發(fā)布時(shí)間：2018-06-20 03:03

  本文選題：類風(fēng)濕性關(guān)節(jié)炎 + 蛋白質(zhì)激酶��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文

【摘要】：類風(fēng)濕性關(guān)節(jié)炎(rheumatoid arthritis,RA)是一種全身性自身免疫性疾病,也是臨床上常見的慢性疾病。這種疾病的特征在于持續(xù)關(guān)節(jié)滑膜炎和全身性炎癥,小關(guān)節(jié)處是RA患者中常見的病變部位,發(fā)病初期的癥狀一般表現(xiàn)為關(guān)節(jié)腫脹、疼痛、發(fā)僵。然而,滑膜炎反復(fù)發(fā)作并且持續(xù)存在,這會(huì)導(dǎo)致患者關(guān)節(jié)軟骨和骨的不可逆損傷,嚴(yán)重影響其正�；顒�(dòng),給其生活帶來極大的不便。病情發(fā)展嚴(yán)重時(shí),對(duì)患者可致畸致殘,也存在引起心血管或是其他并發(fā)癥的可能性。關(guān)于RA的發(fā)病機(jī)制,至今尚無明確而系統(tǒng)的闡述。在已有的研究基礎(chǔ)上,人們比較認(rèn)同的觀點(diǎn)是:各種各樣復(fù)雜的內(nèi)因和外因共同作用,致使患者體內(nèi)滑膜細(xì)胞和軟骨細(xì)胞行為異常,許多細(xì)胞因子過量產(chǎn)生。這些細(xì)胞因子通過不同的方式激活關(guān)鍵的信號(hào)轉(zhuǎn)導(dǎo)通路,形成一系列炎癥級(jí)聯(lián)反應(yīng),最終導(dǎo)致患者關(guān)節(jié)滑膜結(jié)構(gòu)損傷,表現(xiàn)出RA的典型臨床癥狀。正是由于RA發(fā)病機(jī)制的復(fù)雜性,對(duì)其治療的方案也多種多樣,但目前都未能達(dá)到將RA完全治愈的理想效果,人們只能以短期治療來追求長(zhǎng)期緩解。不同的治療手段在RA的治療方面都發(fā)揮著重要的作用,而各種有效的治療手段中最為重要的當(dāng)數(shù)藥物治療,非留體抗炎藥、改善病情抗風(fēng)濕藥和糖皮質(zhì)激素三大類藥物一度成為治療RA的經(jīng)典藥物,在市場(chǎng)上占有重要的地位。隨著人們對(duì)RA研究的日漸深入,具有新的作用機(jī)制且治療效果更好的藥物成為了 RA治療市場(chǎng)上的新起之秀。近年來,化學(xué)小分子蛋白質(zhì)激酶抑制劑(PKi)在疾病治療方面的應(yīng)用吸引了眾多醫(yī)藥工作者的注意力,它在RA治療領(lǐng)域的應(yīng)用也成為了人們的迫切期待。Janus激酶(JAK)、脾酪氨酸激酶(Syk)和p38激酶等成為了治療RA的熱門蛋白質(zhì)激酶靶點(diǎn)。其中靶向JAK的藥物研發(fā)已經(jīng)取得成功,輝瑞公司研發(fā)的Tofacitinib和因賽特與禮來公司聯(lián)合研發(fā)的Baricitinib就是這類藥物的代表。但Tofacitinib和Baricitinib對(duì)JAK的亞型激酶抑制作用沒有單一的選擇性,它們的臨床試驗(yàn)結(jié)果表明,單一亞型的JAK抑制劑或許會(huì)在保持良好的RA治療效果的同時(shí),避免或減小不必要毒副作用的產(chǎn)生,提高其應(yīng)用的臨床安全性。為了尋找并研發(fā)具有JAK亞型激酶抑制選擇性的化合物分子,本課題選擇以Baricitinib分子為先導(dǎo)化合物,參考已經(jīng)報(bào)道的Tofacitinib分子與JAK各亞型間的作用關(guān)系,同時(shí)還借鑒在研的具有單一亞型選擇性抑制作用的JAK抑制劑Filgotinib (作用靶點(diǎn)為JAK1)和Decernotinib (作用靶點(diǎn)為JAK3)的分子結(jié)構(gòu),利用生物電子等排、優(yōu)勢(shì)分子片段拼接等策略進(jìn)行分子結(jié)構(gòu)優(yōu)化和改造。在目標(biāo)化合物的化學(xué)合成過程中,本課題探索并改進(jìn)了合理的合成路線,針對(duì)關(guān)鍵步驟的反應(yīng)條件和關(guān)鍵中間體的合成方法進(jìn)行了的優(yōu)化,提升了目標(biāo)化合物實(shí)驗(yàn)室放大合成實(shí)驗(yàn)操作的可行性。最后使用毛細(xì)管電泳方法通過檢測(cè)底物肽段磷酸化轉(zhuǎn)化率,測(cè)定了目標(biāo)化合物對(duì)3種JAK亞型激酶(JAK1、JAK2、JAK3)的半抑制濃度(halfmaximal inhibitory concentration,IC50)值(由于與 TYK2 的主要相關(guān)疾病是牛皮癬和炎癥性腸病而并非類風(fēng)濕性關(guān)節(jié)炎,因此并未測(cè)定目標(biāo)化合物對(duì)TYK2的抑制活性),以此來評(píng)價(jià)所設(shè)計(jì)并合成的目標(biāo)化合物體外JAK抑制活性和選擇性。在目標(biāo)化合物分子的設(shè)計(jì)過程中,本課題先對(duì)先導(dǎo)化合物分子進(jìn)行了母核的改造,用C-3位取代的7-氮雜吲哚結(jié)構(gòu)替換Baricitinib分子中的7H-吡咯并[2,3-d]嘧啶結(jié)構(gòu),以保證目標(biāo)分子在JAK鉸鏈區(qū)的有效固定,并進(jìn)一步對(duì)酰胺尾部進(jìn)行了優(yōu)化,重點(diǎn)考察了不同的取代酰胺對(duì)化合物體外酶抑制活性的影響,設(shè)計(jì)得到此類化合物J-01～J-10, 10個(gè)目標(biāo)化合物結(jié)構(gòu)均未見報(bào)道。通過體外JAK抑制活性測(cè)試,發(fā)現(xiàn)此類目標(biāo)化合物相比于Baricitinib活性有所下降,但都表現(xiàn)出了激酶亞型抑制選擇性,尤其是化合物J-01和J-04分別對(duì)JAK2和JAK1表現(xiàn)出了較好的抑制選擇性。并且發(fā)現(xiàn)尾部的酰胺取代基對(duì)這類化合物抑制選擇性的影響較大,小的脂肪環(huán)取代基有增加JAK2選擇性的作用,而芳環(huán)取代基則更有利于提高目標(biāo)化合物對(duì)JAK1的抑制選擇性。另外,改構(gòu)所得的此類分子中,氰基結(jié)構(gòu)與激酶間的作用效果不易被其他基團(tuán)替代,將氰基結(jié)構(gòu)變換為氨基或三氟乙胺基后,目標(biāo)化合物的體外JAK抑制活性有顯著降低。另一方面,本課題又對(duì)化合物分子母核上的連接基團(tuán)部分進(jìn)行了片段拼接,借鑒Filgotinib分子中的苯環(huán)連接片段,將其拼接至化合物分子中的7-氮雜吲哚骨架上得到目標(biāo)化合物J-11～J-14,也嘗試著直接將該片段拼接至Baricitinib分子中的7H-吡咯并[2,3-d]嘧啶結(jié)構(gòu)上得到目標(biāo)化合物J-15～J-19,設(shè)計(jì)得到了9個(gè)未見報(bào)道的新結(jié)構(gòu)目標(biāo)化合物。體外JAK抑制活性試驗(yàn)結(jié)果顯示,化合物J-11～J-14的激酶抑制活性下降的較為明顯,而且并未表現(xiàn)出理想的抑制選擇性。然而目標(biāo)化合物J-15～J-19的酶抑制活性雖然也有降低,但其中目標(biāo)化合物J-16和J-19都對(duì)JAK2表現(xiàn)出了較好的抑制選擇性。在目標(biāo)化合物的化學(xué)合成過程中,我們分別選擇以3-溴-7-氮雜吲哚和4-氯-7H-吡咯并[2,3-d]嘧啶為起始原料,經(jīng)過氨基保護(hù)、Suzuki芳基偶聯(lián)、Michael加成、親核取代、水解、催化縮合、氨基去保護(hù)等關(guān)鍵步驟來合成目標(biāo)化合物分子,并通過核磁氫譜進(jìn)行了化合物的結(jié)構(gòu)鑒定。最后還對(duì)Suzuki芳基偶聯(lián)反應(yīng)的最佳實(shí)驗(yàn)條件及關(guān)鍵中間體7a～7g的合成路線、方法進(jìn)行了探索改進(jìn),合理地避開了化學(xué)性質(zhì)不穩(wěn)定的中間體,以獲得理化性質(zhì)較穩(wěn)定的關(guān)鍵中間體,為目標(biāo)化合物在實(shí)驗(yàn)室放大合成的工藝改進(jìn)和優(yōu)化奠定了基礎(chǔ)。本課題中,我們?cè)O(shè)計(jì)并成功合成了共19個(gè)目標(biāo)化合物,其結(jié)構(gòu)未見文獻(xiàn)報(bào)道,并對(duì)其體外Janus激酶抑制活性進(jìn)行了評(píng)價(jià),結(jié)果表明化合物J-01、J-04、J-16和J-19為選擇性JAK抑制劑,為該課題的進(jìn)一步深入研究奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R914;R96

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本文編號(hào)：2042640