he Bcl-2 protein family plays a critical role in regulating apoptosis, making it a key target for cancer therapy. In this study, a series of novel Bcl-2 inhibitors have been designed, synthesized, and evaluated. To disrupt the interactions between anti-apoptotic Bcl-2 and pro-apoptotic proteins, compounds were developed based on essential pharmacophoric features. Among the tested compounds, R4, R14, R17, and R23 demonstrated potent anticancer activity with sub-micromolar IC50 concentrations across various Bcl-2 expressing human cancer cell lines (IC50 ranges: 1.46-7.67 mu M for cancer cells). ELISA binding assays further validated the efficacy of R4, R14, and R23, showcasing their potency with IC50 values ranging from 0.25 to 0.63 mu M, compared to gossypol and ABT-199 (venetoclax), with IC50 values of 0.6 and 0.038 mu M, respectively. Furthermore, the R23 revealed a significant induction of late and early apoptosis and cell cycle arrest at G1 phase. Noteworthy, R23 emerged as a promising candidate with unique computational analysis, showing superior displacement of hydration sites and higher Delta G values in WaterMap studies. Moreover, molecular dynamics simulations reveal low root mean square deviation fluctuations, indicating strong and stable interactions with Bcl-2. These findings underscore the therapeutic potential of R23 as a Bcl-2 inhibitor.