Existing studies on PROTAC technology frequently rely on modifications and validation of already marketed drugs, which limits the exploration of non-drug-forming proteins and targets that do not have existing therapeutic agents. In contrast, utilizing easily synthesized combinatorial chemical databases that offer a broad selection of compounds presents a promising strategy for discovering new PROTAC molecules. In this study, virtual screening was conducted on a small molecule database created using combinatorial chemistry, which contains a total of 12,904 active molecules. A potential CDK6 inhibitor, compound 10, was identified and subsequently validated for its activity. The primary aim of the study was to explore its anti-tumor mechanism and explore its potential for PROTAC modification. The results showed that compound 10 effectively targeted CDK6, leading to the modification of its derivative 10PL8, which had a DC50 value of 26.67 ± 5.09 μM for targeting CDK6. Further structural modifications led to the synthesis of additional derivatives and PROTAC molecules, ultimately identifying a more potent CDK6 inhibitor, compound 2f, with an IC50 of 0.56 ± 0.06 μM in AGS cells. Moreover, the PROTAC molecule 2fPL8 demonstrated a DC50 value of 4.80 ± 0.79 μM for CDK6. The experimental findings above suggest that employing virtual screening of target proteins through combinatorial chemical databases to identify potential ligands and degradation agents is a feasible and valuable strategy for future PROTAC studies.
