Molecular docking studies of some new imidazole derivatives for antimicrobial properties

2.1 Materials and methods

Melting points were determined by open capillary method and were uncorrected. The IR spectra (in KBr pellets) were recorded on a JASCO FT/IR-4100 spectrophotometer. ¹H NMR and ¹³C NMR spectra were recorded (DMSO-d₆) on a Bruker (400 MHz) using TMS as the internal standard. Chemical shift values are given in δ (ppm) scales. The mass spectra were recorded on a JEOL JMS-D 300 spectrometer operating at 70 eV. Elemental analyses were performed on a Flash EA 1112 series CHNS-O Analyzer. The completion of the reaction was checked by thin layer chromatography (TLC) on silica gel coated aluminum sheets (silica gel 60 F254) obtained from Merck. Commercial grade solvents and reagents were used without further purification (see Scheme 1).

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Scheme 1

Synthetic route for the compounds 2a,b and 4a–d.

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2.2 General procedure for the synthesis of new derivatives of 2,4,5-trisubstituted imidazoles (2a,b)

A mixture of 3-aryl-1H-pyrazole-4-carbaldehyde 1a,b (0.01 mol), benzil (0.01 mol) and ammonium acetate (0.05 mol) in acetic acid (50 mL) was refluxed for 6–7 h at 120 °C. After completion of the reaction, the reaction mixture was allowed to cool and filtered to remove any precipitate. 300 mL of ice-water was added to the filtrate and the precipitated product was collected by filtration. The crude product was recrystallized using ethanol–DMF mixture (Vijesh et al., 2011).

2.3 General procedure for the synthesis of methyl(2Z)-[3-({(E)-[3-aryl-1H-pyrazol-4-yl]methylidene}amino)-5-oxo-2-thioxoimidazolidin-4-ylidene]ethanoate (4a–d)

An equimolar mixture of 3-aryl-1H-pyrazole-4-carbaldehyde thiosemicarbazone 3a–d (0.01 mol) and dimethylacetylenedicarboxylate (DMAD) (0.01 mol) in methanol (20 mL) was refluxed for 1 h. After completion of the reaction, the reaction mixture was allowed to cool. The solid thus separated was collected by filtration and recrystallized using ethanol–DMF mixture (Vijesh et al., 2011).

2.4 Antibacterial studies

The antibacterial activity of newly synthesized compounds 2a–d and 4a–j was determined by well plate method (Arthington-Skaggs et al., 2000) in Mueller–Hinton Agar. The in vitro antibacterial activity was carried out against 24 h old cultures of bacterial strains. In this work, Escherichia coli, Bacillus subtilis, Salmonella typhimorium, Clostridium profingens and Pseudomonas aeruginosa were used to investigate the activity. The test compounds were dissolved in dimethyl sulfoxide (DMSO) at concentration of 1 and 0.5 mg/mL. Twenty milliliters of sterilized agar media was poured into each pre-sterilized Petri dish. Excess of suspension was decanted and plates were dried by placing in an incubator at 37 °C for an hour. About 60 μL of 24 h old culture suspension was poured and neatly swabbed with the pre-sterilized cotton swabs. Six millimeter diameter well was then punched carefully using a sterile cork borer and 30 μL of test solutions of different concentrations were added into each labeled well. The plates were incubated for 24 h at 37 °C. The inhibition zone that appeared after 24 h, around the well in each plate were measured as zone of inhibition in mm. Experiments were triplicates and standard deviation was calculated. The antibacterial results were compared with streptomycin and summarized in Table 2.

2.5 In silico molecular docking studies

The ligands were drawn in ChemDraw Ultra 6.0 (ChemOffice package) assigned with proper 2D orientation and the structure of each compound was analyzed for connection error in bond order. OSIRIS, an ADMET based Java library layer that provides reusable cheminformatics functionality which is an entirely in-house developed drug discovery informatics system was used to predict the total drug score via in silico (Sander et al., 2009). Energy of the molecules was minimized using Dundee PRODRG2 server (Schuttelkopf and Aalten, 2004). The energy minimized compounds were then read as input for AutoDock 4.2, in order to carry out the docking simulation (Morris et al., 1998). All the heteroatoms were removed from the2VF5.pdb, to make complex receptor free of any ligand before docking. The Graphical User Interface program “AutoDock Tools” was used to prepare, run, and analyze the docking simulations. Kollman united atom charges, solvation parameters and polar hydrogen’s were added to the receptor for the preparation of protein in docking simulation. Since ligands are not peptides, Gasteiger charge was assigned and then non-polar hydrogens were merged. AutoDock requires pre-calculated grid maps, one for each atom type, present in the ligand being docked as it stores the potential energy arising from the interaction with macromolecule. This grid must surround the region of interest (active site) in the macromolecule. In the present study, the binding site was selected based on the amino acid residues, which are involved in binding with glucosamine-6-phosphate of GlcN-6-P synthase as obtained from PDB with ID 2VF5 which would be considered as the best accurate active region as it is solved by experimental crystallographic data (Mouilleron et al., 2008). Therefore, the grid was centered at the region including all the 12 amino acid residues (Ala602, Val399, Ala400, Gly301, Thr302, Ser303, Cys300, Gln348, Ser349, Thr352, Ser347 and Lys603) that surround active site as in Fig. 2. The grid box size was set at 70, 64, and 56 Å for x, y and z respectively, and the grid center was set to 30.59, 15.822 and −3.497 for x, y and z respectively, which covered all the 12 amino acid residues in the considered active pocket. Docking software AutoDock 4.2 Program supplied with AutoGrid 4.0 and AutoDock 4.0 was used to produce grid maps. The spacing between grid points was 0.375 angstroms. The Lamarckian Genetic Algorithm (LGA) was chosen to search for the best conformers. During the docking process, a maximum of 10 conformers was considered for each compound. All the AutoDock docking runs were performed in Intel Centrino Core2Duo CPU @ 2.20 GHz of IBM system origin, with 2 GB DDR2 RAM. AutoDock 4.0 was compiled and run under Microsoft Windows XP operating system.

3.1 Synthesis of imidazole derivatives and characterization

3-Substituted-1H-pyrazole-4-carbaldehydes (1a–d) were synthesized by the Vilsmayer Haack reaction of semicarbazones (Lebedev et al., 2005). 2,4,5-Trisubstituted imidazoles (2a–b) were obtained in excellent yields by refluxing 3-substituted-1H-pyrazole-4-carbaldehydes with 1,2-diketone (Benzil) and ammonium acetate in acetic acid for 6–7 h via Debus reaction (Vijesh et al., 2011). The imidazolones (4a–d) were obtained in good yield by refluxing substituted thiosemicarbazones (3a–d) with dimethylacetylenedicarboxylate (DMAD) in methanol for 1 h (Vijesh et al., 2011). The starting material 3a–d in turn was synthesized by refluxing equimolar amount of 3-aryl-1-H-pyrazole-4-carbaldehyde with thiosemicarbazide in the presence of anhydrous sodium acetate in ethanol. The reaction pathway has been summarized in Scheme 1. Newly synthesized compounds (2a,b and 4a–d) were characterized by IR, NMR, mass spectral and C, H, N elemental analyses.

Formation of 4-(4,5-aryl-1H-imidazol-2-yl)-3-[substituted]-1H-pyrazole (2a,b) and methyl (2Z)-[3-({(E)-[3-(substituted)-1H-pyrazol-4-yl]methylidene}amino)-5-oxo-2-thioxoimidazolidin-4-ylidene]ethanoate (4a–d) was confirmed by recording their IR, ¹H NMR, ¹³C NMR and mass spectra. All compounds were characterized after recrystallization from appropriate solvents. IR spectrum of compound 4c showed absorption at 3121 cm⁻¹ which is due to the NH stretching. Bands at 1711, 1636 cm⁻¹ are due to C⚌O of ester and cyclic amide respectively. Band at 1598 cm⁻¹ is due to C⚌N, band at 1096 cm⁻¹ is due to C⚌S group. The C–O stretching frequency of ester appeared at 1240 cm⁻¹ and 1188 cm⁻¹ further confirms the structure. The ¹H NMR spectrum of 4c showed a singlet at δ 2.55 is due to SCH₃ protons. A singlet at δ 3.79 is due to OCH₃ protons. A singlet at δ 6.65 is due to C⚌CH. Aromatic protons appeared as multiplet at δ 7.40–7.77. Pyrazole-5H appeared as a singlet at δ 8.48. Similarly a singlet appeared at δ 8.64 is due to –N⚌CH protons. Two singlets at δ 12.75 and δ 13.48 are due to pyrazole–NH and imidazole–NH respectively further confirms the structure. The mass spectrum of 4c showed molecular ion peak at m/z = 402.0 (M+1), which is in agreement with the molecular formula C₁₇H₁₅N₅O₃S₂. Similarly the spectral values for all the compounds and C, H, N analyses are given in the experimental part and the characterization is provided in Table 1.

3.2 Antibacterial studies

The in vitro antibacterial activity of newly synthesized compounds 2a,b and 4a-d was determined by well plate method. The antibacterial screening revealed that some of the tested compounds showed good inhibition against various tested microbial strains. The result indicated that among the tested compounds, 4c showed excellent activity against P. aeruginosa at concentrations of 1 and 0.5 mg/mL compared to standard drug streptomycin. 4c showed similar activity as that of standard, against C. profingens, at 1 and 0.5 mg/mL concentrations. Results of antibacterial studies have been presented in Table 2.

As regards the relationships between the structure of the heterocyclic scaffold and the detected antibacterial properties, it showed varied biological activity. Compound 4c has thioanisyl moiety on pyrazole ring, which is accounted for the enhanced antibacterial activity. However in the first series, compounds showed moderate antimicrobial activity. Imidazole and pyrazole nucleus which is present in both the series are responsible for the biological activity. However the presence of other substituents is responsible for the varied biological activity of the compounds.

3.3 Molecular docking studies

Considering the well obtained in vitro results, it was thought worthy to perform molecular docking studies, hence screening the compounds, inculcating both in silico and in vitro results. Considering GlcN-6-P synthase as the target receptor, comparative and automated docking studies with newly synthesized candidate lead compounds was performed to determine the best in silico conformation. The Lamarckian genetic algorithm, inculcated in the docking program AutoDock 4.2, was employed to satisfy the purpose. Fig. 3 shows the native crystal structure of GlcN-6-P synthase (X chain) in complex with glucosamine-6-phosphate was obtained from Protein Data Bank (http://www.pdb.org/pdb/home/home.do) with the PDB ID 2VF5 which was resolved at 2.90 Å using X-ray diffraction (Mouilleron et al., 2008) (Fig. 1).

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Figure 1

Crystal structure of X chain of GlcN-6-P synthase in complex with glucosamine-6-phosphate.

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Figure 2

PDBsum’s ligplot results for 2VF5, showing all 12 amino acid residues of active pocket.

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Figure 3

The best and stable conformations of all the synthesized molecules including the considered standard drug molecule. (A) 4a forming 1H bond with Ser347. (B) 4b forming 2H bond with Thr352 and Glu488. (C) 4c forming 2H bond with Thr352 and Lys603. (D) 4d forming 1H bond with Thr352. (E) 2a forming 0H bond. (F) 2b forming 0H bond. (G) Streptomycin forming 5H bond with Val399, Cys300, Asp354 and Ala602 (2H bonds).

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The docking of receptor GlcN-6-P with newly synthesized candidate ligands exhibited well established bonds with one or more amino acids in the receptor active pocket. The active pocket was considered to be the site where glucosamine-6-phosphatecomplexes in GlcN-6-Pof 2VF5. The active pocket consisted of 12 amino acid residues as Ala602, Val399, Ala400, Gly301, Thr302, Ser303, Cys300, Gln348, Ser349, Thr352, Ser347 and Lys603 as shown in Fig. 2. The synthesized ligand molecules having 2D structure were converted to energy minimized 3D structures and were further used for in silico protein–ligand docking. All the six synthesized molecules were docked. Fig. 3 shows the docked images of selected candidate ligands including the considered standard drug i.e. Streptomycin. Table 3 shows the Binding Energy and Inhibition Constant of seven compounds including the standard. In silico studies revealed all the synthesized molecules showed good binding energy toward the target protein ranging from −8.01 to −6.91 kJ mol⁻¹.