Synthesis, antitumor evaluation and molecular modeling study of novel benzimidazoles and pyrazinobenzimidazoles

Some novel benzimidazole and pyrazinobenzimidazole derivatives 5 - 8 was designed for evaluation of their in vitro cytotoxicity studies using MTT-based assay against three cancer cell lines namely, human hepatoma cell line (HepG2), human breast cancer cell line (MCF-7) and kidney of African green monkey (Vero B). Compounds 5a and 5c-e exhibit the highest and broad spectrum activities against all of the three cell lines tested when compared with reference drug 5-Fluorouracil (5-FU). 1-(1 H -Benzimidazol-2-yl)-3-phenylprop-2-en-1-one 5a showed superior and great potency and lethal effect against HepG2, MCF-7 and Vero B cell lines with IC 50 values of 2, 1.8 and 3.5µg/ml, respectively, comparable to 5-FU (IC 50 values of 62, 12 and 13µg/ml, respectively). Moreover, compound 7b showed potent activity against MCF-7 and Vero B cell lines with IC 50 values of 2 and 2.5µg/ml, respectively. Docking study of compounds 5a and 7b into the ATP binding site of epidermal growth factor receptor (EGFR) revealed comparable binding manner to an EGFR inhibitor, Erlotinib.


INTRODUCTION
Cancer is the result of uncoordinated and uncontrolled growth of cells.The major cause of death from cancer is metastasis (Vogelstein et al., 2013, Su et al., 2015)where cancer cells detach themselves from the parent neoplasm, invade the circulation system and spread to other body sites through several pathways (Bagi, 2002, Balmer andValley, 2002).Cancer is continuing to be a major threat to public health and the severity of the problem, led to an impressive progress in discovering potent anticancer agents.In the scope of recognising different chemical compounds which may act as a lead for designing new antitumor agents, we are interested in this study with benzimidazole derivatives.Considerable attention has been focused on benzimidazole family as one of the bioactive heterocyclic compounds that exhibited a range of biological and clinical applications.They are structural isosters of purine based nucleic acids and can interact with biological macromolecules such as protein, enzymes and receptors (Bansal and Silakari, 2012).Thus benzimidazole nucleus has been confirmed as an important pharmacophore in drug discovery of new antitumor agents (Hida et al., 1994, Neff et al., 2007, Styskala et al., 2008, Shaharyar et al., 2010, Hranjec et al., 2011, Abdel-Mohsen et al., 2010, Rahman and Siddiqui, 2010, Luo et al., 2011, Ramla et al., 2007, Ng et al., 2007, Zhong et al., 2009, Refaat, 2010, Ramla et al., 2006, Thimmegowda et al., 2008, Gowda et al., 2009).It is well known that small molecules such as pyridopyrimidines (Cockerill, et al., 2001), aminoquinazolines (El-Azab et al., 2010, Hamed et al., 2013) and benzimidazoles (Soni et al., 2012, Boschelli et al., 1999) are potent epidermal growth factor receptor-tyrosine kinase (EGFR-TK) inhibitors.Tyrosine kinase is an enzyme that can transfer a phosphate group from ATP to a protein and this is an important mechanism in regulating cell activity such as cell division.The epidermal growth factor receptors (EGFR) are over-expressed in a considerable number of human cancer cells (Cockerill andLackey, 2002, Wakeling, 2002).
Therefore, inhibition of EGFR-TK represented a rational approach to cancer therapy.In view of the previous findings, our research aimed at the synthesis of different substituted benzimidazolechalcones and pyrazinobenzimidazole derivatives in an attempt to reach an active antitumor agent with potentiated activity and selectivity toward cancerous cells.Computer docking methodology plays an effective role in the drug design and in the mechanistic examination by putting a molecule into the binding site of the target receptor in a noncovalent manner (Kontoyianni et al., 2004).So, the most active compounds were docked into the ATP binding site of EGFR to estimate if these compounds have comparable binding manner to an EGFR inhibitor, Erlotinib (Stamos et al., 2002).

Chemistry
Melting points were recorded using Fisher-Johns apparatus and are uncorrected.IR spectra (KBr) were determined on Mattson 5000 FT-IR spectrometer.Proton magnetic resonance ( 1 HNMR) spectra were recorded on FT-NMR spectrometer (200 MHz) Gemini Varian using DMSOd 6 relative to tetramethylsilane (TMS) as internal standard (chemical shifts in δ ppm).Mass spectra (MS) were measured on JEOL JMS-600H spectrometer.Elemental analysis was carried out for C, H and N at the Microanalytical Centre of Cairo University.All reagents were purchased from the Aldrich Chemical Company.

Cytotoxicity screening
The cytotoxicity screening was done by employing tetrazolium salt MTT assay (Mosmann et al., 1983, Denizot andLang, 1986).Serial dilutions (60 µL) of the test compounds and 5florouracil dissolved in 0.05% DMSO were given to 120 µL of the suspended cells (50,000 cells/mL) in wells of 96-well plates.The viability of the cells was measured by the colorimetric MTT assay after 5 days of incubation according to the reported procedures.The absorbance of the purple formazan solution was measured at λ 540 nm by microplate reader (ELx800 Absorbance Microplate Reader, BioTek) against DMSO as a negative control.The cytotoxicity of the tested compounds was expressed as the concentration that resulted in a 50% inhibition of growth (IC 50 ) compared to the untreated cells (DMSO without the tested compounds) and 5-FU.Qualitative morphological study by MTT assay was done for compounds 5c and 7i against MCF-7 and HepG2, respectively using high dose of 37 µg/ml and low dose of 4 µg/ml for compound 5c and doses of 37, 12 and 4 µg/ml for compound 7i.Images were taken using Gx microscope (GXMGXD202 Inverted Microscope) (10x Eyepiece) and DMSO was used as a negative control.

MOLECULAR DOCKING METHODOLOGY
Docking studies have been carried out using MOE 2008.10 (MOE 2008.10 of Chemical Computing Group. Inc.).The crystal structure of EGFR with Erlotinib (Tarceva TM ) (PDB code: 1M17) were obtained from protein data bank (PDB).Docking study was proceeded according to the standard official procedure of MOE 2008.10 (MOE 2008.10 of Chemical Computing Group. Inc, Halgren, 1996)and the MOE's Pose Viewer utility (El-Azab et al., 2010).

Chemistry
The reaction sequence used to synthesize the desired compounds is depicted in Schemes 1 and 2.

Synthesis of compounds 2-5 (Scheme 1)
The reported 2-(1-hydroxyethyl)-1H-benzimidazole2 was obtained in a high yield via cyclization of o-phenylenediamine with lactic acid in 4N-HCl.Oxidation of 2-(1-hydroxyethyl)-1Hbenzimidazole 2 with potassium dichromate in concentrated sulphuric acid gave 2-acetyl-1H-benzimidazole 3. Claisen-Schmidt condensation is the most convenient method for preparation of α,β-unsaturated carbonyl compounds in which an equimolar quantity of aromatic aldehyde and aliphatic aldehyde or ketone was condensed in the presence of 10-60% aqueous alcoholic alkali.Hence, condensation of 2-acetyl-1H-benzimidazole3 and a variety of aromatic aldehydes 4a-f in aqueous ethanolic solution of sodium hydroxide 10% gave 1-(1H-benzimidazol-2-yl)-3arylprop-2-en-1-ones 5a-f.The structures of the prepared compounds were substantiated by elemental and spectral analyses.For example, in 1 H NMR spectrum of compounds 5b and 5e, the vinylic protons resonated as two doublets within the aromatic region with J value 15 Hz and 16 Hz, respectively and this observation indicated the trans configuration (Silverstein et al.,1991).

Synthesis of compounds 7and 8 (Scheme 2)
N-Alkylation of benzimidazoles was successfully achieved via substitution reaction with alkyl halides.Elaboration to compounds 7a-j was performed by the reaction of compounds 5a-f with the appropriate α-bromophenacyl derivatives 6a,b in dry acetone in the presence of anhydrous K 2 CO 3 .The structures of compounds 7a-j were confirmed by IR that revealed the presence of two different carbonyl bands due to propenone and oxoethyl residues at about 1660-1655 cm -1 and 1694-1691 cm -1 , respectively.Also, 1 HNMR spectra were characterized by the presence of methylene protons which resonate in aliphatic area in the range of 6.30-5.66ppm.Moreover, the vinylic protons appeared as two doublets within the aromatic region in the 1 H NMR spectra of compounds 7d, 7f,7h and 7j with high values of J coupling constant.Hence, it can be concluded that the isomeric form of vinylic group is trans.Cyclization of the diketo compounds 7a-j was achieved via refluxing with ammonium acetate in acetic acid to afford the corresponding pyrazino derivatives 8a-j.The chemical structures of the isolated products 8a-j were confirmed by their spectral and microanalytical data.In IR spectra, the notable feature was the disappearance of the stretching vibrations of the carbonyl groups.In addition, they were confirmed by MS and microanalytical data.

In vitro cytotoxicity screening
The in vitro cytotoxicity studies of the newly synthesized compounds were performed on three different cancer cell lines, namely human hepatoma cell line (HepG2), human breast cancer cell line (MCF-7) and kidney of African green monkey (Vero B).The quantitative evaluation of the cytotoxicity screening was done by employing tetrazolium salt MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay.MTT assay is applied to assess cytotoxicity and viability of the cells.It is a colorimetric metabolic activity assay that measures the ability of viable cells to reduce a yellow tetrazolium salt to purple insoluble formazan in the mitochondria.Hence, formation of the purple formazan depends on the metabolic activity of the cells and directly related to the number of viable cells.However, the cytotoxicity of the tested compounds affects the metabolic activity of the cells and in turn, decreases the production of formazan.By comparing the amount of the formazan resulted from cells treated with the tested compounds with the amount of formazan resulted from untreated control cells, the cytotoxic effects of the target compounds can be concluded.The coloured formazan solution was measured using a spectrophotometer and the correspondent compound concentrations were calculated.Linear regression analysis was used to define dose-response curves and to compute the concentration of the tested compounds needed to reduce absorbance of the formazan by 50%.So, the cytotoxicity of the tested compounds was expressed as the concentration that resulted in a 50% inhibition of growth (IC 50 ) compared to the untreated cells (DMSO without the tested compounds) and 5-FU as shown in Table 1.For the preliminary characterization of the expected cytotoxicity of the tested compounds, we first examined the changes in cell morphology induced by the treatment under a phase contrast microscope.The purple formazan should be visible inside the cells before dissolving it in isopropanol/HCl solution.
A qualitative morphological study of cytotoxicity by MTT assay .was applied for compounds 5c and 7i using high and low doses and compared with the untreated cells (negative control).Viable cells exhibited deep purple cell nucleus with rod shaped formazon whereas apoptotic cells lacked formazon growth in a dose dependent manner (Figures 1 and 2).
Among the tested compounds, compounds 5a and 5c-e showed the highest and broad spectrum activity against the three cancer cell lines.Compound 5a, with unsubstituted phenyl ring, exploited great potency and lethal effect over HepG2, MCF-7 and Vero B cell lines with IC 50 values of 2, 1.8 and 3.5 µg/ml, respectively.It is clear that compound 5a (1.8-3.5µg/ml) is almost 7 to 18 folds as 5-FU (12-62 µg/ml).By comparing the most active compound 5a with the other members in the same series, we concluded that, substitution of the phenyl ring or replacing it with a pyridyl ring relatively decreased the activity.With regard to the selectivity against individual cell lines, compound 5a showed selective inhibition against HepG2 and MCF-7 cell lines.However, compound 5c showed effectiveness against Vero B cell line with IC 50 reached 2.5 µg/ml.The results of the cytotoxicity of compounds 7a,band 7e-j revealed that there was a decrease in the activity when compared with N-unsubstituted compounds 5. Compound 7b showed promising antiproliferative activity against MCF-7 and Vero B cell lines at IC 50 values of 2 and 2.5 µg/ml, respectively.In addition, both 7e and 7i showed good growth inhibition against MCF-7 and Vero B cell lines, while the rest of compounds among this series exerted moderate activity with IC 50 ranging from 20-30 µg/ml.However, compound 7a showed activity against only MCF-7 cell line, while compound 7h gave its activity against only HepG2 and MCF-7 cell lines.A notable decrease in the inhibitory activity was observed in the cyclized pyrazino derivative 8b against MCF-7 and Vero B cell lines at IC 50 10 and 18 µg/ml, respectively in comparison with the activity of its open chain analogue 7b against the same cell lines (IC 50 2 and 2.5 µg/ml, respectively).On the other hand, a little or no change in the activity of most of the cyclized pyrazino derivatives was observed in comparison with that of the corresponding uncyclized α, βunsaturated ketone derivatives 7.
The active pyrazino compounds showed good IC 50 in the range of 10-20 µg/ml.The increased inhibitory activity of compounds 8b, 8h and 8j in comparison to compounds 8a, 8g and 8i, respectively, may reflect the positive impact of the lipophilicity of the molecules resulted from replacement of Cl by Br atom.However, compounds 8g and 8i were completely inactive against all of the three cell lines tested.

LIPINSKI'S RULE OF FIVE
Lipinski's rule of five provided very convenient and easily applied guidelines for the selection of compounds with a greater chance of yielding successful drugs that are orally active in human (Lipinski et al., 1997).If the rule of five score is greater than one, the compound is unlikely to be further pursued as a potential drug, because it would likely lack properties important in its ADME (Lipinski, 2004).The results showed that all investigated compounds obeyed these rules and should present good passive oral absorption (Table 2).

DOCKING STUDIES
The promising antiproliferative activities of compounds 5a and7b over breast cancer cells stimulated us to study the molecular docking of these compounds into the active site of EGFR, which is over expressed in breast cancer cells.The aim of this study is to reveal if these compounds have comparable binding manner to EGFR inhibitors such as Erlotinib.We assumed that the active target compounds 5a and 7b might demonstrate antiproliferative activity against MCF-7 cell line through inhibition of EGFR.The automated docking program of MOE 2008.10 was used to dock compounds 5a and7b into the active binding site of EGFR along with the inhibitor Erlotinib.The calculated binding energies of the docked compounds 5a, 7b were -82.10, -71.22 and -114.13 kj/mol, respectively.The carbonyl group of compound 5a showed two hydrogen bonds with Thr-766 (2.98Å) and HOH-10 (3.03Å) mediated hydrogen bonding interaction with Thr-830 side chain (3.33Å), Gln-767 side chain (3.42Å) and Thr-766 side chain (3.38Å) (Figure 3).Moreover, the benzimidazole ring of compound 5a binds to a narrow hydrophobic pocket in the N-terminal domain of EGFR-TK similar to quinazoline ring of Erlotinib inhibitor.So, the results from the molecular docking study confirmed that the active compound 5a may act on the same target receptor as Erlotinib.On the other hand compound7b bound with active binding site of EGFRin a different mode of interaction in which the carbonyl groups of both chalcone and phenacyl moieties showed one hydrogen bond with HOH-10 (2.66 and 3.81Å) mediated hydrogen bonding interaction with Thr-766 side chain (2.71Å) (Figure 3). .This different mode may be attributed to steric clash of the phenacyl moiety during docking procedure.

CONCLUSION
A series of substituted benzimidazole and pyrazinobenzimidazole derivatives were developed and three cancer cell lines including HepG2, MCF-7 and Vero B were used to evaluate the cytotoxic activity of these designed compounds.Among the tested compounds, Compounds 5a and 5c-e showed the highest and broad spectrum activity against the three cancer cell lines with IC 50 range of 1.8-9 µg/ml, comparable to 5-FU with IC 50 range of 12-62 µg/ml.Compound 5a, with unsubstituted phenyl ring, free benzimidazole NH and α, β-unsaturated carbonyl moiety at the 2-position of benzimidazole nucleus, exploited great potency and lethal effect over HepG2, MCF-7 and Vero B cell lines with IC 50 values of 2, 1.8 and 3.5 µg/ml, respectively.Molecular docking studies confirmed the strong cytotoxic activity of compounds 5a and 7b over MCF-7 and the postulation that these active compounds may act on the same enzyme target where EGFR inhibitor, Erlotinib, acts.

Fig. 3 :
Fig. 3: The binding model of compounds 5a (Upper panel in blue) and 7b (Lower panel in red) with EGFR TK complex (PDB ID: 1M17).Left upper and lower panels showed overlay of the docked 5a and 7b with Erlotinib ligand (in green).The hydrogen bonds are shown in green lines.

Table 1 :
In vitro antitumor activities of the designed compounds.

Table 2 :
Calculated Lipinski's rule of five for the biologically active compounds a Molecular weight.b An octanol-water partition coefficient.c Number of hydrogen bond donors.d Number of hydrogen bond acceptor.