【作者】 徐福明；

【导师】 徐文方；

【作者基本信息】 山东大学 ， 药物化学， 2014， 博士

【摘要】 目的：异常的血管生成,包括内皮细胞激活、降解基底膜、内皮细胞的定向运动和增殖、新生微血管、内皮细胞新的基底膜合成、血管腔产生、芽式生长并形成血管襟等一系列步骤,是肿瘤细胞增殖,侵袭和转移的先决条件。Abl和Akt (proteinkinase B, PKB)蛋白激酶是肿瘤细胞和血管内皮细胞内两个重要的信号通路节点,他们可以通过磷酸化下游底物来调节肿瘤细胞和内皮细胞内许多重要的生理过程,包括细胞存活、增殖、迁移和血管生成等。蛋白激酶抑制剂根据作用于激酶结合位点的不同分为三种：作用于ATP结合位点的蛋白激酶抑制剂,作用于调节区的蛋白激酶抑制剂和蛋白激酶底物竞争性变构抑制剂。近几年,多种靶向于蛋白激酶的药物如Sunitinib、Sorafenib、 Pazopanib等被成功推向了市场用于各种肿瘤的治疗。尽管这些肿瘤血管生成抑制剂有着很大的优势,但是实际应用仍存在部分肿瘤对血管的新生依赖性不强、突变及肿瘤信号传导的代偿性导致的耐药、不良反应和毒性等问题。因此进一步开发选择性更强、活性更高且毒性更小的小分子蛋白激酶抑制剂仍然非常必要。随着对Bcr-Ab1和Akt相关信号通路研究的不断深入,Bcr-Abl和／或Akt抑制剂的研究已经成为抗肿瘤药物研究的热点。方法：本研究首先在计算机虚拟筛选的过程中发现了具有明显抗血管生成作用的化合物Hit6a。后期的酶谱筛选工作表明,6a可以选择性的抑制Abl和Akt1两个激酶,靶点非常明确。之后我们设计合成了五个系列基于4-氨基-1-萘酚骨架的6a类似物,并进行了体外抗肿瘤细胞增殖、抗血管生成和蛋白激酶抑制活性研究,一方面对这一类结构骨架化合物进行构效关系(SAR)研究,另一方面也希望获得比Hit6a的抗肿瘤和抗血管生成活性更好的先导结构。结果：本研究共设计、合成了五个系列81个基于4-氨基-1-萘酚骨架的Hit6a类似物,并对所合成的目标化合物通过电喷雾质谱以及核磁共振氢谱等方法进行了结构确证,经查阅文献证实,除个别化合物外所合成的目标化合物均为新型化合物,未见文献报道。我们对合成的化合物进行了体外抗肿瘤细胞增殖活性、体外抗血管生成活性和抑酶活性研究。实验结果表明,合成的4-氨基-1-萘酚类化合物在抑酶活性研究中表现出的构效关系规律与在体外抗肿瘤细胞增殖和体外抗血管生成活性研究中表现出的构效关系规律具有高度的一致性,而且化合物的抗血管生成活性与抑制VEGF/VEGFR无关,这给我们这样的提示,即合成的4-氨基-1-萘酚类化合物是通过抑制Abl和Aktl蛋白激酶的活性来实现其抗肿瘤细胞增殖和抗血管生成活性的。构效关系研究表明,Hit6a的萘酚环(Figure5-1, a部分)可以通过疏水作用或者π-π堆积作用与激酶活性位点的疏水性口袋的结合,将萘环变为较小的苯环后(D系列)会导致化合物与受体的结合力下降,抑酶活性和抗血管活性明显下降,抗肿瘤细胞增殖活性几乎丧失；而在萘环的6,7位引入两个甲氧基基团后(E系列),将增强化合物的疏水性,增强与受体结合能力,进而化合物的抑酶活性、抗血管活性和抗肿瘤细胞增殖活性明显增强。而在7位保持甲氧基不变,6位引入丙基吗啉侧链(33a)时,一方面提高了化合物水溶性,另一方面通过氢键作用增强了化合物与受体的结合力,使化合物能够充分的占据结合口袋的空腔区域,提高了化合物的抑酶活性,抗血管活性和抗肿瘤细胞增殖活性。Hit6a的三氮唑五元芳杂环(Figure5-1, b部分)可以伸向Abl和Aktl激酶的DFG motif,并与DGF in构象(蛋白处于活化状态时的构象)的氨基酸ASP形成氢键作用；当其被5-甲基-1,3,4-噻二唑、1,3,4-噻二唑或者1-甲基-四氮唑环替代后氢键作用消失,同时引起化合物分子在受体结合口袋中结合位置和构象的改变,导致抑酶活性急剧降低,抗血管活性和抗肿瘤细胞增殖活性大大减弱；而当三氮唑五元芳杂环被嘌呤环替换(B系列)后,引起化合物分子在受体结合口袋中结合位置和构象的改变,但是嘌呤环可以与结合口袋中的其他位置氨基酸形成氢键作用,最终抑酶活性稍有降低,抗血管活性和抗肿瘤细胞增殖活性稍有减弱。需要注意的是三氮唑五元杂环被嘌呤环替换后,化合物(比如9n)对激酶选择的特异性会下降,而这并不是我们所期望的。Hit6a的磺酰胺基-SO2NH-(Figure5-1, c部分)可以与Ab1和Akt1激酶活性口袋内铰链区、G-loop或者P-loop区域氨基酸残基形成氢键作用,提高化合物与激酶的结合力,同时该基团可以使化合物保持在一个易于与活性口袋结合的构象；而用-CONH-基团替代后,氢键作用消失,化合物的空间构象也不利于与活性口袋结合(比较Figure4-1中的9d和13q),导致抑酶活性大大降低甚至消失,抗血管活性和抗肿瘤细胞增殖活性大大减弱。Hit6a的R位置2-萘基(Figure5-1, d部分)可以与Abl和Aktl激酶活性口袋内铰链区,该位置不同的芳香基(R)基团对化合物的激酶抑制活性的影响呈现出了一定的规律性。(a)R基团为苯基时要比为较大的基团(如萘基,喹啉基,4-叔丁基苯基和4-(1,1’-联苯)基)或者较长(苯乙烯基)的基团时活性好(13a和13b vs13c和13d,9f和9p vs9a和9g)。(b)R基团为苯基时,苯环上连接吸电子基团(13d/13c的-NO2/-Cl)的化合物要比连接推电子基团的的化合物(13e/13i的-OMe/-Me)活性好。(c)R基团上为双取代基时化合物的抑制活性要比单取代时的抑制活性要好(9e,9k,9n vs9j,9d,9b).基于以上构效关系研究结果,在E系列抑制剂设计中,我们保存了化合物中间骨架萘酚环,磺酰胺基团和三氮唑五元杂环,同时在萘环的6,7位引入两个甲氧基基团,甲氧基的引入能够进一步增强萘酚片段的疏水作用,从而提高对Abl和Akt1激酶的抑制活性,提高其选择性。该系列化合物26a-26g的体外抑酶活性明显好于前四个系列,整体IC50值在1州以下,其中活性最好的化合物26g对Abl和Aktl激酶的IC50值分别为0.16和0.46μM,比Hit6a的抑酶活性要高约10倍；但甲氧基的引入导致化合物的水溶性较差,我们在对26g进行水溶性改善时设计合成了6位氧上含有丙基吗啉侧链的化合物33a,活性测试结果显示33a对Abl和Aktl激酶的ICs0值分别为0.13和0.28州。丙基吗啉侧链的引入既能提高化合物26g的水溶性,又能使化合物的抑酶活性得到保持,所以化合物33a是一个非常有前景的具有良好水溶性和抗肿瘤活性的Abl和Akt1双靶点抑制剂。结论：本研究基于Abl和Akt1晶体结构及Hit6a与活性位点的结合模式,在实验室前期工作基础上,设计、合成了五个系列共81个基于4-氨基-1-萘酚骨架的Hit6a类似物。通过初步的体外生物活性评价,我们发现了几个具有进一步研究开发价值的化合物,如：化合物26g和33a等,并进一步对其进行了以分子对接为主的构效关系研究,探讨了其与Abl和Akt1靶点的结合模式,为今后新型小分子Abl和Akt1抑制剂的设计、合成奠定了基础。直接以内皮细胞为研究靶标,在对血管内皮细胞增殖与抑制的相关分子的深入了解基础上,采用相应的药物阻断其胞内信号传导通路中的关键蛋白Abl和Akt,抑制血管内皮细胞增殖及肿瘤血管生成的手段,已经证明对肿瘤治疗有十分重要的意义。更多还原

【Abstract】 Objective:Aberrant angiogenesis, involving the proliferation of endothelial cells (ECs) and the migration of these endothelial cells to the tumor site to form new capillaries, is a critical step for tumor cell proliferation, invasion and metastasis. Abl and Akt (proteinkinase B, PKB) protein kinases are two important signaling pathway nodes in tumor cells and endothelial cells, they can modulate many important physiological processes by phosphorylation of downstream substrates, including cell survival, proliferation, migration and angiogenesis, etc.There are three kinds of protein kinase inhibitors according to the difference in binding cites of kinases:protein kinase inhibitors acting on the ATP-binding site, protein kinase inhibitors acting in the regulatory region and allosteric substrate competitive protein kinase inhibitors. Up to now, a large number of small-molecule angiogenesis inhibitors have been reported. Among them, the approval of multi-kinase inhibitors, Sunitinib, Pazopanib and Sorafenib, boosts the optimism of antiangiogenic agents as an effective way to control tumor growth. However, severe drug resistance and on-target adverse events such as hypertension, proteinuria, hemorrhage and hypothyroidism are observed during treatment with these angiogenesis inhibitors, indicating persistent effort required in this research field. With the the deepening of research of Bcr-Abl and Akt related signaling pathways, Abl and Akt have become hotspots in anticancer research.Methods:In this study, Hit6a, showing obvious antiangiogenic activity, was discovered by a computer-aided drug design (CADD) process. In the subsequent kinase screening test, Hit6a can selectively inhibit Abl kinase Aktl kinase. Then we designed and synthesized five series of4-aminonaphthalen-l-ols based on the structure of Hit6a. And we have tested the in vitro anti-proliferation activity, in vitro anti-angiogenesis activity and in vitro kinase inhibition activity of all synthesized compounds for SAR studies and for seeking more potent lead compound than Hit6a.Results:In this study, five series of81target compounds were synthesized, and all the structures were identified by ESI-MS, HRMS and1H-NMR. Most of the compounds were novel and have not been reported in the literature. We have tested the in vitro anti-proliferation activity, in vitro anti-angiogenesis activity and in vitro kinase inhibition activity of all synthesized compounds. Experimental results show that the regularities of synthesized compounds in inhibiting Abl and Akt proteins are highly consistent with their anti-proliferation activity and anti-angiogenic activity. The anti-angiogenic activity is not related with inhibiting VEGF/VEGFR. We speculated that4-aminonaphthalen-l-ols can supress the tumor proliferation and tumor-induced angiogenesis by inhibiting the activities of Abl and Aktl proteins.The SAR study indicated that the naphthol ring of Hit6a may interacte with the hydrophobic region of active sites of protein kinase by a combination of hydrophobic interaction and π-π stacking. Triazole structure can stretch to DFG motif and form hydrogen bond with aspartate. The-SO2NH-group can also form hydrogen bonds with amino acid residues from Hinge, G-loop or P-loop to enhance the adhesion with the kinase. Besides, the-SO2NH-group can make the compound form an easy combination with the active sites. Different aromatic groups of R show a certain regularity in inhibiting the kinase activity.Based on the SAR studies, we kept the naphthol ring,-SO2NH-group and triazole structure unchanged, and introduced two methoxy groups to the6and7position of the naphthalene ring to enhance the hydrophobic interaction with the protein, and then to improve the kinase inhibition ability and selectivity. Compounds26a-26g exhibited better kinase inhibition activities than any other compounds of seriess A-D, with the IC50values all below1μM. Compound26g was found to be the most potent compound, of which the IC50values to Abl and Akt1were0.16and0.46μM, respectively, about tenfold better than Hit6a. But the introduction of the methoxy groups resulted in poor water-solublility. In order to improve the water-solublility of compound26g, we introduced a propyl morpholine to the oxygen atom of position6and gained the compound33a.33a can effectively inhibit the Abl and Aktl with the IC50values being0.13and0.28μM, respectively. So compound33a is a promising both Abl and Aktl kinase inhibitor with good water-solubility.Conclusions:Based on the crystal structure of Abl and Akt1, the interaction model Hit6a with Abl and Aktl and the previous work of our lab, five series of81target small molecule peptidomimetics were designed and synthesized as Abl and Aktl inhibitors. By preliminary in vitro bioactivity assay, we found several potencial compounds such as compounds26g and33a, which need to be further developed in the future. The QSAR and binding models of these compounds were also stutied, which would be beneficial for further design and development of novel small molecule Abl and Aktl inhibitors in the future.It has been proven that the strategy, inhibiting endothelial cell proliferation and tumor angiogenesis through the blockade of the key proteins Abl and Akt in cell signaling pathway, contributes more in tumor treatment.更多还原