Design, Synthesis, and Integrated In Silico and In Vitro Evaluation of Chloro-Substituted Salicylaldehyde Benzoylhydrazones as Anticancer Agents

Abstract

Background: Building on earlier work with methoxy-, dimethoxy-, bromo-, and nitro-substituted salicylaldehyde benzoylhydrazones, we synthesized and evaluated new 4-chloro- and 5-chloro derivatives bearing an additional chlorine substituent on the hydrazide-derived phenyl ring to further explore the impact of halogenation on cytotoxic activity. Methods: Chloro-hydrazones were obtained through a one-step condensation of 4- or 5-chlorosalicylaldehyde with benzhydrazide or 2-, 3-, and 4-chlorobenzhydrazides. Their physicochemical, pharmacokinetic, ADME, lead-likeness, and drug-likeness profiles were evaluated in silico using SwissADME, ACD/Labs v9.10, and MDL QSAR v2.2.0.0.446. The synthesized compounds were structurally characterized by IR, 1H NMR, 13C NMR, and HR ESI–MS, and their cytotoxic activity was subsequently assessed by the MTT assay in selected cancer cell lines. Molecular docking was performed against ABL1 tyrosine kinase (ABL1 TK), a potential molecular target relevant to two of the investigated leukemia cell lines, using GOLD v.5.2.2 (CCDC Ltd., Cambridge, UK). Results: The chloro-substituted salicylaldehyde benzoylhydrazones exhibited pronounced, cell-type-dependent cytotoxicity, with leukemia cells being substantially more sensitive than breast carcinoma cells. Most derivatives showed low- to sub-micromolar IC50 values against SKW-3 human T-cell prolymphocytic leukemia, K-562 human chronic myeloid (myelogenous) leukemia, and BV-173 human BCR::ABL1-positive leukemia cells, with K3 and K4 exhibiting the highest activity in SKW-3 cells (IC50 = 0.5 ± 0.1 µM). K5 showed particularly strong activity against K-562 and BV-173 cells (IC50 = 0.7 ± 0.1 and 0.9 ± 0.1 µM, respectively), compared with 26.9 ± 2.4 and 21.5 ± 3.3 µM for imatinib. HL-60 human acute promyelocytic leukemia cells showed intermediate sensitivity (IC50 = 1.3–13.9 µM), whereas the activity against MCF-7 estrogen receptor-positive and MDA-MB-231 triple-negative breast carcinoma cells was more variable (1.5–32.1 and 4.1–62.5 µM, respectively). The derivatives showed high selectivity toward malignant cells relative to non-malignant CCL-1 non-malignant mouse fibroblasts, with lower-bound SI values frequently exceeding 50 and reaching >200 in SKW-3 cells. Conclusions: The chloro-substituted salicylaldehyde benzoylhydrazones exhibited pronounced and selective cytotoxicity, particularly toward leukemia cell lines, identifying this scaffold as a promising starting point for further anticancer drug-discovery studies.