Desing of Novel 1,4-Naphthoquinone Derivatives: Evaluation of Apoptotic Effects and Anticancer Activity Against Triple Negative Breast Cancer Cells with Molecular Docking and Molecular Dynamics Studies

<title>Abstract</title> <p> Worldwide, the number of deaths due to cancer is steadily increasing. Breast cancer is the most common type of cancer in the female population. Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer and has a limited response to current treatments. Naphthoquinone compounds are a class of molecules with high anticancer potential, exhibit various biological activities such as antioxidant, anticancer, and antimicrobial effects. In this study on quinone derivative compounds, <italic>N,S,O-</italic> substituted-1,4-naphthoquinone derivatives were synthesized as a result of reactions carried out between the compound 2,3-dichloro-1,4-naphthoquinone ( <bold>1</bold> ) and nucleophiles ( <bold>2, 3, 6, 7, 10)</bold> . All synthesized compounds were characterized by FT-IR, ¹ H-/ ¹³ C-NMR and MS. It is our first study that the desining of <italic>N,S,O-</italic> substituted-1,4-Naphthoquinone derivatives and we evaluated of their apoptotic effects and anticancer activity against triple negative breast cancer cells with using molecular docking and molecular dynamics method. The anticancer activities of these compounds were determined in MDA-MB-231 triple-negative breast cancer (TNBC) cells using the CCK-8 assay, and their apoptotic activities were evaluated by Annexin V analysis and acridine orange/propidium iodide staining. All derivatives suppressed cell proliferation; however, compounds <bold>4</bold> and <bold>13</bold> showed the greatest effect against TNBC cells, with IC₅₀ = 2.7 µg/mL and IC₅₀ = 3 µg/mL, respectively. Of the derivatives obtained, compounds <bold>4</bold> and <bold>13</bold> induced early apoptotic death, while compounds <bold>11</bold> and <bold>12</bold> led to late apoptotic death in TNBC cells compared to the control group. The AO/PI staining verified the apoptotic morphology of the cells and compounds <bold>9</bold> , <bold>11</bold> , and <bold>13</bold> induced a greater formation of apoptotic bodies in the cells. The interactions of compound <bold>4</bold> and compound <bold>13</bold> with the MCL-1 protein responsible of apoptosis were analyzed using molecular docking followed by molecular dynamics simulations. A stronger docking score was observed for compound 13 (−8.4 kcal/mol) compared to compound <bold>4</bold> (−7.2 kcal/mol). Furthermore, MD simulations were performed to shed light on the molecular dynamics behavior of both complexes. MCL1-4 and MCL1-13 both remained stable during simulations, with RMSD values below 3 Å. Higher positional stability was seen for compound <bold>13</bold> , while compound <bold>4</bold> showed slightly more flexibility within the binding pocket. The protein structure remained stable in both cases, and reduced residue fluctuations were observed around compound <bold>4</bold> , suggesting local stabilization. MM-GBSA results indicated a more favorable binding free energy for compound <bold>4</bold> (−52.18 kcal/mol) compared to compound <bold>13</bold> (−38.76 kcal/mol). </p>