选择性氮杂吲哚类JAK抑制剂的设计、合成及体外活性评价

发布时间：2018-06-20 03:03

本文选题：类风湿性关节炎 + 蛋白质激酶　；参考：《中国人民解放军军事医学科学院》2017年硕士论文

【摘要】：类风湿性关节炎(rheumatoid arthritis,RA)是一种全身性自身免疫性疾病,也是临床上常见的慢性疾病。这种疾病的特征在于持续关节滑膜炎和全身性炎症,小关节处是RA患者中常见的病变部位,发病初期的症状一般表现为关节肿胀、疼痛、发僵。然而,滑膜炎反复发作并且持续存在,这会导致患者关节软骨和骨的不可逆损伤,严重影响其正常活动,给其生活带来极大的不便。病情发展严重时,对患者可致畸致残,也存在引起心血管或是其他并发症的可能性。关于RA的发病机制,至今尚无明确而系统的阐述。在已有的研究基础上,人们比较认同的观点是:各种各样复杂的内因和外因共同作用,致使患者体内滑膜细胞和软骨细胞行为异常,许多细胞因子过量产生。这些细胞因子通过不同的方式激活关键的信号转导通路,形成一系列炎症级联反应,最终导致患者关节滑膜结构损伤,表现出RA的典型临床症状。正是由于RA发病机制的复杂性,对其治疗的方案也多种多样,但目前都未能达到将RA完全治愈的理想效果,人们只能以短期治疗来追求长期缓解。不同的治疗手段在RA的治疗方面都发挥着重要的作用,而各种有效的治疗手段中最为重要的当数药物治疗,非留体抗炎药、改善病情抗风湿药和糖皮质激素三大类药物一度成为治疗RA的经典药物,在市场上占有重要的地位。随着人们对RA研究的日渐深入,具有新的作用机制且治疗效果更好的药物成为了 RA治疗市场上的新起之秀。近年来,化学小分子蛋白质激酶抑制剂(PKi)在疾病治疗方面的应用吸引了众多医药工作者的注意力,它在RA治疗领域的应用也成为了人们的迫切期待。Janus激酶(JAK)、脾酪氨酸激酶(Syk)和p38激酶等成为了治疗RA的热门蛋白质激酶靶点。其中靶向JAK的药物研发已经取得成功,辉瑞公司研发的Tofacitinib和因赛特与礼来公司联合研发的Baricitinib就是这类药物的代表。但Tofacitinib和Baricitinib对JAK的亚型激酶抑制作用没有单一的选择性,它们的临床试验结果表明,单一亚型的JAK抑制剂或许会在保持良好的RA治疗效果的同时,避免或减小不必要毒副作用的产生,提高其应用的临床安全性。为了寻找并研发具有JAK亚型激酶抑制选择性的化合物分子,本课题选择以Baricitinib分子为先导化合物,参考已经报道的Tofacitinib分子与JAK各亚型间的作用关系,同时还借鉴在研的具有单一亚型选择性抑制作用的JAK抑制剂Filgotinib (作用靶点为JAK1)和Decernotinib (作用靶点为JAK3)的分子结构,利用生物电子等排、优势分子片段拼接等策略进行分子结构优化和改造。在目标化合物的化学合成过程中,本课题探索并改进了合理的合成路线,针对关键步骤的反应条件和关键中间体的合成方法进行了的优化,提升了目标化合物实验室放大合成实验操作的可行性。最后使用毛细管电泳方法通过检测底物肽段磷酸化转化率,测定了目标化合物对3种JAK亚型激酶(JAK1、JAK2、JAK3)的半抑制浓度(halfmaximal inhibitory concentration,IC50)值(由于与 TYK2 的主要相关疾病是牛皮癣和炎症性肠病而并非类风湿性关节炎,因此并未测定目标化合物对TYK2的抑制活性),以此来评价所设计并合成的目标化合物体外JAK抑制活性和选择性。在目标化合物分子的设计过程中,本课题先对先导化合物分子进行了母核的改造,用C-3位取代的7-氮杂吲哚结构替换Baricitinib分子中的7H-吡咯并[2,3-d]嘧啶结构,以保证目标分子在JAK铰链区的有效固定,并进一步对酰胺尾部进行了优化,重点考察了不同的取代酰胺对化合物体外酶抑制活性的影响,设计得到此类化合物J-01～J-10, 10个目标化合物结构均未见报道。通过体外JAK抑制活性测试,发现此类目标化合物相比于Baricitinib活性有所下降,但都表现出了激酶亚型抑制选择性,尤其是化合物J-01和J-04分别对JAK2和JAK1表现出了较好的抑制选择性。并且发现尾部的酰胺取代基对这类化合物抑制选择性的影响较大,小的脂肪环取代基有增加JAK2选择性的作用,而芳环取代基则更有利于提高目标化合物对JAK1的抑制选择性。另外,改构所得的此类分子中,氰基结构与激酶间的作用效果不易被其他基团替代,将氰基结构变换为氨基或三氟乙胺基后,目标化合物的体外JAK抑制活性有显著降低。另一方面,本课题又对化合物分子母核上的连接基团部分进行了片段拼接,借鉴Filgotinib分子中的苯环连接片段,将其拼接至化合物分子中的7-氮杂吲哚骨架上得到目标化合物J-11～J-14,也尝试着直接将该片段拼接至Baricitinib分子中的7H-吡咯并[2,3-d]嘧啶结构上得到目标化合物J-15～J-19,设计得到了9个未见报道的新结构目标化合物。体外JAK抑制活性试验结果显示,化合物J-11～J-14的激酶抑制活性下降的较为明显,而且并未表现出理想的抑制选择性。然而目标化合物J-15～J-19的酶抑制活性虽然也有降低,但其中目标化合物J-16和J-19都对JAK2表现出了较好的抑制选择性。在目标化合物的化学合成过程中,我们分别选择以3-溴-7-氮杂吲哚和4-氯-7H-吡咯并[2,3-d]嘧啶为起始原料,经过氨基保护、Suzuki芳基偶联、Michael加成、亲核取代、水解、催化缩合、氨基去保护等关键步骤来合成目标化合物分子,并通过核磁氢谱进行了化合物的结构鉴定。最后还对Suzuki芳基偶联反应的最佳实验条件及关键中间体7a～7g的合成路线、方法进行了探索改进,合理地避开了化学性质不稳定的中间体,以获得理化性质较稳定的关键中间体,为目标化合物在实验室放大合成的工艺改进和优化奠定了基础。本课题中,我们设计并成功合成了共19个目标化合物,其结构未见文献报道,并对其体外Janus激酶抑制活性进行了评价,结果表明化合物J-01、J-04、J-16和J-19为选择性JAK抑制剂,为该课题的进一步深入研究奠定了基础。
[Abstract]:Rheumatoid arthritis (RA) is a systemic autoimmune disease, which is also a common chronic disease in clinical. This disease is characterized by continuous synovitis and systemic inflammation. The small joints are common lesions in the RA patients. The symptoms of the early onset of the disease are generally manifested in joint swelling, pain, and hair. However, the recurrence and persistence of synovitis will lead to irreversible damage to the cartilage and bone of the joint, which seriously affects its normal activities and causes great inconvenience to its life. When the disease is serious, the patients can be deformity, and the possibility of causing cardiovascular or other complications is also present. The pathogenesis of RA, There is no clear and systematic explanation to this day. On the basis of the existing research, people agree that a variety of complex internal and external causes lead to abnormal behavior of synovial cells and chondrocytes in the patient's body and the excessive production of many cytokines. These cytokines activate key signals in different ways. A series of inflammatory cascade reactions lead to a series of inflammatory cascade reactions that eventually lead to the damage of the synovial membrane structure and the typical clinical symptoms of RA. It is because of the complexity of the pathogenesis of RA that the treatment scheme is varied, but at present, the rational effect of the complete cure of RA has not been reached, and people can only pursue long-term slow treatment with short-term treatment. Different treatments are playing an important role in the treatment of RA, and the most important treatments in various effective treatments, the non retention of anti inflammatory drugs, the improvement of the disease resistant and rheumatic drugs and glucocorticoid three major drugs have once become the classic drugs for the treatment of RA. In recent years, the application of chemical small molecule protein kinase inhibitor (PKi) in the treatment of disease has attracted the attention of many medical workers, and its application in the field of RA treatment has also become a people's attention in the field of the treatment of RA. The urgent expectation of.Janus kinase (JAK), splenic tyrosine kinase (Syk) and p38 kinase, has become a popular protein kinase target for the treatment of RA. The drug targeted to JAK has been successfully developed, and the Baricitinib developed by Pfizer Inc and the joint research and development of JAK and Lilly is the representative of these drugs. But Tofacitinib and B. Aricitinib has no single selectivity for JAK subtype kinase inhibition. Their clinical trial results show that a single subtype of JAK inhibitors may avoid or reduce the production of unnecessary side effects and improve the safety of their applications while maintaining good effects of RA therapy. In order to find and develop JAK subtype excitation In this study, Baricitinib molecules are selected as the precursor compounds, referring to the relationship between the reported Tofacitinib molecules and the JAK subtypes. At the same time, the JAK inhibitor, Filgotinib (the target target JAK1) and the Decernotinib (target target), is also used for reference. The molecular structure of JAK3) is used to optimize and reconstruct the molecular structure by using the bioelectronic platoon and the splicing of the dominant molecular fragments. In the process of chemical synthesis of the target compounds, this subject has explored and improved the rational synthetic route, and optimized the reaction conditions of key steps and the synthesis methods of key intermediates. The feasibility of the experimental operation was raised in the laboratory of the target compound. Finally, the semi inhibitory concentration (halfmaximal inhibitory concentration, IC50) of the target compounds to 3 JAK subtypes kinase (JAK1, JAK2, JAK3) was measured by capillary electrophoresis with the detection of the conversion rate of phosphorylation of the substrate peptide segment (the main phase with TYK2. The disease is psoriasis and inflammatory bowel disease rather than rheumatoid arthritis, so the target compound's inhibitory activity to the TYK2 is not measured to evaluate the JAK inhibitory activity and selectivity of the designed and synthesized target compounds in vitro. In the course of the design of the target compounds, the subject first carried out the pilot compound molecules. In order to guarantee the effective immobilization of the target molecules in the JAK hinge region, the structure of the 7H- pyrrole and [2,3-d] pyrimidine in the Baricitinib molecule was replaced with the structure of the 7- nitrogen heteroindole structure substituted by C-3 bit, which was used to replace the 7H- pyrrole and [2,3-d] pyrimidine structure in the Baricitinib molecule. The structure of the 10 target compounds, J-01 ~ J-10, was not reported. Through the test of JAK inhibitory activity in vitro, it was found that this kind of target compounds decreased compared to Baricitinib activity, but all of them showed the inhibitory selectivity of the kinase subtype, especially the compounds J-01 and J-04 showed good inhibition to JAK2 and JAK1, respectively. It is found that the amide substituents in the tail have great influence on the inhibition of selectivity of these compounds. Small fatty ring substituents have the effect of increasing the selectivity of JAK2, while the aromatic ring substituents are more beneficial to the inhibition of the inhibitory selectivity of the target compounds to the JAK1. When the effect is not easy to be replaced by other groups, the JAK inhibitory activity of the target compounds in vitro is significantly reduced after the cyanic structure is transformed into amino or three fluoroethylamine group. On the other hand, the joint group part of the molecular nucleus of the compound is spliced and used for reference to the connection fragment of the benzene ring in the Filgotinib molecule. The target compounds J-11 to J-14 were obtained from the 7- azaindole skeleton in the compound molecules, and the target compound J-15 to J-19 was obtained directly by splicing the fragment into the 7H- pyrrole and [2,3-d] pyrimidine in the Baricitinib molecule. 9 new structure target compounds were not reported. The results showed that the inhibitory activity of the kinase J-11 to J-14 decreased significantly and did not exhibit an ideal inhibitory selectivity. However, the inhibitory activity of the target compounds J-15 to J-19 was also reduced, but the target compounds J-16 and J-19 both showed a better inhibition selectivity to JAK2. In the chemistry of the target compounds. In the process of synthesis, we selected 3- bromine -7- nitrogen hetero indole and 4- chloride -7H- pyrrole and pyrrole as starting materials, through amino protection, Suzuki aryl coupling, Michael addition, nucleophilic substitution, hydrolysis, catalytic condensation, amino de protection and other key steps to synthesize the target compounds. Finally, the best experimental conditions for the Suzuki aryl coupling reaction and the synthesis route of the key intermediate 7a to 7g were also explored and improved. The intermediates with unstable chemical properties were reasonably avoided to obtain the key intermediates with stable physicochemical properties, and the process improvement of the target compound in the laboratory was improved and the synthesis process was improved. In this subject, we have designed and successfully synthesized 19 target compounds. The structure of the compounds has not been reported, and the inhibitory activity of Janus kinase in vitro is evaluated. The results show that compounds J-01, J-04, J-16 and J-19 are selective JAK inhibitors, which lays the foundation for further research on this subject.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914;R96

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