WO2017198178A1 - 噻唑类衍生物及其应用 - Google Patents
噻唑类衍生物及其应用 Download PDFInfo
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- WO2017198178A1 WO2017198178A1 PCT/CN2017/084748 CN2017084748W WO2017198178A1 WO 2017198178 A1 WO2017198178 A1 WO 2017198178A1 CN 2017084748 W CN2017084748 W CN 2017084748W WO 2017198178 A1 WO2017198178 A1 WO 2017198178A1
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- DYNFCHNNOHNJFG-UHFFFAOYSA-N OC(c1c(C=O)cccc1)=O Chemical compound OC(c1c(C=O)cccc1)=O DYNFCHNNOHNJFG-UHFFFAOYSA-N 0.000 description 3
- SRABRRCKNYGQTH-UHFFFAOYSA-N NNc1nc(-c(cccc2)c2Cl)c[s]1 Chemical compound NNc1nc(-c(cccc2)c2Cl)c[s]1 SRABRRCKNYGQTH-UHFFFAOYSA-N 0.000 description 2
- GGRHYQCXXYLUTL-UHFFFAOYSA-N CC(C)(C)C(OCCl)=O Chemical compound CC(C)(C)C(OCCl)=O GGRHYQCXXYLUTL-UHFFFAOYSA-N 0.000 description 1
- MPZFSXLSFDEQFZ-LGJNPRDNSA-N CC(C)(C)C(OCOC(c1c(/C=N/N(C)c2nc(-c3ccccc3Cl)c[s]2)cccc1)=O)=O Chemical compound CC(C)(C)C(OCOC(c1c(/C=N/N(C)c2nc(-c3ccccc3Cl)c[s]2)cccc1)=O)=O MPZFSXLSFDEQFZ-LGJNPRDNSA-N 0.000 description 1
- WHHBCWXWNIPTKD-UHFFFAOYSA-N CC(C)(C)C(OCOC(c1c(C=O)cccc1)=O)=O Chemical compound CC(C)(C)C(OCOC(c1c(C=O)cccc1)=O)=O WHHBCWXWNIPTKD-UHFFFAOYSA-N 0.000 description 1
- RTBHSDAJZHRRQS-UHFFFAOYSA-N CC(C)=NNc1nc(-c(cccc2)c2Cl)c[s]1 Chemical compound CC(C)=NNc1nc(-c(cccc2)c2Cl)c[s]1 RTBHSDAJZHRRQS-UHFFFAOYSA-N 0.000 description 1
- JHYNXXBAHWPABC-UHFFFAOYSA-N CC(C)OC(OCCl)=O Chemical compound CC(C)OC(OCCl)=O JHYNXXBAHWPABC-UHFFFAOYSA-N 0.000 description 1
- PFVUFAZRLARQAG-BRJLIKDPSA-N CC(C)OC(OCOC(c1c(/C=N/N(C)c2nc(-c3ccccc3Cl)c[s]2)cccc1)=O)=O Chemical compound CC(C)OC(OCOC(c1c(/C=N/N(C)c2nc(-c3ccccc3Cl)c[s]2)cccc1)=O)=O PFVUFAZRLARQAG-BRJLIKDPSA-N 0.000 description 1
- WNTATZHUJGFYGI-UHFFFAOYSA-N CC(C)OC(OCOC(c1ccccc1C=O)=O)=O Chemical compound CC(C)OC(OCOC(c1ccccc1C=O)=O)=O WNTATZHUJGFYGI-UHFFFAOYSA-N 0.000 description 1
- PKAZHLRCUZGGQC-WUXMJOGZSA-N CN(C(N)=S)/N=C/c1ccccc1C(O)=O Chemical compound CN(C(N)=S)/N=C/c1ccccc1C(O)=O PKAZHLRCUZGGQC-WUXMJOGZSA-N 0.000 description 1
- KDZVAMPHKPTTNU-WSDLNYQXSA-N CN(C)C(c1c(/C=N/N(C)c2nc(-c3ccccc3Cl)c[s]2)cccc1)=O Chemical compound CN(C)C(c1c(/C=N/N(C)c2nc(-c3ccccc3Cl)c[s]2)cccc1)=O KDZVAMPHKPTTNU-WSDLNYQXSA-N 0.000 description 1
- 0 C[C@@](C*1)[C@]1N Chemical compound C[C@@](C*1)[C@]1N 0.000 description 1
- NDCYSNHDARLKEU-UFFVCSGVSA-N Cc(cc1)cc(/C=N/N(C)c2nc(-c3ccccc3Cl)c[s]2)c1C(O)=O Chemical compound Cc(cc1)cc(/C=N/N(C)c2nc(-c3ccccc3Cl)c[s]2)c1C(O)=O NDCYSNHDARLKEU-UFFVCSGVSA-N 0.000 description 1
- WZWWEVCLPKAQTA-UHFFFAOYSA-N O=C(CBr)c1ccccc1Cl Chemical compound O=C(CBr)c1ccccc1Cl WZWWEVCLPKAQTA-UHFFFAOYSA-N 0.000 description 1
- WYXQIAWWNXCWQY-AFUMVMLFSA-N O=C(c1c(/C=N/Nc2nc(-c3ccccc3Cl)c[s]2)cccc1)OC1CCCCC1 Chemical compound O=C(c1c(/C=N/Nc2nc(-c3ccccc3Cl)c[s]2)cccc1)OC1CCCCC1 WYXQIAWWNXCWQY-AFUMVMLFSA-N 0.000 description 1
- QPOPOGXCFMHHLZ-UHFFFAOYSA-N O=Cc(cccc1)c1C(Cl)=O Chemical compound O=Cc(cccc1)c1C(Cl)=O QPOPOGXCFMHHLZ-UHFFFAOYSA-N 0.000 description 1
- HQTAJLYIUKDSMX-UHFFFAOYSA-N O=Cc1ccccc1C(OC1CCCCC1)=O Chemical compound O=Cc1ccccc1C(OC1CCCCC1)=O HQTAJLYIUKDSMX-UHFFFAOYSA-N 0.000 description 1
- OEWWFQYTNGODQM-UHFFFAOYSA-N O=Cc1ccccc1C(OCc1ccccc1)=O Chemical compound O=Cc1ccccc1C(OCc1ccccc1)=O OEWWFQYTNGODQM-UHFFFAOYSA-N 0.000 description 1
- WVDDGKGOMKODPV-UHFFFAOYSA-N OCc1ccccc1 Chemical compound OCc1ccccc1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 1
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- R 1 is selected from the group consisting of: C 1 -C 6 alkyl (preferably C 1 -C 3 alkyl);
- R 3 is selected from: H, substituted or unsubstituted C 1 -C 6 alkyl (preferably C 1 -C 3 alkyl)
- R 5 and R 6 are independently selected from: H, halogen (preferably F), unsubstituted or halogen (preferably F) substituted C1-C3 alkyl;
- R 7 and R 8 are independently selected from: H, halogen (preferably) Cl); and
- R 9 is H; or,
- R 1 is selected from: H, C1-C6 alkyl, preferably C1-C3 alkyl;
- R 3 is selected from: H, substituted or unsubstituted C1-C6 alkyl (preferred C1-C3 alkyl);
- R 5 is selected from the group consisting of: H, halogen (preferably F), unsubstituted or halogen (preferably F
- the present invention further provides a pharmaceutical composition
- a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier or excipient .
- Methods of administration include, but are not limited to, various methods of administration well known in the art, which can be determined based on the actual circumstances of the patient. These methods include, but are not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, nasal or topical routes of administration.
- the principle of enzyme level activity test is: first, dihydroorotate (DHO) is oxidatively dehydrogenated by DHODH to form orotic acid (Orotate, OA), accompanied by Flavin mononucleotide (FMN). 2H + and 2e - reduced to reduced flavin mononucleotide (FMNH 2 ); then coenzyme Q (C O Q) as a hydrogen acceptor accepts electron and proton reduction of FMNH 2 to reduced coenzyme Q (C O QH 2 ), The reduced coenzyme Q transfers electrons to the chromogenic substrate, diclofenac sodium salt (DCIP), and the final DCIP is reduced.
- DHO dihydroorotate
- Orotate OA
- FMN Flavin mononucleotide
- C O Q coenzyme Q
- C O Q coenzyme Q
- DCIP diclofenac sodium salt
- Figure 2C shows a representative photograph of a rat foot in each of the different treatment groups on day 28 of the experiment. It can be seen from Fig. 2C that the paw swelling of the model group rats is the most serious, and the degree of swelling of the foot after the administration of the positive drug and the two different concentrations of the compound 1 is improved, and the high dose of the compound 1 is improved. The dose is more pronounced.
- results of tissue sectioning also showed that the synovial hyperplasia and inflammatory cell infiltration in rats treated with Compound 1 were greatly improved compared with the model group.
- Compound 1 has an anti-inflammatory effect and is a potential compound for RA treatment with a dose-dependent relationship.
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Abstract
一种作为DHODH抑制剂的噻唑类衍生物及其应用。具体而言,涉及下式I所示的化合物、含有式(I)化合物的药物组合物及所述化合物在制备治疗DHODH介导的疾病或抑制DHODH的药物中的用途。
Description
本发明涉及药物化学领域;具体地说,本发明涉及新型的噻唑类衍生物其合成方法及其作为二氢乳清酸脱氢酶(Dihydroorotate dehydrogenase,DHODH)抑制剂在治疗DHODH介导的疾病中的应用。
二氢乳清酸脱氢酶(Dihydroorotate dehydrogenase,DHODH)是一种含铁的黄素依赖线粒体酶,是核酸中嘧啶合成的关键酶,催化嘧啶从头生物合成途径中的第4步限速反应。人体内获得嘧啶核苷酸的方式有两种,从头合成和补救合成。人体内静息的淋巴细胞主要通过补救合成途径获取细胞代谢所需的嘧啶核苷酸,然而在免疫激活状态下,淋巴细胞对嘧啶核苷酸的需求可达到静息状态下的8倍以上,此时淋巴细胞的增殖和各种免疫功能的完成都需要启动从头合成途径来补充嘧啶核苷酸。因此,通过抑制DHODH,也就抑制了嘧啶从头合成途径,阻断新生嘧啶合成,致使DNA合成障碍,从而抑制快速增殖的人类细胞,如激活的T-淋巴细胞、B-淋巴细胞以及肿瘤细胞。因而,二氢乳清酸脱氢酶(DHODH)抑制剂可被成功开发为抗类风湿关节炎、抗肿瘤、抗器官移植排异、抗牛皮癣等多种自身免疫性疾病的药物。
来自不同生物体的DHODH主要有两大类:I型和II型。I型DHODH存在于原核生物体内,且位于细胞内,这类酶利用一些水溶性分子(如延胡索酸和NAD+)作为氧化还原过程中的电子传递体;II型DHODH主要位于哺乳动物和一些原生动物体内的线粒体内膜上,这类酶利用黄素单核苷酸(Flavin Mononucleotide,FMN)和辅酶Q(Coenzyme,CoQ,也称作ubiquinone)作为电子转移体。与I型DHODH显著不同的是,II型DHODH有一个高度可变的N末端延伸协助酶和线粒体膜的连接,N端形成独立的双α螺旋结构,为醌类提供了结合位点。人类DHODH(hDHODH)和恶性疟原虫的DHODH(Pf DHODH)都属于II型的线粒体相关酶。
动力学研究表明,DHO(二氢乳清酸)到OA(乳清酸)的转化是一个连续的“ping-pong”机制。该过程主要通过两个半反应完成:首先二氢乳清酸在二氢乳清酸脱氢酶的催化下被氧化为乳清酸,同时FMN接受该过程脱下的氢原子被氧化为FMNH2,这个半反应是催化反应的限速步骤;之后在CoQ的作用下,FMNH2发生脱氢作用重新还原为FMN,回到循环当中,如以下反应式所示。
DHODH抑制剂可有效减少免疫激活的T-淋巴细胞和B-淋巴细胞的活性和数量,可被成功开发为肿瘤、类风湿性关节炎等自身免疫性疾病的治疗药物。目前,已上市的典型的DHODH抑制剂主要有来氟米特(leflunomide)、特立氟胺(teriflunomide,A771726)及布奎那(brequinar),它们的结构式如下所示。
1998年上市的来氟米特是DHODH的有效抑制剂,通过阻断嘧啶的从头合成途径和抑制酪氨酸激酶的活性从而阻断细胞信号传导过程,是新型的具有抗炎及免疫抑制作用的异恶唑类衍生物。临床上己用于治疗类风湿性关节炎、红斑狼疮、多种原发性和继发性肾小球疾病,防治移植物排异反应。然而由于来氟米特较长的半衰期导致其长期使用会产生较大的毒副作用,如腹泻、肝酶异常,皮疹等。特立氟胺是来氟米特的有效代谢物。而布奎那也已进入II期临床试验阶段,但受限于其治疗窗口窄,并且布奎那的临床试验研究结果发现,布奎那联合环孢霉素A或顺铂口服给药时,可引起粘膜炎、白血球减少及血小板减少。
因此,本领域急需寻找新型的、更高效、更安全以及成药性更好的DHODH抑制剂。
发明内容
本发明的目的在于提供一种结构全新、DHODH抑制活性高、毒性低、安全性好的化合物。
在第一方面,本发明提供式I所示化合物或其药学上可接受的盐或酯:
式中,
R1选自:H、取代或未取代的C1-C6烷基、C3-C6环烷基;
R2选自:H、卤素、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷氧基、CN、NO2、羟基、NRaRb;
Ra、Rb可独立选自H或C1-C6烷基
R3选自:H、取代或未取代的C1-C6烷基;
R4选自:H、卤素;
m为0-4的整数;
n为0~5的整数;
其中,当R2为H且R3为H时,R1不为H。
在具体的实施方式中,所述化合物如式II所示:
式中,
R5和R6独立选自:H、卤素、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷氧基、羟基、NH2;
R7、R8和R9独立选自:H、卤素;
R1和R3如上所述。
在具体的实施方式中,R1选自:C1-C6烷基(优选C1-C3烷基);R3选自:H、取代或未取代的C1-C6烷基(优选C1-C3烷基);R5和R6独立选自:H、卤素(优选F)、未取代的或卤素(优选F)取代的C1-C3烷基;R7和R8独立选自:H、卤素(优选Cl);R9是H。
在具体的实施方式中,R1选自:H、C1-C6烷基,优选C1-C3烷基;R3选自:H、取代或未取代的C1-C6烷基(优选C1-C3烷基);R5选自:H、卤素(优选F)、未取代的或卤素(优选F)取代的C1-C3烷基;R6选自:H、卤素(优选F)、羟基、NH2;R7和R8独立选自:H、卤素(优选Cl);和R9是H。
在第二方面,本发明提供选自下组的化合物或其药学上可接受的盐或酯:
在具体的实施方式中,本发明提供选自下组的化合物或其药学上可接受的盐或酯:
在第三方面,本发明提供一种药物组合物,所述药物组合物含有本发明第一或第二方面所述的化合物或其药学上可接受的盐或酯,以及药学上可接受的载体或赋形剂。
在优选的实施方式中,所述药物组合物是适于口服的剂型,包括但不限于片剂、溶液剂、混悬液、胶囊剂、颗粒剂、粉剂。
在第四方面,本发明提供本发明第一或第二方面所述的化合物或其药学上可接受的盐或酯的用途,用于制备治疗DHODH介导的疾病的药物。
在具体的实施方式中,所述DHODH介导的疾病包括但不限于:自身免疫疾病、细菌感染、寄生虫和肿瘤。
在具体的实施方式中,所述自身免疫性疾病包括但不限于:类风湿性关节炎、
器官移植排异、银屑病。
在第五方面,本发明提供一种治疗DHODH介导的疾病的方法,所述方法包括给予需要的对象本发明第一或第二方面所述的化合物或其药学上可接受的盐或酯,或本发明第三方面所述的药物组合物。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
图1是Wistar大鼠类风湿性关节炎实验进度安排图(对应化合物1)。
图2是化合物1对CIA模型大鼠体重及关节肿胀程度的影响,其中,A图表示大鼠的体重变化趋势,每三天称量一次,B图表示大鼠关节肿胀评分的变化趋势,每两天进行一次评分,C图表示不同实验组中具有代表性的大鼠足部照片,数据表示为平均值±SEM,##P<0.01,与对照相比;*P<0.05,**P<0.01,与模型相比。
图3是化合物1对CIA模型大鼠组织病理学的影响,为表示关节组织切片HE染色后具有代表性的照片,其中,S,滑膜;C,软骨;Bn,骨,放大倍数为40×。
发明人经过广泛而深入的研究,出乎意料地发现了一系列噻唑类衍生物,它们的结构骨架完全不同于文献报道过的高活性DHODH抑制剂(例如来氟米特、特立氟胺、布奎那)。并且这些化合物在分子、细胞及动物水平测试中显示出显著的DHODH抑制活性,同时毒性较低、安全性较好,具有良好的成药前景。在此基础上完成了本发明。
术语定义
本文中涉及到的一些基团定义如下:
本文中,“烷基”是指碳链长度为1-10个碳原子的饱和的支链或直链烷基,优选的烷基包括长2-8个碳原子、1-6个、1-4个碳原子、1-3个碳原子不等的烷基。烷基的例子包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、庚基等。烷基可以被1个或多个取代基取代,例如被卤素或卤代烷基取代。例如,烷基可以是被1-4个氟原子取代的烷基,或者烷基可以是被氟代烷基取代的烷基。
本文中,“环烷基”是指含有脂环结构的饱和烷基,例如,C3-C6环烷基。在具体的实施方式中,所述环烷基包括但不限于:环丙基、环丁基、环戊基或环己基。本文所述的环烷基可以是取代或未取代的,包括但不限于被一个或多个卤素原子,例如氟原子取代。
本文中,“氨基”是指结构式为“NRxRy”的基团,其中,Rx和Ry可独立选自H或C1-C3烷基或C1-C3卤代烷基。在具体的实施方式中,本文所述的“氨基”是指NH2。
在本文中,“卤素”是指氟、氯、溴和碘。在优选的实施方式中,卤素是氯或氟;更优选氟。
本发明的化合物
本发明人出乎意料地发现了一系列结构骨架全新的噻唑类衍生物,这些化合物在分子、细胞及动物水平测试中显示出显著的DHODH抑制活性,同时毒性较低、安全性较好,具有良好的成药前景。
在具体的实施方式中,本发明的化合物如以下结构通式所示:
式中,
R1选自:H、取代或未取代的C1-C6烷基、C3-C6环烷基;
R2选自:H、卤素、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷氧基、CN、NO2、羟基、NRaRb;
Ra、Rb可独立选自H或C1-C6烷基
R3选自:H、取代或未取代的C1-C6烷基;
R4选自:H、卤素;
m为0-4的整数;
n为0~5的整数;
其中,当R2为H且R3为H时,R1不为H。
在优选的实施方式中,本发明的化合物如式II所示:
式中,
R5和R6独立选自:H、卤素、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷氧基、羟基、NH2;
R7、R8和R9独立选自:H、卤素;
R1和R3如上所述。
在进一步的实施方式中,R1选自:C1-C6烷基(优选C1-C3烷基);R3选自:H、取代或未取代的C1-C6烷基(优选C1-C3烷基);R5和R6独立选自:H、卤素(优选F)、未取代的或卤素(优选F)取代的C1-C3烷基;R7和R8独立选自:H、卤素(优选Cl);和R9是H;或者,R1选自:H、C1-C6烷基,优选C1-C3烷基;R3选自:H、取代或未取代的C1-C6烷基(优选C1-C3烷基);R5选自:H、卤素(优选F)、未取代的或卤素(优选F)取代的C1-C3烷基;R6选自:H、卤素(优选F)、羟基、NH2;R7和R8独立选自:H、卤素(优选Cl);和R9是H。
在上述化合物的基础上,本发明进一步提供一种药物组合物,该组合物含有治疗有效量的本发明化合物或其药学上可接受的盐或酯,以及药学上可接受的载体或赋形剂。
本发明化合物的药学上可接受的盐的例子包括但不限于无机和有机酸盐,例如盐酸盐、氢溴酸盐、硫酸盐、柠檬酸盐、乳酸盐、酒石酸盐、马来酸盐、富马酸盐、扁桃酸盐和草酸盐;以及与碱例如钠羟基、三(羟基甲基)胺基甲烷(TRIS,胺丁三醇)和N-甲基葡糖胺形成的无机和有机碱盐。
本发明化合物的药学上可接受的酯为式I化合物的羧基被酯化的化合物,包括但不限于:式I化合物的取代或未取代的C1-C10烷基或环烷基的酯、基团的酯、基团的酯,其中,所述取代指被选自下组的基团取代:苯基、卤素。代表性地,本发明化合物的药学上可接受的酯选自下组:
虽然每个人的需求各不相同,本领域技术人员可确定本发明药物组合物中每种活性成分的最佳剂量。一般情况下,本发明的化合物或其药学上可接受的盐,对哺乳动物每天口服给药,药量按照约0.0025到50毫克/公斤体重。但最好是每公斤口服给药约0.01到10毫克。例如,单位口服剂量可以包括约0.01到50毫克,最好是约0.1到10毫克的本发明化合物。单位剂量可给予一次或多次,每天为一片或多片,每片含有约0.1到50毫克,合宜地约0.25到10毫克的本发明化合物或其溶剂化物。
本发明的药物组合物可被配制成适合各种给药途径的制剂形式,包括但不限于被配制成用于肠外,皮下,静脉,肌肉,腹腔内,透皮,口腔,鞘内,颅内,鼻腔或外用途径给药的形式,用于治疗肿瘤和其他疾病。给药量是有效地改善或消
除一个或多个病症的药量。对于特定疾病的治疗,有效量是足以改善或以某些方式减轻与疾病有关的症状的药量。这样的药量可作为单一剂量施用,或者可依据有效的治疗方案给药。给药量也许可治愈疾病,但是给药通常是为了改善疾病的症状。一般需要反复给药来实现所需的症状改善。药的剂量将根据病人的年龄,健康与体重,并行治疗的种类,治疗的频率,以及所需治疗效益来决定。
本发明的药物制剂可以给予任何哺乳动物,只要他们能获得本发明化合物的治疗效果。在这些哺乳动物中最为重要的是人类。本发明的化合物或其药物组合物可用于治疗各种DHODH介导的疾病。本文中,DHODH介导的疾病是自身免疫疾病、器官移植排异、细菌感染、寄生虫疾病、或肿瘤。在具体的实施方式中,所述自身免疫疾病是类风湿性关节炎、红斑狼疮、或牛皮癣。
本发明的药物制剂可用已知的方式制造。例如,由传统的混合,制粒,制锭,溶解,或冷冻干燥过程制造。制造口服制剂时,可结合固体辅料和活性化合物,选择性研磨混合物。如果需要或必要时加入适量助剂后,加工颗粒混合物,获得片剂或锭剂芯。
合适的辅料特别是填料,例如糖类如乳糖或蔗糖,甘露醇或山梨醇;纤维素制剂或钙磷酸盐,例如磷酸三钙或磷酸氢钙;以及粘结剂,例如淀粉糊,包括玉米淀粉,小麦淀粉,大米淀粉,马铃薯淀粉,明胶,黄芪胶,甲基纤维素,羟丙基甲基纤维素,羧甲基纤维素钠,或聚乙烯吡咯烷酮。如果需要,可增加崩解剂,比如上面提到的淀粉,以及羧甲基淀粉,交联聚乙烯吡咯烷酮,琼脂,或褐藻酸或其盐,如海藻酸钠。辅助剂特别是流动调节剂和润滑剂,例如,硅石,滑石,硬脂酸盐类,如镁硬脂酸钙,硬脂酸或聚乙二醇。如果需要,可以給锭剂核芯提供可以抵抗胃液的合适包衣。为此,可以应用浓缩糖类溶液。此类溶液可以含有阿拉伯树胶,滑石,聚乙烯吡咯烷酮,聚乙二醇和/或二氧化钛,漆溶液和合适的有机溶剂或溶剂混合物。为了制备耐胃液的包衣,可使用适当的纤维素溶液,例如醋酸纤维素邻苯二甲酸或羟丙基甲基纤维素邻苯二甲酸。可向药片或锭剂核芯的包衣加入染料或色素。例如,用于识别或为了表征活性成分剂量的组合。
因此,本发明还提供一种治疗DHODH介导的疾病的方法,该方法包括给予需要的对象以本发明的化合物或包含本发明化合物的药物组合物。
给药方法包括但不限于本领域周知的各种给药方法,可根据患者的实际情况加以确定。这些方法包括但不限于肠外、皮下、静脉、肌肉、腹腔内、透皮、口腔、鞘内、颅内、鼻腔或外用途径给药。
本发明也包括本发明化合物在制备治疗DHODH介导的疾病用的药物中的用途。
此外,本领域技术人员基于本领域的公知常识和本发明的内容可以知晓,本发明化合物因其中所含的羧基而能形成盐或酯,进而可以形成前药。
本发明的优点
1.本发明首次发现了一系列结构全新的噻唑类衍生物;
2.本发明的化合物是高效、低毒的DHODH抑制剂,从而具备很重要的学术价值与现实意义;
以下结合具体实施案例对本发明的技术方案进一步描述,但以下实施例不构成对本发明的限制,所有依据本发明的原理和技术手段采用的各种施用方法,均属于本发明范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数按重量计算。
实施例1:
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-羧基苄叉基)肼基]噻唑1
2-甲基氨基硫脲(1-1)
称取2.5g(17.3mmol)甲基肼硫酸盐于250ml单口瓶中,加入100ml乙醇,搅拌下加入1.6g(20.8mmol)硫氰酸铵,加热至回流,反应72h后,将反应液冷至室温,抽滤,滤液旋干硅胶柱层析(DCM/MeOH=40:1),分第二个副产物,得白色粉末状固体0.63g,产率34.2%。1H NMR(400MHz,DMSO-d6,ppm)δ7.36(s,2H),4.89(s,2H),3.41(s,3H).GC-MS(EI)calcd for C2H7N3S[M]+105.0,found 105.0.
2-甲基-1-(2-羧基苄差基)氨基硫脲(1-2)
称取80mg(0.76mmol)化合物(1)于50ml单口瓶中,加入20ml乙醇,搅拌下加入邻羧基苯甲醛114mg(0.76mmol),加热至回流,TLC监测反应至原料转化完全,将反应液冷至室温,旋干溶剂,硅胶柱层析(DCM/MeOH=120:1),得白色粉末状固体100mg,产率56%。1H NMR(400MHz,DMSO-d6,ppm)δ13.36(br,1H),
8.56(s,1H),8.51(s,1H),8.31(d,J=7.8Hz,1H),8.25(s,1H),7.88(d,J=8Hz,1H),7.59(t,J=7.2Hz,1H),7.50(t,J=7.2Hz,1H),3.77(s,3H).LC-MS(ESI)calcd for C10H12N3O2S[M+H]+238.1,found 238.1.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-羧基苄叉基)肼基]噻唑1
称取100mg(0.42mmol)化合物(2)于50ml单口瓶中,加入10ml乙醇,搅拌下加入65μL(0.42mmol)2’-氯-2-溴苯乙酮,升温至回流,TLC跟踪反应至原料转化完全,将反应液冷至室温,旋干溶剂,硅胶柱层析(DCM/MeOH=120:1),得黄色粉末状固体106mg,产率67.9%,Mp.210.4-212.0℃。1H NMR(400MHz,DMSO-d6,ppm)δ13.30(s,1H),8.62(s,1H),8.02(d,J=7.6Hz,1H),7.98–7.90(m,2H),7.66(t,J=7.6Hz,1H),7.56–7.49(m,2H),7.47(s,1H),7.43(td,J1=7.4Hz,J2=1.2Hz,1H),7.36(td,J1=7.6Hz,J2=1.6Hz,1H),3.68(s,3H).13C NMR(125MHz,DMSO-d6,ppm)δ168.98,168.57,147.11,136.94,135.17,133.42,132.48,131.51,131.09,130.98,130.75,130.16,129.45,129.19,127.63,126.51,111.41,32.88.HRMS(ESI)calcd for C18H15N3O2SCl[M+H]+372.0574,found 372.0575.
采取类似的方法,本发明人利用相应的起始材料进一步合成了以下化合物:
(E)-4-(2-氯苯基)-2-[1-乙基-2-(2-羧基苄叉基)肼基]噻唑2
Mp.203.3-204.1℃.1H NMR(500MHz,DMSO-d6,ppm)δ13.32(br,1H),8.64(s,1H),7.98–7.91(m,3H),7.66(t,J=7.5Hz,1H),7.56–7.46(m,2H),7.45–7.39(m,2H),7.34(t,J=6.8Hz,1H),4.32(q,J=6.8Hz,2H),1.27(t,J=7.0Hz,3H).13C NMR(125MHz,DMSO-d6,ppm)δ168.56,168.36,147.34,137.00,135.42,133.53,132.50,131.53,131.12,130.92,130.72,130.22,129.42,129.19,127.62,126.62,111.22,40.38,10.33.HRMS(ESI)calcd for C19H17N3O2SCl[M+H]+386.0730,found 386.0728.
(E)-4-(2-氯苯基)-2-[1-丙基-2-(2-羧基苄叉基)肼基]噻唑3
Mp.172.6-173.4℃.1H NMR(400MHz,DMSO-d6,ppm)δ13.35(br,1H),8.65(s,1H),7.98(d,J=8.0Hz,1H),7.95-7.92(m,2H),7.67(t,J=7.2Hz,1H),7.54(d,J=8.0Hz,1H),7.5(t,J=7.6Hz,1H),7.45-7.42(m,2H),7.36(t,J1=7.6Hz,J2=1.6Hz,1H),4.25(t,J=7.2Hz,2H),1.82-1.73(m,2H),0.97(t,J=7.4Hz,1H).13C NMR(100MHz,DMSO-d6,ppm)δ168.54,168.22 146.95,136.63,134.96,133.17,
132.11,131.10,130.74,130.55,130.34,129.82,129.04,128.80,127.26,126.09,110.77,46.31,17.98,11.17.HRMS(ESI)calcd for C20H19N3O2SCl[M+H]+400.0887,found 400.0879.
(E)-4-(2-氯苯基)-2-[1-异丙基-2-(2-羧基苄叉基)肼基]噻唑4
Mp.185.4-187.0℃.1H NMR(400MHz,DMSO-d6,ppm)δ13.35(br,1H),8.91(s,1H),7.99(d,J=8.0Hz,1H),7.95-7.92(m,2H),7.67(t,J=7.6Hz,1H),7.54(d,J=8.0Hz,1H),7.52(t,J=7.6Hz,1H),7.46-7.42(m,2H),7.36(td,J1=7.6Hz,J2=1.6Hz,1H),5.22-5.11(m,1H),1.57(d,J=6.8Hz,6H).13C NMR(100MHz,DMSO-d6,ppm)δ168.22,168.21,146.90,137.32,135.23,133.14,132.07,131.06,130.64,130.58,130.39,129.88,128.97,128.78,127.28,125.91,111.12,49.61,18.07,18.07.HRMS(ESI)calcd for C20H19N3O2SCl[M+H]+400.0887,found 400.0885.
(E)-4-(2-氯苯基)-2-[1-(2-丁基)-2-(2-羧基苄叉基)肼基]噻唑5
Mp.170.5-170.6℃.1H NMR(400MHz,DMSO-d6,ppm)δ13.32(br,1H),8.91(s,1H),7.99(d,J=8.0Hz,1H),7.95-7.90(m,2H),7.67(t,J1=7.6Hz,1H),7.55-7.48(m,2H),7.46-7.42(m,2H),7.36(td,J1=7.6Hz,J2=1.6Hz,1H),5.00-4.93(m,1H),2.35-2.24(m,1H),1.92-1.81(m,1H),1.54(d,J=6.8Hz,3H),0.88(t,J=7.2Hz,3H).13C NMR(100MHz,DMSO-d6,ppm)δ168.66,168.19,146.97,137.03,135.24,133.19,132.13,131.04,130.67,130.58,130.39,129.70,128.98,128.77,127.29,125.91,111.04,55.67,25.26,16.33,11.14.HRMS(ESI)calcd for C21H21N3O2SCl[M+H]+414.1043,found 414.1029.
(E)-4-(2-氯苯基)-2-[1-(2-戊基)-2-(2-羧基苄叉基)肼基]噻唑6
Mp.146.7-147.0℃.1H NMR(400MHz,DMSO-d6,ppm)δ13.35(br,1H),8.91(s,1H),7.98(d,J=7.6Hz,1H),7.94(d,J=8.0Hz,1H),7.91(dd,J1=7.6Hz,J2=1.6Hz,1H),7.67(t,J=7.6Hz,1H),7.53(d,J=8.0Hz,1H),7.49(t,J=7.6Hz,1H),7.46-7.42(m,2H),7.36(td,J1=7.6Hz,J2=1.6Hz,1H),5.14-5.06(m,1H),2.34-2.25(m,1H),1.82-1.73(m,1H),1.53(d,J=6.8Hz,3H),1.34-1.24(m,2H),0.89(t,J=7.4Hz,1H).13C NMR(100MHz,DMSO-d6,ppm)δ168.68,168.20,
146.98,137.04,135.30,133.20,132.14,131.00,130.68,130.59,130.40,129.68,128.99,127.30,125.90,111.09,53.81,34.20,19.46,16.46,13.60.HRMS(ESI)calcd for C22H23N3O2SCl[M+H]+428.1200,found 428.1193.
(E)-4-(2-氯苯基)-2-[1-羟乙基-2-(2-羧基苄叉基)肼基]噻唑7
Mp.187.9-188.9℃.1H NMR(400MHz,DMSO-d6,ppm)δ13.24(br,1H),8.75(s,1H),7.98-7.01(m,3H),7.66(t,J=7.6Hz,1H),7.54(d,J=8.0Hz,1H),7.49(t,J=7.6Hz,1H),7.45-7.41(m,2H),7.36(td,J1=7.6Hz,J2=1.6Hz,1H),4.35(t,J=6.4Hz,2H),3.76(t,J=6.4Hz,2H).13C NMR(100MHz,DMSO-d6,ppm)δ168.64,168.23,146.87,136.89,134.91,133.15,131.99,131.19,130.71,130.46,130.32,130.16,129.04,128.79,127.22,126.28,110.78,56.24,47.46.HRMS(ESI)calcd for C19H17N3O3SCl[M+H]+402.0679,found 402.0678.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(4-三氟甲基-2-羧基苄叉基)肼基]噻唑8
Mp.209.3-210.7℃.1H NMR(400MHz,DMSO-d6,ppm)δ14.02(br,1H),8.66(s,1H),8.21(d,J=8.0Hz,1H),8.17(s,1H),8.02(d,J=8.0Hz,1H),7.95(d,J=7.6Hz,1H),7.56-7.53(m,2H),7.43(t,J=7.6Hz,1H),7.37(t,J=7.6Hz,1H),3.70(s,3H).13C NMR(100MHz,DMSO-d6,ppm)δ168.33,167.17,146.79,138.38,135.01,132.91,131.12,130.70,130.37,129.14,128.57,128.25,128.20,127.26,127.04,125.10,122.40,111.51,32.68.HRMS(ESI)calcd for C19H14N3O2SClF3[M+H]+440.0447,found 440.0433.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(4-甲基-2-羧基苄叉基)肼基]噻唑9
Mp.231.3-232.5℃.1H NMR(400MHz,DMSO-d6,ppm)δ13.30(br,1H),8.59(s,1H),7.96(d,J=7.6Hz,1H),7.92(d,J=8.0Hz,1H),7.75(s,1H),7.54(d,J=7.6Hz,1H),7.49-7.46(m,2H),7.43(t,J=7.6Hz,1H),7.36(t,J=7.6Hz,1H),3.66(s,3H),2.38(s,3H).13C NMR(100MHz,DMSO-d6,ppm)δ168.60,168.35,146.68,138.54,136.64,133.03,132.70,132.07,131.11,130.86,130.67,130.34,129.83,129.00,127.21,126.04,110.84,32.39,20.69.HRMS(ESI)calcd for
C19H17N3O2SCl[M+H]+386.0730,found 386.0738.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-羧基-4-氟苄叉基)肼基]噻唑10
Mp.228.6-229.7℃.1H NMR(400MHz,DMSO-d6,ppm)δ13.28(br,1H),8.14(s,1H),7.93(dd,J1=7.6Hz,J2=1.6Hz,1H),7.61(dd,J1=7.0Hz,J2=1.8Hz,1H),7.55-7.49(m,3H),7.44-7.41(m,1H),7.36(td,J1=7.6Hz,J2=1.6Hz,1H),3.68(s,3H).13C NMR(100MHz,DMSO-d6,ppm)δ168.55,168.01,159.74(d,1J=249Hz),146.67,133.68,133.10,131.50,131.12,130.72,130.32,130.10(d,3J=8.8Hz),129.06,127.23,125.53(d,4J=3.1Hz),121.75(d,2J=11.8Hz),118.76(d,2J=22Hz),111.13,32.28.HRMS(ESI)calcd for C18H14N3O2FSCl[M+H]+390.0479,found 390.0475.
(E)-5-甲基-4-苯基-2-[1-甲基-2-(4-甲基-2-羧基苄叉基)肼基]噻唑11
Mp.243.2-245.2℃.1H NMR(400MHz,DMSO-d6,ppm)δ13.24(br,1H),8.54(s,1H),7.91(d,J=8.0Hz,1H),7.73(s,1H),7.64-7.53(m,2H),7.46-7.42(m,3H),7.33(t,J=7.4Hz,1H),3.60(s,3H),2.44(s,3H),2.38(s,3H).13C NMR(100MHz,DMSO-d6,ppm)δ168.35,165.40,145.43,138.33,135.76,135.16,132.68,132.22,130.86,129.62,128.24,128.24,127.85,127.85,127.06,125.89,119.27,31.85,20.68,12.24.HRMS(ESI)calcd for C20H20N3O2S[M+H]+366.1276,found 366.1273.
(E)-5-甲基-4-苯基-2-[1-甲基-2-(2-羧基苄叉基)肼基]噻唑12
Mp.222.7-224.9℃.1H NMR(400MHz,DMSO-d6,ppm)δ13.32(br,1H),8.58(s,1H),8.01(d,J=8.0Hz,1H),7.92(d,J=7.6Hz,1H),7.68-7.63(m,3H),7.50-7.43(m,3H),7.34(t,J=7.4Hz,1H),3.62(s,3H),2.44(s,3H).13C NMR(100MHz,DMSO-d6,ppm)δ168.24,165.36,145.47,135.66,135.13,134.90,131.98,130.58,129.70,128.59,128.25,128.25,127.86,127.86,127.09,125.94,119.44,31.93,12.24.HRMS(ESI)calcd for C19H16N3O2S[M-H]-350.0963,found 350.0951.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-羧基苄叉基)肼基]噻唑13
Mp.208.8-209.8℃.1H NMR(400MHz,DMSO-d6,ppm)δ13.46(br,1H),8.60(s,1H),8.01(d,J=8.0Hz,1H),7.92(d,J=8.0Hz,1H),7.64(t,J=7.4Hz,1H),7.56(d,J=7.6Hz,1H),7.49-7.39(m,4H),3.56(s,3H),2.15(s,3H).13C NMR(100MHz,DMSO-d6,ppm)δ168.98,166.46,144.56,136.46,135.29,134.54,133.25,132.50,132.31,131.05,130.72,130.26,130.01,129.09,127.49,126.42,121.85,32.46,12.16.HRMS(ESI)calcd for C19H17N3O2SCl[M+H]+386.0730,found 386.0727.
(E)-5-乙基-4-(2-氯苯基)-2-(2-羧基苄叉肼基)噻唑14
Mp.223.9-223.9℃.1H NMR(500MHz,DMSO-d6,ppm)δ12.57(br,2H),8.79(s,1H),8.01(d,J=8.0Hz,1H),7.88(d,J=8.0Hz,1H),7.62(t,J=7.5Hz,1H),7.56–7.51(m,1H),7.46(t,J=7.5Hz,1H),7.44-7.36(m,3H),2.49(q,J=7.5Hz,1H),1.13(t,J=7.5Hz,3H).13C NMR(125MHz,DMSO-d6,ppm)δ169.28,165.92,144.14,140.84,135.89,135.46,133.99,133.03,133.02,131.46,130.81,130.73,130.60,129.73,128.09,127.73,127.01,20.99,17.25.HRMS(ESI)calcd for C19H17N3O2SCl[M+H]+386.0730,found 386.0729.
(E)-5-甲基-4-(2-氯苯基)-2-(2-羧基苄叉肼基)噻唑15
Mp.217.6-217.8℃.1H NMR(400MHz,DMSO-d6,ppm)δ12.7(br,2H),8.77(s,1H),7.99(d,J=7.6Hz,1H),7.87(d,J=7.2Hz,1H),7.62(t,J=7.4Hz,1H),7.562-7.543(m,1H),7.481-7.397(m,4H),2.14(s,3H).13C NMR(125MHz,DMSO-d6,ppm)δ168.65,165.18,144.49,140.16,135.25,134.56,133.21,132.46,130.83,130.14,130.03,129.11,127.45,126.37,119.49,12.15.HRMS(ESI)calcd for C18H15N3O2SCl[M+H]+372.0574,found 372.0569.
(E)-5-甲基-4-苯基-2-(2-羧基苄叉肼基)噻唑17
Mp.215.9-216.0℃.1H NMR(400MHz,DMSO-d6,ppm)δ12.7(br,2H),8.77(s,1H),7.99(d,J=7.6Hz,1H),7.87(dd,J1=8.0Hz,J2=1.2Hz,1H),7.63-7.59(m,3H),7.48-7.43(m,3H),7.33(t,J=7.2Hz,1H),2.43(s,3H).13C NMR(125MHz,DMSO-d6,ppm)δ168.66,164.72,146.00,140.05,135.64,135.24,132.36,130.83,130.12,129.08,128.73,128.73,128.35,128.35,127.51,126.33,117.61,12.74.
HRMS(ESI)calcd for C18H16N3O2S[M+H]+338.0963,found 338.0954.
(E)-4-(2,5-二氯苯基)-2-[1-甲基-2-(2-羧基苄叉基)肼基]噻唑19
Mp.268.4-269.5℃.1H NMR(400MHz,DMSO-d6,ppm)δ13.31(br,1H),8.63(s,1H),8.02-8.00(m,2H),7.93(d,J=7.2Hz,1H),7.67(t,J=7.6Hz,1H),7.63(s,1H),7.58(d,J=7.6Hz,1H),7.50(t,J=7.2Hz,1H),7.43(dd,J1=7.2Hz,J2=2.8Hz,1H),3.68(s,3H).13C NMR(100MHz,DMSO-d6,ppm)δ168.68,168.15,145.13,136.79,134.70,134.21,132.14,132.06,131.85,130.59,130.17,129.76,129.20,128.84,128.58,126.13,112.31,32.49.HRMS(ESI)calcd for C18H14N3O2SCl2[M+H]+406.0184,found 406.0187.
(E)-4-苯基-2-[1-甲基-2-(2-羧基苄叉基)肼基]噻唑20
Mp.205.6-206.8℃.1H NMR(400MHz,DMSO-d6,ppm)δ13.31(br,1H),8.62(s,1H),8.02(d,J=7.6Hz,1H),7.94-7.91(m,3H),7.66(t,J=7.2Hz,1H),7.49(t,J=7.2Hz,1H),7.45-7.40(m,3H),7.31(t,J=7.2Hz,1H),3.71(s,3H).13C NMR(100MHz,DMSO-d6,ppm)δ169.45,168.20,150.18,136.35,134.80,134.49,132.07,130.60,129.71,128.76,128.56,128.56,127.59,126.09,125.54,125.54,105.97,32.51.HRMS(ESI)calcd for C18H16N3O2S[M+H]+338.0963,found 338.0963.
(E)-4-(3-氯苯基)-2-[1-甲基-2-(2-羧基苄叉基)肼基]噻唑21
Mp.244.7-245.4℃.1H NMR(400MHz,DMSO-d6,ppm)δ13.32(br,1H),8.63(s,1H),8.01(d,J=7.6Hz,1H),7.97(s,1H),7.93(d,J=7.6Hz,1H),7.88(d,J=8.0Hz,1H),7.66(t,J=7.6Hz,1H),7.61(s,1H),7.50(t,J=7.6Hz,1H),7.45(t,J=7.6Hz,1H),7.37(d,J=8.0Hz,1H),3.71(s,3H).13C NMR(100MHz,DMSO-d6,ppm)δ169.57,168.18,148.56,136.64,136.50,134.72,133.48,132.06,130.59,130.44,129.76,128.81,127.28,126.12,125.16,124.05,107.56,32.52.HRMS(ESI)calcd for C18H15N3O2SCl[M+H]+372.0574,found 372.0574.
(E)-5-甲基-4-(2-氯苯基)-2-[1-(2-戊基)-2-(2-羧基苄叉基)肼基]噻唑22
Mp.89.9-90.1℃.1H NMR(400MHz,DMSO-d6,ppm)δ13.29(br,1H),8.85(s,1H),7.98(d,J=8.0Hz,1H),7.92(d,J=7.6Hz,1H),7.65(t,J=7.6Hz,1H),7.56-7.54(m,1H),7.51-7.41(m,4H),5.05-4.96(m,1H),2.29-2.22(m,1H),2.16(s,3H),1.75-1.67(m,1H),1.46(d,J=7.2Hz,3H),1.28-1.23(m,2H),0.87(t,J=7.6Hz,3H).13C NMR(100MHz,DMSO-d6,ppm)δ168.23,166.06,144.08,136.14,135.46,134.09,132.79,132.09,131.93,130.58,129.64,129.60,129.48,128.56,126.95,125.73,121.62,53.39,34.01,19.41,16.35,13.58,11.52.HRMS(ESI)calcd for C23H25N3O2SCl[M+H]+442.1356,found 442.1354.
(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)亚肼基)甲基)-6-氟苯甲酸(58)
熔点:211.2-211.7℃.1H NMR(400MHz,DMSO-d6):δ12.40(s,1H),8.18(s,1H),7.86(dd,J1=7.6Hz,J2=1.6Hz,1H),7.69(d,J=8.0Hz,1H),7.55-7.50(m,2H),7.41(t,J=7.6Hz,1H),7.37(s,1H),7.35(t,J=7.6Hz,1H),7.29(t,J=8.8Hz,1H).13C NMR(100MHz,DMSO-d6):167.56,166.14,160.51,158.06,147.64,138.21,138.17,133.94,133.89,133.68,131.54,131.25,130.83,129.53,127.72,122.04,116.51,116.29,109.58.HRMS(ESI)calcd for C17H10N3O2SClF[M-H]-374.0166,found 374.0164.Purity:97.09%(tR 12.05min).
A系列前药的合成
1-(2-丙叉基)氨基硫脲1
称取氨基硫脲(10g,109.7mmol)于500mL三口瓶中,加入无水乙醇200mL,电磁搅拌下加入丙酮(8.1mL,109.7mmol),醋酸2mL,升温至78℃。反应过程中用TLC板检测反应,4h后反应结束,停止加热。反应液静置过夜,析出白色片状固体,抽滤,得产物粗品,用水重结晶,抽滤,得纯品12.1g,产率84%。1H NMR(400MHz,DMSO-d6,ppm):δ9.86(s,1H),7.95(s,1H),7.45(s,1H),1.90(s,3H),1.88(s,3H).
4-(2-氯苯基)-2-(2-丙叉基肼基)噻唑2
称取化合物1(9g,68.6mmol)于500mL三口瓶中,加入无水乙醇200mL,室温搅拌下加入2-溴-2'-氯苯乙酮(17.6g,75.5mmol),随即升温至78℃。反应用TLC跟踪监测,5小时后反应结束,停止加热,反应液静置冷却至室温,析出黄色固体,抽滤,滤饼用乙醇重结晶,静置,抽滤,干燥后得黄色粉末状固体14.2g,产率78%。1H NMR(400MHz,DMSO-d6,ppm):δ7.69(d,J=7.8Hz,1H),7.50(d,J=7.8Hz,1H),7.40(m,2H),6.98(s,1H),2.21(s,3H),2.10(s,3H).
4-(2-氯苯基)-2-肼基噻唑3
称取化合物2(10g,37.7mmol)于250mL三口瓶中,加无水乙醇130mL,加80%水合肼(5.3mL,107.8mmol),氮气保护下加热至回流,升温过程中析出大量固体,停止反应,抽滤,得到化合物3的粗品5g,反应液继续回流反应12h后停止反应,反应瓶放冰箱静置一夜,抽滤,得产物粗品2g,产物粗品用乙醇重结晶,得白色絮状固体5.1g,产率60.7%。1H NMR(400MHz,DMSO-d6,ppm):δ8.58(s,1H),7.86(dd,J1=7.8Hz,J2=2.0Hz,1H),7.46(dd,J1=7.8Hz,J2=1.6Hz,1H),7.38(td,J1=7.8Hz,J2=1.6Hz,1H),7.27(td,J1=7.8Hz,J2=2.0Hz,1H),7.10(s,1H),4.85(s,2H).LC-MS(ESI)calcd for C9H8N3SCl[M+H]+226.01,found 226.10.
(E)-4-(2-氯苯基)-2-(2-羧基苄叉肼基)噻唑A
称取邻羧基苯甲醛(150mg,1mmol)于50mL单口瓶中,加入化合物3(225mg,1mmol),加入无水乙醇30mL,滴入三滴醋酸作为催化剂,氮气保护下室温搅拌,TLC跟踪至反应结束,反应液趁热快速抽滤,滤饼用冰乙醇淋洗三次,抽干滤饼得亮黄色粉末状固体250mg,产率70%。熔点:195-196℃.1H NMR(400MHz,DMSO-d6,ppm):δ13.30(br,1H),12.05(br,1H),8.84(s,1H),8.02(d,J=7.6Hz,1H),7.91(t,J=8.0Hz,2H),7.66(t,J=7.2Hz,1H),7.55(d,J=7.6Hz,1H),7.51(t,J=8Hz,1H),7.44(t,J=7.2Hz,1H),7.38-7.34(m,2H).13C NMR(100MHz,DMSO-d6,ppm):δ168.12,167.26,140.26,135.03,134.55,133.23,131.94,131.42,131.05,130.72,130.34,129.80,129.01,128.81,127.22,125.96,109.26.HRMS(ESI)calcd for C17H13N3O2SCl[M+H]+358.0417,found 358.0419.HPLC purity:96.88%,Retention time=9.34min.
2-甲酰基苯甲酸乙酯1a
称取邻羧基苯甲醛(1.5g,10mmol)于100mL三口瓶中,加入碳酸铯(6.5g,20mmol),N,N-二甲基甲酰胺(DMF)20mL,电磁搅拌下加入碘乙烷(4.68g,30mmol),加热升温至70℃,反应4h,TLC监测至反应结束,停止加热,向反应瓶中加入20mL水,反应液用二氯甲烷和水萃取三次,合并有机相,加入无水硫酸钠干燥,抽滤,滤液加入等质量的硅胶旋干,过柱纯化,得到无色透明液体1.4g,产率79%。1H NMR(400MHz,DMSO-d6,ppm):δ10.41(s,1H),7.92-7.90(m,1H),7.88-7.85(m,1H),7.81-7.77(m,2H),4.40(q,J=7.2Hz,2H),1.34(t,J=7.2Hz,3H).LC-MS(ESI)calcd for C10H10O3[M+H]+179.06,found 179.10.
(E)-4-(2-氯苯基)-2-(2-乙氧基甲酰基苄叉肼基)噻唑A1
称取化合物1a(178mg,1mmol)于50mL两口瓶中,另取化合物3(225mg,1mmol)加入反应瓶中,加入无水乙醇15mL,氮气保护下,电磁搅拌反应4h,TLC检测原料反应完全,抽滤反应液,滤饼用乙醇淋洗,干燥得到近纯产物280mg,过柱纯化后得到淡黄色固体粉末220mg,产率57%。熔点:172-173℃.1H NMR(400MHz,DMSO-d6,ppm):δ13.30(br,1H),12.05(br,1H),8.84(s,1H),8.02(d,J=7.6Hz,1H),7.91(t,J=8.0Hz,2H),7.66(t,J=7.2Hz,1H),7.55(d,J=7.6Hz,1H),7.51(t,J=8Hz,1H),7.44(t,J=7.2Hz,1H),7.38-7.34(m,2H).13C NMR(100MHz,DMSO-d6,ppm):δ167.92,16.70,147.26,141.03,134.55,133.23,131.94,131.82,131.06,130.70,130.34,128.80,128.74,128.60,127.22,125.96,109.26,61.23,14010.HRMS(ESI)calcd for C19H17N3O2SCl[M+H]+386.0730,found 386.0731.HPLC purity:98.88%,Retention time=16.14min.
2-甲酰基苯甲酸丙酯2a
合成方法同1a,得到透明液体1.5g,产率78%。1H NMR(400MHz,DMSO-d6,ppm):δ10.40(s,1H),7.93-7.90(m,1H),7.88-7.84(m,1H),7.81-7.77(m,2H),4.29(t,J=6.6Hz,2H),1.74(h,J=7.1Hz,2H),0.97(t,J=7.4Hz,3H).LC-MS(ESI)calcd for C11H12O3
[M+H]+193.08,found 193.10.
(E)-4-(2-氯苯基)-2-(2-丙氧基甲酰基苄叉肼基)噻唑A2
合成方法同A1,得到淡黄色固体粉末180mg,产率58%。熔点:170-171℃.1H NMR(400MHz,DMSO-d6,ppm):δ12.40(s,1H),8.71(s,1H),8.00(d,J=7.9Hz,1H),7.88(d,J=7.6,2.4Hz,2H),7.67(t,J=7.6Hz,1H),7.52(q,J=7.9Hz,2H),7.46-7.32(m,3H),4.26(t,J=6.5Hz,2H),1.76(h,J=7.1Hz,2H),0.98(t,J=7.3Hz,3H).13C NMR(100MHz,DMSO-d6,ppm):δ167.68,167.03,147.63,140.16,134.95,133.6,132.71,131.53,131.22,130.85,130.59,129.52,129.4,129.38,127.73,126.74,109.35,67.06,21.96,10.87.HRMS(ESI)calcd for C20H18N3O2SCl[M+H]+400.0808,found 400.0888.HPLCpurity:96.95%,Retention time=12.44min.
2-甲酰基苯甲酸异丙酯3a
合成方法同1a,得到黄色透明液体1.7g,产率88%。1H NMR(400MHz,DMSO-d6,ppm):δ10.40(s,1H),7.91-7.83(m,2H),7.80-7.75(m,2H),5.20(hept,J=6.2Hz,1H),1.35(d,J=6.2Hz,6H).LC-MS(ESI)calcd for C11H12O3[M+H]+193.08,found 193.10.
(E)-4-(2-氯苯基)-2-(2-异丙氧基甲酰基苄叉肼基)噻唑A3
合成方法同A1,得到棕黄色固体粉末200mg,产率60%。熔点:159-160℃.1H NMR(400MHz,DMSO-d6,ppm):δ12.40(s,1H),8.69(s,1H),7.99(d,J=7.8Hz,1H),7.90-7.81(m,2H),7.65(t,J=7.6Hz,1H),7.55-7.46(m,2H),7.44-7.32(m,3H),5.16(hept,J=6.3Hz,1H),1.35(d,J=6.1Hz,6H).13C NMR(100MHz,DMSO-d6,ppm):δ205.55,167.21,166.04,147.14,139.59,134.28,133.20,132.07,131.02,130.72,130.35,130.07,129.35,129.00,128.84,127.21,126.11,108.82,68.85,21.53.HRMS(ESI)calcd for C20H18ClN3O2S[M+H]+400.0808,found 400.0868.HPLC purity:97.35%,Retention time=10.44min.
2-甲酰基苯甲酸丁酯4a
合成方法同1a,得到黄色澄清透明液体1.7g,产率86%。1H NMR(400MHz,DMSO-d6,ppm):δ10.41(s,1H),7.93-7.90(m,1H),7.88-7.85(m,1H),7.81-7.77(m,2H),4.33(t,J=6.5Hz,2H),1.71(p,J=8.5,6.6Hz,2H),1.42(h,2H),0.94(t,J=7.4Hz,3H).LC-MS(ESI)calcd for C12H14O3[M+H]+207.09,found 207.10.
(E)-4-(2-氯苯基)-2-(2-丁氧基甲酰基苄叉肼基)噻唑A4
合成方法同A1,得到黄色粉末210mg,产率55%。熔点:144-145℃.1H NMR(400MHz,DMSO-d6,ppm):δ12.40(s,1H),8.71(s,1H),8.00(dd,J=8.0,1.2Hz,1H),7.89-7.84(m,2H),7.67(td,J=7.6,1.3Hz,1H),7.56-7.48(m,2H),7.42(td,J=7.5,1.5Hz,1H),7.39-7.33(m,2H),4.30(t,J=6.6Hz,2H),1.72(p,2H),1.42(h,2H),0.94(t,J=7.4Hz,3H).13C NMR(100MHz,DMSO-d6,ppm):δ167.68,167.02,147.63,140.16,134.95,133.70,132.70,131.53,131.22,130.85,130.58,129.52,129.40,129.37,127.73,126.77,109.35,65.32,30.57,19.23,14.09.HRMS(ESI)calcd for C21H20N3O2SCl[M+H]+414.0965,found 414.1044.HPLC purity:97.15%,Retention time=11.23min.
2-甲酰基苯甲酸-2-丁酯5a
合成方法同1a,得到无色透明液体1.8g,产率91%。1H NMR(400MHz,DMSO-d6,ppm):δ10.39(s,1H),7.91-7.88(m,1H),7.87-7.84(m,1H),7.81-7.76(m,2H),5.06(q,J=6.2Hz,1H),1.75-1.64(m,2H),1.32(d,J=6.3Hz,3H),0.93(t,J=7.4Hz,3H).LC-MS(ESI)calcd for C12H14O3[M+H]+207.09,found 207.10.
(E)-4-(2-氯苯基)-2-(2-(2-丁氧基)甲酰基苄叉肼基)噻唑A5
合成方法同A1,得到浅黄色固体粉末150mg,产率52%。熔点:137-138℃.1H NMR(400MHz,DMSO-d6,ppm):δ12.41(s,1H),8.71(s,1H),8.00(d,J=7.9,1.2Hz,1H),7.90-7.84(m,2H),7.69-7.64(m,1H),7.56-7.48(m,2H),7.42(td,J=7.5,1.5Hz,1H),7.39-7.33(m,2H),5.03(h,J=6.3Hz,1H),1.77-1.63(m,2H),1.33(d,J=6.3Hz,3H),0.93(t,J=7.4Hz,3H).13C NMR(100MHz,DMSO-d6,ppm):δ167.71,166.60,147.64,140.09,134.89,133.70,132.64,131.53,131.22,130.86,130.52,129.74,129.52,129.39,127.73,126.67,109.35,73.65,28.66,19.65,10.07.HRMS(ESI)calcd for C21H20N3O2SCl[M+H]+414.0965,found 414.0988.HPLC purity:98.05%,Retention time=11.14min.
2-甲酰基苯甲酸戊酯6a
合成方法同1a,得到澄清液体1.6g,产率77%。1H NMR(400MHz,DMSO-d6,ppm):δ10.40(s,1H),7.93-7.89(m,1H),7.88-7.84(m,1H),7.81-7.77(m,2H),4.32(t,J=6.6Hz,2H),1.72(p,J=6.8Hz,2H),1.42-1.30(m,4H),0.92-0.87(m,3H).LC-MS(ESI)calcd for C13H16O3[M+H]+221.11,found 221.10.
(E)-4-(2-氯苯基)-2-(2-戊氧基甲酰基苄叉肼基)噻唑A6
合成方法同A1,得到黄色粉末120mg,产率52%。熔点:98-99℃.1H NMR(400MHz,DMSO-d6,ppm):δ12.39(s,1H),8.71(s,1H),7.99(dd,J=8.0,1.2Hz,1H),7.86(ddd,J=7.8,6.1,1.6Hz,2H),7.66(td,J=7.7,1.4Hz,1H),7.55-7.48(m,2H),7.42(td,J=7.5,1.5Hz,1H),7.38-7.33(m,2H),4.29(t,J=6.7Hz,2H),1.74(p,J=6.8Hz,2H),1.42-1.30(m,4H),0.89(t,J=7.0Hz,3H).13C NMR(100MHz,DMSO-d6,ppm):δ167.68,167.02,147.63,140.19,134.95,133.70,132.69,131.52,131.22,130.85,130.57,129.52,129.40,129.38,127.73,126.80,109.33,65.60,28.21,28.14,22.29,14.34.HRMS(ESI)calcd for C22H22N3O2SCl[M+H]+428.1121,found 428.1201.HPLC purity:99.55%,Retention time=7.73min.
2-甲酰基苯甲酸己酯7a
合成方法同1a,得到无色透明液体1.9g,产率81%。1H NMR(400MHz,DMSO-d6,
ppm):δ10.40(s,1H),7.93-7.88(m,1H),7.88-7.83(m,1H),7.82-7.76(m,2H),4.32(t,J=6.6Hz,2H),1.71(p,2H),1.43-1.25(m,6H),0.90-0.83(m,3H).LC-MS(ESI)calcd for C14H18O3[M+H]+235.13,found 235.10.
(E)-4-(2-氯苯基)-2-(2-己氧基甲酰基苄叉肼基)噻唑A7
合成方法同A1,得到淡黄色粉末110mg,产率59%。熔点:92-93℃.1H NMR(400MHz,DMSO-d6,ppm):δ12.45(s,1H),8.76(s,1H),8.05(d,J=8.0Hz,1H),7.96-7.87(m,2H),7.72(t,J=7.7Hz,1H),7.62-7.53(m,2H),7.51-7.37(m,3H),4.34(t,J=6.6Hz,2H),1.79(t,J=7.4Hz,2H),1.49-1.28(m,6H),0.92(t,J=6.7Hz,3H).13C NMR(100MHz,DMSO-d6,ppm):167.56,166.92,147.53,140.10,134.85,133.60,132.56,131.41,131.12,130.74,130.44,129.39,129.30,129.27,127.60,126.71,109.20,65.51,31.27,28.36,25.52,22.36,14.22.HRMS(ESI)calcd for C23H24N3O2SCl[M+H]+442.1278,found 442.1355.HPLC purity:98.60%,Retention time=13.41min.
2-甲酰基苯甲酸环己酯8a
合成方法同1a,得到淡黄色透明液体1.8g,产率77%。1H NMR(400MHz,DMSO-d6,ppm):δ10.41(s,1H),7.93-7.88(m,1H),7.87-7.82(m,1H),7.81-7.75(m,2H),5.05-4.95(m,1H),1.97–1.88(m,2H),1.76-1.66(m,2H),1.61-1.47(m,3H),1.47-1.36(m,2H),1.36-1.25(m,1H).LC-MS(ESI)calcd for C14H16O3[M+H]+233.11,found 233.10.
(E)-4-(2-氯苯基)-2-(2-环己氧基甲酰基苄叉肼基)噻唑A8
合成方法同A1,得到黄色粉末130mg,产率50%。熔点:153-155℃.1H NMR(400MHz,DMSO-d6,ppm):δ12.42(s,1H),8.70(s,1H),8.00(d,J=7.8Hz,1H),7.90-7.83(m,2H),7.66(t,J=7.4Hz,1H),7.56-7.47(m,2H),7.42(td,J=7.5,1.2Hz,1H),7.39-7.33(m,2H),5.00-4.91(m,1H),1.98-1.89(m,2H),1.78-1.68(m,2H),1.64-1.49(m,3H),1.47-1.25(m,3H).13C NMR(100MHz,DMSO-d6,ppm):δ167.72,166.37,140.10,134.84,132.62,131.52,131.22,130.86,130.60,129.80,129.52,129.39,127.73,126.67,109.34,
73.85,31.44,25.34,23.69.HRMS(ESI)calcd for C23H22N3O2SCl[M+H]+440.1121,found 440.1101.HPLC purity:98.03%,Retention time=13.03min.
2-甲酰基苯甲酸特戊酸甲基酯9a
称取邻羧基苯甲醛(1g,6.7mmol)于100mL三口瓶中,加入碳酸铯(4.3g,13.3mmol),碘化钾(1.1g,6.7mmol),N,N-二甲基甲酰胺(DMF)15mL,电磁搅拌下加入特戊酸氯甲酯(1.5g,10mmol),加热升温至70℃,反应20h,TLC检测至反应结束,停止加热,向反应瓶中加入20mL水,将反应液用二氯甲烷和水萃取三次,合并有机相,加无水硫酸钠干燥,抽滤,滤液加入等质量硅胶,过柱纯化,得到澄清透明液体0.9g,产率52%。LC-MS(ESI)calcd for C14H16O5[M+H]+265.08,found 265.10.
(E)-4-(2-氯苯基)-2-(2-特戊酸甲氧基甲酰基苄叉肼基)噻唑A9
合成方法同A1,后处理得到粗品后,用反析法(粗品用适量乙酸乙酯溶解完全,电磁搅拌下加入石油醚,快速搅拌,析出固体,抽滤即可)得到淡黄色粉末180mg,产率49%。熔点:114-115℃.1H NMR(400MHz,DMSO-d6,ppm):δ12.43(s,1H),8.73(s,1H),8.03(d,J=8.0,1.2Hz,1H),7.89-7.83(m,2H),7.74-7.68(m,1H),7.56-7.51(m,2H),7.42(td,J=7.5,1.4Hz,1H),7.39-7.33(m,2H),5.98(s,2H),1.18(s,9H).13C NMR(100MHz,DMSO-d6,ppm):δ167.13,164.91,139.23,135.19,133.04,131.04,130.73,130.35,130.31,129.04,129.02,127.24,127.06,126.51,108.96,80.13,38.26,26.47.HRMS(ESI)calcd for C23H22N3O4SCl[M+H]+472.1020,found 472.1096.HPLC purity:97.79%,Retention time=9.68min.
2-甲酰基苯甲酸异丙基碳酸甲基酯10a
合成方法同9a,得到无色透明液体0.8g,产率45%。LC-MS(ESI)calcd for C13H14O6[M+NH4]+284.08,found 284.10.
(E)-4-(2-氯苯基)-2-(2-异丙基碳酸甲氧基甲酰基苄叉肼基)噻唑A10
合成方法同A9,得到黄色粉末160mg,产率53%。熔点:110-111℃.1H NMR(400MHz,DMSO-d6,ppm):δ12.44(s,1H),8.76(s,1H),8.04(d,J=8.0,1.2Hz,1H),7.93-7.85(m,2H),7.73(t,J=7.6,1.3Hz,1H),7.57-7.51(m,2H),7.43(td,J=7.5,1.5Hz,1H),7.40-7.33(m,2H),5.96(s,2H),4.85(hept,J=6.2Hz,1H),1.26(d,J=6.2Hz,6H).13C NMR(100MHz,DMSO-d6,ppm):δ167.62,165.32,153.25,147.67,139.76,135.83,133.66,131.54,131.23,131.01,130.85,129.54,129.49,127.74,127.30,126.99,109.47,83.02,73.24,21.80.HRMS(ESI)calcd for C22H20N3O5SCl[M+H]+474.0812,found 474.0889.HPLC purity:99.25%,Retention time=12.74min.
2-甲酰基苯甲酸苯甲酯11a
称取邻羧基苯甲醛(1.5g,10mmol)于100mL三口瓶中,加入无水甲苯20mL,外部冰浴,电磁搅拌下加入氯化亚砜(1.79g,15mmol),搅拌十五分钟后,撤去冰浴,将三口瓶转移到油浴锅中,加热升温至110℃,回流反应4h,停止加热,旋干溶剂得到黄色粘稠液体,直接用10mL无水二氯甲烷稀释备用。另取100mL单口反应瓶,向瓶中加入苯甲醇(1.08g,10mmol),三乙胺(1.52g,15mmol),无水二氯甲烷15mL,冰盐浴下电磁搅拌十五分钟,将酰氯的二氯甲烷溶液逐滴加到单口反应瓶中,冰浴反应三十分钟后,转室温反应,TLC检测反应,反应结束后,向反应液中加水搅拌十分钟,静置分层,取有机相,水相用二氯甲烷萃取三次,合并有机相,干燥,旋干溶剂,过柱纯化,得到无色透明液体1.1g,产率46%。1H NMR(400MHz,DMSO-d6,ppm):δ10.42(s,1H),7.95-7.91(m,2H),7.78-7.75(m,2H),7.50-7.48(m,2H),7.42-7.36(m,3H),5.39(s,2H).LC-MS(ESI)calcd for C15H12O3[M+H]+241.08,found 241.10.
(E)-4-(2-氯苯基)-2-(2-苯甲氧基甲酰基苄叉肼基)噻唑A11
合成方法同A1,得到棕黄色粉末80mg,产率46%。熔点:168-169℃.1H NMR(400MHz,DMSO-d6,ppm):δ12.41(s,1H),8.78(s,1H),8.02(d,J=7.9Hz,1H),7.92-7.86(m,2H),7.70-7.65(m,1H),7.56-7.49(m,4H),7.45-7.40(m,3H),7.39-7.34(m,3H),5.38(s,2H).13C NMR(100MHz,DMSO-d6,ppm):δ166.20,147.14,139.61,135.86,134.72,133.20,132.41,131.04,130.73,130.35,130.21,129.01,128.92,128.51,128.37,128.16,128.08,127.22,126.35,108.87,66.59.HRMS(ESI)calcd for C24H18N3O2SCl[M+H]+448.0808,found 448.0888.HPLC purity:98.45%,Retention time=12.14min.
2-甲酰基苯甲酸苯乙酯12a
合成方法同9a,得到透明液体2.5g,产率70%。1H NMR(400MHz,DMSO-d6,ppm):δ10.31(s,1H),7.86-7.81(m,2H),7.79-7.74(m,2H),7.32(d,J=4.4Hz,4H),7.27-7.20(m,1H),4.56(t,J=6.7Hz,2H),3.06(t,J=6.7Hz,2H).LC-MS(ESI)calcd for C16H14O3[M+H]+255.09,found 255.10.
(E)-4-(2-氯苯基)-2-(2-苯乙氧基甲酰基苄叉肼基)噻唑A12
合成方法同A1,得到黄色粉末100mg,产率51%。熔点:116-118℃.1H NMR(400MHz,DMSO-d6,ppm):δ12.46(s,1H),8.79(s,1H),8.05(d,J=7.9Hz,1H),7.94(d,J=7.7Hz,1H),7.84(d,J=7.8Hz,1H),7.71(t,J=7.7Hz,1H),7.60(d,J=7.9Hz,1H),7.54(t,J=7.6Hz,1H),7.48(t,J=7.6Hz,1H),7.46-7.34(m,6H),7.32-7.25(m,1H),4.57(t,J=6.8Hz,2H),3.14(t,J=6.9Hz,2H).13C NMR(100MHz,DMSO-d6,ppm):δ167.58,166.74,147.55,140.03,138.30,134.93,133.61,132.66,131.43,131.13,130.75,130.45,129.41,129.24,129.03,128.78,127.62,126.80,126.67,109.25,65.98,34.61.HRMS(ESI)calcd for C25H20N3O2SCl[M+H]+462.0965,found 462.1041.HPLC purity:98.60%,Retention time=12.24min.
2-甲酰基苯甲酰(N,N-二甲基)胺13a
称取邻羧基苯甲醛(1.5g,10mmol)于100mL三口瓶中,加入30mL无水二氯甲烷,加入无水DMF四滴,外部冰盐浴,电磁搅拌下加入草酰氯(1.9g,15mmol),搅拌三十分钟后,撤冰浴,转室温反应2h,检测反应完全,旋干溶剂得到黄色粘稠液体,直接用10mL无水二氯甲烷稀释备用。另取100mL单口反应瓶,向瓶中加入二甲胺(0.45g,10mmol),三乙胺(1.52g,15mmol),无水二氯甲烷10mL,冰浴下电磁搅拌十五分钟,将新制酰氯的二氯甲烷溶液经恒压滴液漏斗逐滴加到反应瓶中,冰浴反应三十分钟后,转室温反应,点板检测反应,反应结束后,向反应液中加水搅拌十分钟,静置分层,取有机相,水相用二氯甲烷萃取三次,合并有机相,干燥,旋干溶剂,过柱纯化,得到无色透明液体0.5g,产率26%。1H NMR(400MHz,DMSO-d6,ppm):δ9.97(s,1H),7.98-7.94(m,1H),7.75(td,J=7.5,1.4Hz,1H),7.67-7.61(m,1H),7.44-7.39(m,1H),3.03(s,3H),2.72(s,3H).LC-MS(ESI)calcd for C10H11NO2[M+H]+178.08,found 178.10.
(E)-4-(2-氯苯基)-2-((N,N-二甲基)甲酰基苄叉肼基)噻唑A13
合成方法同A1,得到黄色粉末80mg,产率31%。熔点:181-182℃.1H NMR(400MHz,DMSO-d6,ppm):δ12.25(s,1H),8.07(s,1H),7.94-7.89(m,2H),7.61-7.58(m,1H),7.57-7.53(m,1H),7.52-7.46(m,2H),7.44-7.40(m,2H),7.37-7.32(m,1H),3.13(s,3H),2.85(s,3H).13C NMR(100MHz,DMSO-d6,ppm):δ169.33,167.36,147.51,139.24,136.34,133.59,131.42,131.12,130.92,130.74,129.47,129.41,129.32,127.61,127.19,126.02,109.19,38.50,34.64.HRMS(ESI)calcd for C19H17N4OSCl[M+H]+385.0812,found 385.0802.HPLC purity:96.13%,Retention time=4.18min.
B系列前药的合成
2-甲基氨基硫脲4
称取甲基肼硫酸盐(12.5g,86.5mmol)于500mL三口瓶中,加入350mL无水乙醇,
电磁搅拌下加入硫氰酸铵(8g,104.0mmol),升温至回流,反应过程中TLC检测,反应48h后,将反应停止,冷却至室温,抽滤,滤液旋干,加硅胶过柱层析(洗脱剂及比例:DCM:MeOH=100:1v/v),得白色粉末状固体2.2g,产率24%。1H NMR(400MHz,DMSO-d6,ppm):δ7.38(s,2H),4.88(s,2H),3.40(s,3H).LC-MS(ESI)calcd for C2H7N3S[M+H]+106.04,found 106.10.
2-甲基-1-(2-羧基苄叉基)氨基硫脲5
称取化合物4(600mg,5.7mmol)于250mL三口瓶中,加入150mL无水乙醇,电磁搅拌下加入邻甲酰基苯甲酸(855mg,5.7mmol),升温至回流,反应中用TLC监测反应,反应4h,反应结束,停止加热,将反应液冷却至室温,加硅胶旋干溶剂,过硅胶柱层析(洗脱剂及比例:DCM:MeOH=100:1,v/v),得到白色粉末状固体700mg,产率51.9%。1H NMR(400MHz,DMSO-d6,ppm):δ13.32(br,1H),8.54(s,1H),8.50(s,1H),8.33(d,J=7.6Hz,1H),8.24(s,1H),7.90(d,J=7.8Hz,1H),7.60(t,J=7.2Hz,1H),7.52(t,J=7.2Hz,1H),3.75(s,3H).LC-MS(ESI)calcd for C2H7N3S[M+H]+238.06,found 238.10.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-羧基苄叉基)肼基]噻唑B
称取化合物5(700mg,2.94mmol)于250mL三口瓶中,加入80mL无水乙醇,电磁搅拌下加入2-溴-2’-氯苯乙酮(0.46mL,2.94mmol),加热至回流,反应用TLC检测,4h后,反应结束,停止加热将反应液冷却至室温,加硅胶后旋干溶剂,过硅胶柱层析(洗脱剂及比例:DCM:MeOH=100:1,v/v)分离,得到黄色粉末状固体540mg,产率49%。熔点:210-211℃.1H NMR(400MHz,DMSO-d6,ppm):δ13.26(s,1H),8.60(s,1H),8.04(d,J=7.6Hz,1H),7.97-7.89(m,2H),7.64(t,J=7.8Hz,1H),7.55-7.48(m,2H),7.46(s,1H),7.40(td,J1=7.6Hz,J2=1.2Hz,1H),7.32(td,J1=7.6Hz,J2=1.6Hz,1H),3.65(s,3H).13C NMR(100MHz,DMSO-d6,ppm):δ168.96,168.50,147.22,136.91,135.21,133.40,132.46,131.50,131.02,130.88,130.55,130.14,129.46,129.18,127.62,126.50,111.43,32.90.HRMS(ESI)calcd for C18H15ClN3O2S[M+H]+372.0574,found 372.0573.HPLC purity:98.15%,Retention time=9.46min.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-乙氧基甲酰基苄叉基)肼基]噻唑B1
称取化合物B(0.2g,0.54mmol)于50mL两口瓶中,加入碳酸铯(0.35g,1.08mmol),N,N-二甲基甲酰胺(DMF)10mL,电磁搅拌下加入碘乙烷(0.18g,1.08mmol),加热升温至70℃,反应过程中用TLC检测,直至反应5h后原料反应完全,停止加热,向反应瓶中加入20mL水,用二氯甲烷(DCM)萃取反应液,静置分层取有机相,水相用二氯甲烷萃取三次,合并有机相,干燥,旋干溶剂,过柱纯化,得到黄色粉末固体120mg,产率58%。熔点:109-110℃.1H NMR(400MHz,DMSO-d6,ppm):δ8.49(s,1H),8.02(d,J=8.1,1.2Hz,1H),7.96(dd,J=7.7,1.8Hz,1H),7.92-7.88(m,1H),7.69(t,J=7.6,1.4Hz,1H),7.56-7.47(m,3H),7.45-7.40(m,1H),7.37(td,J=7.6,1.8Hz,1H),4.36(q,J=7.1Hz,2H),3.69(s,3H),1.35(t,J=7.1Hz,3H).13C NMR(100MHz,DMSO-d6,ppm):δ168.51,166.58,146.72,136.19,134.55,133.00,132.32,131.12,130.69,130.36,130.36,129.08,129.06,128.88,127.25,126.36,111.10,61.16,32.55,14.04.HRMS(ESI)calcd for C20H18N3O2SCl[M+H]+400.0808,found 400.0886.HPLC purity:98.65%,Retention time=16.44min.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-丙氧基甲酰基苄叉基)肼基]噻唑B2
合成方法同B1,得到黄色粉末固体100mg,产率51%。熔点:96-97℃.1H NMR(400MHz,DMSO-d6,ppm):δ8.49(s,1H),8.02(d,J=8.0,1.2Hz,1H),7.96(dd,J=7.7,1.9Hz,1H),7.91(dd,J=7.9,1.4Hz,1H),7.72-7.67(m,1H),7.57-7.46(m,3H),7.43(td,J=7.5,1.4Hz,1H),7.37(td,J=7.6,1.8Hz,1H),4.27(t,J=6.6Hz,2H),3.68(s,3H),1.75(h,J=7.1Hz,2H),0.98(t,J=7.4Hz,3H).13C NMR(100MHz,DMSO-d6,ppm):δ169.00,167.14,147.22,136.65,135.11,133.49,132.84,131.62,131.19,130.87,130.81,129.59,129.53,129.42,127.76,126.94,111.61,67.09,33.05,21.99,10.89.HRMS(ESI)calcd for C21H20N3O2SCl[M+H]+414.0965,found 414.1042.HPLC purity:99.75%,Retention time=15.44min.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-异丙氧基甲酰基苄叉基)肼基]噻唑B3
合成方法同B1,得到淡黄色粉末140mg,产率57%。熔点:128-130℃.1H NMR(400MHz,DMSO-d6,ppm):δ8.48(s,1H),8.02(d,J=8.0,1.2Hz,1H),7.96(dd,J=7.7,1.8Hz,1H),7.88(d,J=7.8,1.4Hz,1H),7.71-7.65(m,1H),7.56-7.47(m,3H),7.46-7.39(m,1H),7.36(td,J=7.6,1.8Hz,1H),5.21(hept,J=6.3Hz,1H),3.69(s,3H),1.36(d,J=6.2Hz,6H).13C NMR(100MHz,DMSO-d6,ppm):δ168.52,166.09,146.73,136.11,134.45,133.01,132.24,131.12,130.70,130.36,129.43,129.08,128.87,127.25,126.27,111.10,68.75,32.54,21.59.HRMS(ESI)calcd for C21H20N3O2SCl[M+H]+414.0965,found 414.0955.HPLC purity:99.65%,Retention time=12.44min.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-丁氧基甲酰基苄叉基)肼基]噻唑B4
合成方法同B1,得到黄色粉末固体190mg,产率61%。熔点:135-136℃.1H NMR(400MHz,DMSO-d6,ppm):δ8.49(s,1H),8.01(d,J=7.9,1.2Hz,1H),7.96(dd,J=7.7,1.8Hz,1H),7.90(dd,J=7.8,1.3Hz,1H),7.72-7.66(m,1H),7.56-7.46(m,3H),7.43(td,J=7.5,1.4Hz,1H),7.36(td,J=7.6,1.9Hz,1H),4.31(t,J=6.6Hz,2H),3.68(s,3H),1.71(p,2H),1.42(h,2H),0.93(t,J=7.4Hz,3H).13C NMR(100MHz,DMSO-d6,ppm):δ168.98,167.14,147.21,136.62,135.11,133.49,132.81,131.62,131.18,130.86,130.80,129.58,129.50,129.40,127.75,126.95,111.60,65.33,33.03,30.60,19.26,14.06.HRMS(ESI)calcd for C22H22N3O2SCl[M+H]+428.1121,found 428.1131.HPLC purity:98.65%,Retention time=20.44min.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-(2-丁氧基)甲酰基苄叉基)肼基]噻唑B5
合成方法同B1,得到淡黄色粉末160mg,产率56%。熔点:131-132℃.1H NMR(400MHz,DMSO-d6,ppm):δ8.49(s,1H),8.02(dd,J=8.0,1.2Hz,1H),7.96(dd,J=7.7,1.8Hz,1H),7.90(dd,J=7.8,1.4Hz,1H),7.72-7.66(m,1H),7.56-7.46(m,3H),7.43(td,J=7.5,1.4Hz,1H),7.37(td,J=7.6,1.9Hz,1H),5.06(h,J=6.3Hz,1H),3.69(s,3H),1.76-1.62(m,2H),1.33(d,J=6.3Hz,3H),0.93(t,J=7.5Hz,3H).13C NMR(100MHz,DMSO-d6,ppm):δ169.02,166.68,147.22,136.57,135.04,133.49,132.80,131.62,131.19,
130.87,130.79,129.81,129.60,129.43,127.76,126.84,111.62,73.57,33.04,28.71,19.66,10.04.HRMS(ESI)calcd for C22H22N3O2SCl[M+H]+428.1121,found 428.1131.HPLC purity:98.95%,Retention time=11.42min.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-戊氧基甲酰基苄叉基)肼基]噻唑B6
合成方法同B1,得到淡黄色粉末130mg,产率55%。熔点:117-118℃.1H NMR(400MHz,DMSO-d6,ppm):δ8.48(s,1H),8.04-7.85(m,3H),7.69(t,J=7.6Hz,1H),7.59-7.31(m,5H),4.30(t,J=6.5Hz,2H),3.68(s,3H),1.78-1.64(m,2H),1.35(s,4H),0.88(t,J=6.7Hz,3H).13C NMR(100MHz,DMSO-d6,ppm):δ168.99,167.18,147.22,136.69,135.08,133.50,132.80,131.62,131.19,130.87,130.77,129.60,129.57,129.42,127.76,127.01,111.60,65.64,33.05,28.26,28.19,22.28,14.33.HRMS(ESI)calcd for C23H24N3O2SCl[M+H]+442.1278,found 442.1265.HPLC purity:98.21%,Retention time=22.10min.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-己氧基甲酰基苄叉基)肼基]噻唑B7
合成方法同B1,得到浅黄色粉末170mg,产率49%。熔点:88-89℃.1H NMR(400MHz,DMSO-d6,ppm):δ8.48(s,1H),8.02-7.94(m,2H),7.88(dd,J=7.8,1.3Hz,1H),7.72-7.66(m,1H),7.58-7.46(m,3H),7.43(td,J=7.6,1.4Hz,1H),7.36(td,J=7.6,1.9Hz,1H),4.30(t,J=6.6Hz,2H),3.68(s,3H),1.75-1.66(m,2H),1.41-1.25(m,6H),0.88-0.82(m,3H).13C NMR(100MHz,DMSO-d6,ppm):δ237.52,235.49,158.25,145.81,132.69,131.51,131.35,131.07,130.76,129.48,129.31,127.65,126.94,125.32,124.37,91.91,65.55,32.94,31.27,28.41,25.58,22.38,14.24.HRMS(ESI)calcd for C24H26N3O2SCl[M+H]+456.1434,found 456.1410.HPLC purity:97.55%,Retention time=13.12min.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-环己氧基甲酰基苄叉基)肼基]噻唑B8
合成方法同B1,得到淡黄色粉末130mg,产率53%。熔点:134-136℃.1H NMR(100MHz,DMSO-d6,ppm):δ8.50(s,1H),8.02(d,J=8.0,1.3Hz,1H),7.96(dd,J=7.7,1.8Hz,1H),7.90(dd,J=7.9,1.4Hz,1H),7.72-7.66(m,1H),7.56-7.47(m,3H),7.43(td,J=7.5,1.5Hz,1H),7.37(td,J=7.6,1.9Hz,1H),5.03-4.95(m,1H),3.69(s,3H),1.98-1.90(m,2H),1.79–1.65(m,2H),1.61-1.49(m,3H),1.47-1.29(m,3H).13C NMR(100MHz,DMSO-d6,ppm):δ169.02,166.44,147.22,136.65,135.03,133.50,132.81,131.62,131.20,130.87,129.89,129.61,129.43,127.77,126.82,111.62,73.78,33.06,31.51,25.32,23.69.HRMS(ESI)calcd for C24H24N3O2SCl[M+H]+454.1278,found 454.1294.HPLC purity:97.32%,Retention time=11.53min.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-苯乙氧基甲酰基苄叉基)肼基]噻唑B9
合成方法同B1,不同之处为加入等摩尔量的碘化钾,得到黄色粉末100mg,产率46%。熔点:125-126℃.1H NMR(400MHz,DMSO-d6,ppm):δ8.40(s,1H),8.02-7.93(m,2H),7.84-7.80(m,1H),7.71-7.66(m,1H),7.56-7.47(m,3H),7.43(td,J=7.5,1.5Hz,1H),7.37(td,J=7.6,1.9Hz,1H),7.31(d,J=4.3Hz,4H),7.26-7.19(m,1H),4.54(t,J=6.7Hz,2H),3.61(s,3H),3.06(t,J=6.7Hz,2H).13C NMR(100MHz,DMSO-d6,ppm):δ168.88,166.89,147.13,138.34,136.45,135.03,133.42,132.77,131.52,131.10,130.76,130.60,129.49,129.24,128.77,127.65,126.90,126.80,111.49,65.95,34.67,32.92.HRMS(ESI)calcd for C26H22N3O2SCl[M+H]+476.1121,found 476.1201.HPLC purity:99.01%,Retention time=9.75min.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-特戊酸甲氧基甲酰基苄叉基)肼基]噻唑B10
合成方法同B9,得到亮黄色粉末120mg,产率57%。熔点:126-127℃.1H NMR(400MHz,DMSO-d6,ppm):δ8.44(s,1H),8.03(d,J=8.0,1.2Hz,1H),7.96(dd,J=7.7,1.9Hz,1H),7.89-7.85(m,1H),7.77-7.71(m,1H),7.57-7.53(m,2H),7.49(s,1H),7.43(td,J=7.5,1.4Hz,1H),7.37(td,J=7.6,1.9Hz,1H),5.99(s,2H),3.69(s,3H),1.17(s,9H).13C NMR(100MHz,DMSO-d6,ppm):δ176.91,168.94,165.74,147.24,136.17,135.56,133.60,133.48,131.64,131.20,130.97,130.87,129.62,129.56,128.04,127.77,127.18,111.73,89.14,80.79,33.07,26.96.HRMS(ESI)calcd for C24H24N3O4SCl[M+H]+486.1176,found 486.1256.HPLC purity:97.64%,Retention time=13.15min.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-异丙基碳酸甲氧基甲酰基苄叉基)肼基]噻唑B11
合成方法同B9,得到萤黄色粉末90mg,产率54%。熔点:122-123℃.1H NMR(400MHz,DMSO-d6,ppm):δ8.74(s,1H),8.30(d,J=8.0,1.3Hz,1H),8.15(dd,J=8.0,1.4Hz,1H),8.09(dd,J=7.8,1.8Hz,1H),7.74-7.67(m,1H),7.54-7.47(m,2H),7.44-7.36(m,2H),7.36-7.28(m,2H),6.10(s,2H),5.07-5.00(m,1H),3.86(s,3H),1.42(d,J=6.2Hz,6H).13C NMR(100MHz,DMSO-d6,ppm):δ169.03,165.22,153.36,147.30,136.67,134.75,133.43,133.13,131.72,131.21,131.12,130.39,128.36,128.31,126.86,126.71,126.38,110.68,82.11,73.18,32.67,21.57.HRMS(ESI)calcd for C23H22N3O5SCl[M+H]+488.0969,found 488.1046.HPLC purity:95.65%,Retention time=6.77min.
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-(氯甲酰基苄叉基)肼基]噻唑12b
称取化合物B(0.2g,0.54mmol)于50mL两口瓶中,加入二氯甲烷10mL,N,N-二甲基甲酰胺(DMF)2d,冰浴搅拌十分钟,取草酰氯(0.13g,1.08mmol)溶于5mL二氯甲烷中逐滴滴加入反应瓶中。继续冰浴,半小时,撤冰浴,转室温反应2h,检测反应:取反应液于小样品管内加甲醇震荡后加水和乙酸乙酯,静置分层,取上层有机相点板,发现原料点基本消失。说明酰氯制备成功。将反应液溶剂旋干,得到黄色液体。直接用于下一步反应。
(E)-4-(2-氯苯基)-2-[1-甲基-2-(2-((N,N-二甲基)甲酰基苄叉基)肼基]噻唑B12
取50mL单口反应瓶,向瓶中加入二甲胺(24mg,0.54mmol),三乙胺(55mg,0.54mmol),无水二氯甲烷10mL,冰浴下电磁搅拌十五分钟,将新制的酰氯二氯甲烷溶液逐滴加到反应瓶中,冰浴反应三十分钟后,转室温反应10h,用TLC检测反应,反应结束后,向反应液中加水和二氯甲烷各5mL,搅拌十分钟,静置分层,取有机相,水相用二氯甲烷萃取三次,合并有机相,干燥,旋干溶剂,过柱纯化,得到灰白的固体粉末50mg,产率25%。熔点:160-161℃.1H NMR(400MHz,DMSO-d6,ppm):δ7.96(dd,J=8.0,
2Hz,1H),7.87(d,J=7.6Hz,1H),7.73(s,1H),7.55-7.29(m,7H),3.65(s,3H),3.07(s,3H),2.80(s,3H).13C NMR(100MHz,DMSO-d6,ppm):δ169.57,168.91,147.14,136.30,135.65,133.52,131.62,131.34,131.19,130.86,129.58,129.46,127.76,127.59,127.22,111.59,38.62,34.90,32.95.HRMS(ESI)calcd for C20H19N4OSCl[M+H]+399.0968,found 399.1048.HPLC purity:99.75%,Retention time=4.61min.
实施例2:
hsDHODH蛋白的培养与纯化
蛋白的培养:按照常规方法,例如Sambrook等,分子克隆:实验室指南(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,根据GenBank中的hsDHODH基因序列获得hsDHODH蛋白。
将测序正确的重组质粒pET-19b-DHODH转化E.coli BL21(DE3)感受态,涂布于含氨苄青霉素的LB平板上培养,随机挑取菌种接种于含100μM氨苄青霉素的LB培养基中37℃、230rpm摇床培养过夜。按1:200的比例接种于500mL含100μL氨苄青霉素的LB培养基中37℃、230rpm扩大培养。待菌体OD值达到0.8-1时,向培养基中加入IPTG,使IPDG终浓度为0.5mM,25℃过夜诱导表达。于4℃、4000rmp离心收集诱导后菌体,用去离子水洗涤之后再次离心收集菌体沉淀,于-80℃保存。
蛋白的纯化使用裂解液重悬菌体,裂解液中含有50mM HEPES(pH 8.0),0.15M NaCl,10mM咪唑,10%甘油,0.1%Triton X-100,并加入少许大豆蛋白酶抑制剂,重悬混合均匀后超声破碎菌体。破碎液离心30min,取上清和沉淀进行蛋白电泳以确定蛋白存在形式。将上清液加入Ni-NTA层析柱中,收集穿过液,然后用含20mM咪唑的裂解液洗树脂3-4次,最后用含300mM咪唑的裂解液洗脱,收集洗脱后蛋白液。取以上10μL蛋白样进行SDS-PAGE电泳,测定目的蛋白含量。最后,将洗脱后的蛋白液在透析液中透析从而去除咪唑,即得纯品蛋白。
化合物的酶水平活性测试方法
酶水平活性测试的原理是:首先二氢乳清酸(Dihydroorotate,DHO)在DHODH的催化下氧化脱氢生成乳清酸(Orotate,OA),同时伴随黄素单核苷酸(Flavin mononucleotide,FMN)接受2H+及2e-还原为还原型黄素单核苷酸(FMNH2);随后辅酶Q(COQ)作为氢受体接受FMNH2的电子和质子还原为还原型辅酶Q(COQH2),还原型辅酶Q将电子传递给显色底物二氯靛酚钠盐(DCIP),最终DCIP被还原。DCIP在600nm处有最大吸收,而还原态的DCIP在600nm处没有吸收。根据吸光度的减弱程度即可判断底物DHO被氧化的程度。单位时间内底物DHO被氧化程度即为酶促反应初速度。加入抑制剂后,酶促反应初速度降低。
将纯化后的DHODH用测活缓冲液(50mM HEPES,pH 8.0,150mM KCl,0.1
%Triton X-100)稀释至10nM,加入辅酶Q及DCIP,使其终浓度分别为100μM及120μM,混合均匀后加入96孔板,室温孵育5min,加入底物DHO启动反应,DHO终浓度500μM。使用BioTek酶标仪在600nm处读取吸光值,每30s读一次,持续6min。抑制剂的活性测试即在上述反应体系中加入不同浓度的抑制剂,未加抑制剂时酶促反应初速度为V0,,加抑制剂后酶促反应初速度Vi,化合物的抑制率由公式(1-Vi/V0)×100%计算。化合物IC50的计算,使用Origin 8.0,至少测试8个浓度下的抑制率。实验过程采用Brequinar(购买自Sigma-Aldrich试剂公司)为阳性对照,每次实验至少设三个平行。结果见下表:
类似地,本发明化合物的药学上可接受的酯的测试结果如下表所示:
讨论:
从在酶水平进行的本发明化合物的活性检测结果可知,本发明的化合物具备及其优异的DHODH抑制活性,其中一些化合物抑制DHODH的IC50值甚至达到1-10nM的级别。
因此,本发明的化合物具备成为低毒性、高安全性的新型DHODH抑制剂的前景。
实施例3:
化合物1对类风湿性关节炎模型的药效学评价
1.1.动物分组
表A-1 化合物1Wistar大鼠分组情况
1.2.溶液配制
本实验选用的动物模型为用Wistar大鼠建立的II型胶原诱导的关节炎动物模型(CIA)。在实验第0天和第7天对大鼠进行皮下注射牛II型胶原(Bovine II Collgen,CII)和不完全弗氏佐剂(Incomplete Freund’s Adjuvant,IFA)的混合溶液,建立关节炎大鼠模型。
化合物1药效测试部分,化合物1和阳性药甲氨蝶呤均溶解于二甲基亚砜(DMSO)、聚乙二醇400(PEG 400)以及生理盐水以体积比1:10:9组成的溶剂体系中。
1.3.进度安排见图1。
1.4实验结果
1.4.1.关节炎评估指标
在对大鼠给药期间(实验第0天至27天),每三天称量一次大鼠体重,观察其生长情况。大鼠关节开始肿胀发病后,每两天进行一次关节肿胀程度评分,具体评分标准如表B所示。
表B 大鼠关节肿胀评分标准
由于前爪炎症发生率较低,后爪较易发生肿胀且肿胀程度更严重,因此采用两只后爪的评分之和来评价大鼠关节炎发病的情况,最大评分总和为8分。
1.4.2.化合物1对CIA大鼠体重和关节肿胀程度的影响
本实验选用Wistar大鼠建立CIA动物模型,第28天取关节,以进行后续的固定、石蜡包埋、切片、染色及组织病理学观察等实验。实验结果如下:
实验期间,对照组大鼠体重持续增长。跟模型组相比,模型组大鼠体重增长稍显缓慢。给予化合物1和甲氨蝶呤可以改善大鼠状态及体重增长缓慢的情况(图2A)。
实验第10天,即加强免疫后的第三天,造模大鼠陆续开始发病,实验第18天左右模型组大鼠关节肿胀最为严重,此后病情趋于稳定。给予阳性药甲氨蝶呤(0.3mg/kg)可以显著降低大鼠关节肿胀程度的评分,给予化合物1(5mg/kg、30mg/kg)的大鼠,跟模型组相比,关节肿胀程度也有明显减弱,尤其是给予较高剂量(30mg/kg)时肿胀减弱更明显,第28天时,高剂量组与阳性药组关节肿胀程度评分十分相近(图2B)。
图2C显示的是实验第28天时各不同处理组中具有代表性的大鼠足部照片。从图2C中可以看出模型组大鼠的足部肿胀最严重,给予阳性药和两个不同浓度的化合物1后足部肿胀的程度均有所改善,并且高剂量的化合物1改善效果比低剂量的更加明显。
以上体重增长趋势和关节肿胀程度评分的结果表明,化合物1可能具有很强的改善CIA动物模型关节肿胀的作用。
1.4.3.化合物1对CIA大鼠组织病理学的影响
化合物1对关节炎大鼠组织病理学影响的结果如图3所示,模型组大鼠关节组织中有明显炎性细胞浸润的现象,同时伴有严重的软骨损伤和骨侵蚀。给予化合物1处理的大鼠,该组织病理学特征显著减轻,并且剂量较高的一组改善更加明显,这表明化合物1可能具有显著的抗关节炎作用,并且该作用具有一定的剂量依赖关系。
1.5小结
实验利用Wistar大鼠建立了II型胶原诱导的关节炎动物模型,对DHODH抑制剂化合物1对关节炎的治疗效果进行了测试。结果显示,建立关节炎模型并给予化合物1治疗后,可以改善建立模型引起的体重下降情况,并可使大鼠关节肿胀减轻。
此外,组织切片的结果也显示,给予化合物1治疗的大鼠滑膜增生和炎症细胞浸润现象跟模型组相比有很大的改善。
以上结果表明,化合物1具有抗炎的作用,是RA治疗较有潜力的化合物,且具有一定的剂量依赖关系。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附
权利要求书所限定的范围。
Claims (11)
- 如权利要求2所述的化合物或其药学上可接受的盐或酯,其特征在于,R1选自:C1-C6烷基(优选C1-C3烷基);R3选自:H、取代或未取代的C1-C6烷基(优选C1-C3烷基);R5和R6独立选自:H、卤素(优选F)、未取代的或卤素(优选F)取代的C1-C3烷基;R7和R8独立选自:H、卤素(优选Cl);R9是H。
- 如权利要求2所述的化合物或其药学上可接受的盐或酯,其特征在于,R1选自:H、C1-C6烷基,优选C1-C3烷基;R3选自:H、取代或未取代的C1-C6烷基(优选C1-C3烷基);R5选自:H、卤素(优选F)、未取代的或卤素(优选F)取代的C1-C3烷基;R6选自:H、卤素(优选F)、羟基、NH2;R7和R8独立选自:H、卤素(优选Cl);和R9是H。
- 一种药物组合物,其特征在于,所述药物组合物含有权利要求1-6中任一项所述的化合物或其药学上可接受的盐或酯,以及药学上可接受的载体或赋形剂。
- 权利要求1-6中任一项所述的化合物或其药学上可接受的盐或酯的用途,用于制备治疗DHODH介导的疾病的药物。
- 如权利要求8所述的用途,其特征在于,所述DHODH介导的疾病包括但不限于:自身免疫疾病、细菌感染、寄生虫和肿瘤。
- 如权利要求9所述的用途,其特征在于,所述自身免疫性疾病包括但不限于:类风湿性关节炎、器官移植排异、银屑病。
- 一种治疗DHODH介导的疾病的方法,其特征在于,所述方法包括给予需要的对象权利要求1-6中任一项所述的化合物或其药学上可接受的盐或酯,或权利要求7所述的药物组合物。
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WO2021175224A1 (zh) * | 2020-03-02 | 2021-09-10 | 华东理工大学 | 抗rna病毒药物及其应用 |
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