Rational Design, Synthesis, and Biological Evaluation of Third Generation α-Noscapine Analogues as Potent Tubulin Binding Anti-Cancer Agents

Systematic screening based on structural similarity of drugs such as colchicine and podophyllotoxin led to identification of noscapine, a microtubule-targeted agent that attenuates the dynamic instability of microtubules without affecting the total polymer mass of microtubules. We report a new generation of noscapine derivatives as potential tubulin binding anti-cancer agents. Molecular modeling experiments of these derivatives 5a, 6a-j yielded better docking score (-7.252 to -5.402 kCal/mol) than the parent compound, noscapine (-5.505 kCal/mol) and its existing derivatives (-5.563 to -6.412 kCal/mol). Free energy (ΔGbind) calculations based on the linear interaction energy (LIE) empirical equation utilizing Surface Generalized Born (SGB) continuum solvent model predicted the tubulin-binding affinities for the derivatives 5a, 6a-j (ranging from -4.923 to -6.189 kCal/mol). Compound 6f showed highest binding affinity to tubulin (-6.189 kCal/mol). The experimental evaluation of these compounds corroborated with theoretical studies. N-(3-brormobenzyl) noscapine (6f) binds tubulin with highest binding affinity (KD, 38 ± 4.0 µM), which is ~ 4.0 times higher than that of the parent compound, noscapine (KD, 144 ± 1.0 µM) and is also more potent than that of the first generation clinical candidate EM011, 9-bromonoscapine (KD, 54 ± 9.1 µM). All these compounds exhibited substantial cytotoxicity toward cancer cells, with IC50 values ranging from 6.7 µM to 72.9 µM; compound 6f showed prominent anti-cancer efficacy with IC50 values ranging from 6.7 µM to 26.9 µM in cancer cells of different tissues of origin. These compounds perturbed DNA synthesis, delayed the cell cycle progression at G2/M phase, and induced apoptotic cell death in cancer cells. Collectively, the study reported here identified potent, third generation noscapinoids as new anti-cancer agents.基于秋水仙碱（colchicine）与鬼臼毒素（podophyllotoxin）等药物的结构相似性进行系统性筛选，得以鉴定出那可丁（noscapine）——一种可减弱微管动态不稳定性且不影响微管总聚合质量的微管靶向制剂（microtubule-targeted agent）。本研究报道了新一代那可丁衍生物，其作为潜在的微管蛋白（tubulin）结合型抗癌候选药物具有开发价值。对衍生物5a、6a-j进行的分子建模实验显示，其对接得分（docking score）介于-7.252至-5.402 kCal/mol，优于母合物（parent compound）那可丁的-5.505 kCal/mol，亦优于其现有衍生物的-5.563至-6.412 kCal/mol。基于线性相互作用能（linear interaction energy, LIE）经验方程，并结合表面广义Born（Surface Generalized Born, SGB）连续溶剂模型开展的自由结合能（Free energy, ΔGbind）计算，预测得到衍生物5a、6a-j的微管蛋白结合亲和力自由能范围为-4.923至-6.189 kCal/mol。其中化合物6f对微管蛋白展现出最高的结合亲和力（-6.189 kCal/mol）。对上述化合物的实验评估结果与理论研究结果一致。N-(3-brormobenzyl)那可丁（6f）对微管蛋白的结合亲和力最高，其解离常数（dissociation constant, KD）为38 ± 4.0 µM，亲和力约为母合物那可丁（KD为144 ± 1.0 µM）的4.0倍，同时亦优于第一代临床候选药物EM011——9-溴那可丁（KD为54 ± 9.1 µM）。所有受试化合物均对癌细胞展现出显著的细胞毒性，其半最大抑制浓度（half maximal inhibitory concentration, IC50）范围为6.7 µM至72.9 µM；其中化合物6f的抗癌活性尤为突出，在不同组织来源的癌细胞中，其IC50范围为6.7 µM至26.9 µM。此类化合物可扰乱癌细胞的DNA合成，使其细胞周期进程阻滞于G2/M期，并诱导癌细胞发生凋亡性死亡。综上，本研究成功鉴定出具有强效活性的第三代那可丁类衍生物（noscapinoids），将其作为新型抗癌制剂进行开发。