Potent and Selective Benzothiazole-Based Antimitotics with Improved Water Solubility: Design, Synthesis, and Evaluation as Novel Anticancer Agents

Abstract

The design of colchicine site ligands on tubulin has proven to be a successful strategy to develop potent antiproliferative drugs against cancer cells. However, the structural requirements of the binding site endow the ligands with low aqueous solubility. In this work, the benzothiazole scaffold is used to design, synthesize, and evaluate a new family of colchicine site ligands exhibiting high water solubility. The compounds exerted antiproliferative activity against several human cancer cell lines, due to tubulin polymerization inhibition, showing high selectivity toward cancer cells in comparison with non-tumoral HEK-293 cells, as evidenced by MTT and LDH assays. The most potent derivatives, containing a pyridine moiety and ethylurea or formamide functionalities, displayed IC50 values in the nanomolar range even in the difficult-to-treat glioblastoma cells. Flow cytometry experiments on HeLa, MCF7, and U87MG cells showed that they arrest the cell cycle at the G2/M phases at an early time point (24 h), followed by apoptotic cell death 72 h after the treatment. Tubulin binding was confirmed by microtubule network disruption observed via confocal microscopy. Docking studies support favorable interaction of the synthesized ligands at the colchicine binding site. These results validate the proposed strategy to develop potent anticancer colchicine ligands with improved water solubility.

Frentizole, a Nontoxic Immunosuppressive Drug, and Its Analogs Display Antitumor Activity via Tubulin Inhibition

“…These results suggest that the replacement of the phenyl ring by the smaller ethyl groups requires a compensating increase in volume on the BZT moiety, and the observed differences might result from different binding modes. As anticipated, most of the synthesized compounds did not show antiproliferative activity, thus reflecting the strict structural requirements for eliciting antiproliferative activity, which is in good agreement with many previous studies on colchicine-site ligands showing that structural variations resulted in large potency changes [22,24,28,50]. Frentizole, although not the most potent antiproliferative agent of the series, has the broader antiproliferative profile and is, therefore, the optimal candidate for repurposing.…”

“…Accordingly, the docking results place the cis isomer within the AB subpockets and the trans isomer within the AC ones. In the first instance, the BZT ring is located in the B zone and the phenyl ring in the region between the A the C zones, as previously described by us for related ureas [50]. For the trans isomer, the BZT is allocated to the A zone, similar to MI-181 [36], and the phenyl ring toward the C zone.…”

“…The low toxicity of frentizole is a favorable property as toxicity is one of the main drawbacks of tubulin inhibitors for the treatment of cancer. Furthermore, the activity of frentizole against the glioblastoma cell line U87MG with a low micromolar IC 50 value (7.33 µM) compares very favorably with the only available therapy against glioblastoma, temozolomide, which has in vitro IC 50 values against this cell line that are in the millimolar range [50]. Recently, glioblastoma tumor cells have been shown to be especially sensitive to microtubule-targeting agents, and, therefore, the activity here described for frentizole suggests it as a promising option for drug repurposing against glioblastomas [50,64].…”

“…With the aim of testing the hypothesis that frentizole is a tubulin inhibitor, we synthesized frentizole and a series of analogs and evaluated them as antiproliferative agents against HeLa human cancer cells. Small structural changes can dramatically affect the potency of antiproliferative compounds, and colchicine-site binders are not an exception [22,24,28,50]. Therefore testing only a single compound (frentizole) might fail to uncover the desired activity.…”

“…Interestingly, frentizole was, again, the only compound in the whole series to show activity below the threshold concentration against glioblastoma cell line U87MG, although with a higher IC 50 value of 7.33 µM. We recently showed that BZT-based inhibitors of tubulin polymerization have antiproliferative activity against glioblastoma cells, which are known to be especially sensitive to tubulin inhibitors [50]. In the phenyl urea series, the only substituent tolerated at the BZT moiety is the methoxy group present in frentizole, and methylation of the urea is also detrimental to the activity, as shown by the methylation of frentizole (6e).…”