In Vitro: The antiproliferative activity of these compounds was evaluated in two human metastatic melanoma cell lines (A375 and WM164) and three human prostate cancer cell lines (LNCaP, PC-3, and Du 145) using the methods described previously. Colchicine was used as a positive control. The 3-indole compound 6a (VERU-111) showed high activity (3.8 nM; unless specified, the IC50 value for each compound is the average of all five cancer cell lines), identifying this compound as the most potent compound in the ABI family (I/II/III) to date. The bulky phenylsulfonyl group in the 3-indole ring (6b) resulted in 50-fold lower activity (196.2 nM), suggesting the size of the substituent on the indole NH may play an important role in determining activity. Consistent with this hypothesis, the dimethylated 3-indole compound 6c showed very good potency (39.8 nM), indicating that a smaller group on either the imidazole- or indole-NH was well tolerated. The benzofuran compound 6d and benzothiophene compound 6e showed relatively strong activity (41.2 nM, 35.0 nM for 6d and 6e respectively), although it is about 10-fold less active than the 3-indole compound 6a, implying that the replacement of the 3-indole NH with either an oxygen or a sulfur does not provide any beneficial effect on the activity. [1]

Tubulin Polymerization Assay: Bovine brain tubulin (>97% pure) was incubated with compounds 6a-e, at concentrations of 5 and 10 μM. Colchicine at 5 μM was used as a positive control. Compounds 6a-e inhibited tubulin polymerization in a dose-dependent manner (Figure 3). Complete inhibition of tubulin polymerization was observed after 20 min of treatment with 6a (Figure 3A) at 10 μM, while 70% inhibition was achieved by 6a at 5 μM. Compound 6b (Figure 3B) inhibited tubulin polymerization by 67% and 85% at 5 and 10 μM, respectively. Compound 6c (Figure 3C) inhibited 65% and 80% of tubulin polymerization at 5 and 10 μM, respectively, while 30% and 60% inhibition of tubulin polymerization was observed for compound 6d (Figure 3D) at 5 and 10 μM, respectively. Compound 6e (Figure 3E) at 10 μM inhibited tubulin polymerization to an extent of 30%, but had no effect on tubulin polymerization 5 μM (same as control DMSO). These results clearly indicate that the ABI-III compounds are strong inhibitors of tubulin polymerization and the inhibitory effects are roughly proportional to the relative antiproliferative potency for 6a (IC50=3.8 nM), 6b (IC50=196.2 nM), 6c (IC50=39.8 nM), 6d (IC50=41.2 nM), and 6e (IC50=35.0 nM). [1]

In Vivo: VERU-111 (50 μg/mouse; intra-tumorally; 3 times per week for 3 weeks) effectively inhibits tumor growth as compared to vehicle-treated group. None of the mouse showed any apparent toxicity as constant increase of body weight in VERU-111 treated mice. [2]

References:

[1] Discovery of novel 2-aryl-4-benzoyl-imidazole (ABI-III) analogues targeting tubulin polymerization as antiproliferative agents. J Med Chem. 2012 Aug 23;55(16):7285-9.

[2] Therapeutic efficacy of a novel βIII/βIV-tubulin inhibitor (VERU-111) in pancreatic cancer. J Exp Clin Cancer Res. 2019 Jan 23;38(1):29.