Synthesis, Antibacterial, and Anti HepG2 Cell Line Human Hepatocyte Carcinoma Activity of Some New Potentially Benzimidazole-5-(Aryldiazenyl)Thiazole Derivatives

The paper describes the synthesis and biological evaluation of some new benzimidazole derivatives as potent clinical drugs that are useful in the treatment of some microbial infections and tumor inhibition. The starting compound 2-(bromomethyl)-1H-benzimidazole (1) was prepared, and hence underwent interesting functionalization reactions to afford several series of benzimidazole-5-(aryldiazenyl)thiazole derivatives: 3a–c, 7a–c, and 8a–c. The antibacterial activities of the synthesized compounds were evaluated by calculation of the inhibition zone diameter (mm) and the determination of minimum inhibitory concentration (µg/mL) against selected pathogenic bacteria Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria).Noticeable efficiency was found based on in vitro screening for their antioxidant activity and cytotoxicity effect against the human liver cancer cell line (HepG2) and human hepatocyte carcinoma cells at relatively high concentrations.


Introduction
The promising therapeutic potential of benzimidazole derivatives could be traced back to 1944and Woolley, who speculated that benzimidazole could act similar to purines to elicit some biological responses [1].

Results
Benzimidazoles are hybrid heterocyclic compounds that are formed from fused benzene and imidazole rings with broad industrial, biological, and clinical applications. The associated rich literature with benzimidazole moieties prompted us to synthesize new functionalized benzimidazole derivatives and study their potential as bioactive compounds, continuing our previous efforts related to the synthesis of new pharmacological compounds [33][34][35][36][37][38][39][40][41].

Results
Benzimidazoles are hybrid heterocyclic compounds that are formed from fused benzene and imidazole rings with broad industrial, biological, and clinical applications. The associated rich literature with benzimidazole moieties prompted us to synthesize new functionalized benzimidazole derivatives and study their potential as bioactive compounds, continuing our previous efforts related to the synthesis of new pharmacological compounds [33][34][35][36][37][38][39][40][41].

Chemistry
Synthesis of the starting 2-(bromomethyl)-1H-benzimidazole (1) proceeded smoothly according to the reported method by the reaction of o-phenylenediamine with bromoacetic acid in the presence of 4 M hydrochloric acid, as shown in Scheme 1. The IR spectrum of 2-(bromomethyl)-1H-benzimidazole (1) revealed intense IR bands at 3372-3025 cm −1 (NH, =CH aromatic, and -CH aliphatic). The 1 H-NMR spectrum displayed a singlet signal at δ 4.85 ppm due to the methylene protons (2H, CH2-Br), a singlet at δ 10.32 ppm due to the NHbenzimidazole proton, and at δ 7.21-7.60 ppm due to the aromatic protons. The spectral analysis was found to be in good agreement with the literature [42].

Scheme 2.
Synthesis steps for the preparation of benzimidazole derivatives(3a-c).
The structures of the isolated compounds (2 and 3a-c) were secured by their spectral analysis as described in the experimental section, where a characteristic singlet signal at δ 6.90 ppm due to the C4-thiazole proton was displayed.
The reaction of thiourea with α-haloalkyl or α-halocarbonyl compounds often proceed through the sulfur atom due to its high nucleophilicity to give thiouronium salts [44]. Whereas, conducting the reaction under acidic medium (e.g., trifluoroacetic acid) afforded the alkylthiourea compounds [45][46][47]. Thus, the reaction of 2-(bromomethyl)-1H-benzimidazole (1) with thiourea in acidic medium gave the previously reported derivative 1-((1H-benzimidazol-2-yl)methyl)thiourea (4) [45] through the suggested mechanism in Scheme 3. The structure of the obtained compound was secured by its spectral analysis, and was in good agreement with the literature. The structures of the isolated compounds (2 and 3a-c) were secured by their spectral analysis as described in the experimental section, where a characteristic singlet signal at δ 6.90 ppm due to the C4-thiazole proton was displayed.

Antibacterial activity
The antibacterial activities of novel synthesized compounds (100 µg/mL in DMSO), were determined in vitro by using the disc diffusion method [48] against Staphylococcus aureus (S. aureus) ATCC 25923 as Gram-positive bacteria and Escherichia coli (E. coli) ATCC 25922 as Gram-negative bacteria compared with the standard drugs Kanamycin and Ampicillin, respectively. The results of the assessments are plotted in Figure 1.

Antibacterial activity
The antibacterial activities of novel synthesized compounds (100 µg/mL in DMSO), were determined in vitro by using the disc diffusion method [48] against Staphylococcus aureus (S. aureus) ATCC 25923 as Gram-positive bacteria and Escherichia coli (E. coli) ATCC 25922 as Gram-negative bacteria compared with the standard drugs Kanamycin and Ampicillin, respectively. The results of the assessments are plotted in Figure 1.

Antibacterial activity
The antibacterial activities of novel synthesized compounds (100 µg/mL in DMSO), were determined in vitro by using the disc diffusion method [48] against Staphylococcus aureus (S. aureus) ATCC 25923 as Gram-positive bacteria and Escherichia coli (E. coli) ATCC 25922 as Gram-negative bacteria compared with the standard drugs Kanamycin and Ampicillin, respectively. The results of the assessments are plotted in Figure 1.

Minimum Inhibitory Concentration (MIC)
The Minimum Inhibitory Concentration for the synthesized compounds was determined using a spot-on-lawn assay [49]. The collected data are listed in Table 1. The lowest MIC value is the highest antibacterial effect against the target bacterial strain. The antibacterial study showed that some of the tested compounds exhibited variable antimicrobial activities against both bacteria Gram-negative and Gram-positive bacteria at a concentration of 100 µg/mL. The presence of substituted electron withdrawing groups such as nitro and acetyl groups in compounds 3c, 8a, and 8c showed activities against E. coli (Gram-negative bacteria), while most of the synthesized compounds exhibited moderate activity against S. aureus (Gram-positive bacteria).
Compound 8c showed the highest affect against tested bacterial strains compared with the rest of the synthesized benzimidazoles under consideration due to the presence of an electron-withdrawing group (COR group) on the phenyl ring. It should be noted that the thiazolone moiety resulted in a dramatic reduction of the antibacterial efficiency (e.g., compounds 7a-c).

DPPH Radical Scavenging Assay
Antioxidant capacities for the synthesized compounds were screened using a DPPH free radical scavenging assay incorporating a metastable free radical that is capable of accepting hydrogen radicals from antioxidants in solution. The method is based upon different mechanisms to provide complementary insight into the antioxidant activity of the synthesized compounds [50]. The reaction between DPPH and antioxidants can be monitored by the decrease in the absorbance of the violet-colored free radicals. Inhibition percentages of DPPH radical by selected tested compounds were plotted in Figure 2, and the measure of drug potency in terms of half maximal effective concentration (EC 50 ) values were plotted in Figure 3 (i.e., the compound concentration where 50% of its maximal effect is observed). The high antioxidant and scavenging activities of the tested compounds may be due to the resonance delocalization of the nitrogen lone pairs through the double bonds. This structure may lead to radical formation at more than one site, especially on the benzene ring attached to the electron-withdrawing groups (e.g., 3a and 3c: the highest DPPH value and the lowest EC 50 value, respectively). This conclusion is also supported by previously reported results [51]. The antibacterial study showed that some of the tested compounds exhibited variable antimicrobial activities against both bacteria Gram-negative and Gram-positive bacteria at a concentration of 100 µg/mL. The presence of substituted electron withdrawing groups such as nitro and acetyl groups in compounds 3c, 8a, and 8c showed activities against E. coli (Gram-negative bacteria), while most of the synthesized compounds exhibited moderate activity against S. aureus (Gram-positive bacteria).
Compound 8c showed the highest affect against tested bacterial strains compared with the rest of the synthesized benzimidazoles under consideration due to the presence of an electronwithdrawing group (COR group) on the phenyl ring. It should be noted that the thiazolone moiety resulted in a dramatic reduction of the antibacterial efficiency (e.g., compounds 7a-c).

DPPH Radical Scavenging Assay
Antioxidant capacities for the synthesized compounds were screened using a DPPH free radical scavenging assay incorporating a metastable free radical that is capable of accepting hydrogen radicals from antioxidants in solution. The method is based upon different mechanisms to provide complementary insight into the antioxidant activity of the synthesized compounds [50]. The reaction between DPPH and antioxidants can be monitored by the decrease in the absorbance of the violetcolored free radicals. Inhibition percentages of DPPH radical by selected tested compounds were plotted in Figure 2, and the measure of drug potency in terms of half maximal effective concentration (EC50) values were plotted in Figure 3 (i.e., the compound concentration where 50% of its maximal effect is observed). The high antioxidant and scavenging activities of the tested compounds may be due to the resonance delocalization of the nitrogen lone pairs through the double bonds. This structure may lead to radical formation at more than one site, especially on the benzene ring attached to the electron-withdrawing groups (e.g., 3a and 3c: the highest DPPH value and the lowest EC50 value, respectively). This conclusion is also supported by previously reported results [51].

Evaluation of Cytotoxicity Effects of Benzimidazoles and its Derivatives Against the Human
Liver Cancer Cell Line (HepG2).
All of the compounds were screened in vitro for possible potential antitumor molecules against human liver cancer cell line (HepG2) and showed variable anticancer activity. The Neutral Red (NR) technique [52] was used to evaluate the cytotoxic effects of gradient concentrations (100 µg/mL to 500 µg/mL) of the tested compounds on the human liver (HepG2) cell line and in terms of cell viability percentage, which measured the potency of the compound in inhibiting tumor growth (Figure 4).

Evaluation of Cytotoxicity Effects of Benzimidazoles and its Derivatives Against the Human
Liver Cancer Cell Line (HepG2).
All of the compounds were screened in vitro for possible potential antitumor molecules against human liver cancer cell line (HepG2) and showed variable anticancer activity. The Neutral Red (NR) technique [52] was used to evaluate the cytotoxic effects of gradient concentrations (100 µg/mL to 500 µg/mL) of the tested compounds on the human liver (HepG2) cell line and in terms of cell viability percentage, which measured the potency of the compound in inhibiting tumor growth (Figure 4). As shown in Figure 4, the tested compounds exhibited antitumor activities in different concentrations, where increasing the concentration led to less percent viability. The benzimidazole derivatives (3) and (8) in common showed higher activity than the other selected compounds that were tested. The presence of the 4-phenyl function with or without attached electron-withdrawing groups increased the antitumor activity due to their high antioxidant and free radical scavenging ability (e. g., 3a and 8b). This also could be explained by the previously reported facts [51], and is supported by the literature [53]. As shown in Figure 4, the tested compounds exhibited antitumor activities in different concentrations, where increasing the concentration led to less percent viability. The benzimidazole derivatives (3) and (8) in common showed higher activity than the other selected compounds that were tested. The presence of the 4-phenyl function with or without attached electron-withdrawing groups increased the antitumor activity due to their high antioxidant and free radical scavenging ability (e.g., 3a and 8b). This also could be explained by the previously reported facts [51], and is supported by the literature [53].

Chemical Reagents
All of the chemical reagents that were used were of analytical grade or chemically pure and supplied by Sigma Aldrich Co., Darmstadt, Germany.

Micro and Spectroscopic Analysis
All of the corrected melting points are in degree centigrade and measured using a Stuart SMP20 melting point apparatus (Bibby Scientific Limited, Staffordshire, UK). The infrared spectra were recorded on a PerkinElmer Alpha platinum-ATR spectrometer; the 1 H-NMR (300 MHz) and 13 C-NMR (75 MHz) spectra were recorded on a Varian Mercury VXR-300 spectrometer (Varian Inc., Palo Alto, CA, USA) and the chemical shifts were related to that of the solvent DMSO-d 6 using tetramethylsilane (TMS) as an internal standard. MS spectra (HRMS) were acquired on TRACE GC Ultra gas chromatograph mass spectrometry, coupled with a THERMO mass spectrometer detector ISQ Single Quadrupole Mass Spectrometer (THERMO Scientific Corp., Waltham, MA, USA)and were obtained by electron ionization (EI) at 70 eV, using a spectral range of m/z 50-1000.All of the microanalyses and spectral analyses were performed by the Micro Analytical Centers of Taif University-Saudi Arabia (IR spectra, HRMS), Cairo University ( 1 H-NMR, 13 C-NMR), and National Research Center-Egypt (Mass spectra).

Biological Analysis
All of the used kits for biochemical parameters were supplied by the Diagnostic Company, Maidenhead, UK. All of the chemicals that were used were supplied by Gibco ® Life Technologies, Waltham, MA, USA. The biological tests were performed by the biotechnology unit, faculty of Agriculture-Cairo University, Egypt. Human transformed cell lines from liver (HepG2) were obtained from the Egyptian Company for Vaccine and Serum (VACSERA, Cairo, Egypt). The statistical package for social science (SPSS) 1999 program version (IBM, Armonk, NY, USA) was used for all of the analyses [54].

Antibacterial Evaluation
The disc diffusion procedure was used to evaluate the antibacterial activity of the synthesized compounds [53]. Uninoculated agar medium was poured in sterilized Petridishes and left for solidifying. One hundred milliliters of trypticase soy agar + 0.6% yeast extract were melted and cooled to 50 • C, and then inoculated by the culture of the examined microorganism. Inoculated medium was poured over the previous layer and left to solidify at 4 • C (surface layer should be constant in volume and horizontally homogenous). Discs of Whatman No. 1 filter paper (6.0 mm in diameter) were sterilized by autoclaving at 121 • C for 15 min. Each individual tested compound was dissolved in 10% dimethyl sulfoxide (DEMSO) with a final concentration 100 mg/mL. An accurate volume (15 µL) of each tested compound (100 mg/mL) was aseptically added to each disc and left to dry. Each disc was aseptically placed on the middle of an agar plate surface (in triplicate) and left at 4 • C. Fifteen µL of DEMSO (10%) was aseptically added to the disc and used as control. Then, 15 µL of Kanamycin (30 µg/mL) was aseptically added to the disc and used as a positive standard reference for Gram-positive strains, and Ampicillin (10 µg/mL) was used for Gram-negative strains. The inhibition clear zones (mm) were recorded in all of the samples and equal zero in no-inhibition results.

Determination of Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration (MIC) of synthesized compounds was assayed by using a spot-on-lawn assay [49]. Cells of laboratory strains (LAB strains) were grown in de Man, Rogosa & Sharpe MRS broth (Oxoid) supplemented with 0.5% CaCO 3 at 30 • C with shaking (120 strokes per min (SPM)) to the early stationary phase, and the initial pH of the medium was adjusted to pH 6.0. Culture fluid was obtained by removing the cells by centrifugation, and was filtered with a cellulose acetate membrane filter (pore size 0.2 µm; Advantec, Tokyo, Japan). The resulting sample was serially diluted twofold with MRS broth (pH 5.0). Soft nutrient agar (20 mL) was solidified in a sterile dish (140 mm by 100 mm) after the addition of 60 µL of an overnight culture of Listeria monocytogenes SUB635. This strain was used as an indicator in the assay, since a clearer inhibitory zone was obtained with this organism than with B. subtilis. After 30 min of drying, 10 µL of each diluted sample was spotted onto the plate. The resulting plates were incubated at 30 • C overnight, and the titer was defined as the reciprocal of the highest dilution (2 n ) that resulted in the inhibition of the indicator lawn. Thus, the arbitrary unit (AU) of antibacterial activity per milliliter was defined as 2 n × 1000 µL/10 µL.
To examine the spectrum of activity of the purified bacteriocin against various bacterial strains, the test described above was performed with soft agar containing cells of test strains grown in a suitable medium to the early stationary phase. The abilities to inhibit a certain bacterial strain were compared by using the highest dilution that resulted in a growth inhibition zone.

Assay of Cytotoxicity
Neutral Red (NR) assay [55] was used to evaluate the cytotoxicity effects of prepared compounds. 30.000 Cells/well were plated in 96-well plates in quadruplets for each treatment. 24 h later, cells were washed with pre-warmed phosphate-buffered saline (PBS) and 125 µL of media with an appropriate concentration of each synthesized compound. Afterwards, cells were washed with pre-warmed PBS, and 125 µL of the NR reagent was added in each well. The plates were incubated at 37 • C with 5% CO 2 , for a further 5 h. After incubation, plates were taken out, gently washed three times with PBS, and 100 µL of NR desorb solution was added per well. Plates were protected from light and shaken on a shaker at 60 rpm for 30 min, followed by keeping the plates still for 5 min. Plates were taken to a 96-well plate reader, and their absorbance was measured in each well at 540 nm. Each value is based on means of the absorbance values acquired from the quadruplet of treatments and normalized to the mean value of untreated control and expressed as % cell death according to the following equation (Equation (1)): % Cell death = Abs 540 treated sample Abs 540 untreated sample × 100 The data is based on three independent experiments.

Antioxidant Potential
The tested compounds were analyzed for their antioxidant potential according to the reported technique [56]. The stock solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH) was prepared by dissolving 24 mg of DPPH in 100 mL of methyl alcohol (MeOH), and then stored at 20 • C in the dark. The working solution was obtained by diluting 10 mL of stock solution with 45 mL of MeOH, in order to obtain an absorbance of 1.1 ± 0.1 units at 515 nm. A volume of 10 µL of different synthesized compounds' concentrations (20 µg/mL, 50 µg/mL, and 100 µg/mL) was added to 990 µL of 0.094 mM DPPH freshly working solution up to total volume of one mL. Assays were continuously monitored at 515 nm over a one-hour period at 25 • C. Changes in absorbance were minimal for all of the samples after 50 min. The antioxidant abilities were expressed as µM Trolox equivalents. Each sample was analyzed in triplicate. The inhibition percentage of the DPPH radical by the samples was calculated according to Equation (2): where, Ab is the absorption of the blank sample (t = 0 min), and Aa is the absorption of the tested compound or standard substance solution (t = 30 min). The EC 50 value is defined as the concentration of antioxidant in the reactive system that is necessary to decrease the initial DPPH concentration by 50%, and is calculated from the data obtained.

Conclusions
In the development of this work, the newly synthesized series of benzimidazole derivatives 3a-c, 7a-c, and 8a-c derived from 2-(bromomethyl)-1H-benzimidazole were prepared in satisfactory yields and fully characterized. They exhibited in vitro variable antimicrobial activities when subjected to the bacterial strains Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria) compared with Kanamycin and Ampicillin as standard drugs, respectively. Their inhibition effect against DPPH (the lowest EC 50 values) compared to Trolox (EC 50 = 55.13) was shown as well. They were screened for their antitumor activities against human liver cancer cell line (HepG2) to evaluate the cytotoxic effects of gradient concentrations (100-500 µg/mL) in terms of cell viability percentage and EC 50 values. The tested compounds exhibited noticeable efficiency against the tumor cell line under investigation at the higher concentration. The benzimidazole derivatives (3a, c) and (8b) showed higher activity than the other selected compounds that were tested. Presence of electron withdrawing groups increased the antitumor activities as discussed.