Design, Synthesis, and Cytotoxic Evaluation of 1,3,4-Thiadiazolyl- Benzamides as Potent BRAF Inhibitors: Integrating In Vitro and In Silico Approaches

Introduction: Melanoma, an aggressive skin cancer, is frequently associated with the BRAF^V600E mutation, leading to continuous activation of the MAPK/ERK pathway. Although current BRAF inhibitors have improved clinical outcomes, the development of resistance remains a significant challenge. This study aimed to design and evaluate a new series of 1,3,4-thiadiazolyl-benzamide derivatives as potential BRAF kinase inhibitors for melanoma therapy. Methods: The compounds were synthesized via a two-step protocol and characterized by spectroscopic techniques. Molecular docking studies were performed using the BRAF kinase crystal structure (PDB ID: 4LMN) to predict binding affinity and interaction patterns. In silico ADMET analyses were conducted to evaluate pharmacokinetic and drug-likeness properties. Results: Docking studies revealed that several designed derivatives exhibited strong pre-dicted affinities within the BRAF active site, forming key hydrogen bonds and hydrophobic interactions comparable to dabrafenib, the reference inhibitor. Among them, IA25 showed the highest docking score (−9.7 kcal/mol), forming two hydrogen bonds with Asn221 and Phe209. ADMET analyses indicated favorable bioavailability, stability, and low toxicity. All ten synthesized compounds were tested for in vitro cytotoxicity using the MTT assay on M-14 and B16-F1 cell lines. Compounds IA25, IA11, and IA8 exhibited the highest activity, with IC₅₀ values ranging from 33.93 to 40.62 nM, indicating strong anti-proliferative poten-tial. discussion: In contrast, the other tested compounds demonstrated moderate cytotoxic effects on the cell lines. Overall, this study represents a significant step toward the discovery of new BRAF inhibitors for melanoma cancer treatment through in-silico methods. Future work may focus on developing additional derivatives and conducting further in-depth in vivo studies to construct a structure-activity relationship (SAR) for rational drug design. Conclusion: The synthesized thiadiazole–benzamide hybrids demonstrate promising in silico and in vitro activities as potential BRAF-targeted anticancer agents. These findings provide a solid foundation for further optimization and preclinical evaluation.