A B S T R A C T
Clear cell Renal Cell Carcinoma (ccRCC) is the most common subtype of renal malignancy and remains a majorclinical challenge. This challenge underscores the urgent need to identify novel molecules with potential astherapeutic targets in ccRCC. Therefore, in this study, four novel pyrimidine-based derivatives of TG-101,209(3a-d) were designed and synthesized as potential inhibitors of BUB1B (Budding Uninhibited by Benzimidazoles1 Mitotic Checkpoint Serine/Threonine Kinase B), a mitotic checkpoint kinase involved in ccRCC pathogenesis.Complete structural characterization of all synthesized compounds was achieved using Fouriertransforminfrared (FT-IR) spectroscopy, nuclear magnetic resonance (1H and 13C NMR), and mass spectrometry(MS). The crystalline architecture and intermolecular interactions of compound 3a were further elucidatedby single-crystal X-ray diffraction. Theoretical computations were performed using Density Functional Theory(DFT) at the B3LYP/6–311++G(d,p) level of theory to investigate the electronic and structural properties of themost active compound. The computed IR spectrum of compound 3a showed excellent agreement with experimentaldata, supporting the structural findings from X-ray analysis. Hirshfeld surfaces (HS) analyses were carriedout to visualize the intermolecular interactions in the crystal packing of 3a. This analysis highlighted keyN—H⋅⋅⋅O and N—H⋅⋅⋅N hydrogen bonds, which were found to be in excellent agreement with the experimentalsingle-crystal X-ray diffraction data. Multiple noncovalent interactions (N–H⋅⋅⋅O/N, C–H⋅⋅⋅O, C–H⋅⋅⋅π) wereidentified in the solid state and quantitatively examined using Independent Gradient Model based on Hirshfeldpartition (IGMH) analysis, confirming N–H⋅⋅⋅N as the strongest interaction. In vitro cytotoxicity assays on Caki-1cells revealed that compounds 3a, 3c, and 3d exerted significant antiproliferative effects. Compound 3aexhibited superior BUB1B inhibition compared to the other derivatives, eliciting a robust apoptotic response asevidenced by enhanced PARP cleavage and caspase activation. Molecular docking and dynamics studies revealedstable and favorable binding affinity of the compound 3a within the BUB1B active site, consistent with experimentalobservations. The combined experimental and computational findings indicate that these pyrimidinederivatives, particularly compound 3a, act as promising BUB1B inhibitors with potential therapeutic relevance inccRCC therapy.
