PARP-1抑制剂的设计、合成与生物活性评价

发布时间：2018-07-15 10:20

【摘要】：癌症作为严重危害人类健康与生命的全球公共卫生问题,已经引起世界范围的广泛关注。我国作为一个发展中的大国,随着人口老龄化等诸多因素的影响,癌症发病率与死亡率均呈持续上升趋势。乳腺癌和卵巢癌是妇科常见的恶性肿瘤,其中乳腺癌在女性恶性肿瘤中发病率最高,特别是侵袭性强、生存期短的三阴性乳腺癌目前仍无有效治疗手段;卵巢癌死亡率居妇科恶性肿瘤首位,复发和耐药一直是治疗中所面临的难题。流行病学调查发现,约10~40%的三阴性乳腺癌及8~18%的卵巢癌患者中检测出了BRCA1/2基因突变,BRCA1/2基因水平的改变一方面增加了罹患乳腺癌和卵巢癌的风险,同时又为此类癌症的预防及治疗提供了契机。近年来,PARP-1抑制剂单独或与其他药物联合应用于BRCA1/2缺陷的乳腺癌和卵巢癌成为癌症治疗领域研究的热点,给三阴性乳腺癌和复发性卵巢癌的治疗带来了希望。聚腺苷二磷酸核糖聚合酶(Poly(ADP-ribose)polymerases,PARPs)是真核生物细胞中存在的一种核酶,主要参与DNA修复过程并维持基因组的稳定性。PARP家族的18个成员中,PARP-1在细胞内含量最高,研究也最为深入,主要以NAD+为底物催化ADP-核糖单元转移至核受体蛋白,从而形成聚ADP-核糖聚合物进而引导修复酶对DNA缺口进行修复,在碱基切除修复通路中发挥关键作用。在当今药物研发优质靶标极其缺乏的现状下,PARP-1给我们提供了为数不多经过临床验证的抗癌靶点。但其发展历程因受作用机制认识的局限和药物结构的制约等因素而颇为曲折。最初的研究方向是通过与放疗或化疗药物联合应用以增强药物疗效和降低用药剂量,但是这种机制一直受毒性所限,三十余年的研究仍未取得成功。合成致死理论的提出和应用给此类药物的发展带来了转机,PARP-1抑制剂可通过切断同源重组修复缺陷肿瘤细胞的碱基切除修复通路从而发挥靶向作用,因此可单独应用于此类肿瘤,如BRCA1/2缺陷的乳腺癌和卵巢癌等。2014年12月,Olaparib首先被批准用于治疗BRCA1/2缺陷的铂敏感复发性卵巢癌,其单独或联合应用于同源重组修复缺陷的其他适应症如乳腺癌、胰腺癌和前列腺癌等的临床研究也正在进行中。作为21世纪抗肿瘤领域取得的重大突破,合成致死理论促成了Olaparib的成功上市,并开始真正激发PARP-1抑制剂的应用潜能,在其他类型缺陷的肿瘤中也展现出巨大的潜力。在合成致死理论之外,PARP-1抑制剂的研究还存在着诸多挑战与机遇,包括最近发现其与C-Met抑制剂、AKT抑制剂和PD-1抑制剂联用有助于克服肿瘤耐药问题,甚至在实体瘤之外的血液瘤中也已表现出疗效。这些研究大大拓展了PARP-1抑制剂的潜在应用领域和发展空间,具有重大意义。现有PARP-1抑制剂大多以酶天然底物NAD+的烟酰胺部分为结构基础进行设计,其结构差异较大。我们基于靶标和底物的结合特征进行分析,发现PARP-1抑制剂的共同结构特点是具有关键酰胺基团和刚性平面结构,这些关键特征是活性PARP-1抑制剂与蛋白结合的基础。此外,活性位点内部还存在一个大的疏水口袋,可允许PARP-1抑制剂侧链引入不同的基团以增加抑制活性、改善水溶性及其他理化性质,此结构特点给了PARP-1抑制剂非常大的改造空间。本文以进展最快、研究最多的Olaparib和Veliparib为模板,在保持酰胺构象和母核平面结构这些关键特征的基础上,设计了四类不同母核的PARP-1抑制剂,分别为三唑并嘧啶类、噻吩并咪唑类、二氢喹唑啉酮类和单环三嗪酮类;考虑到活性位点内部的大疏水口袋可容纳基团的多样性,进一步在母核的基础上连接脂肪族和芳香族侧链、以及不同的长侧链和短侧链以考查其对活性的影响。同时本文采用分子对接技术,通过分析化合物与PARP-1之间的相互作用,验证化合物设计的合理性、确保所设计目标化合物与PARP-1蛋白之间能够较好结合。在目标化合物的合成中,本文通过合成路线的合理设计和反应条件的不断探索,完成了三唑并嘧啶类、噻吩并咪唑类、二氢喹唑啉酮类三类化合物的合成工作,根据母核连接侧链的不同,本文目标化合物的合成共涉及7条合成路线,主要包括缩合、环合、还原、氨解等反应步骤,并对合成路线中的关键步骤进行改进。第四类单环三嗪酮类化合物在合成过程中遇到一些困难,目前仍在打通合成路线中。本文共合成了56个目标化合物,其结构均经过MS,1H-NMR确证。对所合成化合物进行了酶水平和细胞水平的初步生物活性评价。1.PARP-1酶水平的抑制活性评价中,第一类和第三类化合物只初步测定了化合物在10μM下的抑制率,其中第一类三唑并嘧啶类初筛抑制率均低于10%,可能由于化合物的溶解性较差,考虑将部分化合物做成盐酸盐重新测定。第二类噻吩并咪唑类化合物初筛后选出抑制率大于50%的化合物进行IC50值的测定。令人遗憾的是哌嗪长侧链化合物与相同侧链的阳性药相比活性差距较大,IC50值均在微摩尔级,有幸的是我们获得了意外的发现,随着取代基团体积逐渐减小,活性有所提高,并推测可能由于咪唑环的存在限制了侧链的构象,大基团侧链在噻吩并咪唑母核之上不能较好的与活性位点匹配。随后合成的侧链缩短化合物活性总体得到提高,验证了上述推测,其中化合物27l的活性最好(IC50=43 n M)。2.活性最好的四个化合物(27g,27i,27j,27l)进行了细胞水平的活性评价,PARylation试验结果显示,四个化合物在Hela细胞上抑制活性弱于两个阳性药,其中活性最好的是27i(1.080μM);然而在BRCA-1/2缺陷的HCC1937细胞上,4个化合物的抗增殖活性均优于两个阳性药。推测化合物透膜性等因素可能是其中原因之一,目前正在开展进一步验证。本文通过合理药物设计、目标化合物的合成与生物活性评价,发现了活性较好的噻吩并咪唑全新骨架的PARP-1抑制剂,其中四个化合物(27g,27i,27j,27l)单独作用于BRCA1/2细胞系HCC1937和CAPAN-1的活性优于阳性药,且对于人正常细胞(人胚胎纤维细胞)的毒性低于阳性药,值得进一步研究。初步的构效关系研究对开展PPAR抑制剂的进一步优化设计具有一定的指导意义。本研究也提示,尽管如今的药物设计方法提供了更加合理和多样化的手段,但药物结构对活性的影响尚难以通过现有的这些药物设计手段进行精确预测,药物设计的挑战尚在,同时其不确定性也是药物研究者的希望和动力所在。
[Abstract]:As a global public health problem that seriously endangers human health and life, cancer has attracted worldwide attention. As a big developing country, cancer incidence and mortality are on the rise continuously with the influence of many factors such as population aging. Breast and ovarian cancer are common malignant tumors in gynecology. The incidence of breast cancer in female malignant tumors is the highest, especially the aggressive, and the three negative breast cancer with short survival time is still not effective. The mortality of ovarian cancer is the first in gynecologic malignant tumor, and the relapse and resistance have been the difficult problems in the treatment. The flow disease survey found that about 10~40% of three negative breast cancer and 8 The BRCA1/2 gene mutation was detected in ~18% patients with ovarian cancer. The changes in the BRCA1/2 gene level increased the risk of breast and ovarian cancer, and also provided an opportunity for the prevention and treatment of such cancers. In recent years, PARP-1 inhibitors have been used alone or in combination with other drugs in BRCA1/2 deficient breast and ovarian cancer. Poly (ADP-ribose) polymerases (PARPs), a ribozyme existing in eukaryotic cells, is a ribozyme in eukaryotic cells, which is mainly involved in the DNA repair process and maintains the stability of the genomic stability of the 18.PARP family. Among the members, the content of PARP-1 is the highest in the cell, and the research is also the most in depth. NAD+ is used as the substrate to catalyze the transfer of the ADP- ribose unit to the nuclear receptor protein, thus forming a poly (ADP- ribose) polymer to guide the repair enzyme to repair the DNA gap and to play a key role in the base removal repair pathway. In the absence of the current situation, PARP-1 provides us with a few clinically proven anticancer targets, but its development is rather tortuous due to the limitations of the mechanism of action and the constraints of the drug structure. The initial research direction is to enhance the efficacy and reduce the drug use by combining with radiotherapy or chemotherapy drugs. However, this mechanism has been limited to toxicity, and more than thirty years of research have not been successful. The introduction and application of synthetic lethal theory have brought about the development of this kind of drug. The PARP-1 inhibitor can be used alone by cutting the base excision repair pathway of the defective tumor cells by cutting off the homologous recombination and thus can be used alone. In December, Olaparib was first approved for the treatment of BRCA1/2 deficient platinum sensitive recurrent ovarian cancer in this kind of tumor, such as BRCA1/2 deficient breast and ovarian cancer. Clinical studies on other indications, such as breast cancer, pancreatic cancer and prostate cancer, are also in progress. For the major breakthrough in the field of anti-cancer in twenty-first Century, the synthetic lethal theory contributed to the successful listing of Olaparib, and began to truly stimulate the potential of PARP-1 inhibitors, and showed great potential in other types of tumors. In addition to the synthetic lethal theory, there are many challenges and opportunities for the research of PARP-1 inhibitors. The combination of C-Met inhibitors, AKT inhibitors, and PD-1 inhibitors has recently been found to help overcome the problem of tumor resistance, even in haematoma other than solid tumors. These studies have greatly expanded the potential applications and development space of PARP-1 inhibitors. Most of the existing PARP-1 inhibitors are enzymes. The nicotinamide part of the natural substrate NAD+ is designed for the structure basis, and its structure is different. Based on the analysis of the binding characteristics of the target and substrate, we find that the common structural characteristics of the PARP-1 inhibitors are the key amide group and the rigid plane structure. These key features are the basis of the binding of active PARP-1 inhibitors and proteins. In addition, there is a large hydrophobic pocket inside the active site, which allows the PARP-1 inhibitor side chain to introduce different groups to increase the inhibitory activity, improve the water solubility and other physical and chemical properties. This structure has given the PARP-1 inhibitor a very large transformation space. This paper is the fastest progresses, the most studied Olaparib and Veliparib as the template, On the basis of maintaining the key characteristics of the conformation of amides and the planar structure of the parent nucleus, four kinds of PARP-1 inhibitors with different parent nuclei are designed, which are three azolimidines, thiophenazolidazole, two hydroquinazolones and mono ring three pylozones, which take into account the diversity of the large water pockets within the active site, and further in the mother. On the basis of the nuclear connection, the aliphatic and aromatic side chains, the different long side chains and the short side chains are used to examine the effect on the activity. At the same time, the molecular docking technique is used to verify the rationality of the compound design by analyzing the interaction between the compound and the PARP-1, so as to ensure that the designed target compounds and the PARP-1 protein can be compared. In the synthesis of target compounds, through the rational design of synthetic routes and the continuous exploration of reaction conditions, the synthesis of three zazolidines, thiophenacidazoles and two hydrogen quinazolones is completed. According to the difference of the side chain of the parent nucleus, the synthesis of the target compounds involves 7 synthetic routes. It mainly includes the reaction steps of condensation, cyclization, reduction and ammoniation, and improving the key steps in the synthesis route. The fourth kind of mono ring three mazine compounds have encountered some difficulties in the synthesis process, and are still in the synthesis route. This paper has synthesized 56 target compounds, their structure is confirmed by MS, 1H-NMR. In the evaluation of the inhibitory activity of the initial Bioactivity Evaluation of the enzyme level and cell level, the first and third compounds only preliminarily determined the inhibitory rate of the compound under 10 mu M, of which the first type of three azolimidines was lower than 10%, which may be due to the poor solubility of the compounds. The second class of thiophene and imidazole compounds were selected for the determination of the IC50 value of the compounds with inhibitory rate greater than 50%. It is regrettable that the activity gap between the piperazine long side chain compounds and the same side chain positive drugs is larger, and the IC50 values are at the mole level, and fortunately we have obtained the results. As the volume of the substituent group gradually decreased, the activity increased, and it was presumed that the conformation of the side chain was limited by the presence of the imidazole ring, and the large group side chain was not well matched with the active site on the thiophene and imidazole parent nucleus. It is speculated that the best four compounds (27g, 27i, 27j, 27L) of the best activity of compound 27L (27g, 27i, 27j, 27L) were evaluated at the cell level. The results of PARylation test showed that the inhibitory activity of four compounds on Hela cells was less than two positive drugs, of which the best activity was 27i (1.080 mu). The antiproliferative activity of the 4 compounds on CC1937 cells is better than that of two positive ones. It is presumed that the membrane permeability of the compound may be one of the reasons, and is now being further verified. In this paper, the synthesis and Bioactivity Evaluation of the target compounds and the evaluation of the biological activity of the target compounds have been developed by the rational drug design, and the P of the highly active thiophene and imidazole skeleton has been developed. ARP-1 inhibitors, of which four compounds (27g, 27i, 27j, 27L) are superior to the positive drugs in BRCA1/2 cell lines HCC1937 and CAPAN-1, and the toxicity of the normal cells (human embryonic fibroblasts) is lower than that of the positive drug. It is worth further study. The preliminary structure-activity relationship study on the further optimization of the PPAR inhibitors is designed. This study also suggests that, although the current drug design approach provides a more reasonable and diversified approach, the impact of drug structure on activity is still difficult to accurately predict through existing methods of drug design, the challenge of drug design is still at the same time, and its uncertainty is also the hope of drug researchers. The motivation lies.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R91;R914.5

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