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Design, Synthesis and Biological Evaluation of Novel Bromophenol Derivatives Incorporating Indolin-2-One Moiety as Potential Anticancer Agents

A series of bromophenol derivatives containing indolin-2-one moiety were designed and evaluated that for their anticancer activities against A549, Bel7402, HepG2, HeLa and HCT116 cancer cell lines using MTT assay in vitro. Among them, seven compounds (4g–4i, 5h, 6d, 7a, 7b) showed potent activity against the tested five human cancer cell lines. Wound-healing assay demonstrated that compound 4g can be used as a potent compound for inactivating invasion and metastasis by inhibiting the migration of cancer cells. The structure–activity relationships (SARs) of bromophenol derivatives had been discussed, which were useful for exploring and developing bromophenol derivatives as novel anticancer drugs.


Introduction
Cancer is a significant health problem throughout the world, which is a leading cause of death. It is estimated that 14.1 million new cancer cases and 8.2 million cancer-related deaths occurred in 2012 according to the WHO report. The incidence of cancer has been increasing in most regions of the world, which will further increase to 19.3 million new cancer cases per year by 2025 [1]. Although many effective anticancer agents have been developed, therapies for cancer are less than satisfactory due to side effects. Therefore, novel effective agents for treating cancer disease are urgently needed today.
In view of these observations, a novel series of bromophenols derivatives containing indolin-2-one moiety was designed and evaluated for in vitro anticancer activity against cancer cell lines (Human lung cancer cell line, A549; Human hepatoma cell lines, Bel-7402 and HepG2; human cervical cancer cell line, HeLa; human colon cancer cell line, HCT116) using MTT assay. The most potent activity compound 4g on cancer cell migration was investigated by using the wound-healing assay. Structure-activity relationships (SARs) of these bromophenols analogs are also discussed in this paper.

Chemistry
The synthetic procedures for the preparation of aldehydes (Scheme 1) have been publised in our previous reports [27][28][29][30]. As shown in Scheme 2, a series of bromophenol derivatives were synthesized by Knoevenagel condensation between different substituted indolin-2-one and the corresponding aldehydes in order to explore the SARs of these derivatives and obtain the potencial lead compounds. Firstly, oxindole (1) was reacted with ClSO3H to yield compound 2. Then, compound 2 and amines were heated for 3 h in tetrahydrofuran (THF) at 80 °C to afford 5-substituted-indolin-2-one (3). At last, the reaction between 5-substituted-indolin-2-one (3) and the synthesized aldehydes was performed under the condition of Knoevenagel condensation in ethanol with a catalytic amount of piperidine to give the desired bromophenol derivatives 4-7 in good yields. All of the synthesized derivatives were purified and their structures were characterized by spectroscopic means ( 1 H, 13 C NMR, MS and HRMS).

Antiproliferative Activity
All the synthesized compounds were investigated for their anticancer activity in vitro on five human cancer cell lines, namely against A549, Bel7402, HepG2, HeLa and HCT116 cancer cell lines using MTT assay and sunitinib as a positive control. The results of inhibited ration of comounds 4-7 were listed in Figure 3, and the IC50 values of selective compounds (4g-4i, 5h, 6d, 6e, 7a, 7b) were listed in Table 1.

Inhibitory Effect of Compound 4g on Cancer Cell Migration
Cancer cell invasion, a hallmark of cancer, plays a critical role during the carcinomas arising from epithelial tissues progressed to higher pathological grades of malignancy [31]. To further investigate the anticancer effect of 4g, we studied the inhibitory effect on cancer cell migration; an invasive process often observed in cancer by using the wound-healing assay [32]. We found that the migration of HepG2 cells was significantly inhibited by 48 h treatment with 4g at 3 μg/mL concentration compared to the untreated control ( Figure 4A). The migration width (the distances of migrated cells) of cancer cells treated with/without 4g was summarized in Figure 4B. It was demonstrated that ~20% of cell migration was inhibited by treatment with 4g at 3 μg/mL concentration for 48 h. These results suggested that 4g might be used as a potent compound for inactivating invasion and metastasis by inhibiting the migration of cancer cells. To a 100 mL flask charged with chlorosulfonic acid (25 mL), 2-oxindole (50 mmoL) at 0 °C was added slowly. After the addition, the reaction mixture was stirred at room temperature for 1.5 h. Then, the reaction mixture was heated to 68 °C for 1 h, cooled, and poured into ice water (200 mL). The precipitate was washed with water and dried in a vacuum oven to give 5-chlorosulfonyl-2-oxindole (2) which was used without further purification.

Experimental Section
A mixture of 5-chlorosulfonyl-2-oxindole (5 mmoL) and appropriate amine (10 mmoL) in tetrahydrofuran (THF, 50 mL) was heated to refluxing and stirred for 3 h. Then, this mixture was concentrated under reduced pressure, and HCl (pH = 3, 25 mL) was added and stirred for 15 min. The crude product was filtered, washed with ice water (100 mL) and dried in a vacuum oven to give 5-sulfamoyl-2-oxindole (3) which was used without further purification.

Wound-Healing Assay
The HepG2 cells (1 × 10 5 cells/well) were cultured in 6-well plates rinsed with PBS and then starved overnight in 2% FBS medium until they reached 95% confluence. A single wound was then scratched in the center of the cell monolayers with a 200 μL sterile plastic pipette tip. The wounded monolayers were washed twice to remove the non-adherent cells and were incubated with various concentrations of compound 4g in the presence of 1 μg/mL of mitomycin C. To measure the length of the endothelial cells that had migrated from the edge of the injured monolayer, images were obtained immediately after wounding and after a 24, 48 h incubation period, using a phase-contrast microscope (Olympus, Tokyo, Japan). The length was measured by the Image-Pro Plus v 6.0 (Media Cybernetics, Inc., Bethesda, MD, USA). Each experiment was repeated at least three times.

Statistical Analysis
All the experiments were performed at least three times, and the data are presented as mean ± SD values. Differences between the mean values were assessed using one-way analysis of variance. For all the analyses, p < 0.05 was considered significant. Statistical analyses were performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA).

Conclusions
In summary, a series of bromophenols derivatives containing indolin-2-one moiety were designed and evaluated for their anticancer activities against A549, Bel7402, HepG2, HeLa and HCT116 cancer cell lines using MTT assay in vitro. The preliminary SAR analysis (summarized in Figure 5) reveals that: (i) the hydrophobic parameter may affect their anticancer activity; (ii) the steric hindrance may also affect their activity; (iii) the number of bromine atoms on phenol moiety could affect the anticancer activities of these hybrid derivatives; (iv) the types of amino groups at 5-position of indolin-2-one could influence the activities; (v) the bromophenol moiety played a crucial role in maintaining anticancer activities of the conjugated derivatives. Among them, seven compounds (4g-4i, 5h, 6d, 7a, 7b) showed potent activity. Wound-healing assay demonstrated that compound 4g can be used as a potent compound for inactivating invasion and metastasis by inhibiting the migration of cancer cells. These active derivatives targeting properties needs to be further investigated. Potentially, this finding may aid in the design of novel agents for the intervention of cancer.