Synthesis, antibacterial activity, synergistic effect

Computational Biology and Chemistry 76 (2018) 1–16

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Computational Biology and Chemistry journal homepage: www.elsevier.com/locate/cbac

Synthesis, antibacterial activity, synergistic effect, cytotoxicity, docking and molecular dynamics of benzimidazole analogues

T

Ritika Srivastava, Sunil K. Gupta, Farha Naaz, Anuradha Singh, Vishal K. Singh, Rajesh Verma, ⁎ Nidhi Singh, Ramendra K. Singh Bioorganic Research Laboratory, Department of Chemistry, University of Allahabad, Allahabad, 211002, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Benzimidazoles Minimum inhibitory concentration (MIC) Fractional inhibitory concentration (FIC) Cytotoxicity Docking and molecular dynamics

A series of 2-Cl-benzimidazole derivatives was synthesized and assessed for antibacterial activity. Antibacterial results indicated that compounds 2d, 2e, 3a, 3b, 3c, 4d and 4e showed promising activity against B. cerus, S. aureus and P. aeruginosa (MIC: 6.2 μg/mL) and excellent efficacy against E. coli (MIC: 3.1 μg/mL). Furthermore, compounds 3d and 3e displayed better activity (MIC: 3.1 μg/mL) than the reference drugs chloramphenicol and cycloheximide against gram positive and gram negative bacterial strains. The compounds 3d–e also showed better activity than the reference drug paromomycin against B. cerus and P. aeruginosa and showed similar inhibition pattern against S. aureus and E. coli. (MIC: 3.1 μg/mL). Studies on fractional inhibitory concentration (FIC) determination of compounds 1a–e, 2a–c, 4a–c and the reference antibiotic via combination approach revealed a synergistic effect as the MIC values were lowered up to 1/8th to 1/33rd of the original MIC. In-vitro cytotoxicity study indicated that 2-Cl-benzimidazole derivatives showed less toxicity than the reference used against PBM, CEM and Vero cell lines. Docking studies and MD simulations of compounds on bacterial protein (eubacterial ribosomal decoding A site, PDB: 1j7t) have been conducted to find the possible mode of action of the molecules. In silico ADMET evaluations of compounds 3d and 3e showed promising results comparable to the reference drugs used in this study.

1. Introduction Multi-drug resistant (MDR) gram +ve and gram −ve bacteria represent a severe public health concern all over the world not only by causing the present day drugs to fail but also by inviting other opportunistic infections (Levy and Marshall, 2004; Walker et al., 2009; ElGohary and Shaaban, 2017). The protection shield of antimicrobial drugs becomes thinner day by day because of wide use and misuse of antibiotics and this contributes enormously in the development of drug resistance in bacterial pathogens. Thus, the development of drug resistance in bacterial strains warrants the discovery of new antibiotics stand alone or as a new cocktail of two different antibiotics directed to a single target or different targets (Azevedo et al., 2015). Bacterial protein synthesis, one of the several targets, has been the most successful one for developing effective antibacterial agents and has been used for efficient structure-based interventions against antibiotic resistance. Most of the naturally occurring clinically approved antibiotics including macrolides, tetracyclines, chloramphenicol and aminoglycosides target the ribosome (Brown and Wright, 2016). These antibiotics bind to one of the functional sites in the ribosome and inhibit the process of protein

⁎

Corresponding author. E-mail address: [email protected] (R.K. Singh).

https://doi.org/10.1016/j.compbiolchem.2018.05.021 Received 8 April 2018; Received in revised form 18 May 2018; Accepted 24 May 2018 Available online 24 May 2018 1476-9271/ © 2018 Elsevier Ltd. All rights reserved.

synthesis by blocking the formation of peptide bond (McCoy et al., 2011). In bacterial protein synthesis, the mutational propensity of ribosome is low because of the redundancy of ribosomal RNA (rRNA) gene in bacterial genome and, therefore, it is an ideal target for antibacterials (Gagnon et al., 2016). The 16S rRNA, a constituent of 30S subunit of bacterial 70S ribosome, is the site for mRNA binding and protein synthesis initiation and the molecules studied have been docked into the binding pocket of this16S rRNA (Chellat et al., 2016). In order to avoid drug resistance, there is an imperious requirement of discovery of new antibacterial agents with minimum adverse effects. In search of some new antibiotics, we have focussed on benzimidazole nucleus, which has a great significance in the area of medicinal chemistry and drug development and has been used as a core substituent of antimicrobial agents (Goker et al., 2002; Kus et al., 2008; Madalageri and Kotresh, 2012; Petkar et al., 2013; El-Gohary and Shaaban, 2014; Mahalakshmi and Chidambaranathan, 2015). Several substituted benzimidazole derivatives have been reported as potent antibacterial agents (Yadav and Ganguly, 2015; Akhtar et al., 2017). In this pursuance, some new antibacterial agents of 2-Cl-benzimidazole analogues 1(a–e) – 4(a–e) have been designed (Fig. 1) and synthesized.


R. Srivastava et al.

Fig. 1. Design consideration of 2-Cl-benzimidazole derivatives, 1(a–e) – 4(a–e), for enhanced biological activity.

in Scheme 2.

These molecules were tested for their antibacterial properties and methods have been suggested for establishing possible mechanism of their mode of action. Literature survey revealed that compounds 1a–c, 2a and 4a already existed but synthetic route and biological studies on these analogues were quite different than reported here (Iemura et al., 1986; Mokrushina et al., 1988; Chermova et al., 1991; Galy et al., 1997; Rao et al., 2013).

2.2. Analysis of physicochemical properties of benzimidazole analogs The physicochemical data of all compounds and standard antibacterial agents (chloramphenicol, cycloheximide and paromomycin) were calculated online using Molinspiration and ChemDraw softwares. The physicochemical properties of molecules, like lipophilicity and steric descriptors – partition coefficient, number of hydrogen bond acceptor and donor, molecular weight and total polar surface area were studied using Lipinski’s rule of five (Lipinski et al., 2012). Data have been presented in Table 1. It revealed that all molecules possessed the drug-like characteristics like the standard antibiotics used. All compounds behaved as H-bond acceptors because of the presence of CeN and CeX dipole. The compounds having eOH group in their structure (compound 1d–e, 2d–e, 3d–e and 4d–e) behaved as H-bond donors. The lipophilicity of all compounds, reported as logP, indicated that compounds had no problem to passage through cell membrane. Lower TPSA values of all compounds proved their good cell internalization, similar to reference antibiotics. So, all compounds were expected to possess drug like properties as shown in Table 1.

2. Results and discussion 2.1. Chemistry Substituted benzimidazoles (substitution on 5,6 positions) have been synthesized from compound 1 (2-Cl-1H-benzimidazole). Compound 2 (2-chloro-5-nitro-1H-benzimidazole) and compound 4 (2chloro-5-6-dinitro-1H-benzimidazole) were synthesized by nitration of compound 1 with a mixture of concentrated nitric acid and concentrated sulphuric acid at reflux. Bromination of compound 1 in MeOH was performed using a solution of Br2/MeOH as a brominating agent followed by addition of H2O at room temperature to synthesize compound 3 (5,6-dibromo-2-chloro-1H-benzimidazole). The synthetic route to obtain the compounds 2–4 is outlined in Scheme 1. A suspension of compound 1 (or 2/3/4) and anhydrous potassium carbonate in acetonitrile was stirred at room temperature. Alkyl halide (a/b/c/d/e) was added drop wise. The reaction mixture was heated at 40–50 °C for 3–4 h. After completion of reaction, the reaction mixture was dried over rotavapour and partitioned between EtOAc and aqueous NaHCO3 solution. The organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum to give the desired crude product 1(a–e) – 4(a–e). All compounds were purified by column chromatography. The structures of 2-Cl-benzimidazole analogues were characterized by 1H NMR, 13C NMR, mass spectra and elemental analysis. The synthetic way to obtain the compounds 1(a–e) – 4(a–e) is outlined

2.3. Antibacterial assay The broth dilution method was used to assess the antibacterial activity of compounds. All compounds were screened against selected gram-positive bacteria, viz. B. cerus and S. aureus and gram-negative bacteria, viz. E. coli (wild type) and P. aeruginosa and gram-negative bacteria mutant type, viz. Escherichia coli (U-621) for measuring the minimum inhibitory concentration (MIC) using chloramphenicol, cycloheximide and paromomycin as references. All compounds showed similar pattern of inhibition towards gram-positive bacteria, viz. B. cereus, S. aureus and different patterns of inhibition against gram-

Scheme 1. Reagents & conditions: (i) Conc. HNO3, Conc. H2SO4, reflux for 1 h; (ii) Br2/MeOH, H2O, rt, 18 h; (iii) Conc. HNO3, Conc. H2SO4, reflux for 4–5 h. 2



Scheme 2. Reagents & conditions: Alkyl halide, K2CO3, acetonitrile, Temp 40–50 °C for 3–4 h.

for H-bonding and hydrophilic interactions. In the next instance, introduction of eBr group at 5th and 6th position of compounds 3a–e showed the highest antibacterial activity when polar sustituents were present at N-1 position as in compounds 3d–e (MIC: 3.1 μg/mL against all bacterial strains for both compounds) in comparison to compounds 3a–c (MIC: 6.2 μg/mL against B. cerus, S. aureus and P. aeruginosa and 3.1 μg/mL against E. coli for each compound) having non polar groups. Dibromo groups are more hydrophobic than mononitro and di-nitro group, therefore, compounds 3a–c have more chances for hydrophobic or lipophilic interactions as well as H-bonding due to ring nitrogen atom. However, compounds 3d–e have higher chances of H-bonding due to the presence of polar group at N-1 position, in addition to hydrophobic interaction. Compounds 4a–e, due to the presence of eNO2 group at both fifth and sixth positions, showed similar antibacterial activity as compounds 2a–e (bearing the same substituents at N-1 position and mono-nitro group at fifth position). It revealed that the addition of an extra nitro group did not affect the MIC value much and showed similar activity. Among all compounds 1(a–e) – 4(a–e), compounds 2d–e, 3a–e and 4d–e showed the highest antibacterial activity. These compounds exhibited excellent inhibitory activity against the

negative bacteria, viz. E. coli and P. aeruginosa as shown in Table 2. SAR of compounds 1a–e showed that higher antibacterial activity was observed when polar sustituents were present at N-1 position as in compounds 1d–e (MIC: 12.5 μg/mL against B. cerus, S. aureus and E. coli and 25 μg/mL against P. aeruginosa for both compounds) in comparison to compounds 1a–c (MIC: 25 μg/mL against B. cerus, S. aureus and E. coli and 50 μg/mL against P. aeruginosa for each compound) having non polar groups. Compounds 1a–c have no chances of any interaction except hydrophobic or lipophilic ones, whereas the polar groups present in compounds 1d–e have chances for both H-bonding and lipophilic interaction. Similarly, in the case of compounds 2a–e, significant pattern of inhibition was observed when eNO2 group was present at 5th position of 2-Cl-1H-benzimidazole with polar sustituent at N-1 position as in compounds 2d–e (MIC: 6.2 μg/mL against B. cerus, S. aureus and P. aeruginosa and 3.1 μg/mL against E. coli for both compounds) in comparison to compounds 2a–c (MIC: 12.5 μg/mL against B. cerus, S. aureus and P. aeruginosa and 6.2 μg/mL against E. coli for each compound) having non polar groups at N-1 position. In this case, the eNO2 group at fifth position seems to be responsible for reduction of MIC values. Because of the eNO2 group, all compounds have chances Table 1 Physicochemical data of compounds 1 (a–e)–4 (a–e). Compound

MW

Log P

TPSA

H-A

H-D

VLR5

Rotatable bonds

Log S

Log Kp

SAS

Rule 1a 1b 1c 1d 1e 2a 2b 2c 2d 2e 3a 3b 3c 3d 3e 4a 4b 4c 4d 4e Chloramphenicol Cycloheximide Paromomycin

< 500 166.60 180.64 194.66 196.64 210.66 211.61 225.63 239.66 241.63 255.66 324.40 338.43 352.46 354.43 368.46 256.61 270.63 284.66 286.63 300.66 323.13 281.35 615.63

≤5 2.50 2.87 3.37 1.86 2.13 2.43 2.81 3.31 1.80 2.07 4.02 4.39 4.90 3.39 3.66 2.32 2.69 3.20 1.69 1.96 0.73 0.76 −6.04

≤140 17.83 17.83 17.83 38.05 38.05 63.65 63.65 63.65 83.88 83.88 17.83 17.83 17.83 38.05 38.05 109.47 109.47 109.47 129.70 129.70 115.38 83.47 347.34

≤10 2 2 2 3 3 5 5 5 6 6 2 2 2 3 3 8 8 8 9 9 7 5 19

≤5 0 0 0 1 1 0 0 0 1 1 0 0 0 1 1 0 0 0 1 1 3 2 18

≤1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3

≤10 0 1 2 2 3 1 2 3 3 4 0 1 2 2 3 2 3 4 4 5 7 3 8

≤5 −2.27 −2.73 −3.28 −2.06 −2.44 −2.48 −2.88 −3.29 −2.32 −2.73 −3.06 −3.18 −3.45 −2.57 −2.79 −1.85 −2.25 −2.65 −1.69 −2.09 −2.39 −2.38 6.39

< −5 −5.63 −5.48 −5.19 −4.87 −4.68 −5.68 −5.88 −5.59 −4.55 −4.15 −5.14 −5.18 −5.23 −4.31 −4.08 −5.96 −5.69 −5.28 −4.89 −4.77 −7.46 −7.63 −16.49

≤10 1.38 1.50 1.62 1.72 1.74 1.95 2.03 2.12 2.15 2.19 1.76 1.84 1.94 2.00 2.05 2.18 2.25 2.34 2.35 2.40 2.78 3.17 7.36

MW = Molecular weight, TPSA = Total polar surface area, H-A = no. of H-bond acceptors, H-D = no. of H-bond donor, VLR5 = violence of Lipinski’s rule of five, Log S = predicted aqueous solubility, Log Kp = predicted skin permeability coefficient, SAS (Synthetic accessibility score) = from 1 (very easy) to 10 (very difficult). 3



Table 2 Antibacterial activity and cytotoxicity of compounds 1(a–e) – 4 (a–e) against Gram +ve and Gram −ve bacteria. Compound

(Wild strain)

(Mutant Strain) Gram −ve

Gram +ve

1a 1b 1c 1d 1e 2a 2b 2c 2d 2e 3a 3b 3c 3d 3e 4a 4b 4c 4d 4e Chloramphenicol Cycloheximide Paromomycin Average Standard Deviation Standard Error

Cytotoxicity: IC50 μg/mL (μM)

Gram −ve

B. cerus MIC

S. aureus MIC

E. coli MIC

P. aeruginosa MIC

E. coli MIC

PBM

CEM

VERO

25 25 25 12.5 12.5 12.5 12.5 12.5 6.2 6.2 6.2 6.2 6.2 3.1 3.1 12.5 12.5 12.5 6.2 6.2 6.2 12.5 6.2 10.848 6.536 1.363

25 25 25 12.5 12.5 12.5 12.5 12.5 6.2 6.2 6.2 6.2 6.2 3.1 3.1 12.5 12.5 12.5 6.2 6.2 6.2 12.5 3.1 10.713 6.667 1.390

25 25 25 12.5 12.5 6.2 6.2 6.2 3.1 3.1 3.1 3.1 3.1 3.1 3.1 6.2 6.2 6.2 3.1 3.1 6.2 12.5 3.1 8.126 7.355 1.534

50 50 50 25 25 12.5 12.5 12.5 6.2 6.2 6.2 6.2 6.2 3.1 3.1 12.5 12.5 12.5 6.2 6.2 6.2 12.5 6.2 15.196 14.921 3.111

> 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 50 50 50 50 50 25 25 > 100 > 100 > 100 50 50 25 100 25 – – –

5.93 (35.6) > 18.06 (> 100) > 19.46 (> 100) > 19.66 (> 100) > 21.06 (> 100) > 21.16 (> 100) 13.81 (61.2) 16.82 (70.2) > 24.16 (> 100) > 25.56 (> 100) > 32.44 (> 100) 28.96 (85.6) 18.81 (53.4) > 35.44 (> 100) > 36.84 (> 100) 23.94 (93.3) 11.58 (42.8) 19.87 (69.8) > 28.66 (> 100) > 30.06 (> 100) – 0.25 (0.9) – – – –

> 16.66 (> 100) > 18.06 (> 100) > 19.46 (> 100) > 19.66 (> 100) > 21.06 (> 100) > 21.16 (> 100) 8.73 (38.7) 6.35 (26.5) > 24.16 (> 100) > 25.56 (> 100) 11.67 (36.0) 6.39 (18.0) 7.50 (21.3) 21.72 (61.3) 23.65 (64.2) 17.53 (68.3) 9.66 (35.7) 8.85 (31.1) > 28.66 (> 100) > 30.06 (> 100) – 0.056 (0.2) – – – –

> 16.66 (> 100) > 18.06 (> 100) > 19.46 (> 100) > 19.66 (> 100) > 21.06 (> 100) > 21.16 (> 100) > 22.56 (> 100) > 23.96 (> 100) > 24.16 (> 100) > 25.56 (> 100) 25.11 (77.4) > 33.84 (> 100) 22.73 (64.5) > 35.44 (> 100) > 36.84 (> 100) ≥25.66 (≥100) > 27.06 (> 100) > 28.46 (> 100) 3.32 (11.6) 8.36 (27.8) – 0.056 (0.2) – – – –

MIC (in μg/mL) = Minimum Inhibitory Concentration; IC50 = 50% Index of cytotoxicity.

reference drug used. Compounds 1a–e showed higher cytotoxicity against all three cell lines. Among compounds 2a–e, only compounds 2a (IC50 = 21.16 μg/mL) and 2d–e (IC50 = 24.16 μg/mL and 25.56 μg/ mL, respectively) exhibited less toxicity against all cells, whereas compounds 2b (IC50 = 22.56 μg/mL) and 2c (IC50 = 23.96 μg/mL) showed less toxicity against Vero cells only and were mild toxic against PBM and CEM cell lines. Compounds 3a–b (IC50 = 32.44 μg/mL, 28.96 μg/mL on PBM cells and 25.11 μg/mL, 33.84 μg/mL on Vero cells) and 3d–e (IC50 = 35.44 μg/mL and 36.84 μg/mL) were found to be less toxic against PBM and Vero cells. Additionally, compound 3c (IC50 = 22.73 μg/mL) showed less toxicity against Vero cells only. Compounds 4a–c (IC50 = 25.66 μg/mL, 27.06 μg/mL and 28.46 μg/mL) exhibited less toxicity against Vero cells as compared to PBM and CEM cells. Further, compounds 4d–e (IC50 = 28.66 μg/mL and 30.06 μg/mL) were less toxic on PBM and CEM cells only. Thus, the cytotoxicity assay clearly suggested that compounds having hydrophobic substituents at 5,6 – positions in the benzimidazole ring and polar groups at N-1 position showed less toxicity against PBM, Vero and CEM cell lines.

entire set of tested microorganisms than the reference drug cycloheximide. The compounds 2d–e, 3a–c and 4d–e showed antibacterial activity against B. cerus, S. aureus and P. aeruginosa comparable to the reference drug chloramphenicol and against B. cerus and P. aeruginosa to the reference drug paromomycin. These compounds were found to be more active than the reference drug chloramphenicol and equally active as reference drug paromomycin against E. coli bacterial strain. However, the compounds 3d–e showed better activity than the reference drug chloramphenicol against all bacterial strains used in antibacterial assay. Furthermore, compounds 3d–e showed better activity than paromomycin against B. cerus and P. aeruginosa and similar inhibition pattern against S. aureus and E. coli. Analysis of the antibacterial results of compounds suggested that the hydrophobic or hydrophilic groups at fifth and sixth positions and polar group (eOH group) at N-1 position in 2-Cl-benzimidazole, played an important role in determining the antibacterial activity. It is confirmed by compounds 2d–e, 3a–e and 4d–e as shown in Fig. 2(A) & (B). All Compounds were also tested for their antibacterial activity against E. coli mutant type strain and results are presented in Table 2. It was found that compounds 2d–e, 3a–c and 4d–e showed better activity (MIC: 50 μg/mL) as compared to reference antibiotics cycloheximide (MIC: 100 μg/mL). Compounds 3d and 3e were found to be equipotent in activity as compared to reference drug chloramphenicol and paromomycin (MIC: 25 μg/mL).

2.5. Determination of fractional inhibitory concentration (FIC) or combination assay Once the antibacterial activity (MIC) of all compounds was determined, another study was performed for evaluating the synergistic effect of compounds 1a–e, 2a–c, 4a–c and the reference antibiotic (chloramphenicol) on the antibacterial property in the form of fractional inhibitory concentration (FIC). Only the compounds having MIC value higher than the MIC value of reference drug (chloramphenicol) were undertaken for this study (Moody, 1992). A synergistic effect is noticed for a test compound used in combination when FIC value is ≤0.25. When the FIC value is > 2, there is antagonism between the compounds in combination whereas FIC values between 0.25–2

2.4. Cytotoxicity evaluation of benzimidazole analogs Cytotoxicity studies were done using three different cell lines, i.e., human PBM and CEM and Vero (African green monkey kidney) cell lines and the reference drug cycloheximide. The results showing the cytotoxicity of compounds 1(a–e) – 4(a–e) are summarized in Table 2. In cytotoxic evaluation of compounds, we noticed that all compounds were found to be less toxic against all the three cell lines than the 4



Fig. 2. (A) Structure – activity – relationship diagram of benzimidazole analogs on the basis of physical and biological results. (B) Antibacterial activity (MIC in μg/ mL) of compounds 1(a–e) – 4(a–e) and reference antibiotics.

reduced up to 1/8 (1.5 μg/mL) against gram +ve bacteria whereas in the case of gram −ve bacteria, the combined MIC values were reduced to approximately 1/33 (0.375 μg/mL) and 1/4 (6.2 μg/mL) of their original MIC, respectively. So, the greater extent of synergism was observed in the case of compounds 1d–e against each bacterial strain, which was much pronounced in E. coli. In combination with reference drug, compounds 2a–c and 4a–c were found to be more active against gram +ve bacterial strains at the concentration of 0.375 μg/mL (MIC reduced up to 1/33 of its original MIC) and 1.5 μg/mL (MIC reduced up to 1/8 of its original MIC), respectively. On the other hand, in case of P. aeruginosa the combined MIC of all these compounds was found at the concentration of 0.75 μg/mL (approximately 1/16 of its original MIC) (Table 3). Thus, it was inferred that the synergistic effect of the compounds and reference drug has lowered the combined MIC to a much greater extent in all cases (Fig. 3). Some combinations are more effective against gram +ve and others against gram −ve bacteria. This clearly indicated that the development of potential antibiotics using a combination approach may be the choice of research in days to come.

indicates absence of synergism and antagonism (Rajamuthiah et al., 2015; Pieroni et al., 2015). The synergistic effect between the compounds and the reference was expressed as the fractional inhibitory concentration (FIC) and calculated as follows: FIC = MIC of test compound in combination with reference/MIC of test compound alone For this purpose, a combination approach was followed where ½ MIC (3.1 μg/mL) of reference drug and varying concentration (25 μg/ mL–0.187 μg/mL) of compounds were used. All selected compounds with ½ MIC of reference drug in combination were tested by two-dimensional broth micro dilution method to evaluate the extent of synergism. The FIC value of all selected compounds was observed between 0.03–0.24 against the tested bacterial strains and results are presented in Table 3. Due to the combination effect of compounds 1a–c and reference, the combined MIC values were lowered to approximately ¼ (6.2 μg/mL) against B. cerus and S. aureus and 1/8 (6.2 μg/mL) against P. aeruginosa and 1/16 (1.5 μg/mL) against E. coli of their respective original MIC values. In the case of compounds 1d–e, the value of combined MIC was 5



Table 3 Fractional Inhibitory Concentration (FIC) of the compounds 1a–e, 2a–c and 4a–c showing synergistic effect via combination approach. Compound

MIC (combination)

FIC Gram −ve strain

Gram +ve strain

1a + ref 1b + ref 1c + ref 1d + ref 1e + ref 2a + ref 2b + ref 2c + ref 4a + ref 4b + ref 4c + ref

Gram −ve strain

Gram +ve strain

B. cerus

S. aureus

E. coli

P. aeruginosa

B. cerus

S. aureus

E. coli

P. aeruginosa

6.2 6.2 6.2 1.5 1.5 0.375 0.375 0.375 1.5 1.5 1.5

6.2 6.2 6.2 1.5 1.5 0.375 0.375 0.375 1.5 1.5 1.5

1.5 1.5 1.5 0.375 0.375 ND ND ND ND ND ND

6.2 6.2 6.2 6.2 6.2 0.75 0.75 0.75 0.75 0.75 0.75

0.24 0.24 0.24 0.12 0.12 0.03 0.03 0.03 0.12 0.12 0.12

0.24 0.24 0.24 0.12 0.12 0.03 0.03 0.03 0.12 0.12 0.12

0.06 0.06 0.06 0.03 0.03 – – – – – –

0.12 0.12 0.12 0.24 0.24 0.06 0.06 0.06 0.06 0.06 0.06

Ref = ½ MIC of Chloramphenicol (Antibiotic), MIC (in μg/mL) = Minimum Inhibitory Concentration, ND = Not done, FIC (Fractional inhibitory concentration) = MIC of test compound in combination with ref/MIC of test compound alone.

formed by the eOH group with Urd27 and the other one was formed by the N-atom of 2-Cl-benzimidazole of both compounds with Cyt28 residue at the distance of 3.1 Å inside the active site of bacterial protein. Three inter atomic H-bonds (3.0, 2.7 Å with Ade29 and 6.1 Å with Cyt28) were formed between compound 4d, having eNO2 group at fifth and sixth position and eOH group at N-1 position, and Cyt28 and Ade29. Additionally, compound 4d interacted with Gua26 and formed two π − + interactions at a distance of 5.3 and 6.6 Å. One H-bond (2.8 Å) with Urd27 as well as one π–π interaction (4.9 Å) with Gua26 was seen in case of compound 4e. Docking interactions of potential compounds at the eubacterial ribosomal decoding A site of bacterial protein are shown in Fig. 4. Scoring of ligand is the best way to evaluate the true conformation and the binding tightness of ligand docked within active site of a target protein receptor (Kitchen et al., 2004). We have analysed the correct pose of protein-ligand complexes through the scoring functions, like PLP-1, PLP-2, Lig_Internal_Energy, Binding energy, Dock score and Ludi 2 values. Scoring values of all potential molecules are presented in Table 5. PLP-1 and PLP-2 are the two different types of function of piecewise linear potential by which we explain the binding of ligand with the target protein in a better way. Higher value of PLP functions shows the stronger binding of ligand to the protein receptor (Gehlhaar et al., 1995). From Table 5, it was observed that compounds 3d and 3e showed

2.6. Molecular docking studies and scoring of benzimidazole analogs In order to illustrate antimicrobial mechanism of potential compounds 2d, 2e, 3d, 3e, 4d and 4e theoretically, further docking and molecular dynamics simulation studies were performed to investigate the binding pattern at the eubacterial ribosomal decoding A site of bacterial protein. Molecular docking studies were performed using DS 2.5 on 3D X-ray crystal structure of bacterial protein (1j7t: PDB, www. rcsb.org) for this study. The results were explained on the basis of hydrogen bonding and non-covalent π–π, π − + interactions, which stabilized the ligand – protein complexes (Gallivan and Dougherty, 1999; Chen et al., 2006). Docking results of compounds with bacterial protein are shown in Table 4. The compounds interacted with Cyt25, Gua26 and Urd27 residues on the B – chain of eubacterial rRNA just like the reference drug. Additionally, some compounds showed interaction with Cyt28 and Ade29 residues, which was not observed with the reference drug. Docking studies revealed that introduction of −NO2 group at fifth position of 2-Cl-benzimidazole ring of compounds 2d and 2e and eOH group at N-1 position, stabilized the ligand-protein complexes through the H-bonding. Both these compounds interacted with active site of bacterial protein and formed three H-bonds with Cyt25, Urd27 and Cyt28 (Table 4). Compounds 3d and 3e (showing potent antibacterial activity) having eBr group at fifth and sixth position of 2-Cl-benzimidazole, exhibited two H-bonds: one H-bond (2.9 Å for both 3d and 3e)

Fig. 3. Fractional inhibitory concentration (FIC) of selected compounds in combination with reference antibiotic against gram +ve and gram −ve bacterial strains. 6



Table 4 Docking interaction of active compounds 2(d–e) - 4 (d–e) with eubacterial ribosomal decoding A site in ligand – receptor docked complex. Compound

No. of H-B

2d

3

2e

3

3d

2

3e

2

4d

3

4e ref.

1 4

Nucleotide in H-B

Cyt 25 Cyt 28 Urd 27 Cyt 25 Cyt 28 Urd 27 Cyt 28 Urd 27 Cyt 28 Urd 27 Ade 29 Cyt 28 Urd 27 Gua 13 Cyt 25 Gua 26 Urd 27

Position of H-B

B: Cyt 25: N4 − 2d: O12 B: Cyt 28: N4 − 2d: N7 2d: O16 − B: Urd 27: OP2 B: Cyt 25: N4 − 2e: O12 B: Cyt 28: N4 − 2e: N7 2e: O16 − B: Urd 27: OP2 B: Cyt 28: N4 − 3d: N7 3d: O13 − B: Urd 27: OP2 B: Cyt 28: N4 − 3e: N7 3e: O14 − B: Urd 27: OP2 B: Ade 29: N6 − 4d: O18 4d: O13- B: Ade 29: OP2 B: Cyt 28: N4 − 4d: O18 4e: O20 − B: Urd 27: OP2 ref: O10 − A: Gua 13: OP2 B: Cyt 25: OP2 − ref: O40 ref: N42 − B: Gua 26: O6 ref: O28 − B: Urd 27: OP2

D(A°)

2.8 2.9 3.1 2.9 2.9 2.7 3.0 2.8 3.1 2.8 3.0 2.7 3.0 2.8 3.1 2.9 2.8 2.7

D-A

N4 N4 O16 N4 N4 O17 N4 O13 N4 O14 N6 O13 N4 O20 O10 OP2 N42 O28

A-A

O12 N7 OP2 O12 N7 OP2 N7 OP2 N7 OP2 O18 OP2 O18 OP2 OP2 O40 O6 OP2

No. of π − B

Nucleotide in π −B

‘ π- π’ monitor Bond

D(A°)

End1

End2

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

2

Gua 26

B: Gua 26 − 4d B: Gua 26 − 4d

5.3 6.6

Gua 26 Gua 26

4d 4d

1 –

Gua 26 –

B: Gua 26 − 4e –

4.9 –

Gua 26 –

4e –

HeB = Hydrogen bond; D = Distance (A°); DeA = Donor Atom; AeA = Acceptor Atom; πeB = Pi bond, ref. = Paromomycin (Antibiotic).

(1j7t) with compounds 3d–e (showing the highest antibacterial activity) and reference drug paromomycin for investigating the stability, interaction and binding mode of these compounds. Three independent MD simulations were used for this study. The MD results of paromomycin were used as a control.

the highest PLP scores, i.e., PLP-1 (56.48 and 56.78 for 3d and 3e, respectively) and PLP-2 (54.81 and 56.55 for 3d and 3e, respectively). However, compounds 2d and 2e showed PLP values (PLP-1 – 54.52 and 55.61 for 2d and 2e and PLP-2 – 42.15 and 46.49 for 2d and 2e, respectively) close to the reference drug paromomycin (56.47- PLP-1 and 45.13- PLP-2). Rest of the compounds exhibited relatively lower value of PLP function with respect to the standard antibiotic used in molecular docking simulation. The calculated internal ligand energy (LIG) of each pose of different ligands, obtained from the docking simulation as a sum of the van der Walls (vdW) and electrostatic energy, was taken up for studying efficiency of binding of ligands to the protein. Compounds 3d and 3e exhibited a little higher stabilization energy than the standard drug whereas the LIG value of compounds 2d and 2e was very close to the standard drug (Table 5). Binding free energy of protein-ligand complex of a particular ligand suggests the suitability of a ligand for further studies (Lagos et al., 2008). Binding free energy of all compounds and paromomycin (reference) was obtained using docking experiment. Docking results illustrated that compounds 3d (4.49 kcal/mol) and 3e (4.51 kcal/mol) showed higher binding free energy than the reference (4.04 kcal/mol) while other compounds showed a little lower values. Ludi_2 is empirical scoring function derived from the Ludi algorithm and used to score the refined conformations of protein – ligand complex. The highest Ludi_2 score of ligand pose was selected as the best conformation. From Ludi_2 scoring function, binding energy of targeted compounds was predicted (Kumar et al., 2010). From docking simulation on targeted compounds, it was established that higher value of binding energy was associated with higher affinity towards stable protein – ligand complex. Poses of ligand were evaluated and prioritized on the basis of Dock Score function. It was confirmed from Table 5 that compounds 2d, 2e, 3d and 3e showed the significant docking scores ranging between 51.70–57.30. Dock Scores of compounds 4d and 4e were found relatively lower than the reference (56.92) but all molecules had similar pattern of binding to the RNA active site and also had significant binding affinity with RNA core, just like the reference drug.

2.7.1. Root mean square deviation (RMSD) and radius of gyration To investigate the stability of eubacterial RNA, eubacterial RNA:paromomycin, eubacterial RNA:compound 3d–e complexes, the RMSD values of these three complexes and free eubacterial RNA were calculated (Fig. 5). From Fig. 5, it was clear that there was a gradual increase in the RMSD value of free eubacterial RNA up to 6.9 Å at 3350 ps, and then it decreased up to 3.1 Å at 6123 ps. After 9179 ps, it finally raised up to 7.1 Å and at 10,000–20,000 ps, there was no significant change in RMSD values (3.9 Å–5.2 Å) of free eubacterial RNA. In the case of eubacterial RNA:paromomycin complex, the RMSD value raised up to 6.5 Å at 6117 ps and then decreased up to 5.0 Å at 6517 ps. Afterward, the RMSD value was approximately constant at 7000–18,000 ps. At 19113 ps, it further decreased up to 4.1 Å. In both cases – eubacterial RNA:compound 3d and eubacterial RNA:compound 3e complexes, the RMSD values followed the similar pattern of up and down as the reference drug. An increase in RMSD values up to 5.2 Å in eubacterial RNA:compound 3d complex and up to 5.5 Å in eubacterial RNA:compound 3e complex was found at 6495 and 6509 ps, respectively. Then these values slightly went down up to 3.5 Å at 6966 ps and 3.7 Å at 6975 ps for both eubacterial RNA:compound 3d and eubacterial RNA:compound 3e complexes, respectively. The RMSD profiles of all three complexes, i.e., eubacterial RNA:paromomycin, eubacterial RNA:compound 3d–e complexes at 7000–20,000 ps were approximately similar to each other except for a sudden enhancement up to 6.9 Å at 18,325 ps in eubacterial RNA:compound 3d complex. During 20 ns (20,000 ps) MD simulations, radius of gyration for the whole eubacterial RNA was calculated and analyzed to see the effects of binding mode of ligand on structural features of eubacterial RNA backbone (Fig. 6). For free eubacterial RNA, radius of gyration increased up to 20.8 Å at 3973 ps and then decreased to 17.2 Å at 8810 ps. Further, it enhanced up to a maximum of 21.1 Å at 12,730 ps and remained almost constant (19 ± 1 Å) till 20,000 ps. In the case of eubacterial RNA:paromomycin complex, the radius of gyration initially increased up to 20.5 Å at 3247 ps and then decreased up to 17.6 Å at 5124 ps. Further, an enhancement was found in radius of gyration up to 21.2 Å

2.7. MD simulations on eubacterial RNA with compounds 3d–e and reference drug (Paromomycin) MD simulations were performed on active site of bacterial protein 7



Fig. 4. Molecular docking interaction of compounds 2d, 2e, 3d, 3e, 4d and 4e with eubacterial ribosomal decoding A site.

RNA:compound 3d complex. On the other hand, the maximum increase in radius of gyration of eubacterial RNA:compound 3e complex was observed up to 20.8 Å at 8010 ps. Just like the reference drug, both eubacterial RNA:compound 3d and eubacterial RNA:compound 3e complexes showed the constant radius of gyration around 19 ± 1 Å till 20,000 ps. On the basis of MD simulations it can be inferred that the ligands under study formed stable complexes with eubacterial RNA like the reference drug. This result pointed towards the enormous potential of these compounds as broad spectrum antibacterial agents (Fig. 7).

at 8090 ps. After that there was no significant variation till 20,000 ps, i.e., a constant value 19 ± 1 Å was observed. In both eubacterial RNA:compound 3d and eubacterial RNA: compound 3e complexes, similar mode of an increase and decrease was found in radius of gyration as the reference drug. A gradual increase in radius of gyration values up to 21.1 Å in eubacterial RNA:compound 3d and up to 20.6 Å in eubacterial RNA:compound 3e complexes was found at 4851 and 4388 ps, respectively. Then these values slightly decreased up to 19.0 Å at 6764 ps and 17.5 Å at 5156 ps for both eubacterial RNA:compound 3d and eubacterial RNA:compound 3e complexes, respectively. After 7682 ps, the radius of gyration finally raised up to 21.6 Å in eubacterial 8



2.7.2. Binding free energy calculations of inhibitor:eubacterial RNA complexes In order to corroborate our results, a combination of MM-PBSA and MD method was used to estimate the binding free energy of eubacterial RNA:paromomycin and eubacterial RNA:compound 3d–e complexes. For analysis of the binding free energy, the snap shots were extracted from the last 10 ns of MD simulation of each component. All the energy components of MD simulations are listed in Table 6. In Table 6, the binding free energy (ΔGbinding) of eubacterial RNA:paromomycin, eubacterial RNA:compound 3d and 3e complexes was −53.1153, −51.5804 and −52.7294 Kcal/mol, respectively. The results indicated that both compounds have almost similar calculated binding energy as the reference drug. The calculated van der Waals

Table 5 Docking scores of potential compounds 2 (d–e) - 4 (d–e) in ligand – receptor (bacterial protein) docked complexes. Compound

-PLP1

-PLP2

LIG

LUDI 2

ΔG (kcal/ mol)

DS

2d 2e 3d 3e 4d 4e Paromomycin

54.52 55.61 56.48 56.78 49.74 49.96 56.47

42.15 46.49 54.81 56.55 41.32 41.39 45.13

−3.89 −3.96 −4.87 −4.96 −2.83 −2.92 −3.01

269 276 316 318 245 253 285

−3.82 −3.91 −4.49 −4.51 −3.47 −3.59 −4.04

51.72 53.94 56.97 57.29 48.47 48.86 56.92

-PLP = Piecewise linear potential, LUDI 2 = Empirical scoring functions, LIG = Ligand_Internal_Energy, ΔG = Binding energy, DS = Dock score.

Fig. 5. RMSD values of free eubacterial RNA (black line), eubacterial RNA: paromomycin complex (blue line) and complexes of compounds 3d and 3e with eubacterial RNA (red line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 9



Fig. 6. Radius of gyration of free eubacterial RNA (black line), eubacterial RNA: paromomycin complex (blue line) and complexes of compounds 3d and 3e with eubacterial RNA (red line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

DS 2.5 software and presented in Table 7 in order to support biological results. Intestinal absorption has been predicted to be more than 80% for compounds 3d (87.429) and 3e (87.467), which is greater than reference drugs Paromomycin (25.047%) and chloramphenicol (65.262%). Metabolism plays a vital role in the bioavailability of drugs and cytochrome CYP450 enzymes are most important class to study this effect. Compounds were studied either to act as substrate or inhibitors of CYPs. Both compounds 3d and 3e were found to be the inhibitor of CYP1A2. All the ADMET parameters were found to be favourable for both compounds 3d and 3e and even better in some sense than that of reference antibiotics.

contributions (ΔEvdw) to the binding free energy in the eubacterial RNA:compound 3d (−274.9593 Kcal/mol) and 3e (−263.1321 Kcal/ mol) complexes are higher than that for the eubacterial RNA:paromomycin complex (−256.4825 Kcal/mol). The contributions of solvation energy (ΔEsolv), van der Waals energy (ΔEvdw) and electrostatic energy (ΔEelect) are most effective for binding of ligand to the protein. These three energies favoured the binding of compounds 3d, 3e and the reference drug to binding site of eubacterial RNA and also indicated the probability of these molecules working as protein synthesis inhibitors. 2.8. In silico ADMET prediction of potential compounds 3d and 3e In silico ADMET (Adsorption, Distribution, Metabolism, Excretion and Toxicity) parameters of compounds 3d and 3e are predicted using 10



Fig. 7. Binding mode of compounds 3d and 3e with eubacterial ribosomal decoding A site after 20 ns MD simulation. The Hydrogen bonds are shown by the green dashed lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Table 6 Molecular energy data of eubacterial RNA:paromomycin, eubacterial RNA:compound 3d and eubacterial RNA:compound 3e complexes. Energy (kcal/mol)

Eubacterial RNA: paromomycin

Eubacterial RNA: compound 3d

Eubacterial RNA: compound 3e

ΔEvdw ΔEelect ΔEsolv ΔGbinding

−256.4825 ± 0.4782 −118.6027 ± 0.8356 −171.6238 ± 0.2963 −53.1153 ± 0.4657

−274.9593 ± 1.7071 −105.7977 ± 1.5826 −172.7285 ± 0.6834 −51.5804 ± 0.0851

−263.1321 ± 4.8561 −106.2184 ± 1.8028 −178.0841 ± 0.8254 −52.7294 ± 0.0756

ΔEvdw = van der Waals energy; ΔEelect = Electrostatic energy; ΔEsolv = Solvation energy; ΔGbinding = Binding energy. Table 7 In Silico ADMET predictions of compounds 3d and 3e. Compound

3d 3e Ref 1 Ref 2

Absorption

Distribution

Metabolism

Excretion

Toxicity

Total Clearance (log/ml/ min/kg)

Oral rat chronic toxicity (LOAEL) (log mg/ kg_bw/ day)

Hepatotoxicity (Yes/No)

0.412 0.402 0.390 0.287

1.226 1.253 4.389 2.016

No No No No

Water solubility (log mol/ L)

Intestinal absorption (human) (% absorbed)

Blood brain barrier Permeability (log BB)

CNS permeability (log PS)

CYP 2D6 3A4 Substrate (Yes/No)

1A2 2C19 2C9 2D6 Inhibitor (Yes/No)

3A4

−2.566 −2.671 −2.407 −2.826

87.429 87.467 25.047 65.262

0.467 0.132 −2.271 −1.387

−2.544 −2.541 −6.887 −3.060

No No No No

Yes Yes No No

No No No No

No No No No

No No No No

No No No No

No No No No

Ref 1 = Paromomycin; Ref 2 = Chloramphenicol; Water Solubility = < −4 soluble; Intestinal absorption = Below 30% indicates poor absorbance; Blood brain barrier Permeability = < −1considered poorly distributed to the brain; CNS (Central Nervous System) permeability = > −2 considered to penetrate the CNS; Total Clearance (logCLtot) = Lower value indicates high drug half lifetime; LOAEL (Lowest Observed Adverse Effect) = Lower value predicts minimum toxicity.

3. Conclusion

compounds having hydrophobic groups at positions- 5,6 and polar hydrophilic groups at position N-1 in 2-Cl-benzimidazole nucleus enhanced the inhibitory activity. Contrary to this, the hydrophilic groups at positions 5,6 and non polar hydrophobic groups at position- N-1 in 2Cl-benzimidazole nucleus had lower inhibitory activity against bacterial strains studied. A deep investigation of in-vitro activity supported by docking results, MD simulations and In silico ADMET results clearly suggested that compounds 3d and 3e can work as lead inhibitors of bacterial protein synthesis. The study, thus, reflected that the molecules under consideration in this manuscript can be developed as broad spectrum antibacterial agents.

In summary, analogues of 2-Cl-benzimidazole 1(a–e) – 4(a–e) bearing H/NO2/Br groups at fifth and sixth position and alkyl groups at N-1 position, developed on the basis of physiochemical studies, exhibited different pharmacological profiles during antibacterial screening. Compounds 2d–e, 3a–c and 4d–e displayed promising efficacy toward B. cerus, S. aureus and P. aeruginosa. Further, all compounds showed the highest efficacy against E. coli except compounds 1a–e, and compounds 3d and 3e showed much better antibacterial activity against all gram +ve and gram −ve strains. The analogues 1d–e, 2a–c and 4a–c exhibited high synergistic effect when tested in combination with the reference drug chloramphenicol and the MIC values were lowered by 1/8th to 1/33rd of the original MIC. Cytotoxicity assay against PBM, Vero and CEM cell lines proved that the active analogues were less cytotoxic. SAR studies confirmed the significance of the substituents at fifth, sixth and N-1 positions of 2-Cl-benzimidazole nucleus for antibacterial activity. The structure activity relationship study revealed that

4. Experimental 4.1. Chemistry All chemicals and reagents were purchased from Sigma Aldrich 11



4.1.4. General synthesis of 2-chloro-1H-benzimidazole derivatives 1(a–e) – 4(a–e) A solution of compound 1 (or 2/3/4) (1 mmol) and anhydrous potassium carbonate (2 mmol) in acetonitrile (5 mL) was stirred at room temperature for 20 min. Alkyl halide a/b/c/d/e (1.5 mmol) was added drop wise. The reaction mixture was heated at 40–50 °C for 3–4 h. TLC analysis indicated the complete consumption of compound 1 (or 2/3/ 4). The reaction mixture was dried and partitioned between EtOAc and aqueous NaHCO3 solution. The organic layer was collected and dried over anhydrous Na2SO4 and the solvent removed under vacuum to give crude product. The crude product was purified by column chromatography using silica gel column and ethyl acetate and hexane as solvent.

Chemical Company, USA and E. Merck India Ltd, India. All reactions were carried out in oven dried apparatus using dried and distilled solvents. Column chromatography was carried out on silica gel (100–200 mess). Reactions were monitored by TLC, using silica gel 60F254 aluminium plates and visualized under ultraviolet light at 254 nm. Melting points were recorded on electro thermal apparatus. NMR spectra were recorded on BRUKER-AV400 spectrometer (Bruker Co., Faellanden, Switzerland) in DMSO-d6 (1H at 400 MHz and 13C at 100 MHz). Chemical shifts (δ) are expressed in parts per million (ppm) and J (Coupling constant) values are expressed in Hz. Multiplicities are indicated by s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet) and br (broad spectrum). Mass spectra were recorded on Micromass Q-Tof (ESI-HRMS).The elemental analyses were performed for all compounds on a Perkin-Elmer 240-C analyses equipment.

4.1.4.1. 2-Chloro-1-methyl-1H-benzimidazole (1a). Yield 69%, yellow solid; m.p. 190–192 °C; Rf − 0.57 (EtOAc:Hexane: 2:8); 1H NMR (400 MHz, DMSO-d6) δppm: 7.60–7.57 (m, 2H, Ar-H), 7.32–7.22 (m, 2H, Ar-H), 3.79 (s, 3H, eCH3); 13C NMR (100 MHz, DMSO-d6) δppm: 141.1 (C8), 139.2 (C2), 134.3 (C9), 122.5 (C5), 122.1 (C6), 118.4 (C4), 110.2 (C7), 14.4 (CH3); HRMS m/z (M+Na)+: 189.0195, Calcd. for C8H7ClN2: 189.0194; Anal. Calcd. for C8H7ClN2: C, 50.65; H, 3.68; Cl, 18.73; N, 14.73, Found: C, 50.67; H, 3.65; Cl, 18.79; N, 14.75.

4.1.1. Synthesis of 2-chloro-5-nitro-1H-benzimidazole (2) A suspension of compound 1 (2-chloro-1H-benzimidazole; 6.55 mmole, 1 g) in concentrated nitric acid (5 mL) was cooled in a 100 mL round bottomed flask, maintaining the temperature at 5–10 °C. Concentrated sulphuric acid (2.5 mL) was added. The yellow-red coloured mixture was stirred at 10 °C for 5 min and then refluxed for 1 h. After refluxing, the reaction mixture was cooled and poured onto 100 g of ice. The resulting yellow coloured solid was collected by filtration and crystallized from EtOH. Yield 73%, light yellow solid; m.p. 221–222 °C; Rf − 0.62 (EtOAc:Hexane: 6:4); 1H NMR (400 MHz, DMSO-d6) δppm: 14.04 (br, s, 1H, −NH), 8.43 (s, 1H, Ar-H), 8.15 (dd, 1H, J = 8.8, 2.0 Hz, Ar-H), 7.72 (d, 1H, J = 8.8 Hz, Ar-H); 13C NMR (100 MHz, DMSO-d6) δppm: 142.3 (C5), 139.5 (C8), 138.2 (C9), 137.4 (C2), 118.9 (C6), 116.6 (C7), 112.8 (C4); HRMS m/z (M+H)+: 198.0073, Calcd. for C7H4ClN3O2: 198.0070; Anal. Calcd. for C7H4ClN3O2: C, 42.54; H, 2.05; Cl, 17.92; N, 21.25, Found: C, 42.56; H, 2.09; Cl, 17.96; N, 21.27.

4.1.4.2. -Chloro-1-ethyl-1H-benzimidazole (1b). Yield 65%, yellow viscous oil; Rf − 0.64 (EtOAc:Hexane: 2:8); 1H NMR (400 MHz, DMSO-d6) δppm: 7.63–7.59 (m, 2H, Ar-H), 7.29–7.22 (m, 2H, Ar-H), 4.28 (q, 2H, J = 7.2 Hz, eCH2), 1.31 (t, 3H, J = 7.2 Hz, eCH3); 13C NMR (100 MHz, DMSO-d6) δppm: 141.2 (C8), 139.4 (C2), 134.5 (C9), 122.9 (C5), 122.3 (C6), 118.6 (C4), 110.4 (C7), 38.9 (CH2), 14.7 (CH3); HRMS m/z (M+Na)+: 203.0351, Calcd. for C9H9ClN2: 203.0352; Anal. Calcd. for C9H9ClN2: C, 53.12; H, 4.45; Cl, 17.38; N, 13.75, Found: C, 53.14; H, 4.48; Cl, 17.42; N, 13.78. 4.1.4.3. 2-Chloro-1-propyl-1H-benzimidazole (1c). Yield 62%, yellow viscous oil; Rf − 0.68 (EtOAc:Hexane: 2:8); 1H NMR (400 MHz, DMSO-d6) δppm: 7.63–7.59 (m, 2H, Ar-H), 7.31–7.21 (m, 2H, Ar-H), 4.21 (t, 2H, J = 6.8 Hz, eCH2), 1.80–1.71 (m, 2H, eCH2), 0.85 (t, 3H, J = 7.2 Hz, eCH3); 13C NMR (100 MHz, DMSO-d6) δppm: 141.1 (C8), 139.7 (C2), 135.0 (C9), 122.9 (C5), 122.3 (C6), 118.6 (C4), 110.6 (C7), 45.3 (CH2), 22.2 (CH2), 10.8 (CH3); HRMS m/z (M+Na)+: 217.0508, Calcd. for C10H11ClN2: 217.0508; Anal. Calcd. for C10H11ClN2: C, 55.12; H, 5.12; Cl, 16.31; N, 12.85, Found: C, 55.14; H, 5.16; Cl, 16.34; N, 12.86.

4.1.2. Synthesis of 5,6-dibromo-2-chloro-1H-benzimidazole (3) To a solution of compound 1 (2-chloro-1H-benzimidazole; 6.55 mmole, 1 g) in MeOH (30 mL), a solution of Br2/MeOH (1.5 mL/15 mL) was added drop wise. The resulting suspension was stirred at room temperature for 6 h, added H2O (30 mL) and stirring continued at room temperature for 18–20 h. The reaction mixture was filtered and the precipitate was washed thoroughly with cold H2O until the washing was neutral. The white coloured solid was air dried and crystallized from MeOH. Yield 72%, white solid; m.p. 227–228 °C; Rf − 0.52 (EtOAc:Hexane: 3:7); 1H NMR (400 MHz, DMSO-d6) δppm: 13.79 (br, s, 1H, −NH), 7.95 (s, 2H, Ar-H); 13C NMR (100 MHz, DMSO-d6) δppm: 140.2 (C8–C9), 138.7 (C2), 121.5 (C4-C7), 120.9 (C5–C6); HRMS m/z (M+H)+: 308.8434, Calcd. for C7H3Br2ClN2: 308.8430; Anal. Calcd. for C7H3Br2ClN2: C, 27.05; H, 0.95; Br, 51.45; Cl, 11.42; N, 9.01, Found: C, 27.07; H, 0.96; Br, 51.48; Cl, 11.44; N, 9.05.

4.1.4.4. 2-(2-Chloro-1H-benzimidazol-1-yl)ethan-1-ol (1d). Yield 75%, white solid; m.p. 138–139 °C; Rf − 0.53 (EtOAc:Hexane: 5:5); 1H NMR (400 MHz, DMSO-d6) δppm: 7.60-7.58 (m, 2H, Ar-H), 7.30–7.21 (m, 2H, Ar-H), 4.97 (t, 1H, J = 5.6 Hz, eOH), 4.31 (t, 2H, J = 5.2 Hz, eCH2), 3.75–3.71 (m, 2H, eCH2); 13C NMR (100 MHz, DMSO-d6) δppm: 141.1 (C8), 140.2 (C2), 135.4 (C9), 122.3 (C5), 122.1 (C6), 118.4 (C4), 110.8 (C7), 59.1 (CH2), 46.7 (CH2); HRMS m/z (M+H)+: 197.0482, Calcd. for C9H9ClN2O: 197.0482; Anal. Calcd. for C9H9ClN2O: C, 54.95; H, 4.54; Cl, 18.06; N, 14.23, Found: C, 53.14; H, 4.48; Cl, 17.42; N, 13.78.

4.1.3. Synthesis of 2-chloro-5-6-dinitro-1H-benzimidazole (4) Compound 1 (2-chloro-1H-benzimidazole; 6.55 mmole, 1 g) was added in small portions to a nitrating mixture consisting of concentrated nitric acid (5 mL) and concentrated sulphuric acid (2.5 mL) in cold. The reaction mixture was refluxed for 4–5 h, cooled and poured on to 100 g of ice. The yellow coloured precipitate was filtered, dried and crystallized from dioxane. Yield 65%, yellow solid; m.p. 196–197 °C; Rf − 0.67 (EtOAc:Hexane: 6:4); 1H NMR (400 MHz, DMSO-d6) δppm: 11.76 (br, s, 1H, −NH), 8.35 (s, 2H, Ar-H); 13C NMR (100 MHz, DMSOd6) δppm: 147.9 (C8–C9), 139.4 (C2), 138.5 (C5–C6), 114.6 (C4–C7); HRMS m/z (M+Na)+: 264.9740, Calcd. for C7H3ClN4O4: 264.9741; Anal. Calcd. for C7H3ClN4O4: C, 34.62; H, 1.23; Cl, 14.57; N, 23.08, Found: C, 34.64; H, 1.27; Cl, 14.64; N, 23.12.

4.1.4.5. 3-(2-Chloro-1H-benzimidazol-1-yl)propan-1-ol (1e). Yield 55%, white viscous oil; Rf − 0.56 (EtOAc:Hexane: 5:5); 1H NMR (400 MHz, DMSO-d6) δppm: 7.61–7.58 (m, 2H, Ar-H), 7.32–7.22 (m, 2H, Ar-H), 4.31 (t, 2H, J = 6.8 Hz, eCH2), 3.75 (s, 1H, eOH), 3.43 (t, 2H, J = 6.0 Hz, eCH2), 1.91–1.85 (m, 2H, eCH2); 13C NMR (100 MHz, DMSO-d6) δppm: 141.1 (C8), 139.8 (C2), 135.1 (C9), 122.9 (C5), 122.4 (C6), 118.6 (C4), 110.5 (C7), 57.6 (CH2), 41.2 (CH2), 32.1 (CH2); HRMS m/z (M+H)+: 211.0656, Calcd. for C10H11ClN2O: 211.0638; Anal. Calcd. for C10H11ClN2O: C, 57.05; H, 5.24; Cl, 16.86; N, 13.27, Found: C, 57.07; H, 5.29; Cl, 16.88; N, 13.28. 4.1.4.6. 2-Chloro-1-methyl-5-nitro-1H-benzimidazole (2a). Yield 75%, 12



light yellow solid; m.p. 88–92 °C; Rf − 0.59 (EtOAc:Hexane: 6:4); 1H NMR (400 MHz, DMSO-d6) δppm: 8.50 (d, 1H, J = 2.0 Hz, Ar-H), 8.23 (dd, 1H, J = 9.2, 2.0 Hz, Ar-H), 7.85 (d, 1H, J = 8.8 Hz, Ar-H), 3.87 (s, 3H, eCH3); 13C NMR (100 MHz, DMSO-d6) δppm: 148.3 (C9), 144.1 (C5),139.5 (C2), 139.2 (C8), 118.3 (C6), 112.6 (C4), 110.8 (C7), 31.2 (CH3); HRMS m/z (M+Na)+: 234.0046, Calcd. for C8H6ClN3O2: 234.0046; Anal. Calcd. for C8H6ClN3O2: C, 40.94; H, 2.56; Cl, 15.11; N, 17.92, Found: C, 40.97; H, 2.58; Cl, 15.16; N, 17.96.

72%, white viscous oil; Rf − 0.56 (EtOAc:Hexane: 5:5); 1H NMR (400 MHz, DMSO-d6) δppm: 8.18 (s, 1H, Ar-H), 7.98 (s, 1H, Ar-H), 4.27 (q, 2H, J = 7.2 Hz, eCH2), 1.28 (t, 3H, J = 7.2 Hz, eCH3); 13C NMR (100 MHz, DMSO-d6) δppm: 141.8 (C8), 141.4 (C2), 134.7 (C9), 121.5 (C4), 117.5 (C7), 116.8 (C5), 115.4 (C6), 39.5 (CH2), 14.4 (CH3); HRMS m/z (M+H)+: 336.8744, Calcd. for C9H7Br2ClN2: 336.8743; Anal. Calcd. for C9H7Br2ClN2: C, 31.95; H, 2.08; Br, 47.22; Cl, 10.42; N, 8.26, Found: C, 31.97; H, 2.11; Br, 47.24; Cl, 10.46; N, 8.27.

4.1.4.7. 2-Chloro-1-ethyl-5-nitro-1H-benzimidazole (2b). Yield 73%, light yellow viscous oil; Rf − 0.65 (EtOAc:Hexane: 6:4); 1H NMR (400 MHz, DMSO-d6) δppm: 8.50 (d, 1H, J = 2.4 Hz, Ar-H), 8.22 (dd, 1H, J = 9.2, 1.6 Hz, Ar-H), 7.91 (d, 1H, J = 8.8 Hz, Ar-H), 4.38 (q, 2H, J = 7.2 Hz, eCH2), 1.35 (t, 3H, J = 7.2 Hz, eCH3); 13C NMR (100 MHz, DMSO-d6) δppm: 144.2 (C5), 140.1 (C9),139.5 (C2), 139.2 (C8), 118.4 (C6), 112.5 (C4), 110.5 (C7), 37.2 (CH2), 14.2 (CH3); HRMS m/z (M +Na)+: 248.0204, Calcd. for C9H8ClN3O2: 248.0203; Anal. Calcd. for C9H8ClN3O2: C, 43.45; H, 3.26; Cl, 14.19; N, 16.92, Found: C, 43.48; H, 3.29; Cl, 14.21; N, 16.98.

4.1.4.13. 5,6-Dibromo-2-chloro-1-propyl-1H-benzimidazole (3c). Yield 78%, white viscous oil; Rf − 0.56 (EtOAc:Hexane: 5:5); 1H NMR (400 MHz, DMSO-d6) δppm: 8.23 (s, 1H, Ar-H), 8.02 (s, 1H, Ar-H), 4.22 (t, 2H, J = 7.2 Hz, eCH2), 1.78–1.67 (m, 2H, eCH2), 0.85 (t, 3H, J = 7.6 Hz, eCH3); 13C NMR (100 MHz, DMSO-d6) δppm: 142.2 (C8), 141.3 (C2), 135.3 (C9), 122.9 (C4), 117.5 (C7), 116.8 (C5), 115.6 (C6), 45.7 (CH2), 22.1 (CH2), 10.7 (CH3); HRMS m/z (M+H)+: 350.8899, Calcd. for C10H9Br2ClN2: 350.8899; Anal. Calcd. for C10H9Br2ClN2: C, 34.05; H, 2.58; Br, 45.22; Cl, 10.02; N, 7.96, Found: C, 34.07; H, 2.59; Br, 45.26; Cl, 10.07; N, 7.98.

4.1.4.8. 2-Chloro-5-nitro-1-propyl-1H-benzimidazole (2c). Yield 68%, light yellow viscous oil; Rf − 0.66 (EtOAc:Hexane: 6:4); 1H NMR (400 MHz, DMSO-d6) δppm: 8.50 (d, 1H, J = 2.0 Hz, Ar-H), 8.22 (dd, 1H, J = 9.2, 2.0 Hz, Ar-H), 7.92 (d, 1H, J = 8.8 Hz, Ar-H), 4.32 (t, 2H, J = 7.2 Hz, eCH2), 1.84–1.74 (m, 2H, eCH2), 0.87 (t, 3H, J = 7.6 Hz, eCH3); 13C NMR (100 MHz, DMSO-d6) δppm: 144.2 (C5), 140.1 (C9),139.5 (C2), 139.2 (C8), 118.5 (C6), 112.6 (C4), 110.5 (C7), 49.2 (CH2), 22.2 (CH2), 11.2 (CH3); HRMS m/z (M+Na)+: 262.0358, Calcd. for C10H10ClN3O2: 262.0359; Anal. Calcd. for C10H10ClN3O2: C, 45.75; H, 3.86; Cl, 13.52; N, 16.02, Found: C, 45.76; H, 3.85; Cl, 13.55; N, 16.05.

4.1.4.14. 2-(5,6-Dibromo-2-chloro-1H-benzimidazol-1-yl)ethan-1-ol (3d). Yield 80%, white solid; m.p. 140–141 °C; Rf − 0.67 (EtOAc:Hexane: 6:4); 1H NMR (400 MHz, DMSO-d6) δppm: 8.14 (s, 1H, Ar-H), 8.02 (s, 1H, Ar-H), 4.94 (t, 1H, J = 5.6 Hz, eOH), 4.31 (t, 2H, J = 5.2 Hz, eCH2), 3.73–3.69 (m, 2H, eCH2); 13C NMR (100 MHz, DMSO-d6) δppm: 142.7 (C8),) 141.4 (C2), 135.9 (C9), 122.7 (C4), 117.2 (C7), 116.6 (C5), 115.9 (C6), 59.1 (CH2), 47.2 (CH2); HRMS m/z (M +H)+: 352.8691, Calcd. for C9H7Br2ClN2O: 352.8692; Anal. Calcd. for C9H7Br2ClN2O: C, 30.55; H, 1.96; Br, 45.02, Cl, 10.01; N, 7.92, Found: C, 30.56; H, 1.98; Br, 45.05, Cl, 10.05; N, 7.95. 4.1.4.15. 3-(5,6-Dibromo-2-chloro-1H-benzimidazol-1-yl)propan-1-ol (3e). Yield 77%, white viscous oil; Rf − 0.65 (EtOAc:Hexane: 6:4); 1H NMR (400 MHz, DMSO-d6) δppm: 8.14 (s, 1H, Ar-H), 8.02 (s, 1H, Ar-H), 3.75 (s, 1H, eOH), 4.31 (t, 2H, J = 6.8 Hz, eCH2), 3.42 (t, 2H, J = 6.0 Hz, eCH2), 1.91–1.85 (m, 2H, eCH2); 13C NMR (100 MHz, DMSO-d6) δppm: 142.5 (C8),) 139.7 (C2), 135.9 (C9), 122.5 (C4), 117.1 (C7), 116.3 (C5), 115.8 (C6), 58.3 (CH2), 45.2 (CH2), 32.2 (CH2); HRMS m/z (M + H)+: 366.8849, Calcd. for C10H9Br2ClN2O: 366.8848; Anal. Calcd. for C10H9Br2ClN2O: C, 32.65; H, 2.46; Br, 43.35, Cl, 9.62; N, 7.62, Found: C, 32.67; H, 2.48; Br, 43.37, Cl, 9.65; N, 7.65.

4.1.4.9. 2-(2-Chloro-5-nitro-1H-benzimidazol-1-yl)ethan-1-ol (2d). Yield 61%, light yellow solid; m.p. 99–102 °C; Rf − 0.67 (EtOAc:Hexane: 6:4); 1H NMR (400 MHz, DMSO-d6) δppm: 8.49 (d, 1H, J = 2.0 Hz, ArH), 8.21 (dd, 1H, J = 9.0, 2.0 Hz, Ar-H), 7.85 (d, 1H, J = 8.8 Hz, 1H, Ar-H), 5.00 (t, 1H, J = 5.6 Hz, eOH), 4.40 (t, 2H, J = 5.2 Hz, eCH2), 3.78–3.74 (m, 2H, eCH2); 13C NMR (100 MHz, DMSO-d6) δppm: 144.4 (C5), 143.1 (C9),140.1 (C2), 139.9 (C8), 118.3 (C6), 114.6 (C4), 111.8 (C7), 59.1 (CH2), 47.5 (CH2); HRMS m/z (M+H)+: 242.0336, Calcd. for C9H8ClN3O3: 242.0332; Anal. Calcd. for C9H8ClN3O3: C, 44.75; H, 3.36; Cl, 14.62; N, 17.32, Found: C, 44.76; H, 3.38; Cl, 14.65; N, 17.35.

4.1.4.16. 2-Chloro-1-methyl-5,6-dinitro-1H-benzimidazole (4a). Yield 59%, yellow solid; m.p. 134–136 °C; Rf − 0.69 (EtOAc:Hexane: 7:3); 1 H NMR (400 MHz, DMSO-d6) δppm: 8.14 (s, 1H, Ar-H), 8.02 (s, 1H, ArH), 3.71 (s, 3H, eCH3); 13C NMR (100 MHz, DMSO-d6) δppm: 145.7 (C8), 141.2 (C9), 139.8 (C2), 137.5 (C5–C6), 112.5 (C4–C7), 31.3 (CH3); HRMS m/z (M+H)+: 257.0078, Calcd. for C8H5ClN4O4: 257.0078; Anal. Calcd. for C8H5ClN4O4: C, 37.46; H, 1.95; Cl, 13.81; N, 21.85, Found: C, 37.48; H, 1.97; Cl, 13.84; N, 21.88.

4.1.4.10. 3-(2-Chloro-5-nitro-1H-benzimidazol-1-yl)propan-1-ol (2e). Yield 63%, light yellow viscous oil; Rf − 0.69 (EtOAc:Hexane: 6:4); 1H NMR (400 MHz, DMSO-d6) δppm: 8.50 (d, 1H, J = 2 Hz, Ar-H), 8.22 (dd, 1H, J = 9.2, 1.6 Hz, Ar-H), 7.91 (d, 1H, J = 8.8 Hz, Ar-H), 4.32 (t, 2H, J = 6.8 Hz, eCH2), 3.44–3.41 (m, 3H, eCH2, OH), 1.91–1.85 (m, 2H, eCH2); 13C NMR (100 MHz, DMSO-d6) δppm: 144.3 (C5), 140.1 (C9),139.7 (C2), 139.5 (C8), 118.3 (C6), 112.6 (C4), 110.8 (C7), 58.1 (CH2), 43.5 (CH2), 32.5 (CH2); HRMS m/z (M +H)+: 256.0488, Calcd. for C10H10ClN3O3: 256.0489; Anal. Calcd. for C10H10ClN3O3: C, 46.85; H, 4.36; Cl, 13.82; N, 16.32, Found: C, 46.87; H, 4.39; Cl, 13.87; N, 16.35.

4.1.4.17. 2-Chloro-1-ethyl-5,6-dinitro-1H-benzimidazole (4b). Yield 56%, yellow solid; m.p. 116–118 °C; Rf − 0.62 (EtOAc:Hexane: 7:3); 1 H NMR (400 MHz, DMSO-d6) δppm: 8.14 (s, 1H, Ar-H), 8.02 (s, 1H, ArH), 3.99 (q, 2H, J = 8.5 Hz, eCH2), 1.23 (t, 3H, J = 8.2 Hz, eCH3); 13C NMR (100 MHz, DMSO-d6) δppm: 145.7 (C8), 141.2 (C9), 139.8 (C2), 137.5 (C5–C6), 112.5 (C4–C7), 37.3 (CH2), 14.1 (CH3); HRMS m/z (M +H)+: 271.0235, Calcd. for C9H7ClN4O4: 271.0234; Anal. Calcd. for C9H7ClN4O4: C, 39.93; H, 2.64; Cl, 13.14; N, 20.72, Found: C, 39.95; H, 2.67; Cl, 13.17; N, 20.76.

4.1.4.11. 5,6-Dibromo-2-chloro-1-methyl-1H-benzimidazole (3a). Yield 79%, white solid; m.p. 104–106 °C; Rf − 0.52 (EtOAc:Hexane: 5:5); 1 H NMR (400 MHz, DMSO-d6) δppm: 8.14 (s, 1H, Ar-H), 8.00 (s, 1H, ArH), 3.78 (s, 3H, eCH3); 13C NMR (100 MHz, DMSO-d6) δppm: 141.6 (C8), 141.2 (C2), 134.5 (C9), 121.4 (C4), 117.4 (C7), 116.6 (C5), 115.2 (C6), 14.2 (CH3); HRMS m/z (M+H)+: 322.8586, Calcd. for C8H5Br2ClN2: 322.8586; Anal. Calcd. for C8H5Br2ClN2: C, 29.65; H, 1.57; Br, 49.28; Cl, 10.92; N, 8.66, Found: C, 29.67; H, 1.58; Br, 49.31; Cl, 10.95; N, 8.69. 4.1.4.12. 5,6-Dibromo-2-chloro-1-ethyl-1H-benzimidazole

4.1.4.18. 2-Chloro-5,6-dinitro-1-propyl-1H-benzimidazole (4c). Yield 55%, yellow viscous oil; Rf − 0.62 (EtOAc:Hexane: 7:3); 1H NMR (400 MHz, DMSO-d6) δppm: 8.17 (s, 1H, Ar-H), 8.09 (s, 1H, Ar-H), 4.04 (t, 2H, J = 6.8 Hz, eCH2), 1.74–1.70 (m, 2H, eCH2), 0.86 (t, 3H, J = 7.8 Hz, eCH3); 13C NMR (100 MHz, DMSO-d6) δppm: 145.7 (C8),

(3b). Yield 13



different concentrations ranging from 25 μg/mL to 0.187 μg/mL. Half MIC of the reference antibacterial (chloramphenicol) was added to the test compounds in serial dilution except negative control (without antibiotic and bacteria) and positive control (without antibiotic but contained bacteria). The rest of the method involving addition of bacteria and measurement of growth was carried out as described in measurement of MIC.

141.2 (C9), 139.8 (C2), 137.5 (C5–C6), 112.5 (C4–C7), 49.3 (CH2), 22.3 (CH2), 11.4 (CH3); HRMS m/z (M+H)+: 285.0390, Calcd. for C10H9ClN4O4: 285.0391; Anal. Calcd. for C10H9ClN4O4: C, 42.23; H, 3.22; Cl, 12.46; N, 19.65, Found: C, 42.26; H, 3.25; Cl, 12.48; N, 19.68. 4.1.4.19. 2-(2-Chloro-5,6-dinitro-1H-benzimidazol-1-yl)ethan-1-ol (4d). Yield 51%, yellow viscous oil; Rf − 0.69 (EtOAc:Hexane: 7:3); 1H NMR (400 MHz, DMSO-d6) δppm: 8.53 (s, 1H, Ar-H), 8.42 (s, 1H, Ar-H), 5.00 (t, 1H, J = 5.6 Hz, eOH), 4.40 (t, 2H, J = 4.8 Hz, eCH2), 3.78–3.74 (m, 2H, eCH2); 13C NMR (100 MHz, DMSO-d6) δppm: 145.7 (C8), 141.2 (C9), 139.8 (C2), 137.5 (C5–C6), 112.5 (C4–C7), 58.3 (CH2), 48.3 (CH2); HRMS m/z (M+H)+: 287.0183, Calcd. for C9H7ClN4O5: 287.0183; Anal. Calcd. for C9H7ClN4O5: C, 37.73; H, 2.45; Cl, 12.35; N, 19.65, Found: C, 37.77; H, 2.48; Cl, 12.37; N, 19.68.

4.4. Cytotoxicity assay To evaluate cytotoxic activity of test compounds, MTT assay was performed. The human PBM cell lines and CEM and Vero (African green monkey kidney) cell lines were used for cytotoxic evaluation. The human PBM cells (5 × 104 cells per well) and CEM and Vero cells (African green monkey kidney) (∼3 × 105 cells per well) were seeded in 96-well plates in the presence of increasing concentrations of the tested compounds and incubated in an incubator at 37 °C with 5% CO2. After 5-day incubation for human PBM cells and CEM cells (African green monkey kidney), cell viability was measured by addition of 20 μL MTT (5 mg/mL per well) and incubated at 37 °C for 3–5 h followed by addition of MTT solvent (100 μL per well). The toxicity of the compounds in Vero cells was assessed after 3 days of treatment with MTT as described earlier in case of human PBM cells and CEM cells (Hamada et al., 2013).

4.1.4.20. 3-(2-Chloro-5,6-dinitro-1H-benzimidazol-1-yl)propan-1-ol (4e). Yield 53%, yellow viscous oil; Rf − 0.68 (EtOAc:Hexane: 7:3); 1H NMR (400 MHz, DMSO-d6) δppm: 8.14 (s, 1H, Ar-H), 8.02 (s, 1H, Ar-H), 4.31 (t, 2H, J = 9.8 Hz, eCH2), 3.75 (s, 1H, eOH), 3.43 (t, 2H, J = 6.0 Hz, eCH2), 1.91-1.85 (m, 2H, eCH2); 13C NMR (100 MHz, DMSO-d6) δppm: 145.7 (C8), 141.2 (C9), 139.8 (C2), 137.5 (C5–C6), 112.5 (C4–C7), 58.3 (CH2), 43.4 (CH2), 32.4 (CH2); HRMS m/z (M +H)+: 301.0341, Calcd. for C10H9ClN4O5: 301.0340; Anal. Calcd. for C10H9ClN4O5: C, 39.93; H, 3.05; Cl, 11.79; N, 18.65, Found: C, 39.95; H, 3.07; Cl, 11.84; N, 18.69.

4.5. Molecular docking studies 4.2. Antibacterial assay Molecular docking studies were performed using Discovery Studio 2.5 (DS 2.5, Accelrys Ltd., UK). The standard procedures were used for preparing, docking and scoring of protein and molecular dynamics (MD) simulation of protein. The 3D X-ray crystal structure of protein (1j7t: PDB, www.rcsb.org) with ligand (paromomycin) docked at the eubacterial ribosomal decoding A site (E. coli 16S rRNA A site) was used for this study (Singh et al., 2012).

The broth dilution method or micro dilution susceptibility test in nutrient broth was used to assess the antibacterial activity as recommended by the National Committee for Clinical Laboratory Standards with minor modifications (Singh et al., 2017). All compounds were screened against selected gram-positive bacteria, viz. Bacillus cerus (NCIM-2156), Staphylococcus aureus (NCIM-2079) and gram-negative bacteria, viz. Escherichia coli (wild type) (NCIM-2065) and Pseudomonas aeruginosa (NCIM-2036) and gram-negative bacteria mutant type, viz. Escherichia coli (U-621) for measuring the value of minimum inhibitory concentration (MIC, μg/mL) of the compounds. Chloramphenicol, cycloheximide and paromomycin were used as reference antibacterial agents. Stock solutions of the test compounds and reference drugs were prepared in dimethyl sulphoxide (DMSO) at a concentration of 1000 μg/mL. Eight tubes of the given concentration were prepared in duplicate with the second set being used as MIC reference control (16–24 h visual). After preparation of stock solutions, the controls were incubated at 37 °C for 24 h to observe the macroscopic growth or antibacterial activity in a humid chamber. Tubes 2–7 contained 0.8 mL of nutrient broth, tube 1 (negative control) contained 1 mL of nutrient broth and tube 8 (positive control) contained 0.9 mL of nutrient broth. The negative control, i.e., tube 1 did not have bacteria or antibiotic where as the positive control, i.e., tube 8 contained bacteria but not antibiotic. The test compound (0.1 mL of stock solution) was serially diluted in tubes 2–7 as 100, 50, 25, 12.5, 6.2 and 3.1 μg/mL. After this, 0.1 mL of bacterial strain having concentration 0.5 McFarland scale (9 × 108 cells/mL) was added to tubes 2–7 and each tube was incubated at 37 °C for 24 h at 150 rpm on a rotary shaker in a humid chamber. After 24 h, the lowest concentration of the substance was recorded as the MIC value that gave no visible turbidity, due to macroscopic growth of bacteria.

4.5.1. Preparation of receptor To prepare the receptor, the target protein docked with ligand was taken, extracted the ligand and corrected the bond order. Added the hydrogen atoms and optimized their position on DS 2.5 using the allatom CHARMm forcefield with Adopted Basis set Newton Raphson (ABNR) minimization algorithm. The optimization was performed until the root mean square (r.m.s.) gradient for potential energy was < 0.05 kcal/mol/Å (Brooks et al., 1983). The protein, e.g., eubacterial ribosomal decoding A site (1j7t: PDB) was minimized and defined as receptor using the ‘Binding Site’ tool in DS 2.5. The binding site was defined as the space covered by the ligand in the receptor and an input site sphere of a radius of 5 Å was defined over the binding site. The central part of the sphere was created as the centre of the binding site. The receptor created from the protein was used for docking simulation. 4.5.2. Ligand setup A series of ligands 1(a–e) – 4(a–e) was built using the built-and-edit module of DS 2.5 and optimized their position using the all-atom CHARMm forcefield parameter and then minimized using ABNR method. A stimulated annealing MD approach was used to make a conformational search of the ligands. The ligand was heated up to 700 K and then annealed up to 200 K. Thirty such cycles were carried out. The conformation obtained after thirty cycles, was further subjected to local energy minimization using the ABNR method. The 30 energy-minimized conformations were then superimposed and the conformation having the lowest energy in the major cluster was taken to be the most probable conformation.

4.3. Combination assay or determination of fractional inhibitory concentration (FIC) To evaluate the synergistic effect of compounds 1a– e, 2a–c and 4a–c, a combination screening of selected compounds with reference antibacterial (chloramphenicol) was performed. Stock solutions of the test compounds were prepared in dimethyl sulphoxide (DMSO) at

4.5.3. Docking and scoring The docking process involves the computational prediction of the structures of ligand-protein complexes within a targeted binding site 14



of ligand

from the conformations of the unbound ligand and protein molecules (Kitchen et al., 2004). The DS 2.5 Ligandfit protocol combined a shape comparison filter with a Monte Carlo transformational search was used for the docking of ligand with targeted protein (Venkatachalam et al., 2003). Dreiding forcefield was used to refine the docked poses by rigid body minimization of the ligand with respect to the grid based calculated interaction energy (Mayo et al., 1990). During docking, the targeted protein receptor conformation was kept fixed. The minimization of the docked poses was performed using all-atom CHARMm forcefield and smart minimization method until the r.m.s. gradient for potential energy was < 0.05 kcal/mol/Å. In the process of minimization, the ligands and protein having binding site were kept flexible using simulation method (Molecular dynamics, energy minimization and Monte Carlo simulation). The scoring (-PLP1, −PLP2, Lig_Internal_Energy, Binding Energy, Dock Score and Ludi 2 value) of ligands has been described in the result and discussion section. Determination of binding energy of ligands in stable ligand-protein complex required for binding of ligands with the receptor was calculated by ‘Calculate Binding Energy protocol’ in DS 2.5 using the default settings (Bohm, 1994; Wang et al., 2003; Prathipati and Saxena, 2006).

Declaration of interest The authors have no conflict of interest. Acknowledgement The author Ritika Srivastava acknowledges financial support in the form of Research Fellowship from the Research grant, University of Allahabad, Allahabad. References Akhtar, W., Khan, M.F., Verma, G., Shaquiquzzaman, M., Rizvi, M.A., Mehdi, S.H., Akhter, M., Alam, M.M., 2017. Therapeutic evolution of benzimidazole derivatives in the last quinquennial period. Eur. J. Med. Chem. 126, 705–753. Azevedo, M.M., Teixeira-Santos, R., Silva, A.P., Cruz1, L., Ricardo, E., Pina-Vaz, C., Rodrigues, A.G., 2015. The effect of antibacterial and non-antibacterial compounds alone or associated with antifungals upon fungi. Front. Microbiol. 6, 1–5. Bohm, H.J., 1994. The development of a simple empirical scoring function to estimate the binding constant for a protein- ligand complex of known three-dimensional structure. J. Comput. Aided Mol. Des. 8, 243–256. Brooks, B.R., Bruccoleri, R.E., Olafson, B.D., States, D.J., Swaminathan, S., Karplus, M., 1983. CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 4, 187–217. Brown, E.D., Wright, G.D., 2016. Antibacterial drug discovery in the resistance era. Nature 529, 336–343. Chellat, M.F., Raguz, L., Riedl, R., 2016. Targeting antibiotic resistance. Angew. Chem. Int. Ed. 55, 6600–6626. Chen, H., Lyne, P.D., Giordanetto, F., Lovell, T., Li, J., 2006. On evaluating moleculardocking methods for pose prediction and enrichment factors. J. Chem. Inf. Model. 46, 401–415. Chermova, E.Yu., Mokrushina, G.A., Chupakhin, O.N., Kotovaskaya, S.K., Il’enko, V.I., Andreeva, O.T., Boreko, E.I., Vladyko, G.V., Korobchenko, L.V., Garagulya, A.D., Dukhovnaya, V.M., 1991. Synthesis and biological activity of 5,6-dinitro derivatives of benzimidazole. Khim-Farm. Zh. 25, 50–52. El-Gohary, N.S., Shaaban, M.I., 2014. Antimicrobial and antiquorum-sensing studies. Part 2: synthesis, antimicrobial, antiquorum-sensing and cytotoxic activities of new series of fused [1,3,4]thiadiazole and [1,3]benzothiazole derivatives. Med. Chem. Res. 23, 287–299. El-Gohary, N.S., Shaaban, M.I., 2017. Synthesis and biological evaluation of a new series of benzimidazole derivatives as antimicrobial, antiquorum-sensing and antitumor agents. Eur. J. Med. Chem. 131, 255–262. Gagnon, M.G., Roy, R.N., Lomakin, I.B., Florin, T., Mankin, A.S., Steitz, T.A., 2016. Structures of proline-rich peptides bound to the ribosome reveal a common mechanism of protein synthesis inhibition. Nucleic. Acid. Res. 1–12. Gallivan, J.P., Dougherty, D.A., 1999. Cation-pi interactions in structural biology. Proc. Natl. Acad. Sci. U. S. A. 96, 9459–9464. Galy, J.P., Hanoun, J.P., Pique, V., Jagerovic, N., Elguero, J., 1997. Pyridinium betaines derived from thiazolo and imidazoacridinones. J. Heterocyclic. Chem. 34, 1781–1787. Gehlhaar, D.K., Verkhivker, G.M., Rejto, P.A., Sherman, C.J., Fogel, D.B., Fogel, L.J., Freer, S.T., 1995. Molecular recognition of the inhibitor AG-1343 by HIV-1 protease: conformationally flexible docking by evolutionary programming. Chem. Biol. 2, 317–324. Goker, H., Kus, C., Boykin, D.W., Yildiz, S., Altanlar, N., 2002. Synthesis of some new 2substituted-phenyl-1H-benzimidazole-5-carbonitriles and their potent activity against Candida species. Bioorg. Med. Chem. 10, 2589–2596. Hamada, M., Roy, V., McBrayer, T.R., Whitaker, T., Urbina-Blanco, C., Nolan, S.P., Balzarini, J., Snoeck, R., Andrei, G., Schinazi, R.F., Agrofoglio, L.A., 2013. Synthesis and broad spectrum antiviral evaluation of bis(POM) prodrugs of novel acyclic nucleosides. Eur. J. Med. Chem. 67, 398–408. Iemura, R., Kawashima, T., Fukuda, T., Ito, K., Tsukamoto, G., 1986. Synthesis of 2-(4substituted-l-piperazinyl) benzimidazoleass H1-antihistaminicAgents. J. Med. Chem. 29, 1178–1183. Kitchen, D.B., Decornez, H., Furr, J.R., Bajorath, J., 2004. Docking and scoring in virtual screening for drug discovery: methods and applications. Nat. Rev. Drug. Discov. 3, 935–949. Kumar, M., Vijayakrishnan, R., Rao, G.S., 2010. In silico structure-based design of a novel class of potent and selective small peptide inhibitor of Mycobacterium tuberculosis dihydrofolate reductase, a potential target for anti-TB drug discovery. Mol. Divers. 14, 595–604. Kumari, R., Kumar, R., Lynn, A., 2014. g_mmpbsa – a GROMACS tool for high-throughput MM-PBSA calculations. J. Chem. Inf. Model. 54 (7), 1951–1962. Kus, C., Kilcgil, G.A., Ozbey, S., Kaynak, F.B., Kaya, M., Coban, T., Eke, B.C., 2008. Synthesis and antioxidant properties of novel N-methyl-134-thiadiazol- 2-amine and 4-methyl-2H-124-triazole-3(4H)-thione derivatives of benzimidazole class. Bioorg. Med. Chem. 16, 4294–4303. Lagos, C.F., Caballero, J., Gonzalez-Nilo, F.D., Pessoa-Mahana, C.D., Perez-Acle, T., 2008. Docking and quantitative structure-activity relationship studies for the

4.6. Molecular dynamics simulations setup and parameters The Molecular Dynamics simulations for the free eubacterial rRNA unit and RNA – ligand complexes were performed using Sander Program provided with the AMBER 9 package. The FF99SB variant of the AMBER force field was used to explain the RNA unit and generalised amber force field (GAFF) was used to parameterize the ligands using Amber tools package in AMBER. The addition of counter ions for neutralization and hydrogen atoms to the RNA unit was done using LEAP module in AMBER 9. The systems were implanted within a rectangular box of TIP3P water molecules such that no protein atom was within 8 Å of any box edge. Periodic boundary conditions were imposed, and the particle-mesh Ewald method integrated in Amber 9 with a direct space and vdW (vander Wall) cut-off of 10 Å, was used for treatment of long-range electrostatic interactions. The energy minimization process was performed for 2500 steps of steepest descent followed by 2500 steps of conjugate gradient minimization with a restraint potential of 500 kcal/mol Å2 applied to the solute. Further, 5000 steps of unrestrained conjugate gradient minimization of the system were then carried out. Gradual heating from 0 to 300 K and a Langevin thermostat with a random collision frequency of 2/ps was used for canonical ensemble (NVT) MD simulations carried out for 50 ps. The system was now equilibrated at 300 K in the NPT ensemble for 250 ps with the restraint potential of 500 kcal/mol Å2 to the solute, followed by NPT equilibration for 50 ps and without any restraint. A Berendsen barostat was used to maintain the pressure at 1 bar. A Langevin thermostat was used for MD simulation in canonical (NVT) ensemble for 20 ns at 300 K. Hydrogen bonds were constrained using the SHAKE algorithm. MD runs were recorded by a time lapse of 1 fs. The PTRAJ module and MMPBSA program implemented in AMBER 9 were used for coordinates saving and trajectories analysis every 1 ps (Moonsamy et al., 2016). PYMOL software was used to generate the figures. 4.7. MM/PBSA binding free energy calculation of inhibitor:eubacterial RNA complexes Molecular Mechanics-Poisson Boltzmann Surface Area (MM/PBSA) software was used to calculate the binding free energy (Kumari et al., 2014). In drug design, MM/PBSA algorithm is utilized as a scoring function. The free energy of binding is calculated using the following formula: Gbinding = Gcomplex − (Gprotein + Gligand) free energy of protein-ligand where,Gcomplex = total complexGprotein = total free energy of proteinGligand = total free energy 15



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