Tubulin polymerization-IN-6 (compound 5f) is a potent inhibitor of tubulin polymerization, with an IC50 of 1.09 μM. It not only inhibits cell migration and tube formation but also has anti-angiogenic properties. Additionally, Tubulin polymerization-IN-6 has been found to effectively hinder tumor growth in HT29 xenograft Balb/c nude mice [1].

Tubulin polymerization-IN-6 (compound 5f) (0-20 μM, 24 h) shows a broad spectrum of anti-proliferation activity against cancer cell lines [1]. Tubulin polymerization-IN-6 (0-100 nM, 24 h) inhibits tumor cells colony formation, up-regulates the expression of Ac-α-tubulin and DeY-α-tubulin [1]. Tubulin polymerization-IN-6 (0-5 μM, 1 h) competes with colchicine and directly binds to the colchicine binding site, thus inhibit tubulin polymerization [1]. Tubulin polymerization-IN-6 (0-250 nM, 24 h) possesses a favorable anti-migration activity against cancer cells [1]. Tubulin polymerization-IN-6 (0-50 nM, 24 h) has the ability to inhibit the angiogenesis of HUVEC cells [1]. Tubulin polymerization-IN-6 (0-100 nM, 24 h) induces cell cycle arrest by regulating associated proteins, induces apoptosis by regulating associated proteins and down-regulating mitochondrial membrane potential, and dose-dependently promotes the production of ROS in HT29 cells [1]. Cell Proliferation Assay Cell Line: HT29, MCF-7, HeLa, MDA-MB-231, A549 [1] Concentration: 0-20 μM Incubation Time: 24 h Result: Had a broad spectrum of anti-proliferation activity against cancer cell lines (MCF-7, MDA-MB-231, A549, Hela, and HT29), with IC 50 values of 0.14 ± 0.03, 0.10 ± 0.00, 0.24 ± 0.03, 0.035 ± 0.002, and 0.023 ± 0.001 μM, respectively; and showed moderate anti-proliferative activity against drug resistant cancer cells (MCF-7/TxR and A549/TxR), with IC 50 values of 0.18 ± 0.02 and 0.31 ± 0.08 μM, and DRI (drug-resistant index) of 1.3 and 1.2, respectively. Western Blot Analysis Cell Line: HT29 cells [1] Concentration: 0, 25, 50, and 100 nM Incubation Time: 24 h Result: Up-regulated the expression of Ac-α-tubulin (acetyl-α-tubulin) and DeY-α-tubulin (detyrosinated-α-tubulin); regulated the expressions of the proteins involved in cell cycle such as cdc25c, cdk7, cyclin B1, and cdc2; down-regulated the level of Bim and up-regulated the levels of Bcl-2, p-Bcl-2, and Bax, decreased the expression of p-Histone H3(Ser10) and increased the expression of cleaved-Caspase-9, cleaved-Caspase-3, PARP, and cleaved-PARP. Immunofluorescence Cell Line: HT29 cells [1] Concentration: 0, 25, 50, and 100 nM Incubation Time: 6 h Result: Dose-dependently depolymerized the tubulin polymers into oligomers, and caused the microtubule network to collapse in HT29 cells. Cell Cycle Analysis Cell Line: HT29 cells [1] Concentration: 0, 12.5, 25, 50, and 100 nM Incubation Time: 24 h Result: Induced a dose dependent G2/M phase arrest, increased the proportion of G2/M phase cells from 20.9% to 87.5% at 100 nM. Apoptosis Analysis Cell Line: HT29 cells [1] Concentration: 0, 25, 50, and 100 nM Incubation Time: 24 h Result: Induced apoptosis, increased the percentages of total apoptosis cells, down-regulated mitochondrial membrane potential.

Tubulin polymerization-IN-6 (compound 5f) (HT29 xenograft Balb/c nude mice, 0-10 mg/kg, IP, once every two days, for three weeks) dose-dependently inhibits the tumor growth [1]. Tubulin polymerization-IN-6 (SD rats, 10 mg/kg, IV, once) shows the better pharmacokinetic properties [1]. Pharmacokinetic Parameters of Tubulin polymerization-IN-6 in SD rats [1]. Parameters 5f t 1/2 (h) 1.73 AUC (μg/L·h) 5.67 MRT (h) 1.92 CL (L/h/kg) 1.76 T max (h) 0.14 C max (ng/mL) 6.88 Animal Model: Immunodeficient Balb/c nude mice (HT29 xenograft, 5-week-old, 36 mice, six groups) [1] Dosage: 0, 5, 7.5, 10 mg/kg Administration: IP, once every two days, for three weeks Result: Dose-dependently inhibited the tumor growth, inhibits the tumor weight growth by 75.5% at 10 mg/kg. Animal Model: SD rats (5-week-old) [1] Dosage: 10 mg/kg Administration: IV, once (Pharmacokinetic Analysis) Result: Showed the better pharmacokinetic properties, exhibited an eight-fold half-life and a two-fold AUC improvement.