Synthesis, Characterization, Docking study and Anti-Bacterial Evaluation of Benzimidazole Derivatives as Biotin Carboxylase Inhibitors

 

K. Hemalatha, D. Niranjan Raj, M. Fousia Begam, Sharanya. V.K., K. Girija

Department of Pharmaceutical Chemistry, College of Pharmacy, Mother Theresa Post Graduate and Research Institute of Health Sciences, (A Government of Puducherry Institution), Indira Nagar, Gorimedu,

Puducherry-06., India

 

ABSTRACT:

A series of some novel mannich bases substituted Benzimidazole derivatives were synthesized by condensing Benzimidazole with various aromatic aldehydes and different amino compounds in the presence of ethanol. The purity of the compounds were checked by thin Layer Chromatography and Melting point determination method. The functional group of the compounds were characterized by FT-IR spectroscopy. The Structures of the synthesized compounds were optimized with Auto Dock 4.0.1 to investigate the interaction between the target ligand and the amino acid residues of Biotin Carboxylase to determine the probable model. The title compounds were subjected to preliminary QSAR study using Molinspiration software. All the newly synthesized benzimidazole derivatives were evaluated for their invitro anti-bacterial activity by disc diffusion method by measuring the zone of inhibition and the results were comparable to standard.

 

 

 

INTRODUCTION:

Microbial drug resistance is a serious issue, especially as increasing numbers of strains are becoming resistant to multiple antimicrobial agents, with some bacteria now being resistant to all available antibiotics. There is thus a critical need to develop new drugs with novel mechanisms of action. However, the investment available for such development is frequently lower than the required level.

 

The development of new drug entities is hampered by several issues, notably the high cost and length of time required, as well as the logistical and regulatory challenges of performing the necessary clinical evaluations across multiple geographical areas. Therefore, a few new classes of antimicrobials have been developed since the late 1980s^1,2, and much research has focused only on the chemical modification of existing drugs to improve their potency and/or ability to overcome antibiotic resistance mechanisms. Inhibition of fatty acid biosynthesis could be one of the best ways to control the microbial agents. Fatty acid biosynthesis is a multi-component system comprising of biotin carboxyl carrier protein (BCCP or AccB). Biotin carboxylase (AccC) is an excellent target for anti-bacterial agents, which involves in first step of fatty acid biosynthesis^3,4. The catalytic reaction of ACCase is mainly divided into two half reactions (Fig. 1). Benzimidazole⁵ nucleus is a constituent of many bioactive heterocyclic compounds that are of wide interest because of their diverse biological and clinical applications like antimicrobial⁶ antisecretory⁷, anticancer⁸, antiHIV⁹, antihypertensive¹⁰, antitumor¹¹, anthelmintic¹², antidiabetic¹³, antioxidant¹⁴, antifungal¹⁵, analgesic¹⁶ & anti inflammatory¹⁷ and anti protozoal¹⁸ activities. In view of pharmacological significance of benzimidazole derivatives, it is planned to design and synthesize some new mannich bases substituted benzimidazole derivatives and screened for their enzyme inhibitory activity by insilico studies (Docking study) and invitro antibacterial activity by paper diffusion method.

 

Fig. 1: Pictorial representation of Biotin Carboxylase pathway

 

MATERIALS AND METHODS:

Melting points were determined using an open ended capillary tube method and are uncorrected. FT-IR spectra were recorded on a Perkin-Elmer 1800 FT-IR in KBr disc. Completion of the reaction and purity of the compounds were checked by TLC using Silica gel G as stationary phase using chloroform and methanol (9:1) as mobile phase and the spot were visualized by UV-Chamber.

 

General procedure for the synthesis of Mannich bases of benzimidazole derivatives (4a-4f):

To a solution containing benzimidazole (1) in 20 ml of ethanol, aromatic aldehyde (2) and compound containing aromatic primary amino group (3) were added with constant stirring for 1 hr. Then the reaction mixture was refluxed for 10 hrs .On ice cooling, the product formed was filtered, dried in vacuum and recrystallized with ethanol.¹⁹ The scheme for the synthesis of title compounds were shown in Scheme 1. The physic chemical properties were shown in table 1.

 

4a: 4-{[1H-benzimidazol-1-yl (2- hydroxyphenyl) methyl]amino}benzene sulphonamide:

To a solution containing benzimidazole (1) (1.18g, 10 mmol), in 20 ml of ethanol, 1.40g (10 mmol) of salicylaldehyde and 1.37g(10 mmol) of sulphanilamide were added with constant stirring for 1 hr. Then the reaction mixture was refluxed for 10 hrs .On ice cooling, the product formed was filtered, dried in vacuum and recrystallized with ethanol.  IR (KBr):  3342 (C-OH), 3246 (C-H, Ar), 2362 (C-H), 1618 (C=N), 1572 (C=C, Ar) Elemental analysis: calculated: C (60.90%), H (4.60%), N(14.20%), O(12.17%), S(8.13%).

 

4b: 4-{[1H-benzimidazol-1-yl (2-hydroxyphenyl) methyl]amino}benzene sulfonic acid:

To a solution containing benzimidazole (1) (1.18g,10 mmol), in 20 ml of ethanol, 1.40g     (10 mmol) of salicylaldehyde and 1.37g(10 mmol) of sulphanilicaicd were added with constant stirring for 1 hr. Then the reaction mixture was refluxed for 10 hrs .On ice cooling, the product formed was filtered, dried in vacuum and recrystallized with ethanol. IR (KBr): 3304 (C-OH), 3452 (C-H, Ar), 2831 (C-H), 1618 (C=N), 1573 (C=C, Ar); Elemental analysis: calculated: C(60.75%), H(4.33%), N(10.63%), O(16.18%), S(8.11%)

 

4c: 4-{[1H-benzimidazol-1-yl(phenyl)methyl]amino} benzene sulfonamide

To a solution containing benzimidazole (1) (1.18g,10 mmol), in 20 ml of ethanol, 1.40g(10 mmol) of benzaldehyde and 1.37g(10 mmol) of sulphanilamide were added with constant stirring for 1 hr. Then the reaction mixture was refluxed for 10 hrs .On ice cooling, the product formed was filtered, dried in vacuum and recrystallized with ethanol. IR (KBr):  3375 (C-OH), 3344 (C-H, Ar), 2542 (C-H), 1627 (C=N), 1597 (C=C, Ar); Elemental analysis: calculated: C (63.47%), H(4.79%), N(14.80%), O(8.46%), S(8.47%)

 

4d: 4-{[1H-benzimidazol-1-yl (2-chlorophenyl) methyl]amino}benzene sulfonamide:

To a solution containing benzimidazole (1) (1.18g,10 mmol), in 20 ml of ethanol, 1.40g(10 mmol) of o-chlorobenzaldehyde and 1.37g(10 mmol) of sulphanilamide were added with constant stirring for 1 hr. Then the reaction mixture was refluxed for 10 hrs .On ice cooling, the product formed was filtered, dried in vacuum and recrystallized with ethanol. IR (KBr):   3462 (C-OH), 3246 (C-H, Ar), 2570 (C-H), 1637 (C=N), 1599 (C=C, Ar); Elemental analysis: calculated: C(58.18%), H(4.15%), Cl(8.59%), N(13.57%), O(7.75%), S(7.77%)

 

4e: 4-{[1H-benzimidazol-1-yl (4-nitrophenyl)methyl] amino}benzene sulfonamide:

To a solution containing benzimidazole (1) (1.18g,10 mmol), in 20 ml of ethanol, 1.40g(10 mmol) of p-nitro benzaldehyde and 1.37g(10mmol) of sulphanilamide were added with constant stirring for 1 hr. Then the reaction mixture was refluxed for 10 hrs .On ice cooling, the product formed was filtered, dried in vacuum and recrystallized with ethanol. IR (KBr): 3441 (C-OH), 3421 (C-H, Ar), 2964 (C-H), 1600 (C=N), 1523 (C=C, Ar); Elemental analysis: calculated: C(56.73%), H(4.05%), N(16.54%), O(15.11%), S(7.57%)

 

4f: 4-{[1H-benzimidazol-1-yl (4-nitrophenyl)methyl] amino}benzene sulfonic acid:

To a solution containing benzimidazole (1) (1.18g,10 mmol), in 20 ml of ethanol, 1.40g(10 mmol) of p-nitro benzaldehyde and 1.37g(10 mmol) of sulphanilic acid were added with constant stirring for 1 hr. Then the reaction mixture was refluxed for 10 hrs .On ice cooling, the product formed was filtered, dried in vacuum and recrystallized with ethanol.; IR (KBr):   3452 (C-H, Ar), 3100 (N-H stretch in Amine), 2982 (C-H), 1623 (C=N), 1562 (C=C, Ar), 696 (C-Cl); Elemental analysis: calculated: C(56.60%), H(3.8%), N(13.20%), O(18.85%), S(7.55%).

 

 

Molinspiration Cheminformatics was used for calculating important drug like properties like logP, Polar surface area, Number of hydrogen bond donors, Number of hydrogen bond acceptors, Number of rotatable bonds, Volume, Number of violations from rule of five. It was also used to predict bioactive scores against important drug targets like GPCR ligand, Kinase inhibitors, Ion channel modulators, nuclear receptors, Protease inhibitors, Enzyme inhibitors. The results were shown in table 2 and 3.

 

Scheme 1

 

Table 1: Physical data of the synthesized benzimidazole derivatives:

Sl. No	Compound code	Molecular Formula	Molecular weight (gms)	Melting point (ºC)	% yield
1	4a	C20H18N4O3S	394.43	192-194	75
2	4b	C20H17N3O4S	395.43	130-132	82
3	4c	C20H18N4O2S	378.45	115-118	73
4	4d	C20H17ClN4O2S	412.89	85-87	85
5	4e	C20H17N5O4S	423.45	280-282	70
6	4f	C20H16N4O5S	424.43	270-272	73

 

Table 2: Lipinski rule of five properties of synthesized _{Benzimidazole} compounds:

Sl. No.	Compound code	Log P	TPSA	Molecular Weight (in gms)	HBA	HBD	No. of rotatable bonds
1	4a	3.183	110.246	394.43	7	4	5
2	4b	1.54	84.223	395.43	6	2	5
3	4c	3.243	90.018	378.45	6	3	5
4	4d	3.921	90.018	412.89	6	3	5
5	4e	3.202	135.842	423.45	9	3	6
6	4f	1.499	130.047	424.43	9	2	6

HBA: Hydrogen bond acceptors; HBD: Hydrogen bond donors; TPSA: Topological surface area

 

_{Table 3: Drug likeness score of the synthesized Benzimidazole derivatives:}

Sl. No	Compound code	GPCR ligand	Ion channel modulator	Kinase inhibitor	Nuclear receptor ligand	Protease inhibitor	Enzyme inhibitor
1	4a	-0.26	-0.34	-0.11	-0.45	-0.13	-0.07
2	4b	-0.11	-0.21	-0.23	-0.60	-0.13	-0.10
3	4c	-0.30	-0.38	-0.15	-0.58	-0.16	-0.12
4	4d	-0.29	-0.37	-0.16	-0.58	-0.19	-0.15
5	4e	-0.39	-0.38	-0.27	-0.61	-0.26	-0.20
6	4f	-0.23	-0.23	-0.24	-0.62	-0.23	-0.18

 

Molecular Docking study:

Protein Preparation:

The crystallographic structure of Biotin carboxylase (Figure2) which was retrieved from the RCSB Protein Data Bank (PDB code 3JZI) serves as docking receptor and all the designed compounds are selected as ligand molecules. Before docking the screened ligands into the protein active site, the protein was prepared by deleting the substrate cofactor as well as the crystallographically observed water molecules and then protein was defined for generating the grid.

 

Ligand Preparation:

ChemSketch, the chemically intelligent drawing interface freeware (http://www.acdlabs.com/download)   was used to draw the structures of Benzimidazole derivatives, followed by generation of 3Dstructure in PDB format using Marvin sketch.  Automated docking was used to locate the appropriate binding orientations and conformations of various inhibitors into the 3JZI binding pocket. To perform the task, the powerful genetic algorithm method implemented in the program Auto Dock 4.0.1 was employed. Grid maps were generated by Auto Grid program. Each grid was centered at the crystal structure of the corresponding 3JZI. Lamarckian Genetic Algorithm was employed as the docking algorithm. The grid dimensions were 60Å X 60Å X 60Å with points separated by 0.375Å. For all ligands, random starting positions, random orientations and torsions were used. During docking, grid parameters were specified for x, y and z axes as 40, 40 and 40 respectively. The Docking parameters, Number of Genetic Algorithm (GA) runs: 10, Population size: 150, Maximum number of evaluation: 2,500,000, Maximum number of generation: 27,000 were used for this study. The structure with the lowest binding free energy and the most cluster members was chosen for the optimum docking conformation. The Interactions of the synthesized compounds with aminoacids at the active site of the protein (BC) were shown in table 4 and 5.

 

Fig:2 Crystal structure ofBiotin carboxylase (AccC), (PDB ID: 3JZI)

 

Table 4: Interactions of the synthesized compounds with aminoacids at the active site of the protein (BC)

S. No	Synthesized compounds	No. of Hydrogen bonds formed	Amino acid involved in Hydrogen bond interactions	Distance between Donor and Acceptor (Å)	Aminoacid involved in van der waals interactions
1	4a	1	Lys 159(H)	1.838	Ile 120, Phe286, Val131, Glu 276, Glu 288, Ile 276, Ile 287, His209, Lys 116, Tyr 199, Glu 201, Lys 159, Lys 160, Gly 165
2	4b	1	Arg 292	2.033	Lys 238, Gly 165, Glu 288, Lys 116, Asn 290, Gly 114, Glu 87, Gly 164, Gly 83
3	4c	0	-----	------	Leu 278, His 209, Ile 437, Ile 287, Glu 276, Glu 288, Lys 159, Gly 163, Gly 164,  Gly 165, Met 169, Arg 167
4	4d	2	Glu 276 (O) Ala 160 (O)	2.66 2.829	Ile 287, Glu 288, Tyr 199, Lys 116, Gly 163, Gly 164, Gly 165, Lys 159, His 266, Ile 437, His 236,  Met 169, Gly 166
5	4e	2	Arg 292 (H) Arg 292 (H)	1.855 2.204	Glu 288, glu 276, Ile 287, Lys 159, Gly 165, Leu 278, His 209, Ile 437, Gln 233, His 236
6	4f	2	Arg 292(H) Gln 294(H)	1.908 2.201	Gly 165, His 236, Met 169, Lys 159, Gln 233, Glu 288, Glu 276, Ile287, His 209,Ile 437

Table 5: Energy Minimization

Sl. No.	Compound code	No. of Hydrogen bond formed	Binding Energy(kcal/mol)	Inhibitory constant (μM)	Vdw. Desolvation Energy	Intermolecular Energies
1	4a	1	-2.1	2.64	-2.57	-3.12
2	4b	1	-2.48	2.83	-4.82	-3.57
3	4c	0	0.27	1.21	0.53	0.32
4	4d	2	-3.34	3.12	-4.52	-1.23
5	4e	2	-3.62	3.22	-4.92	-5.11
6	4f	2	-3.41	3.19	-4.72	-4.9

 

_{Figure 3: Broken line (Yellow) represents the hydrogen bonds realized by the ligand with amino acid residue of the protein active site, viewed through chimera Software}

 

Table 6 Invitro Antibacterial Activity of the Synthesized Compounds

Sl. No	Compound code	Microorganism used	Zone of Inhibition (in mm)
			Std	50 µg/ml	100 µg/ml	200µg/ml
1	4a	E.coli	14	8	10	10
		S.aureus	13	7	9	11
2	4b	E.coli	12	7	8	10
		S.aureus	13	6	9	11
3	4c	E.coli	13	7	9	10
		S.aureus	13	7	9	10
4	4d	E.coli	10	5	7	9
		S.aureus	10	5	7	9
5	4e	E.coli	14	5	7	9
		S.aureus	13	5	7	9
6	4f	E.coli	11	5	7	9
		S.aureus	11	6	8	10

 

_{Anti-bacterial activity19:}

All the synthesized compounds were screened for their anti-bacterial activity against Staphylococcus aureus (Gram positive bacteria) and Escherichia coli (Gram negative bacteria) by paper disc diffusion technique using Ampicillin (20 µg/disc) as a standard. The sterilized (autoclaved at 120 oC for 30 min) medium was inoculated with the suspension of the microorganism and poured into a petridish to give a depth of 3-4 mm. The paper impregnated with the test compounds (50, 100 and 200 µg/ml in DMSO) was placed on the solidified medium. The plates were pre-incubated for 1 hr at room temperature and incubated at 37^oC for 24 hrs using Ampicillin as standard at a concentration of 20 µg/ml. The antimicrobial activity of the synthesized compounds was recorded in table 6.

 

CONCLUSION:

Six Benzimidazole derivatives were synthesized and characterized for their physical data and IR spectral data. Lipinski's rule of five was calculated for all the synthesized compounds (Table 2) that satisfy the 'rule of- 5' and it was found that all the compounds satisfied the rule for potent promotors. The synthesized compounds were evaluated for the drug likeness score using Molinspiration software (www.molinspiration.com). The derivatives were act as a ligand for various receptors like G-Protein Coupled Receptor (GPCR), Ion Channel Modulator, Kinase receptor and neuron receptor. The results were within the limits (-3 to +3). (Table 3). Docking study showed that compounds 4d, 4e and 4f exhibited good hydrogen bond interactions between the atoms of the synthesized compounds and amino acid residues of Biotin Carboxylase receptor. The synthesized compounds were screened for in vitro antibacterial activity at a concentration of 50, 100 and 200 µg/ml in DMSO by paper disc diffusion method against Staphylococcus aureus and Escherichia coli. All the compounds showed significant anti-bacterial activity compared to standard Ampicillin (20 µg/ml).

 

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Received on 01.04.2017       Accepted on 11.06.2017     

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