Synthesis, Structural Characterization, and Antiproliferative Evaluation of Cyclosenegalin B Analogues in Breast Cancer Cells

Triple-negative breast cancer (TNBC), which represents approximately 10–20% of all breast cancer cases worldwide, is considered one of the most aggressive and difficult-to-treat subtypes because it lacks ER, PR, and HER2 expression while exhibiting high genomic instability and strong resistance to therapy. Despite its structurally rigid cyclic peptide framework and enhanced stability against enzymatic degradation, Cyclosenegalin B isolated from Annona species exhibits only modest anticancer potential in its native form. To improve its therapeutic prospects, a multidisciplinary workflow combining computational modeling, chemical synthesis, and biological evaluation was conducted to develop and investigate 36 newly designed Cyclosenegalin B analogs. Each designed analog was initially constructed using ChemDraw followed by three-dimensional structure generation in Chem3D prior to geometric refinement through MM2 minimization and DFT calculations at the B3LYP/3-21G level. Electronic characteristics were subsequently analyzed to identify candidates possessing optimal electronic stability and reactivity profiles. Binding affinity analysis toward multiple breast cancer–related targets, including ERα (PDB ID: 3ERT ), PR (2OVH), EGFR kinase (2ITY), and the NF-κB/IκB-α complex (1NFI), revealed that analogs B9 and B27 demonstrated the most favorable interaction profiles, characterized by strong binding energies and extensive non-covalent interactions involving hydrogen bonding and hydrophobic contacts. These selected peptide candidates were subsequently prepared through Fmoc solid-phase peptide synthesis, followed by purification using reverse-phase HPLC and structural confirmation by ESI-MS analysis (Cyclosenegalin B9: calcd 905.13 Da, obs m/z 903–905; Cyclosenegalin B27: calcd 784.91 Da, obs m/z 782–784). Biological evaluation by MTT assay demonstrated preferential cytotoxicity toward TNBC cells. Cyclosenegalin B9 exhibited IC₅₀ values of 304.52 ± 9.7 µg/mL (luminal T-47D) and 179.82 ± 7.8 µg/mL (MDA-MB-231), confirming subtype selectivity aligned with EGFR/NF-κB–driven binding predictions. These findings validate a rational design approach for Cyclosenegalin B macrocycles and position B9 as a promising lead for further structural refinement, mechanistic studies, and early in vivo assessment.