Clear cell Renal Cell Carcinoma (ccRCC) is the most common subtype of renal malignancy and remains a major clinical challenge. This challenge underscores the urgent need to identify novel molecules with potential as therapeutic 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 Benzimidazoles 1 Mitotic Checkpoint Serine/Threonine Kinase B), a mitotic checkpoint kinase involved in ccRCC pathogenesis. Complete structural characterization of all synthesized compounds was achieved using Fourier-transform infrared (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 elucidated by 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 the most active compound. The computed IR spectrum of compound 3a showed excellent agreement with experimental data, supporting the structural findings from X-ray analysis. Hirshfeld surfaces (HS) analyses were carried out to visualize the intermolecular interactions in the crystal packing of 3a. This analysis highlighted key N—H···O and N—H···N hydrogen bonds, which were found to be in excellent agreement with the experimental single-crystal X-ray diffraction data. Multiple noncovalent interactions (N–H···O/N, C–H···O, C–H···π) were identified in the solid state and quantitatively examined using Independent Gradient Model based on Hirshfeld partition (IGMH) analysis, confirming N–H···N as the strongest interaction. In vitro cytotoxicity assays on Caki-1 cells revealed that compounds 3a, 3c, and 3d exerted significant antiproliferative effects. Compound 3a exhibited superior BUB1B inhibition compared to the other derivatives, eliciting a robust apoptotic response as evidenced by enhanced PARP cleavage and caspase activation. Molecular docking and dynamics studies revealed stable and favorable binding affinity of the compound 3a within the BUB1B active site, consistent with experimental observations. The combined experimental and computational findings indicate that these pyrimidine derivatives, particularly compound 3a, act as promising BUB1B inhibitors with potential therapeutic relevance in ccRCC therapy.