Microtubules composed of α-tubulin and β-tubulin heterodimers are an organelle widely present in cells, with multiple biological functions such as cell mitosis, maintenance of cell shape, intracellular transportation, and cell signaling. Increasing reports have considered microtubules a promising target for anti-tumor drugs by disrupting their polymerization dynamics [1,2]. Microtubule targeting agents (MTAs) are a group of molecules that exert their anti-tumor activity via binding to tubulin, with several binding sites including Paclitaxel site, Vinca site, Laulimalide site, Pironetin site, Maytansine site, and Colchicine site (Fig. 1) [[3], [4], [5]]. Currently, most MTAs used clinically such as taxanes and Vinca alkaloids have obvious drawbacks including complex structure, poor aqueous solubility, dose-limiting toxicity, and the development of multi-drug resistance (MDR). In contrast, Colchicine binding site inhibitors (CBSIs) have received extensive research and attention for their potential ability to overcome the aforementioned problems due to their simpler structures, favorable pharmacokinetic properties, and non-P-gp substrate nature [6,7]. To date, no CBSIs have been approved for the treatment of cancer, necessitating further structural optimization and more in-depth structure-activity relationship (SAR) studies. Moreover, cancer immunotherapy (e.g., anti-PD-1/PD-L1, anti-CTLA-4) harnesses the body's immune system to recognize and eliminate cancer cells, representing a transformative advance in oncology. Unlike traditional chemotherapy or radiotherapy, which directly kill tumor cells, immunotherapy activates or reinvigorates anti-tumor immune responses. For example, checkpoint inhibitors block immunosuppressive pathways such as PD-1/PD-L1 to restore T cell-mediated tumor clearance. Although this approach has improved clinical outcomes in multiple cancer types, challenges including low response rates and primary or acquired resistance persist, underscoring the need for novel therapeutic strategies—such as combining tubulin inhibitors with immunotherapeutic agents—to enhance treatment efficacy. Therefore, we have designed a library of novel imidazo[1,2-a]pyridine analogues, aiming to develop potential tubulin polymerization inhibitors and enhanced immune potentiation for cancer treatment.