中, 再加入50 mL的80%水合肼, 60 ℃搅拌4 h。冷却至室温, 抽滤, 滤饼用少量水洗, 干燥, 得乳白色固体4.6 g。1H NMR (DMSO-d6, 400 MHz) δ: 7.45 (s, 1H), 7.24 (d, J = 6.0 Hz, 1H), 7.16 (m, 1H), 6.74 (m, 1H), 6.67 (m, 1H), 6.58 (d, J = 6.0 Hz, 1H), 3.85 (s, 3H); HR-MS (ESI) m/z: 293.102 6 [M+H]+

1.5 N1-{3-氟-4-[(6-甲氧基吡啶-3-基)氧基]苯基}-N4-苯亚甲基缩氨基脲(e)的合成

将中间体d (0.4 g, 1.37 mmol)和苯甲醛(0.17 g, 1.64 mmol)加入至4 mL异丙醇中, 再加入1滴冰乙酸, 回流2 h。冷却至室温, 抽滤, 得白色固体0.43 g, 收率84.2%。1H NMR (DMSO-d6, 400 MHz) δ: 9.02 (s, 1H), 8.58 (s, 1H), 7.96 (s, 1H), 7.53~7.58 (m, 3H), 7.47 (s, 1H), 7.34~7.39 (m, 2H), 7.28 (d, J = 6.0 Hz, 1H), 7.16 (m, 1H), 6.76 (m, 1H), 6.67 (m, 1H), 6.62 (d, J = 6.0 Hz, 1H), 3.86 (s, 3H); HR-MS (ESI) m/z: 381.134 7 [M+H]+

1.6 N1-{3-氟-4-[(6-甲氧基吡啶-3-基)氧基]苯基}- N3-(4-氧代-2-苯基噻唑啉-3-基)脲(f)的合成

将中间体e (0.43 g, 1.13 mmol)加入至5 mL干燥的二氯甲烷中, 依次加入巯基乙酸(0.52 g, 5.65 mmol)和0.5 mL四氯化硅, 加毕, 回流5 h。向反应液中加入5 mL冰水, 10%氢氧化钠水溶液调pH至9, 抽滤, 固体经柱色谱(二氯甲烷-甲醇30:1)分离纯化得白色固体0.24 g, 收率46.8%。1H NMR (DMSO-d6, 400 MHz) δ: 8.85 (s, 1H), 8.64 (s, 1H), 7.54~7.58 (m, 3H), 7.46 (s, 1H), 7.33~7.39 (m, 2H), 7.27 (d, J = 6.0 Hz, 1H), 7.17 (m, 1H), 6.74 (m, 1H), 6.65 (m, 1H), 6.60 (d, J = 6.0 Hz, 1H), 5.89 (s, 1H), 3.85 (s, 3H), 3.81 (m, 2H); HR-MS (ESI) m/z: 455.117 8 [M+H]+

1.7 N1-{3-氟-4-[(6-氧代-1, 6-二氢吡啶-3-基)氧基]苯基}-N3-(4-氧代-2-苯基噻唑啉-3-基)脲(g1)的合成

将中间体f (0.2 g, 0.44 mmol)加入至2 mL干燥的乙腈中, 再加入三甲基氯硅烷(0.48 g, 4.4 mmol)和碘化钠(0.20 g, 1.32 mmol), 回流2 h。向反应液中加入10%水溶液至pH为8, 乙酸乙酯萃取(2×5 mL), 无水硫酸钠干燥, 蒸干, 柱色谱(二氯甲烷-甲醇20:1)分离纯化的白色固体0.11 g, 收率56.7%。1H NMR (DMSO-d6, 400 MHz) δ: 11.37 (br, 1H), 9.05 (br, 1H), 8.77 (s, 1H), 7.52~7.58 (m, 3H), 7.37~7.47 (m, 5H), 7.11~7.14 (m, 1H), 7.02~7.07 (m, 1H), 6.45 (d, 1H), 5.86 (s, 1H), 3.80~3.94 (m, 2H); 13C NMR (DMSO-d6, 100 MHz) δ 169.58, 161.10 (2C), 154.14, 153.93, 151.53, 140.66, 140.35, 139.33, 136.29, 135.04, 131.53, 127.26, 120.41, 119.15, 115.47, 112.43, 112.36, 104.59, 56.41, 29.53; HR-MS (ESI) m/z: 441.099 6 [M+H]+

1.8 化合物g2~g13的合成

采用类似于化合物g1的合成方法制备化合物g2~g13

N1-{3--4-[(6-氧代-1, 6-二氢吡啶-3-)氧基]苯基}-N3-[4-氧代-2-(3, 4, 5-三氟苯基)噻唑啉-3-](g2)   白色固体, 收率47.3%。1H NMR (DMSO-d6, 400 MHz) δ: 11.34 (br, 1H), 9.07 (br, 1H), 8.63 (s, 1H), 7.61~7.68 (m, 1H), 7.44~7.53 (m, 2H), 7.32~7.37 (m, 2H), 7.07~7.11 (m, 1H), 6.97~7.03 (m, 1H), 6.41 (d, 1H), 5.82 (s, 1H), 3.74~3.93 (m, 2H); 13C NMR (DMSO-d6, 100 MHz) δ 169.67, 161.13 (2C), 154.13, 153.98, 151.53, 140.68, 139.48, 139.35, 138.37, 135.03, 131.55, 127.20, 120.43, 119.25, 115.31, 112.54, 112.35, 104.72, 56.41, 29.60; HR-MS (ESI) m/z: 495.072 6 [M+H]+

N1-{3--4-[(6-氧代-1, 6-二氢吡啶-3-)氧基]苯基}-N3-[4-氧代-2-(2, 4, 6-三氟苯基)噻唑啉-3-](g3)  白色固体, 收率41.4%。1H NMR (DMSO-d6, 400 MHz) δ: 11.33 (br, 1H), 9.08 (br, 1H), 8.72 (s, 1H), 7.67~7.74 (m, 1H), 7.49~7.55 (m, 3H), 7.28~7.36 (m, 2H), 7.14~7.19 (m, 1H), 7.08~7.11 (m, 1H), 6.98~7.04 (m, 1H), 6.41 (d, 1H), 6.01 (s, 1H), 3.76~3.93 (m, 2H); 13C NMR (DMSO-d6, 100 MHz) δ 169.68, 161.12 (2C), 154.14, 153.99, 151.55, 140.68, 139.49, 139.36, 138.39, 135.05, 131.57, 127.21, 120.43, 119.26, 115.32, 112.54, 112.36, 104.71, 56.43, 29.60; HR-MS (ESI) m/z: 495.071 9 [M+H]+

N1-{3--4-[(6-氧代-1, 6-二氢吡啶-3-)氧基]苯基}-N3-[4-氧代-2-(2, 4-二氟苯基)噻唑啉-3-](g4)   白色固体, 收率52.9%。1H NMR (DMSO-d6, 400 MHz) δ: 11.34 (br, 1H), 9.13 (br, 1H), 8.78 (s, 1H), 7.67~7.72 (m, 1H), 7.49~7.53 (m, 3H), 7.28~7.38 (m, 3H), 7.14~7.18 (m, 1H), 7.09~7.12 (m, 1H), 6.98~7.03 (m, 1H), 6.40 (d, 1H), 6.02 (s, 1H), 3.76~3.90 (m, 2H); 13C NMR (DMSO-d6, 100 MHz) δ 169.67, 161.11 (2C), 154.14, 153.97, 151.55, 140.68, 139.47, 139.36, 136.26, 135.01, 131.56, 127.22, 120.43, 119.26, 115.30, 112.54, 112.33, 104.71, 56.42, 29.59; HR-MS (ESI) m/z: 477.081 7 [M+H]+

N1-{3--4-[(6-氧代-1, 6-二氢吡啶-3-)氧基]苯基}-N3-[4-氧代-2-(3-氟苯基)噻唑啉-3-](g5)   白色固体, 收率48.4%。1H NMR (DMSO-d6, 400 MHz) δ: 11.34 (br, 1H), 9.07 (s, 1H), 8.69 (s, 1H), 7.49~7.53 (m, 1H), 7.31~7.46 (m, 5H), 7.17~7.22 (m, 1H), 7.08~7.11 (m, 1H), 6.98~7.02 (m, 1H), 6.40 (d, 1H), 5.85 (s, 1H), 3.74~3.92 (m, 2H); 13C NMR (DMSO-d6, 100 MHz) δ 169.67, 161.11 (2C), 154.05, 153.96, 151.54, 140.68, 139.34, 139.33, 136.37, 136.28, 135.01, 131.15, 131.07, 127.23, 120.42, 119.26, 116.30, 116.09, 56.42, 29.59; HR-MS (ESI) m/z: 459.089 8 [M+H]+

N1-{3--4-[(6-氧代-1, 6-二氢吡啶-3-)氧基]苯基}-N3-[4-氧代-2-(2, 6-二氟苯基)噻唑啉-3-](g6)   白色固体, 收率50.1%。1H NMR (DMSO-d6, 400 MHz) δ: 11.35 (br, 1H), 9.04 (br, 1H), 8.73 (s, 1H), 7.67~7.75 (m, 1H), 7.49~7.56 (m, 3H), 7.28~7.35 (m, 2H), 7.14~7.19 (m, 1H), 7.08~7.12 (m, 1H), 6.98~7.05 (m, 1H), 6.41 (d, 1H), 5.96 (s, 1H), 3.76~3.94 (m, 2H); 13C NMR (DMSO-d6, 100 MHz) δ 169.65, 161.10 (2C), 154.13, 153.95, 151.55, 140.69, 139.46, 139.37, 136.25, 135.03, 131.55, 127.23, 120.43, 119.28, 115.29, 112.53, 112.32, 104.72, 56.44, 29.58; HR-MS (ESI) m/z: 477.084 9 [M+H]+

N1-{3--4-[(6-氧代-1, 6-二氢吡啶-3-)氧基]苯基}-N3-[4-氧代-2-(4-氟苯基)噻唑啉-3-](g7)   白色固体, 收率46.2%。1H NMR (DMSO-d6, 400 MHz) δ: 11.33 (br, 1H), 8.99 (br, 1H), 8.59 (s, 1H), 7.49~7.57 (m, 3H), 7.32~7.38 (m, 2H), 7.21~7.25 (m, 2H), 7.07~7.10 (m, 1H), 6.97~7.02 (m, 1H), 6.42 (d, 1H), 5.82 (s, 1H), 3.74~3.90 (m, 2H); 13C NMR (DMSO-d6, 100 MHz) δ 169.68, 161.11 (2C), 154.07, 153.96, 151.54, 140.68, 139.37, 139.34, 136.38, 136.28, 135.03, 131.15, 131.09, 127.25, 120.42, 119.26, 116.30, 116.08, 56.43, 29.59; HR-MS (ESI) m/z: 459.089 7 [M+H]+

N1-{3--4-[(6-氧代-1, 6-二氢吡啶-3-)氧基]苯基}-N3-[4-氧代-2-(2-氟苯基)噻唑啉-3-](g8)   白色固体, 收率43.4%。1H NMR (DMSO-d6, 400 MHz) δ: 11.33 (br, 1H), 9.01 (br, 1H), 8.71 (s, 1H), 7.60~7.64 (m, 1H), 7.49~7.53 (m, 1H), 7.40~7.46 (m, 1H), 7.33~7.38 (m, 2H), 7.23~7.28 (m, 2H), 7.06~7.10 (m, 1H), 6.98~7.03 (m, 1H), 6.42 (d, 1H), 6.04 (s, 1H), 3.77~3.90 (m, 2H); 13C NMR (DMSO-d6, 100 MHz) δ 169.67, 161.10 (2C), 154.04, 153.96, 151.53, 140.68, 139.33, 139.31, 136.37, 136.28, 135.01, 131.16, 131.09, 127.23, 120.42, 119.25, 116.31, 116.08, 56.43, 29.58; HR-MS (ESI) m/z: 459.089 3 [M+H]+

N1-{3--4-[(6-氧代-1, 6-二氢吡啶-3-)氧基]苯基}-N3-[4-氧代-2-(3, 4-二氟苯基)噻唑啉-3-](g9)   白色固体, 收率47.2%。1H NMR (DMSO-d6, 400 MHz) δ: 11.33 (br, 1H), 9.06 (br, 1H), 8.66 (s, 1H), 7.62~7.68 (m, 1H), 7.43~7.53 (m, 2H), 7.33~7.38 (m, 3H), 7.07~7.10 (m, 1H), 6.98~7.02 (m, 1H), 6.42 (d, 1H), 5.83 (s, 1H), 3.74~3.94 (m, 2H); 13C NMR (DMSO-d6, 100 MHz) δ 169.66, 161.12 (2C), 154.13, 153.94, 151.53, 140.69, 139.46, 139.38, 136.25, 135.05, 131.57, 127.23, 120.52, 119.28, 115.29, 112.52, 112.34, 104.72, 56.43, 29.59; HR-MS (ESI) m/z: 477.084 7 [M+H]+

N1-{3--4-[(6-氧代-1, 6-二氢吡啶-3-)氧基]苯基}-N3-[4-氧代-2-(2-氯苯基)噻唑啉-3-](g10)   白色固体, 收率42.9%。1H NMR (DMSO-d6, 400 MHz) δ: 11.34 (br, 1H), 9.09 (s, 1H), 8.78 (s, 1H), 7.64~7.66 (m, 1H), 7.49~7.53 (m, 2H), 7.33~7.46 (m, 4H), 7.08~7.11 (m, 1H), 7.00 (m, 1H), 6.42 (d, 1H), 6.14 (s, 1H), 3.77~3.90 (m, 2H); 13C NMR (DMSO-d6, 100 MHz) δ 169.67, 161.12 (2C), 154.05, 153.95, 151.53, 140.68, 139.33, 139.31, 136.38, 136.27, 135.01, 132.23, 131.09, 127.23, 120.43, 119.25, 116.32, 116.07, 56.42, 29.58; HR-MS (ESI) m/z: 475.065 6 [M+H]+

N1-{3--4-[(6-氧代-1, 6-二氢吡啶-3-)氧基]苯基}-N3-[4-氧代-2-(4-氯苯基)噻唑啉-3-](g11)   白色固体, 收率45.5%。1H NMR (DMSO-d6, 400 MHz) δ: 11.33 (br, 1H), 9.01 (br, 1H), 8.61 (s, 1H), 7.45~7.54 (m, 5H), 7.32~7.38 (m, 2H), 7.04~7.09 (m, 2H), 6.97~7.02 (m, 1H), 6.40 (d, 1H), 5.82 (s, 1H), 3.75~3.90 (m, 2H); 13C NMR (DMSO-d6, 100 MHz) δ 169.67, 161.11 (2C), 154.07, 153.95, 151.54, 140.68, 139.34, 139.31, 136.38, 136.27, 135.02, 132.23, 131.10, 127.25, 120.43, 119.25, 116.34, 116.07, 56.42, 29.59; HR-MS (ESI) m/z: 475.064 9 [M+H]+

N1-{3--4-[(6-氧代-1, 6-二氢吡啶-3-)氧基]苯基}-N3-[4-氧代-2-(3, 4, 5-三氟苯基)噻唑啉-3-](g12)   淡黄色固体, 收率44.8%。1H NMR (DMSO-d6, 400 MHz) δ: 11.40 (br, 1H), 9.11 (br, 1H), 8.70 (s, 1H), 7.70 (s, 1H), 7.54~7.57 (m, 2H), 7.32~7.35 (m, 2H), 7.25~7.28 (m, 1H), 6.94 (d, 1H), 6.41 (d, 1H), 5.83 (s, 1H), 3.73~3.97 (m, 2H)。13C NMR (DMSO-d6, 100 MHz) δ 169.85, 161.13(2C), 154.09, 151.93, 149.15, 140.13, 136.44, 135.40, 131.19, 127.56, 123.50, 119.87, 119.44, 119.13, 113.15, 112.86, 112.69, 112.65, 61.34, 29.55; HR-MS (ESI) m/z: 511.043 9 [M+H]+

N1-{3--4-[(6-氧代-1, 6-二氢吡啶-3-)氧基]苯基}-N3-[4-氧代-2-(3, 4, 5-三氟苯基)噻唑啉-3-](g13)   淡黄色固体, 收率48.9%。1H NMR (DMSO-d6, 400 MHz) δ: 9.23 (br, 1H), 8.83 (s, 1H), 7.85 (m, 1H), 7.53~7.57 (m, 2H), 7.29~7.34 (m, 3H), 6.93 (d, 1H), 6.41~6.43 (m, 1H), 5.83 (s, 1H), 3.72~3.96 (m, 2H)。13C NMR (DMSO-d6, 100 MHz) δ 169.83, 161.14 (2C), 154.13, 151.91, 149.18, 140.11, 136.41, 135.40, 131.16, 127.58, 123.51, 119.82, 119.41, 119.17, 113.12, 112.90, 112.71, 112.65, 61.33, 29.55; HR-MS (ESI) m/z: 554.997 1 [M+H]+

2 体外抗肿瘤活性实验[20]

所用细胞株分别为人非小细胞肺癌A549、人结直肠癌细胞HT-29和人乳腺癌细胞MDA-MB-231, 阳性对照药为cabozantinib。采用MTT细胞毒测试法对目标化合物进行体外抗肿瘤活性测试。将处理好的肿瘤细胞加入至96孔板中, 每孔100 μL, 细胞数量为每孔1×104个, 置于37 ℃、5% CO2温箱培养24 h使细胞贴壁。将配制好的药液分别加入96孔板中, 每孔加170 μL, 每浓度加3个孔, 置于37 ℃、5% CO2培养箱中培养72 h。将0.5% MTT按照每孔100 μL加入96孔板, 置于37 ℃、5% CO2培养箱中培养4 h。吸出96孔板中液体, 每孔加入100 μL DMSO, 置于磁力震荡器上震荡3 min, 使结晶物充分溶解, 在酶标仪上用双波长法(490 nm和630 nm)测量各孔的吸光度值。根据吸光度用Bliss法计算出每个药物的半数抑制浓度IC50

3 激酶活性测试

采用迁移率检测实验评价化合物g2对c-Met、KDR、c-Kit、c-Src、ALK和HER-2等6种激酶的抑制活性。将激酶加入至96孔板中, 每孔50 μL。用100% DMSO将化合物配成500 μmol·L-1的溶液, 另取一96孔板, 每孔加入10 μL上述溶液和90 μL的激酶缓冲液(50 mmol·L-1 HEPES、10 mmol·L-1 MgCl2、0.001 5% Brij-35和2 mmol·L-1 DTT)。每孔取5 μL加入至384孔板中, 再加入FAM-标记肽、ATP和激酶溶液等, 室温培养10 min, 每孔加入10 μL上述肽溶液, 28 ℃培养一段时间后加入25 μL终止缓冲液(100 mmol·L-1 HEPES、0.015% Brij-35和50 mmol·L-1 EDTA)终止反应。测量转化值, 再将其转换成抑制率, 拟合抑制率曲线, 得出IC50值。

4 实时动态活细胞成像实验[18]

将处理好的HT-29细胞加入至96孔板中, 每孔加入100 μL含10% FBS的DMEM, 细胞数为每孔5×103个, 置于37 ℃、5%CO2培养箱中, 隔天换液, 传2代后铺板, 细胞数量为每孔5×103个, 细胞贴壁后加入化合物g2和阳性药(共3个浓度, 起始浓度为10.0 μg·mL-1, 3倍稀释, 每个浓度设置3个平行实验, 同时设置空白孔), 按照实时动态活细胞成像仪的标准操作方法进行实验。采用绿光观察药物对细胞的杀伤作用, 白光观察细胞的增殖情况, 实验时间为72 h, 拍照频率为每小时1张, 每次每孔自动拍照3张, 10倍镜。

5 Annexin V-FITC/PI双染色法检测细胞凋亡[21]

将每孔接种1×105个HT-29细胞于48孔板中(设阴性对照组、阳性对照组和5个浓度的实验组, 均设置3个平行实验), 37 ℃、5% CO2培养箱中, 细胞贴壁后, 相应孔中加入阳性药及不同浓度的化合物g2。作用72 h后的细胞, 以4 ℃ PBS洗涤2次, 加入5 μL Annexin V-FITC后避光孵育30 min, 上机前加入10 μL PI孵育15 min, FACS进行检测并分析数据。

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