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

Chemistry

For the synthesis of target compounds, 5,5′-methylenebis(2-hydroxybenzaldehyde) (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 (¹H NMR, ¹³C NMR, IR, MS, and HRMS) which were provided in the experimental part. The ¹H 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 ¹³C 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 C₂₃H₁₇O₄.

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 ¹H-NMR, ¹³C-NMR, IR, ESI–MS and HRMS analytical techniques. The assigned structures of compound (4a) are based on the detailed spectroscopic analysis. The ¹H 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. ¹³C 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 C₂₉H₂₁N₂O₃.

In the proton NMR spectrum of (5-(3-(5-fluoro-1H-benzo[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 ¹³C 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 C₂₉H₂₀FN₂O₃.

The IR spectrum characterization of compound (5-(3-(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)-4-hydroxybenzyl)benzofuran-2-yl)(phenyl) methanone (4g) 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 ¹³C 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 C₃₁H₂₅N₂O₃.

In the proton NMR spectrum of (5-(3-(5-bromo-3H-imidazo[4,5-b]pyridin-2-yl)-4-hydroxybenzyl)benzofuran-2-yl)(4-chlorophenyl)methanone (4u), the singlet at δ 13.29 and 12.35 ppm was assigned to the NH of the imidazo pyridine and hydroxyl group proton of the phenolic moiety. The thirteen aromatic protons of the compound (4u) have resonated at δ 8.20–7.05 ppm as multiplet. The mass spectrum characterization of the compound (4u) exhibits an ion peak at 558.9 m/z which can be designated as the M+1 ion peak and HRMS spectra showed the obtained peak at m/z = 557.02621 ([M+H]⁺) consequent to a molecular formula C₂₉H₁₉O₃N₂BrCl.

In vitro antibacterial activity

Almost all the synthesized compounds (4a–u) were found to be active against all investigated pathogenic bacterial strains. As shown in Table 1, it is cleared that the compounds (4d), (4f), (4g), (4k), (4l), (4o) and (4u) have superior significant antibacterial potency to the reference drug. In case of Staphylococcus aureus, B. megaterium, E. coli, E. aerogens, P. mirabilis, compounds (4d) and (4f) exhibits the highest antibacterial potential with MIC 25–75 μg/mL, it may be attributed that the presence of more electronegative fluoro and dichloro substituent on phenyl ring. The compound (4u) displayed significant inhibitory potential with MIC 25 μg/mL against P. mirabilis, P. vulgarise and threshold activity against B. megaterium, and E. coli with MIC 120–175 μg/mL, it may have a basic moiety of the bromo pyridine group. In case of E. aerogens compounds with methyl (4c), dichloro (4d) and 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

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 IC₅₀ 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 IC₅₀ 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 IC₅₀ were shown in Figs. 2 and 3, respectively. Both the compounds were found non-toxic at very low concentrations (10, 25, 50 and 75 ng), however, the concentration of drugs in about 500 ng was extremely cytotoxic (> 80%). The IC₅₀ values at 299.009 and 505.618 ng concentrations in Hela cell lines were shown by the compounds (4f) and (4u) respectively. The IC₅₀ value of compound (4f) is 278.73 ng and (4u) is 499.903 ng in Supt1 cells. Although the anticancer activity of (4f) and (4u) is not comparable as standard drug activity, the functional group modifications at the substituted benzimidazole and pyrido imidazole can guide to upgrade the drug leads for further investigations from this laboratory.

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 co-crystalized 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 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 co-ordination 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.

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 ¹H 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. ¹³C NMR spectra were recorded with a Bruker Advance 400 (100 MHz) spectrometer. Mass spectra on Agilent LC–MS instrument giving only (M⁺+H) values.

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 [26–28].

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 CaCl₂ 44.1 mg/mL (final concentration 0.4 mM), 2 mM l-glutamine at 37 °C under a 5% CO₂ 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.