Novel Benzothiazole, Benzimidazole and Benzoxazole Derivatives as Potential Antitumor Agents: Synthesis and Preliminary in Vitro Biological Evaluation

In a previous hit-to-lead research program targeting anticancer agents, two promising lead compounds, 1a and 1b, were found. However, the poor solubility of 1a and 1b made difficult further in vivo studies. To solve this problem, a lead optimization was conducted through introducing N-methyl-piperazine groups at the 2-position and 6-position. To our delight, the optimized analogue 1d showed comparable antiproliferative activity in vitro with better solubility, compared with 1a. Based on this result, the replacement of the benzothiazole scaffold with benzimidazole and benzoxazole moieties afforded 1f and 1g, whose activities were fundamentally retained. In the preliminary in vitro biological evaluation, the immunofluorescence staining of HCT116 cells indicated that 1d, 1f and 1g led to cytosolic vacuolization which was not induced by 1a at low micromolecular concentrations. These results suggest that these optimized compounds might potentially constitute a novel class of anticancer agents, which merit further studies.


Introduction
Cancer is the leading cause of death in developed countries and the second leading cause of death in developing countries [1]. Although surgical resection is potentially curative, the risk of recurrence remains very high. Besides surgical resection, treatment strategies for high risk patients are still mainly based on adjuvant or neoadjuvant use of chemotherapeutic agents alone or in combination with radiotherapy. Unfortunately, the use of the above standard therapeutic protocols only results in a moderate decline in mortality and the risk of sustaining a recurrence of disease remains high. Thus, it is urgent to develop novel chemotherapeutic agents for the treatment of cancer. In a recent hit-to-lead research of anticancer agents [2], lead compounds 1a and 1b ( Figure 1) with benzothiazole scaffolds, showed promising anticancer in vitro activities. However, the solubility of 1a and 1b was far from satisfactory for further in vivo research. As a result, a lead optimization of 1a and 1b was conducted. The N-methyl-piperazine group as found in imatinib is a commonly used motif in lead optimization [3]. Introduction of N-methylpiperazine moieties at the 2-position and 6-positions of the benzothiazole scaffold afforded compounds 1c, 1d and 1e. To determine the role of the benzothiazole scaffold, the benzoxazole analogue 1f and benzimidazole analogue 1g were also designed ( Figure 2). Herein, we report the synthesis of 1c-g, preliminary structure-activity relationships (SARs) and biological evaluation of these anticancer agents.

Chemistry
The synthetic route of compounds 1c-g using a modified literature method [4,5] is outlined in Scheme 1. Taking 1d for example, the key intermediate 2a, 2-mercapto-6-aminobenzothiazole, was alkylated with side chain 3b under basic conditons to give 4a. Subsequent acylation of 4a with 2-methoxybenzoic acid yielded the target compound 1d. Besides the optimization of side chains, the replacement of the benzothiazole scaffold with benzimidazole and benzoxazole units (Table 1), afforded 1f and 1g which were prepared using the same synthetic strategy.  The key intermediate 2c was readily prepared from commercially available 2-amino-5-nitrophenol following cyclization and subsequent reduction of the nitro group according to the method represented in Scheme 2. Side chain 3b was prepared from 4-fluoronitrobenzene condensed with N-methylpiperazine, subsequent reduction and acylation as shown in Scheme 3.

The MTT Assay
With 1c-g at hand, we evaluated their anti-proliferative activities (IC 50 ) on HepG2 and HCT116 cells, using the MTT assay ( Table 1). As shown in the Table, introduction of an N-methylpiperazine substituent at the 2-position (compound 1d) in the R1 group was well tolerated. To our surprise, replacement of the benzyl group in R1 with an N-methylpiperazine-phenyl group (compound 1c) led to almost total loss of activity. The introduction of N-methylpiperazine substituent at the 6-position (1e) in the R2 group had a detrimental effect on the potency, that is, about 20-50-fold decrease in potency, compared with that of 1a. After identifying that the N-methylpiperazine-phenyl substituent is well tolerated at the 2-position in the R1 group, we next turned to examining the role of the benzothiazole scaffold. Replacement of the benzothiazole ring with benzimidazole and benzoxazole yielded 1f and 1g with a slightly decrease in potency for HCT-116 cells compared with 1a, but showed no clear SAR trends. It was noteworthy that analogue 1d exhibited comparable inhibitory activity against HepG2 and HCT-116 cells.
To further study the cytotoxic profile, compound 1d was selected for evaluation of inhibitory activities against a panel of different types of human cancer cell lines: Prostate cancer cell line PC-3, human cervical carcinoma cell line Hela, ovarian cancer cell line SKOV-3, lung cancer cell line A549, melanoma cell line A375, nonsmall cell lung cancer cell line H460 and epithelial cancer cell line A431 ( Table 2). As shown in Table 2, compound 1d also showed broad-spectrum inhibitory activity which was exhibited in previous research [2]. Some IC 50 values were better than 1a, such as against Hela and SKOV-3 cells.

The Solubility Assay
The optimized analogues 1d, 1f and 1g showed comparable anti-proliferative activities in vitro compared with 1a. To conduct a preliminary assessment of the absorption in vivo, solubility was further determined ( Table 3). As shown in Table 3, 1d, 1f and 1g showed better solubility both in ethanol and water after the introduction of N-methylpiperazine group.

Immunofluorescence Staining
In our previous research, it was found that 1a could induce apoptosis of cancer cells. To further study the antitumor mechanisms of 1a, 1d, 1f and 1g in vitro, an immunofluorescence staining of HCT116 cells was conducted ( Figure 3). As shown in Figure 3, the tubulin of HCT-116 cells was intact after the treatment with 1d, 1f and 1g. However, cytosolic vacuolization appeared, which was't observed for 1a. It has been reported that this kind of cytosolic vacuolization is related with the inhibition of HSP-90 protein or the proteasome-promoted protein ubiquitination [6]. Further mechanistic studies are currently in progress.

General Procedure for Preparing Compounds 4a-d
Compound 3a-b (13.76 mmol) was added to a mixture of 2a-c (16.51 mmol) and potassium carbonate (20.64 mmol) in acetone (25 mL) while refluxing with stirring. The resulting mixture was stirred under reflux for 4 h, then cooled to ambient temperature. After removal of acetone on a rotary evaporator, the solid was dissolved in ethyl acetate (100 mL) and washed with water (2 × 10 mL), saturated solution of sodium bicarbonate (2 × 20 mL) and sodium chloride (2 × 20 mL). Then, the organic phase was dried over Na 2 SO 4 and concentrated in vacuo to give a solid product. The title compounds 4a-d were thus obtained, and could be used directly for the next step without further purification. The yields of 4a, 4b, 4c and 4d, were 92%, 90%, 90% and 93%, respectively.

Cell Proliferation Assay (MTT Assay)
Cells (2 × 10 3 /well) were seeded in 96-well plates and cultured for 24 h, followed by compounds which dissolved in dimethylsulfoxide (DMSO) treatment for 48 h. Ten microliters of 10 mg/mL MTT was added and cultured for 4 h, the medium was removed and 150 μL of DMSO was added to dissolve formazan crystals. Absorbance was measured at 570 nm using an SpectraMAX M5 microplate spectrophotometer (Molecular Devices). The proliferation inhibitory effects of the compounds on cancer cells were expressed as IC 50 .

Immunofluorescence
Cells (1 ×10 5 ) were plated in 6-well plates. Following drug treatment for 24 h, cells were washed with PBS, punched by Triton X-100. Subsequently cells were probed by the primary antibodies. Then cells were incubated with the secondary antibody for 30 min at room temperature and examined under a fluorescence microscope [8].

Conclusions
In this paper, five novel benzothiazole-2-thiol, benzimidazole-2-thiol and benzoxazole-2-thiol derivatives were synthesized for lead optimization, and their in vitro antitumor activity was evaluated. To our delight, compounds 1d, 1f and 1g showed comparable antitumor activities and better solubility compared with the lead compound 1a. In a preliminary antitumor activity mechanism study, we found a novel phenomenon, that is, cytosolic vacuolization after treatment with these compounds which was not induced by 1a. Thus, it indicates that compounds 1d, 1f and 1g might possess different anticancer mechanism, which deserves further study.