Synthesis and Antiproliferative Activities of Benzimidazole-Based Sulfide and Sulfoxide Derivatives

The design, synthesis, and in vitro antiproliferative activity of a novel series of sulfide (4a–i) and sulfoxide (5a–h) derivatives of benzimidazole, in which different aromatic and heteroaromatic acetamides are linked to benzimidazole via sulfide (4a–i) and sulfoxide (5a–h) linker, are reported and the structure-activity relationship is discussed. The new derivatives were prepared by coupling 2-(mercaptomethyl)benzimidazole with 2-bromo-N-(substituted) acetamides in dry acetone in the presence of anhydrous potassium carbonate. With very few exceptions, all of the synthesized compounds showed varying antiprolific activities against HepG2, MCF-7, and A549 cell lines. Compound 5a was very similar in potency to doxorubicin as an anticancer drug, with IC50 values 4.1 ± 0.5, 4.1 ± 0.5, and 5.0 ± 0.6 µg/mL versus 4.2 ± 0.5, 4.9 ± 0.6, and 6.1 ± 0.6 µg/mL against HepG2, MCF-7, and A549 cell lines, respectively. In contrast, none of the compounds showed activity against human prostate PC3 cancer cells. Additionally, the sulfoxide derivatives were more potent than the corresponding sulfides.


Introduction
The high mortality caused by cancer puts it as the number one cause of death worldwide. This represents a great impact on human health, society, and the global economy. Taking into consideration the commercially available cancer therapies, chemotherapy has turned out to be one of the most significant treatments in cancer management [1]. Although a large number of potent chemotherapeutic anticancer agents has been successfully identified, clinical treatments still suffer from many toxic side effects of the drugs such as bone marrow suppression, gastrointestinal tract lesions, nausea, hair loss, drug resistance, and so on [2]. Therefore, the development of novel, efficient, and less toxic anticancer agents which selectively kill or inhibit cell growth of neoplastic cells without affecting non-cancerous host tissues is still of utmost importance.
Benzimidazole derivatives are commonly used chemical scaffolds because they play an important role in medicinal chemistry. They have earned an essential place in the list of chemotherapeutic agents. During the past 5-10 years, many condensation products of benzimidazole have been reported for a variety of biological activities, such as antiinflammatory [3][4][5], antiviral [6,7], and antifungal [8,9]. Extensive biochemical and pharmacological studies have confirmed that benzimidazoles are effective against various strains of microorganisms [10,11]. Importantly, the synthesis of benzimidazoles has received much attention due to their antitumor and antiproliferative activities [12][13][14].
Benzimidazole conjugated with other aliphatic, aromatic, or heterocyclic moieties have resulted in compounds with pronounced antitumor profiles. The chemical structures of some of these conjugates are displayed in Fig. 1. Some of these compounds are in preclinical testing while others are still in the early laboratory investigation phase, for example, FB642 [15][16][17], A-62022 [18], Hoechst-33258 [19,20], Nocodazole (NSC-238189) [21,22], and ABT-888 (Veliparib). The latter is a potential antitumor drug. It inhibits poly(ADP-ribose) polymerase (PARP)-1 and -2, thereby inhibiting DNA repair and potentiating the cytotoxicity of DNA-damaging agents [23][24][25][26]. On June 26 th 2014, AbbVie announced the initiation of a Phase III clinical trial evaluating the safety and efficacy in patients with advanced breast cancer. Bendamustine hydrochloride is a benzimidazolebased antitumor drug and is being marketed under the commercial name Treanda. It is made by Cephalon Inc. (Frazer, PA. USA). It is an alkylating drug indicated for the treatment of patients with chronic lymphocytic leukemia (CLL), a slowly progressing blood and bone marrow disease [27][28][29][30].
As a contribution to this field and in continuation of our previous work on the synthesis and evaluation of new compounds as anticancer agents [31][32][33], we report herein the synthesis, spectroscopic identification, and in vitro antitumor activities of two novel series of benzimidazole-methyl sulfide and benzimidazole-methyl sulfoxide conjugated with aromatic and heteroaromatic acetamide moieties. Data on their antitumor properties is also presented.

Chemistry
Initially, we focused on the preparation of compounds 3a-i as previously described in the literature [34][35][36][37][38][39][40][41], starting with the corresponding amine and bromoacetyl bromide in the presence of a base such as triethylamine or potassium carbonate in dry solvent such as dichloromethane (DCM), tetrahydrofuran (THF), or N,N-dimethylformamide (DMF) at room temperature. However, the obtained yields were low, which could be attributed to the instability of bromoacetyl bromide due to its rapid decomposition and/or the possibility of the double alkylation of the starting amine under the basic reaction conditions. Consequently, to avoid these difficulties, we prepared compounds 3a-i by reacting the appropriate amine (1a-i) with bromoacetic acid (2) in the presence of N,Ndicyclohexylcarbodiimide (DCC) in dry THF or DMF with stirring at room temperature to afford compounds 3a-i in high yields (Sch. 1). The purity of compounds 3a-i was checked by TLC and their melting points were in good agreement with the literature.
Next, 2-(mercaptomethyl)benzimidazole (BISH) was prepared according to an adaptation of the Phillips method [42]. Acid-catalyzed condensation of o-phenylenediamine with thioglycolic acid at reflux temperature in 4 N HCl afforded the key product after basifying it with ammonium hydroxide. Compound BISH was coupled with 3a-i in acetone in the presence of finely powdered anhydrous potassium carbonate at room temperature to furnish compounds 4a-i in good yields, (Sch. 2). The 1 H-NMR spectra of 4a-i were characterized by three distinct and common proton types: (1) the aliphatic protons of the linker which showed two singlets appearing around δ 3.3 and 4.1 ppm assigned to acetamido-methylene and benzimidazolyl-methylene, respectively; (2) the aromatic protons appearing at δ 6.2-7.8 ppm; and (3) two D 2 Oexchangeable protons appearing at about δ 5.6 and < 8.5 ppm assigned to the benzimidazole NH and the amide NH, respectively.
Finally, the target sulfoxides (5a-h) were obtained by chemoselective oxidation of the corresponding sulfides 4a-h (Sch. 2). Many methods have been reported in the literature for the synthesis of sulfoxides [43]. However, double oxidation of the sulfides to sulfones can be a significant issue. Therefore, the reaction conditions such as the molar ratio of the oxidizing agents, temperature, and time must be controlled to prevent over-oxidation. Some commercially available oxidizing agents are inexpensive and efficient. For instance, hydrogen peroxide in the presence of vanadium compounds [44], potassium hydrogen persulfate (oxone) [45], periodate, persulfate, and permanganate are all inorganic oxidizing reagents. However, handling our substrates under aqueous conditions was a major problem. On the other hand, m-chloroperbenzoic acid (m-CPBA) appeared to be an excellent oxidizing agent with high chemoselectivity. Compounds (4a-h) were transformed smoothly to sulfoxide (5a-h) analogues using m-CPBA in dry dichloromethane at −20 °C. The reaction was stirred further at room temperature to effect the transformation. The transformation of 4i failed due to its insolubility under the reaction conditions.
The structures of all novel compounds were confirmed by 1 H-and 13 C-NMR spectra, mass spectrometry, and microanalyses techniques. Complete and unambiguous assignments for all 1 H and 13 C resonances were achieved on the basis of chemical shift considerations and J-coupling information. Interestingly, in the 1 H-NMR spectra of sulfoxides (5a-h), the chemical shifts and the coupling constants of the methylene protons of the linker showed a completely different pattern from the one appeared for the corresponding sulfides (4a-h).
In the sulfoxide, each proton showed a doublet with the coupling constant J = 13.4 Hz and the chemical shift was shifted downfield. This could be attributed to the diastereotopic nature of the linker protons affected by the pyramidal chiral sulfoxide group [46. 47]. Also, the chemical shifts of the benzimidazole NH protons (δ 5.6 ppm) for 4a-i were also moved to the downfield region (δ 9.5 ppm), whereas the amide proton showed a broad singlet around δ 12.5 ppm for compounds 5a-h.

Antiproliferative Activity
The antiproliferative activities were expressed by the median growth inhibitory concentration (IC 50 ). As shown in Tab. 1, the antiproliferative activities of the synthesized compounds were evaluated against human liver HepG2, breast MCF-7, lung A549, and prostate PC3 cancer cell lines using the sulforhodamine B stain (SRB) assay, in comparison with doxorubicin as a reference drug.
None of the compounds exerted any activity against human prostate PC3 cancer cells.
The tumor cell line showed normal growth in our culture system and DMSO did not seem to have any noticeable effect on cellular growth. A gradual decrease in viability of cancer cells was observed with increasing concentration of the tested compounds in a dosedependent inhibitory effect.

SAR Analysis
The structure-activity relationship (SAR) investigation of the compounds used in this study gives an understanding of the essential structural requirements for boosting the antiproliferative activities of this class of compounds. The data in Tab. 1 revealed some significant observations: (1) it is noticed that the sulfoxides (5a-h) were more potent than the sulfides (4a-h) towards all cell lines with 4h as an exception. (2) The significantly high potency of the latter compound could be attributed to the polar nature of the sulfonamide group as well as the heterocyclic thiazole ring which contributes to the antiprolific effect.
(3) Also, the nature of the N-substituent on the acetamide was found to affect the activity of these compounds. The heterocyclic substituent had a high impact on the potency as shown by the thiazole, antipyrine, and sulfathiazole moieties, as in, 5f, 5h, and 4i, respectively. (4) The high potency of the p-substituted phenyl groups in 5a and 5c compared to 5b could be referred to the polarity and the size of the acetyl and the bromo compared to the fluoro substituent. (5) The substituent in the m-position in 4d, 4e, 5d, and 5d as well as the benzyl group contributes negatively to the antiprolific effect of these compounds. This information on SAR explored in the present study could be helpful in further structural modification and development of new benzimidazole-acetamide hybrids as potent antitumor agents.

Conclusion
A novel series of sulfide (4a-i) and sulfoxide (5a-h) derivatives of benzimidazole were synthesized. The synthesized compounds were tested against four different tumor cell lines. The tested compounds exerted antitumor activity in liver HepG2, breast MCF-7, and lung A549 cancer cell lines by reducing cell proliferation and resulted in significant growth inhibitory. Also, the present study revealed that MCF-7 cells were more sensitive to the tested compounds than both HepG2 and A549 cancer cells.

Experimental
All chemicals were purchased from common commercial suppliers and used without further purification. Melting points (m.p.) were determined on a Gallenkamp Melting Point Apparatus and were uncorrected. 1 H-and 13 C-NMR spectra were recorded on a Jeol EX-Spectrometer at 500 and 125 MHz, respectively, in DMSO-d 6 as a solvent at the National Research Centre. Mass spectra were recorded on the Thermo Finnigan SSQ 7000 Advantage spectrometer in EI ionization mode. Microanalyses were performed at the Microanalytical Center in Cairo University. All reactions were performed in air. The reaction progress was monitored using thin-layer chromatography (TLC) which was performed on silica gel 60 F254 aluminum plates (E. Merck, layer thickness 0.2 mm). 4-Amino-N-(thiazol-2-yl)benzenesulfonamide (1i) was prepared according to published procedures [48,49].
Method B: To the mixture of 1i (2.5 g, 10.0 mmol) and triethylamine (1.4 mL, 11.0 mmol) in THF (20 mL) cooled at −20°C was added a solution of bromoacetyl bromide in THF (5 mL) over 30 min. The ice bath was removed after the addition and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated on a rotary evaporator, resuspended in ethyl acetate (50 mL), and then filtered through a pad of silica gel. The filtrate was then concentrated to provide the product as pale yellow powder.

General procedure for the synthesis of 2-{[(1H-benzo[d]imidazol-2-yl)methyl]thio}-Nsubstituted-acetamides (4a-i)
In a 100-mL Erlenmeyer flask with a standard ground top was successively added BISH (328 mg, 2 mmol), 2-bromo-N-substituted-acetamide (2 mmol), finely ground K 2 CO 3 (552 mg, 4 mmol), and 15 mL dry acetone. The closed reaction vessel was set at room temperature while the reactants were allowed to stir overnight. After the reaction was complete (inspected by TLC), the vessel content was poured onto crushed ice water and the mixture was stirred for 30 min. The crude product was filtered over a sintered-glass Buchner funnel (porosity grade 4) and the product was washed with cold ether, then hot ether, and dried in air.

General Procedure for the Chemoselective Oxidation of 4a-h
A solution of m-CPBA (1.2 mmol) in DCM (10 mL) was added dropwise to 4a-h (1 mmol) dissolved in DCM (35 mL) cooled to −20°C for 30 min. The reaction mixture was stirred for an additional 2h. The temperature was raised gradually to room temperature and the reaction was stirred overnight. After complete consumption of the starting materials as indicated by TLC, water was added and the organic product was extracted with ethyl acetate. The combined organic extract was collected, dried over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure to afford pure sulfoxide products 5a-h in high yields.

In Vitro Cytotoxicity Assay
The antiproliferative activity was measured in vitro using the SRB assay according to the previously reported standard procedure [50]. Cells were inoculated in a 96-well microtiter plate (10 4 cells/well) for 24 h before treatment with the tested compounds to allow attachment of cells to the wall of the plate. The test compounds were dissolved in DMSO at 1 mg/mL immediately before use and diluted to the appropriate volume just before the addition to the cell culture. Different concentrations of test compounds (0-100 µg/mL) and doxorubicin were added to the cells. Four wells were prepared for each individual dose. Monolayer cells were incubated with the compounds for 48 h at 37°C and in an atmosphere of 5% CO 2 . After 48 h cells were fixed, washed, and stained for 30 min with 0.4% (w/v) SRB, then dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and the attached stain was recovered with Tris-EDTA buffer. Color intensity was measured in an ELISA reader at wavelength 540 nm. The relation between the surviving fraction and drug concentration was plotted to get the survival curve for each cell line after the specified time. The concentration required for 50% inhibition of cell viability (IC 50 ) was calculated and the results are given in Tab. 1.

Statistical Analysis
The results are reported as the mean ± standard error (S.E.) for at least four experiments.