Tubulin-IN-63 is a potent inhibitor of tubulin polymerization, targeting the colchicine binding site with an IC50 value of 6.03 µM. It disrupts microtubule dynamics, induces G2/M phase arrest, and triggers apoptosis, thereby inhibiting cancer cell proliferation. Tubulin-IN-63 impairs the capillary network formation in human umbilical vein endothelial cells (HUVEC) and demonstrates antitumor activity in vivo in the B16-F10 mouse model. This compound is useful for research on cancers such as melanoma, lung cancer, and liver cancer.

Tubulin-IN-63 (compound 4a) exhibits significant anti-proliferative activity against various cancer cell lines (B16-F10, MCF-7, Hela, and HepG-2) with IC₅₀ values of 0.052, 0.045, 0.076, and 0.036 µM, respectively, while showing lower cytotoxicity toward normal cell lines (HUVEC, MCF-10A, and LO2) with IC₅₀ values of 751, 903, and 579 nM, indicating selectivity. It inhibits proliferation in A549 and A2780 cells with IC₅₀ values of 85.3 nM and 42.4 nM and remains effective against Paclitaxel-resistant cells (A549/T and A2780/T) with IC₅₀ values of 172.5 nM and 61.8 nM, indicating its potential to overcome Paclitaxel-related resistance. At concentrations of 10-100 nM over 24 hours, Tubulin-IN-63 persistently suppresses cancer cell growth and proliferation in HepG-2 cells, resulting in dose-dependent decreases in clonogenic survival after 10-14 days of drug-free culture. In HepG-2 cells, it induces dose-dependent G2/M cell cycle arrest and apoptosis at concentrations of 10-100 nM over 48 hours， inhibits migration in a concentration-dependent manner over 0-24 hours， and causes dose-dependent microtubule network disruption over 12 hours. Additionally, Tubulin-IN-63 disrupts vascular network formation in HUVECs at 10-100 nM over 6 hours.

Tubulin-IN-63 (compound 4a), administered intraperitoneally at a dose of 10 mg/kg once daily for 14 consecutive days, significantly inhibits tumor growth in the B16-F10 mouse model. Furthermore, it exhibits a synergistic effect when used in combination with the PD-L1 inhibitor NP19.