Herein, a new series of cyclopentaquinoline derivatives (6a–g) were designed and synthesized based on the pharmacophoric features acquired by some reported DNA intercalators and topoisomerase II inhibitors. The anti-proliferative properties of the newly synthesized compounds were investigated and both 6d and 6f compounds revealed promising anticancer activities against the five different cancer cell lines. Compound 6d exhibited very strong to strong anticancer activities against HepG-2, MCF-7, HCT-116, MDA-231, and Caco-2 cell lines with IC₅₀ values of 7.06, 11.61, 6.28, 8.32, and 18.76 μM, respectively, compared to those of doxorubicin as a reference standard (4.50, 4.17, 5.23, 3.18, and 12.49 μM, respectively). However, compound 6f showed superior anticancer activities to doxorubicin against HepG-2, HCT-116, and Caco-2 cell lines with IC₅₀ values of 2.31, 3.67, and 9.83 μM, respectively. Also, compound 6f showed very strong anticancer activities against MCF-7 and MDA-231 cell lines with IC₅₀ values of 6.83 and 4.78 μM, respectively. Moreover, it was revealed that compounds 6d and 6f efficiently inhibited the topoisomerase II activity with IC₅₀ values of 2.26 and 0.97 μM, respectively, and close to that attained by etoposide as a reference standard (IC₅₀ = 0.34 μM). Moreover, compounds 6d and 6f were investigated to have apoptosis percentages of 28.05% and 35.56% which are more than the control (0.95%) by approximately 27- and 37-fold, respectively. Besides, both compounds induced a significant cell-cycle arrest in S and G1 phases, respectively. Furthermore, the molecular docking studies of compounds 6d and 6f against the DNA–topoisomerase II complex revealed their binding scores to be −5.75 and −6.62 kcal mol⁻¹ compared to both the co-crystallized inhibitor (EVP) and doxorubicin as two reference standards (−10.52 and −8.92 kcal mol⁻¹, respectively). Also, ADMET in silico studies were performed to describe the physicochemical, pharmacokinetics, and toxicity parameters of the synthesized derivatives. Finally, a structure–activity relationship study (SAR) was accomplished so that any changes in their antiproliferative activities can be disclosed upon further structural modifications in the future.