靶向秋水仙碱结合位点的新型抗紫杉醇耐药微管蛋白抑制剂的设计与合成

The indenopyrazol derivatives LL-01 and LL-02 are the newly discovered potent tubulin inhibitors targeting the colchicine binding site. Through the inhibiton of tubulin polymerization, they act as both antimitotic and vascular disrupting agents (VDAs), inhibit the proliferation of a variety of tumor cells, including those multi-drug resistant (MDR), Pgp-over-expressing and taxol–resistant, with IC50 values in the nM level. “Enhancing protein backbone binding” is a fruitful concept for combating drug-resistance caused by the target protein mutation. Following this drug design strategy and based on the structure of LL01 and its interactions with the three zones in the tubulin colchicine site, and by the full consideration of electronic parameters, hydrophobic properties, steric effects and hydrogen-bondings, etc., in this research, we will make rational modifications and optimizations at the A-, B-, and C-rings, to design and construct the structurally diverse indenopyrazole, benzofuropyrazole and pyrazoloindole series of compound libraries. By introducing hydrogen bond donors and acceptors, as well as the polar functional groups, these molecules are expected to make multiple interactions with the backbones in the active site and thus increase their binding potency to the tubulin. As a result, the antitumor and anti-drug resistance activities, water solubility and drug-like properties will be improved accordingly. Through the systematic pharmacological studies and the preliminary toxicological and pharmacodynamic evaluations, we will make a full understanding of their action mechanisms and structure-activity-relationships (SARs). We hope to find novel high efficient, low toxic tubulin inhibitors that can overcome paclitaxel resistance and have good water solubity and drugability.

茚并吡唑衍生物LL-01和LL-02是我们最近发现的、靶向秋水仙碱结合位点的强力微管蛋白抑制剂。它们通过抑制微管蛋白的聚合，发挥抗有丝分裂和破坏肿瘤血管的双重作用，对包括多药耐药、紫杉醇耐药在内的多种肿瘤细胞具有强抑制作用（nM）。“增强与活性中心蛋白骨架的结合”是克服因靶点蛋白变异而造成耐药的有效方法。基于这一药物设计策略，我们以LL-01的结构为基础，针对它与秋水仙碱结合位点的三个结合区域，综合考虑电性、疏水性、立体性、氢键结合等因素，拟在A、B和D环进行合理的结构修饰和优化，构建结构多样性茚并吡唑，苯并呋喃并吡唑和吡唑并吲哚三个系列化合物库。通过氢键受体、供体和极性基团的引入，增强它们与微管蛋白的多位点结合，提高其活性、水溶性和成药性。通过系统抗肿瘤药理学研究、初步毒性学、药代动力学评价等，阐明该类化合物的作用机制和构效关系，发现高效低毒、抗紫杉醇耐药、成药性好的新型微管蛋白抑制剂。

以紫杉醇为代表的微管蛋白抑制剂广泛用于癌症的治疗，但这些药物水溶性差，存在剂量依赖性的骨髓抑制和神经毒性，并在临床应用中出现了多药耐药问题。为提高微管蛋白抑制剂的水溶性，降低其毒性和不良反应，克服肿瘤多药耐药和紫杉醇耐药等问题，本研究以微管蛋白秋水仙碱结合位点的蛋白晶体结构为基础，采用“增强与活性中心蛋白骨架的结合”的药物设计策略，在计算机辅助药物设计的指导下，基于与秋水仙碱结合位点三个区域的特点，进行合理的药物设计、结构修饰与优化，设计合成了茚并吡唑衍生物82个，苯并呋喃并吡唑及其开环吡唑胺衍生物各6个，苯并吡唑类（吲唑类）衍生物23个。通过抗肿瘤细胞增殖抑制活性评价，发现了18个高活性茚并吡唑衍生物LL01—LL18和2个高活性吲唑类衍生物BP01和BP02，对多种肿瘤细胞株（A549, HepG2, HCT116, MCF-7）等的抑制活性（IC50）均在纳摩尔水平，优于紫杉醇或与紫杉醇活性相当。这些高活性化合物靶向秋水仙碱结合位点，抑制微管蛋白的聚合。在细胞水平，这些高活性化合物能够抑制或干扰微管和微丝的形成，通过调控相关细胞周期蛋白，使细胞周期停滞于G2/M期；通过caspase 9、PARP 的活化，诱导肿瘤细胞凋亡。这些高活性化合物还能有效抑制HUVEC小管形成，具有潜在抗新生血管生成的作用。化合物LL01具有良好的肝微粒体代谢稳定性和药代动力学性质，口服生物利用度较好（41%）。在非小细胞肺癌、P-gp高表达的KB/V肿瘤模型、HepG2肿瘤模型和HCT116结肠癌模型中，这些高活性微管蛋白抑制剂具有良好的抗肿瘤活性，并对动物的体重等没有明显的影响。通过上述研究，明确了茚并吡唑衍生物的构效关系，提出了茚并吡唑衍生物与秋水仙碱结合位点的新型结合模式，并与该类化合物的构效关系完全吻合。更重要的是，这些高活性化合物不是P-gp底物，对紫杉醇耐药和多药耐药肿瘤细胞有效，解决了紫杉醇耐药这一科学问题；部分茚并吡唑衍生物盐酸盐，如LL04、LL17和LL18，水溶性好，解决了大多数微管蛋白抑制剂水溶性差的问题，有望作为候选药物进行临床前评价。