Selective elimination of tumors has always been the mainstay of oncology research. The on-going research underlying the cellular apoptotic mechanisms reveal caspases activation, especially the key effector caspase-3, as a personalized tumor-selective therapeutic strategy. Our continued research protocol has exploited new optimized Passerini α-acyloxy carboxamides as efficient apoptotic inducers via caspase-3/7 dependent mechanism with highly selective anticancer profiles. The adopted design rationale relied on excluding structural alerts of previous leads, while merging various pharmacophoric motifs of natural and synthetic caspase activators via optimized one-pot Passerini reaction conditions. The prepared compounds resulting from Passerini reaction were screened for their cytotoxic activities against colorectal Caco-2 and liver HepG-2 cancer cells compared to normal fibroblasts utilizing MTT assay. Notably, all compounds exhibited promising low-range submicromolar IC₅₀ against the studied cancer cell lines, with outstanding tumor selectivity (SI values up to 266). Hence, they were superior to 5-fluorouracil. Notably, 7a, 7g, and 7j conferred the highest potencies against Caco-2 and HepG-2 cells and were selected for further mechanistic studies. Caspas-3/7 activation assay of the hit compounds and flow cytometric analysis of the treated apoptotic cancer cells demonstrated their significant caspase activation potential (up to 4.2 folds) and apoptotic induction capacities (up to 58.7%). Further assessment of Bcl2 expression was performed being a physiological caspase-3 substrate. Herein, the three studied Passerini adducts were able to downregulate Bcl2 in the treated Caco-2 cells. Importantly, the mechanistic studies results of the three hits echoed their preliminary MTT antiproliferative potencies data highlighting their caspase-3 dependent apoptotic induction. Finally, the in silico predicted physicochemical and pharmacokinetic profiles, as well as ligand efficiency metrics were drug-like.

选择性消除肿瘤一直是肿瘤学研究的支柱。正在进行的细胞凋亡机制基础研究揭示了 caspases 激活，尤其是关键效应子 caspase-3，作为一种个性化的肿瘤选择性治疗策略。我们的持续研究方案利用具有高选择性抗癌特性的 caspase-3/7 依赖机制，将新优化的 Passerini α-酰氧基甲酰胺作为有效的细胞凋亡诱导剂。采用的设计原理依赖于排除先前线索的结构警报，同时通过优化的一锅 Passerini 反应条件合并天然和合成半胱天冬酶激活剂的各种药效团基序。与正常成纤维细胞相比，使用 MTT 测定法筛选由 Passerini 反应制备的化合物对结直肠 Caco-2 和肝 HepG-2 癌细胞的细胞毒性活性。值得注意的是，所有化合物都表现出有前途的低范围亚微摩尔 IC₅₀对抗研究的癌细胞系，具有出色的肿瘤选择性（SI 值高达 266）。因此，它们优于 5-氟尿嘧啶。值得注意的是，7a、7g和7j赋予了针对 Caco-2 和 HepG-2 细胞的最高效力，并被选择用于进一步的机制研究。命中化合物的 Caspas-3/7 激活测定和处理过的凋亡癌细胞的流式细胞术分析证明了它们显着的半胱天冬酶活化潜力（高达 4.2 倍）和凋亡诱导能力（高达 58.7%）。作为生理性 caspase-3 底物，对 Bcl2 表达进行了进一步评估。在此，研究的三种 Passerini 加合物能够下调经处理的 Caco-2 细胞中的 Bcl2。重要的是，这三个命中的机制研究结果与它们的初步 MTT 抗增殖效力数据相呼应，突出了它们的 caspase-3 依赖性细胞凋亡诱导。最后，计算机预测的物理化学和药代动力学特征以及配体效率指标与药物类似。