Synthesis, anti-microbial activity, cytotoxicity of some novel substituted (5-(3-(1H-benzo[d]imidazol-2-yl)-4-hydroxybenzyl)benzofuran-2-yl)(phenyl)methanone analogs

Background There is a dire need for the discovery and development of new antimicrobial agents after several experiments for a better resistance of microorganisms towards antimicrobial agents become a serious health problem for a few years in the past. As benzimidazole possess various types of biological activities, it has been synthesized, in the present study, a new series of (5-(3-(1H-benzo[d]imidazol-2-yl)-4-hydroxybenzyl)benzofuran-2-yl)(phenyl)methanone analogs by using the condensation and screened for its in vitro antimicrobial activity and cytotoxicity. Results The synthesized (5-(3-(1H-benzo[d]imidazol-2-yl)-4-hydroxybenzyl) benzofuran-2-yl)(phenyl)methanone analogs were confirmed by IR, 1H and 13C-NMR, MS spectra and HRMS spectral data. The synthesized compounds were evaluated for their in vitro antimicrobial potential against Gram-positive (Bacillus subtilis, Bacillus megaterium, Staph aureus and Streptococcus pyogenes), Gram-negative (Escherichia coli, Proteus vulgaris, Proteus mirabilis and Enterobacter aerogenes) bacterial and fungal (Aspergillus niger, Candida albicans, Fusarium oxysporum, Fusarium solani) strains by disc diffusion method and the minimum inhibitory concentration (MIC) in which it has been recorded in microgram per milliliter in comparison to the reference drugs, ciprofloxacin (antibacterial) and nystatin (antifungal). Further, the cytotoxicity (IC50 value) has also been assessed on human cervical (HeLa), Supt1 cancer cell lines by using MTT assay. Conclusions The following screened compounds (4d), (4f), (4g), (4k), (4l), (4o) and (4u) were found to be the best active against all the tested bacterial and fungal strains among all the demonstrated compounds of biological study. The MIC determination was also carried out against bacteria and fungi, the compounds (4f) and (4u) are found to be exhibited excellent potent against bacteria and fungi respectively. The compounds (4f) and (4u) were shown non-toxic in nature after screened for cytotoxicity against the cancer cell lines of human cervical (HeLa) and Supt1. Additionally, structure and antibacterial activity relationship were also further supported by in silico molecular docking studies of the active compounds against DNA topoisomerase. Electronic supplementary material The online version of this article (10.1186/s13065-017-0364-3) contains supplementary material, which is available to authorized users.


Background
The innovation and the growth of new antimicrobial and anticancer inhibitory agents are the fundamental goals in medicinal chemistry. According to WHO, number of people affected by cancer will rise from 14 million in 2012 to 22 million within the next 20 years [1]. Most of the cancer cells are defined by unrestrained growth of the cells without differentiation due to the deregulation of essential enzymes and other proteins controlling cell division and proliferation [2,3]. Clinically, many chemotherapeutic drugs provide a satisfactory response, but they origin a variety of side effects to the patients despite curing the main problem, when they are first exposed to the tumors. Cancer cells have become invulnerable; therefore, there is an urgent need for potential, selective anticancer drugs in contemporary oncology [4]. On the other hand, typhoid, cholera and pneumonia are common worldwide bacterial diseases caused by Gramnegative bacteria. When comparing Gram-positive and Gram-negative bacteria, several species of Gram-negative bacteria are pathogenic. This potential is usually associated with confident components of the walls of Gram-negative cell while exacting the lipopolysaccharide layer [5].
Benzimidazole is a privileged pharmacophore encountered in a number of fundamental cellular components and bioactive molecules. Indeed, a number of important drugs used in different therapeutic areas contain a benzimidazole moiety [6]. Examples are proton pump inhibitor omeprazole, anti-hypertensive drugs candesartan and telmisartan, anthelmintics albendazole and mebendazole, as well as several other kinds of investigational therapeutic agents including antitumor and anticancer. The substituted benzimidazoles have received considerable interest during preceding two decades as they are endowed with a variety of biological activities and have wide range of therapeutic properties [7]. Benzimidazoles are one of the most proficient heterocyclic moieties, which have active sites in treating various diseases [8]. Frequent reports were published on benzimidazole fragment and its analogues competent to exhibit anticancer as well as antimicrobial activities [9,10]. The basic moiety of telmisartan (reported as cytotoxicity agent in prostate cancer cell line) is also bis-benzimidazole scaffold. [11]. Literature survey revealed that of the compounds attitude benzimidazole moites reported to possess a number of attractive biological activities such as anti tubercular, anticancer, antihelmintic, anti allergic [12], antihistaminic [13], antifungal [14][15][16] and anti-inflammatory [17]. Recently, Thomas et al. reported that some novel 2-phenyl benzimidazole were shown cell based assays for cytotoxicity and antiviral activity against the panel of RNA and DNA virus molecules which have been found to be more potent [18]. Benzimidazole related molecules are exhibited to antimicrobial agents in search of the new chemical entities, as there is another approach in which the combination of two or more heterocyclic pharmacophore in the single entity results in more potent activity with different modes of action [19,20].
In this present study some novel benzofuran bearing benzimidazole derivatives have been synthesized and their antimicrobial and cytotoxic activities have been established. It was considered worthwhile that to synthesize convinced new chemical entities include two active pharmacophore such as benzimidazoles and benzofuran nucleus single molecular frame work and to get them evaluated for their antimicrobial activity. To the preeminent of our knowledge, this is the best report on the synthesis of benzimidazoles derivatives bearing benzofuran ring. Among the several chemical classes that boast the potential to display the antimicrobial activity [21], cancer activity [22] structures of few benzimidazole possessing significant in vitro antimicrobial (A, B) and cytotoxicity activity (C, D) are presented in Fig. 1.

Chemistry
For the synthesis of target compounds, 5,5′-methylenebis(2hydroxybenzaldehyde) (2) [23] was prepared in good yield by electrophilic substitution reaction of salicylaldehyde (1) with 1,3,5-trioxane(formaldehyde trimer) in glacial acetic acid in the presence of a catalytic amount of concentrated sulfuric acid. Aldehyde (2) was subjected to condense cyclisation with substituted phenacyl bromide in the presence of potassium carbonate at the room temperature to obtain the corresponding 5-[(2-benzoylbenzofuran-5-yl)methyl]-2-hydroxybenzaldehyde (3a) [24,25] (Scheme 1). The structures of compound (3a) were confirmed by their spectroscopic data ( 1 H NMR, 13 C NMR, IR, MS, and HRMS) which were provided in the experimental part. The 1 H NMR spectrum of compound (3a) showed two singlet signals at 9.85 and 10.98 ppm corresponding to aldehyde and hydroxyl groups, respectively. A singlet signal was suitable to the bridged methylene protons at δ 3.98 ppm, in addition to down field singlet signal due to benzofuran proton and aromatic protons in the region 8.03-6.95 ppm. The conformation regards the structure of the compound (3a) is executed by the 13 C NMR data varying between δ 191.3 and δ 42.09 ppm. The carbon atoms of the two carbonyl groups present at aldehyde and keto appeared more downfield at 191.3 and 182.1 ppm. The two carbon of the -C-O-C-linkage in benzofuran nucleus exhibited the absorption peaks at δ 159.2 and δ 151.4 ppm, respectively. The carbon atoms aromatic ring was observed to exhibit between absorption peaks at δ 154.0-112.1 ppm. The presence of the bridged methylene group between the two aromatic rings was observed to exhibit an absorption peak at δ 42.0 ppm. The IR spectra of the products showed the absorption bands of C=C str , HC=O str and -OH str in the region 1650, 1710 and 3550 cm −1 respectively. Further confirmation of HRMS spectra showed the obtained peak at m/z = 357.11204 ([M+H] + ); resultant to a molecular formula C 23 H 17 O 4 .
The synthesis of various compounds (4a-u) were carried out by condensation of the 5-((2-benzoylbenzofuran-5-yl)methyl)-2-hydroxybenzaldehyde (3a) with various substituted ortho phenylenediamine in the presence of glacial acetic acid under conventional reflux temperature in good yield (Scheme 1). The structures of all the synthesized compounds (4a-u) were thoroughly analyzed by using 1 H-NMR, 13 C-NMR, IR, ESI-MS and HRMS analytical techniques. The assigned structures of compound (4a) are based on the detailed spectroscopic analysis. The 1 H NMR spectrum of compound (4a) showed singlets of the hydroxyl proton at δ 12.23 and signal of -NH of imidazole ring appeared as singlet at δ 13.18, which was further corroborated through a sharp band at 3420 cm −1 in its IR spectrum. 13 C NMR spectrum of compound (4a) showed resonance at δ 182.3 ppm attributed to carbonyl group of benzofuran ring, which was further confirmed by IR spectrum through band at 1740 cm −1 . The mass spectrum of compound (4a) showed peak at m/z 445.08 (M+H) + . Further confirmation HRMS spectra showed peak obtained at m/z = found 445.180745 ([M+H] + ); consequent to a molecular formula C 29 H 21 N 2 O 3 .
In the proton NMR spectrum of (5-(3-(5-fluoro-1Hbenzo[d]imidazol-2-yl)-4-hydroxybenzyl)benzofuran-2-yl)(phenyl)methanone (4f), the signals at δ 13.21 and 12.52 ppm value can be assigned to NH of benzimidazole and OH proton of phenolic moiety, respectively. They multiple at δ 8.02-6.89 ppm in the proton NMR spectrum of compound (4f) accounts for three aromatic protons of benzimidazole and phenyl moiety out of the twelve protons. The methylene protons singlet peak assigned at δ 4.01 ppm. In the 13 C NMR spectrum of compound (4f), the chemical signal δ 182.1 and 159.1 ppm were assigned to the carbonyl and hydroxyl aromatic ring respectively. The chemical signals at δ 42.1 were attributed to the bridged methylene carbon. The mass spectrum characterization of the compound (4f) exhibits an ion peak at 463.1 m/z which can be designated as the M+1 ion peak and HRMS spectra showed the obtained peak at m/z = 463.14142 ([M+H] + ), corresponding to a molecular formula C 29 H 20 FN 2 O 3 .
The IR spectrum characterization of compound indicated by a shift of absorption band from 3413 cm −1 , corresponding to the NH group stretching. This can be correlated to the ring stretching of benzimidazole moiety. In the proton NMR spectrum of compound (4g), the NH proton has resonated as a singlet at δ 12.95 ppm, OH proton showed as a singlet at δ 9.81 and the multiplet at δ 8.01-6.95 ppm value can be attributed to the aromatic protons of compound (4g). The singlet at δ 4.10 and 2.31 ppm was assigned to the bridged methylene protons and two methyl protons respectively. In the 13 C NMR spectrum of compound (4g), the chemical signal δ 183.0 and 151.5 ppm were assigned to the carbonyl and hydroxyl attached carbon respectively. The chemical signals at δ 156.4-112.1 ppm were attributed to the aromatic ring carbons and chemical signals at δ 45.2 and 21.0 ppm bridged methylene protons and two methyl protons respectively. The mass spectrum characterization of the compound (4g) exhibits an ion peak at 473.2 m/z which can be designated as the M+1 ion peak and HRMS spectra showed the obtained peak at m/z = 473.18597 ([M+H] + ) and corresponding to a molecular formula C 31 H 25 In the proton NMR spectrum of (5-(3-(5-bromo-3H-imidazo [4,5-b]pyridin-2-yl)-4-hydroxybenzyl) benzofuran-2-yl) (

In vitro antibacterial activity
The in vitro antibacterial studies of various compounds (4a-u) were assayed with concentrations (900 μg/mL) against Gram-positive and Gram-negative bacteria by disc diffusion assay [26]. The Gram-positive strains used were Bacillus subtilis, Bacillus megaterium, Staph aureus, Streptococcus pyogenes and Gram-negative bacteria were Escherichia coli, Proteus vulgaris, Proteus mirabilis and Enterobacter aerogenes. The results were recorded for each weathered compound diameter of inhibition zones of microbial growth around the disks (in mm). The values of zone of inhibition for bacterial are revealed in Table 1. The minimum inhibitory concentration (MIC, μg/mL) is the lowest concentration of a chemical that prevents visible growth of bacterium in microgram per milliliter of active compounds (Table 1) was determined by the microorganism's susceptibility tests in nutrient and potato dextrose broths were used for the determination of MIC [27]. The results revealed that seven organic compounds displayed better inhibitory effects on the growth of the tested bacterial strains. It was interesting to note that the compounds with substituent on the 3rd position of imidazole ring displayed outstanding antibacterial activity and almost equal to that of standard. However, these trends were transformed closely to antifungal activity. Almost all the synthesized compounds (4a-u) were found to be active against all investigated pathogenic bacterial strains. As shown in Table 25) nitro (4e) displayed moderate to be good antibacterial activity with MIC 25 μg/mL. However, the compounds of (4a), (4k); (4c), (4m); (4e), (4o) and (4g), (4r) were not showed any activity against S. aureus, streptococcus pyogenes, and B. subtilis, respectively; it may be rational owing to the presence of low polar substituents.

Antifungal activity
The compounds (4d), (4f), (4g), (4k), (4l), (4o) and (4u) were tested against four reference fungal strains such as Aspergillus niger, Candida albicans, Fusarium oxysporum, Fusarium solani by disc diffusion method. Minimum inhibitory concentration (MIC) in microgram per milliliter of compounds exhibiting activity ( Table 2) was determined by the microorganism's susceptibility tests in nutrient and potato dextrose broths were used for the determination of MIC. The evaluated seven compounds were found to exert a prominent antifungal activity against pathogenic fungal strains. The compounds (4l) and (4u) exhibited a significant inhibitory activity against A. niger and F. oxysporum with MIC 25-50 μg/ mL, whereas, the compounds with floro (4f) and dichloro (4d) also exhibited maximum activity with MIC > 25. In case of all fungal strains, compound (4o) revealed moderate to good activity with MIC 25-100 μg/mL. The compounds (4g) and (4k) exhibit less potent inhibitory potential against A. niger with an absence of MIC and all the results are given in Table 2. From the obtained in vitro antimicrobial results, it was observed that substitution of electron withdrawing groups at 3rd position of benzimidazole ring leads to increase in both antifungal and antibacterial activity.

In vitro cytotoxicity
MTT assays determine the ability of viable cells to convert a soluble yellow tetrazolium salt (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) into insoluble purple formazan crystals by the mitochondrial dehydrogenase enzymes. Cells were exposed to 0.5 mg/mL of MTT for 3 h at 37 °C in an appropriate complete medium. The medium and the MTT were removed after solubilisation in dimethyl sulfoxide (DMSO) and the amount of insoluble formazan crystals was evaluated by measuring the optical density at 550 nm. Each condition was performed in triplicate. Each measurement was corrected from the optical density of MTT alone and expressed relative to the non-treated conditions. Determination of the inhibiting concentration of 50% cell viability and IC 50 was performed. In short, the fraction of cell affected (Fa) and the fraction of cell unaffected (Fu) relative to one were determined from the viability assay. The log of (Fa/Fu) was plotted against the log of concentration for each compound. Log of IC 50 was determined at the y-intercept. Standard error was evaluated through the 95% confidence interval.
The appreciable results were obtained from the previous biological studies, specifically, anti-microbial activity for the derivatives of more microbial active compounds, which encouraged in the present research work to test their cytotoxicity against a human cancer cells (HeLa), Supt1 cell lines. In all the above cases, the activity of the compound (4f) and (4u) was found to be significantly similar than the controller of the DMSO solvent. To determine the relative cytotoxicity of compounds in the Hela, Supt1 cells were incubated with increasing concentrations (10, 25, 50, 75, 100, 250, 500 and 1000 ng) of the drugs and the survival of the cell was estimated by MTT assay and their IC 50 were shown in Figs. 2 and 3, respectively. Both the compounds were found nontoxic at very low concentrations (10, 25, 50 and 75 ng), however, the concentration of drugs in about 500 ng was extremely cytotoxic (> 80%). The IC 50 values at 299.009

Molecular docking
The crystal structure of DNA type IIA topoisomerase (pdb id: 2XCT) [28] was retrieved from the Protein Data Bank to understand the interaction between new series of benzimidazole derivatives and type II topoisomerase. The devised software, GLIDE 5.6 [29] was used for molecular docking studies. Protein was prepared by applying default parameters of wizard Maestro 9.0; a grid was generated around the active site by selecting the cocrystalized ligand. Receptor van der Waals scaling for non-polar atoms was kept at 0.9 [30]. The molecules were built by using Maestro build panel and prepared by the Fig. 2 Cell viability assay: hela cells were exposed to increasing concentrations (10, 25, 50, 75, 100, 250, 500 and 1000 ng) of (4f) and (4u) drugs for 24 h, after which cell viability was determined by MTT assay and their IC 50 values application of Lig Prep. Low energy confirmation of the ligands were selected and docked into the grid generated for the protein using the docking mode of extra precision (XP) [31]. Dock pose of each ligand was analyzed for interactions with the receptor. The best and similar interactions with the protein active site were shown from those active molecules, (4d), (4f) and (4g). The molecules were deeply embedded into the hydrophobic active site pocket and they were occupied the similar position as represented in Fig. 4. The docking pose and ligand interaction diagrams of (4d), (4f) and (4g) (pulm in color, green in color and orange in color) are shown in Fig. 5.
To get more insights into the structural basis for its activity, all synthesized compounds were docked into DNA type IIA topoisomerase (2XCT) and the most active compounds (4d), (4f) and (4g) were analyzed in more detail. Docking studies of these compounds showed similar interactions (hydrogen bonds with DC 12 G; DT 10 H, π-π stacking interactions with DG F 8, DG H 9, π-cation interactions with Arg D 458 and metal co-ordination bond with Mn) with the target.
Docking analysis of the best active compounds in the active site of 2XCT revealed that the hydrogen bond, π-π stacking, π-cation and metal co-ordination interactions are important for the activity of these compounds. The obtained result from docking study was well in agreement with experimental results: compound (4d) (pulm in color, Fig. 5a) showed only one hydrogen bond with DC G 12 (bond length of 1.893 Å), one π-cation interaction with ARG D 458, two π-π stacking interactions with DG F 8, DG H 9 and metal co-ordination interaction with Mn; whereas compound (4f) (green in colour, Fig. 5b) showed two hydrogen bond interactions with DC G 12 (bond length of 1.863 Å), DT H 10 (bond length of 1.916 Å), one π-cation interaction with ARG D 458, three π-π stacking interactions with DG F 8, DG H 9 and metal coordination interaction with Mn; compound (4g) (orange in color, Fig. 5c) showed two hydrogen bond interactions with DC G 12 (bond length of 1.944 Å), DT H 10 (bond length of 1.905 Å), one π-cation interaction with ARG D 458, two π-π stacking interactions with DG H 9, ARG B 1122 and metal co-ordination interaction with Mn.
From the above results, compound 4d with one hydrogen bond showed less activity, whereas compounds 4f, 4g with two hydrogen bonds showed more activity. These docking results clearly coincided with that of experimental antibacterial activity. Hence, more number of hydrogen bonding interactions with target type II topoisomerase, will affects on more activity. Docking score, glide energy and emodel energies are tabulated in Table 3. Predicted ADME (drug-likeness) properties of all synthesized compounds are provided in Additional file 1.

Chemistry
Melting points are uncorrected and were find out in open capillary tubes in sulphuric acid bath. TLC was carrying out on silica gel-G, and spotting was done using UV light. IR spectra were recorded using Perkin-Elmer 1000 instrument in KBr phase, The 1 H NMR spectra were record on a Varian as 400 MHz instrument in DMSO, chemical shifts are given in δ ppm relative to TMS, and coupling constants (J) are expressed in hertz (Hz). Combinations of the following abbreviations are used to describe NMR spectra: s-singlet; d-doublet; t-triplet; m-multiplet. 13 C NMR spectra were recorded with a Bruker Advance 400 (100 MHz) spectrometer. Mass spectra on Agilent LC-MS instrument giving only (M + +H) values.

General procedure of the synthesized benzimidazole derivatives (4a-u)
o-Phenylenediamine, 0.068 g (0.632 mmol), was slowly added to a solution of 0.150 g (0.421 mmol) of 5-((2-benzoylbenzofuran-5-yl)methyl)-2-hydroxybenzaldehyde (3a) in glacial acetic acid, and the mixture was refluxed (70 °C) for 4-6 h under N 2 atmosphere, the progress of the reaction being monitored by TLC. The mixture was cooled to room temperature, then, the mixture was poured into ice cold water and neutralised with sodium bicarbonate solution, after the mixture was washed with water and DCM for two times, the DCM layer was separated and dried over anhydrous sodium sulphate. Fatherly DCM solvent was removed in vacuo, the residue was purified by column chromatography on silica gel to give the corresponding products 4a. The other compounds 4b-u was also prepared by the similar procedure.

In vitro antimicrobial assay
Antimicrobial activity was evaluated using agar well diffusion method. The activity was determined by measuring the diameter of the inhibition zone (in mm). Samples of the tested compounds (50 μL, 1 mg/mL concentration) were loaded into the wells on the plates. All solutions were prepared in DMSO and pure DMSO was loaded as control. The plates were kept for incubation at 35 °C for 1-5 days and then were examined for the formation of inhibition zone. Each inhibition zone was measured three times to get an average value. The test was performed three times for each bacterium culture [26][27][28].

Minimal inhibitory concentration (MIC) measurement
The microorganism's susceptibility tests in nutrient and potato dextrose broths were used for the determination of MIC. Stock 1000 μg/mL solutions of the tested compounds, ciprofloxacin and nystatin were prepared in DMSO followed by dilutions to 250-25 μg/mL concentrations. Inoculated microorganism suspensions were incubated at 37 °C for 1-5 days for MIC determination. The microorganism suspensions were inoculated into the concentrations of corresponding compounds and control experiments and listed in Tables 1 and 2 respectively.

Evaluation of cell cytotoxicity
Hela, Supt1 cancer cell lines were used to evaluate the impact on cell viability of each compound. Hela cells were maintained in DMEM, Supt1 cells in RPMI, all the mediums were supplemented with 10% fetal bovine serum (FBS) cells were maintained in keratinocyte serum free media with 0.1 ng/mL human recombinant EGF, 0.05 mg/mL bovine pituitary extract and additional CaCl 2 44.1 mg/mL (final concentration 0.4 mM), 2 mM l-glutamine at 37 °C under a 5% CO 2 atmosphere. For each cell line, 70% confluent cell culture flask was trypsinized and were seeded cells in a 96-well plate at a density of 5000 cells by well in the appropriate complete media; These cells were treated with increasing concentrations of drugs, and incubated for 24 h. The cells were washed with media and resuspended in new medium. To this, 20 mL of 5 mg/mL MTT (Sigma-Aldrich) was added and incubated for 4f. The medium was removed from cells, and dissolved in DMSO (DMSO 0.1% in phosphate saline buffer) and read in an ELISA micro plate reader at 570 nm; 48 h after treatment, viability was accessed by MTT assay.

Conclusions
In conclusion, the present work offers the promotion of a simple procedure in an inexpensive route for the synthesis of benzimidazole derivatives (4a-u) via condensation of 5-((2-benzoylbenzofuran-5-yl)methyl)-2hydroxybenzaldehyde with ortho phenylenediamine under conventional heating. The biological evolution exhibited that these molecules (4a-u) were good and selective against bacterial and fungal strains in the micro molar range. The experimental antimicrobial studies resulted that the compounds (4f) and (4u) are good inhibitors for antibacterial and antifungal activities respectively. The compounds of (4f) and (4u) have proven potential against cancer cell lines. Interestingly, the compound concentration increases nature of cytotoxicity also increasing. Molecular docking study revealed that not only hydrogen bonding interactions, π-π stacking, π-cation and Mn metal co-ordination interactions were also favourable for its antibacterial activity.