药学学报  2014, Vol. 49 Issue (12): 1694-1698   PDF    
C-3噁二唑硫乙酰腙取代的氟喹诺酮类似物的合成、抗肿瘤活性及构-效关系
高留州1, 谢玉锁1, 李涛1, 黄文龙2, 胡国强1     
1. 河南大学化学生物学研究所, 河南 开封 475001;
2. 中国药科大学新药研究中心, 江苏 南京 210009
摘要:为寻找氟喹诺酮由抗菌活性转化为抗肿瘤活性的有效策略, 基于药效团拼合原理, 用噁二唑替代培氟沙星C-3位羧基, 功能酰腙基为其修饰基团, 设计合成了C-3噁二唑硫乙酰腙目标化合物7a7o, 目标化合物结构经元素分析、1H NMR、MS确证.采用MTT法评价了目标化合物对人肝癌Hep-3B细胞株的体外增殖抑制活性.结果表明, 15个目标化合物活性均显著高于对照培氟沙星的活性, 其中带吸电子取代基化合物的活性高于供电子基团化合物的活性, 尤其是含羧基取代基化合物的活性与对照抗肿瘤药物阿霉素相当, 表明芳环羧基修饰基的存在有利于提高抗肿瘤活性.
关键词氟喹诺酮     噁二唑     酰腙     抗肿瘤活性     构-效关系    
Synthesis, antitumor activity and SAR of C-3 oxadiazole sulfanylacetylhydrazone-substituted fluoroquinolone analogues
GAO Liu-zhou1, XIE Yu-suo1, LI Tao1, HUANG Wen-long2, HU Guo-qiang1     
1. Institute of Chemistry and Biology, Henan University, Kaifeng 475001, China;
2. Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, China
Abstract: To explore an efficient strategy for the conversion of antibacterial fluoroquinolones into antitumor fluoroquinolones, an azole heterocyclic ring of oxadiazole instead of the C-3 carboxylic acid group with a functionalized hydrazone group as a modified side-chain, fifteen novel 2-(fluoroquinolon-3-yl)-oxadiazole-5-sulfanylacetylhydrazone derivatives 7a-7o were designed and synthesized on the basis of the pharmacophore hybridization principle from pefloxacin, separately. The structures for fifteen title compounds were characterized by elemental analysis, 1H NMR and MS, and their in vitro antitumor activity against Hep-3B cell line was evaluated by a MTT assay. The results showed that the title compounds exhibited more significantly inhibitory activity than that of the parent pefloxacin, in which compounds with electron-withdrawing group attached on aryl ring had more potency than that of compounds with electron donating group, especially compounds with a carboxylic substituent were comparable to comparison doxorubicin. It suggests that it is favorable for an improvement of antitumor activity to remain a carboxylic acid unit at the aromatic ring.
Key words: fluoroquinolone     oxadiazole     acylhydrazone     antitumor activity     SAR    

新药的研发起源于先导物的发现,基于机制的药物设计是发现先导物的有效途径,而对先导化合物的优化是促进其向成药性发展的关键环节[1]。拓扑异构酶不但是抗菌氟喹诺酮的作用靶点[2],同时也是抗肿瘤药物的重要靶点[3]。基于此,转化氟喹诺酮的抗菌活性到抗肿瘤活性备受关注[4]。近期研究发现,氟喹诺酮C-3羧基虽是抗菌活性必需的药效团,但并非是抗肿瘤活性所必要的,可被其等排体如酰胺、酰腙、杂环或稠杂环等替代[5, 6, 7],扩展了设计发现抗肿瘤氟喹诺酮先导物的新思路。为继续抗肿瘤氟喹诺酮课题的研究,一方面考虑到1,3,4-噁二唑类杂环化合物因具有抗肿瘤[8]、抗病毒[9]、抗菌[10]、抗炎[11]等多种生物活性已成为许多药物设计中的常用的药效团之一; 另一方面,酰腙结构中有N、O等供电子原子易与配体形成氢键,可提高与靶点结合的亲和力,而且酰腙的活泼亚胺 (CH=N) 双键能与大分子配体中的亲核基团如巯基和氨基发生亲核加成反应产生细胞毒作用,这可进一步提高其抗肿瘤活性[12]。另更重要的是,噁二唑杂环和酰腙均可作为氟喹诺酮C-3羧基的等排体,但在一个分子中同时存在对抗肿瘤活性有何影响仍值得探索。为此,基于药物设计的生物电子等排及药效团拼合原理,用噁二唑作为培氟沙星1的C-3羧基的等排体,酰腙为其修饰基,并以硫醚链相连接,设计合成了C-3噁二唑硫乙酰腙类目标化合物7a7o,进而实现了药效团的叠加,希望活性得到进一步提高。

培氟沙星1直接肼解可得其相应的酰肼2,然后与二硫化碳缩环合得氟喹诺酮C-3噁二唑硫醇4。硫醇4与氯乙酸乙酯用熔融的乙酸钠作缚酸剂在乙醇中可高收率制得酯化物5,接着在乙醇中进行肼解得噁二唑硫乙酰肼65在肼中反应可引起噁二唑的开环和脱硫反应,得不到预期的产物6。中间体6与各种芳香醛易发生缩合反应到目标物7。目标化合物的合成见合成路线1。

R: H (7a); 4-CH3O (7b); 2-CH3O (7c); 2-OH (7d); 3,4-(OCH2O) (7e); 3-OCH3-4-OH (7f); 3,4,5-(OCH3)3 (7g); 4-CH3 (7h); 4-Cl (7i); 4-F (7j); 4-O2N (7k); 3-O2N (7l); 4-HOOC (7m); 2-HOOC (7n); 4-NC (7o) Scheme 1 Synthetic route of title compounds 7a-7o from pefloxacin
结果与讨论 1 化学部分

目标化合物的结构经 1H NMR、MS及元素分析确证,产物的收率、物理常数及波谱数据见表 12。目标化合物7a7o1H NMR在低场δ 11.40处出现的单质子化学位移单峰可归属为侧链酰胺的N-H质子,δ 9.00~8.50处的2个单质子单峰化学位移可分别归属为喹啉骨架C2-H和侧链酰腙的亚胺CH=N质子,羧基质子化学位移出现在低场δ 13.00处,喹啉骨架的C5-H和C8-H质子及苯环质子的化学位移出现在正常范围内。另外,元素分析结果和MS也进一步表明目标化合物与设计的预期结构相一致。

Table 1 Physical constants and spectral data of target compounds (7a-7o)

Table 2 Spectral data of the target compounds 7a-7o
2 抗肿瘤活性

对合成的15个噁二唑硫乙酰腙目标化合物7a7o及对照1 (pefloxacin,PF) 和抗肿瘤药物阿霉素(doxorubicin,DOX) 进行抗人肝癌Hep-3B细胞株活性筛选,各供试化合物对实验肿瘤细胞的半数抑制浓度 (IC50) 见表 3

Table 3 Growth inhibitory activities of title compounds against Hep-3B tumor cell line. n = 3,± s

15个目标物的IC50值均在40.0 μmol·L-1以下 (表 3),低于母体培氟沙星 (IC50 > 100 μmol·L-1) 的值。构-效关系表明,当修饰基酰腙相连的苯环带有游离的酚羟基如化合物7d7f或较小体积的F原子如化合物7j时 ,其抗肿瘤活性显著增强; 更有意义的是,当苯环带有吸电子取代基如羧基、硝基、氰基时,其抗肿瘤活性高于供电子基化合物的活性,尤其是带羧基的化合物如7m7n,其活性与抗肿瘤对照药阿霉素相当,具有发展为先导物的前景。

实验部分

熔点用WK-1B数字熔点仪 (上海精密科学仪器厂),毛细管法,温度未校正; AM-400型核磁共振仪 (德国Bruker公司),DMSO-d6为溶剂,TMS为内标; Esquire LC型质谱仪 (德国Bruker公司); 2400-Ⅱ元素分析仪 (美国PE公司)。所用培氟沙星1为市售商品,C-3噁二唑硫醇中间体4参照文献[13]的方法制备,其他试剂均为分析纯。

1 化学合成 1.1 中间体酯化物5和酰肼物6的合成

中间体4(10.0 g,26.0 mmol) 和熔融的无水乙酸钠 (3.2 g,39.0 mmol) 加入到无水乙醇 (200 mL) 中,搅拌滴加氯乙酸乙酯 (4.9 g,39.0 mmol),回流反应24 h。滤除产生的氯化钠固体,滤集析出的固体,用无水乙醇重结晶,干燥,得无色C-3噁二唑硫乙酸乙酯固体5,收率87.0%,mp 172~174 ℃; MS (m/z): 476 [M+H]+; 计算值 (C22H26FN5O4S): 475.55。

中间体5 (10.0 g,21.0 mmol) 悬浮于无水乙醇(100 mL) 中,加入80% 水合肼(2.0 g,32.0 mmol)。反应混合物搅拌回流6 h,放置过夜。滤集产生的固体,无水乙醇重结晶,干燥,得C-3噁二唑硫乙酰肼6,收率84%,mp 178~180 ℃; 元素分析 (C20H24FN7O3S 计算值)/%: C 52.18 (52.05),H 5.02 (5.24),N 21.38 (21.24)。1H NMR (DMSO-d6) δ: 11.33 (s,1H,CONH),8.66 (s,1H,2-H),7.83 (d,J = 13.2 Hz,1H,5-H),7.05 (d,J = 6.8 Hz,1H,8-H),4.57 (s,2H,NH2),4.46 (q,J = 7.0 Hz,2H,CH2),4.17 (s,2H,SCH2),3.43~3.04 (m,8H,piperazine-H),2.33 (s,3H,N-CH3),1.38 (t,J = 7.0 Hz,3H,CH3); MS (m/z): 462 [M+H]+; 计算值: 461.52。

1.2 目标物1-乙基-6--7-(4-甲基哌嗪-1-)-3-[5-(芳甲叉肼甲酰基) 甲硫基-[1,3,4]-噁二唑-2-]-4 (1H)-喹啉酮 (7a~7o) 的合成

中间体6 (0.5 g,1.1 mmol)与等物质量的取代苯甲醛(1.1 mmol) 在无水乙醇 (10 mL) 中回流反应10 h,放置冰箱中冷冻过夜。滤集析出的固体,用无水乙醇洗涤,干燥,用乙醇-DMF重结晶制得各自的目标化合物7a7o

2 抗肿瘤活性筛选

合成的15个噁二唑硫乙酰腙目标化合物7a7o及对照1 (pefloxacin,PF) 和抗肿瘤药物阿霉素 用DMSO配成1.0×10-2 μmol∙L-1浓度的储备液,用RPMI-1640稀释到所需浓度。取对数生长期的人肝 癌Hep-3B细胞株,以每孔7 000个细胞的密度接种 于96孔板,随后加入不同浓度的上述化合物。48 h后每孔加入5 g·L-1噻唑蓝 (MTT) 溶液10 μL,继 续培养4 h后加入10% 十二烷基磺酸钠 (SDS) 溶液100 μL培养过夜,用酶标仪在570 nm处测其吸收度。按公式计算各组对肿瘤细胞的抑制率: 细胞抑制率 (%) = (1 - 实验组吸收度 / 对照组吸收度) × 100。然后以各药物浓度对数值对各浓度下的抑制率作线性回归得剂量-效应方程,依此计算出各供试化合物对实验肿瘤细胞的半数抑制浓度 (IC50)。

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