Synthesis and Invitro Anti-Inflammatory activity of some 1, 2-disubstituted benzimidazoles
Praneetha V*, Sreerama Usha Rani, K Srinivas
Department of Pharmaceutical Chemistry, Sri Vasavi Institute of Pharmaceutical Sciences,
Pedatadepalli, Tadepalligudem, Andhra Pradesh, 534101.
*Corresponding Author E-mail: vpranee4u@gmail.com
ABSTRACT:
Fused heterocyclic compound, Benzimidazole formed by fusion of benzene and imidazole. Often the benzo derivative of imidazole is referred to as benzimidazole. Although it is the commonest name of the parent compound of the series, other names such as benzimidazole and 1, 3-benzodiazole are often used have wide range of diverse therapeutic applications. The synthesis of substituted benzimidazole derivatives thought remarkably effective compounds as anti-ulcers, anti-cancers, anti-malarials, anti-convulsants, anti-virals, anti-fungal and much more which have been reported in literature. A practical and convenient synthetic method which has been developed for the facile synthesis of 1, 2-disubstituted benzimidazoles is reported in the paper. The method described has the benefits of operational simplicity, excellent yields, and high chemo selectivity. In this present study, in the first step, 2-substituted benzimidazoles were synthesized which have been prepared by reaction of o-phenylenediamine with various carboxylic acids in presence of suitable solvent. Later, in the second step, the formed 2-substituted benzimidazoles reacts with chloroacetyl chloride in presence of base gives desired title compounds of 1, 2-disubstituted benzimidazole derivatives. All the structures of synthesized compounds have been purified by recrystalisation and thin layer chromatography which were further elucidated by elemental analysis, IR and 1H NMR spectral data. All the four synthesized compounds have been screened for their invitro anti-inflammatory activity using HRBC membrane stabilization method by comparing with standard drug Diclofenac sodium in the concentrations of 25 µg/mL, 50 µg/mL and 100 µg/mL. All the tested compounds exhibit significant invitro anti-inflammatory activity when compared with standard drug.
KEYWORDS: Fused heterocyclic system, 1, 2-disubstituted Benzimidazoles, Chloroacetyl chloride, invitro Anti-inflammatory activity, HRBC membrane stabilization method.
In this 21st century, fused heterocyclic chemistry comprises of major research worldwide. These play an important role in medicinal chemistry and also emerge as a pharmacophore. Heterocycles comprising nitrogen, sulfur and oxygen have been under investigation for a long time due to their crucial medicinal properties. There has been increasing interest in the role played by benzimidazoles, benzothiazole, pyrazole like five membered ring condensed with other heterocycles because of their broad pharmacological activities.
Fused heterocyclic system, benzimidazoles consists of fusion of benzene and imidazole ring system. The benzimidazole ring is of much greater interest as it occurs in Vitamin B12 1 and in many biologically active compounds. Recent observations imply that synthesis of substituted benzimidazoles have easy interaction with the biopolymers, possess potential activity with lower toxicities in chemotherapeutic approach in man and befit as major focus in medicinal chemistry. Benzimidazole ring carrying different substituent’s are associated with a wide range of therapeutic activities such as antiviral 2, antimicrobial3, analgesic 4, anticancer 5, anti-inflammatory 6, antifungal 7, anti-helminthic 8, anti-convulsant 9, anti-hypertensive 10 and anti-malarial 11 and anti-ulcer 12. The literature has revealed that positions 1, 2 and 5 substitution is crucial for exhibiting major pharmacological activities 13.
Apart from present work substituted benzimidazole derivatives possess anti-inflammatory and anti-helminthic activity which was well documented in literature. They also possess variety of biological activities as mentioned earlier. This wide range of activity exhibited by benzimidazole derivatives provokes the attention of many researches who are involved in search for new potent molecule possessing anti-inflammatory activity. The HRBC membrane stabilization has been used as one of the well known method to study the invitro anti-inflammatory activity. The NSAID’s act either by inhibiting these lysosomal enzymes or by stabilizing the lysosomal membrane. So, the prevention of hypotonicity induces HRBC membrane lysis is taken as a measure of anti-inflammatory activity of 1, 2-disubstituted benzimidazole derivatives. Keeping this in mind, we synthesized some disubstituted benzimidazole derivatives and evaluated them for invitro anti-inflammatory activity, which is now being reported in this paper.
EXPERIMENTAL WORK:
Melting points, (m.p.) were determined in open capillaries on a Thiel’s tube apparatus and are uncorrected. The purity of compounds was checked by TLC, Merck pre-coated, Silica Gel G and spots are exposed to iodine vapours / UV light and Pet. Ether : ethyl acetate 3:1. IR spectra were recorded on a BRUKER-FTIR spectrophotometer as KBr pellets and the wave numbers were given in cm-1. The 1H NMR spectra were recorded in DMSO-d6 on AVANCE-300. All the synthesized compounds gave satisfactory C, H, O and N analysis. Starting materials were purchased from Finar chemicals limited or S.D. Fine Chem. Ltd. and used without further purification. All solvents were of analytical grade and freshly distilled prior to use.
Synthesis of 1H-benzoimidazole (1a):
o-phenylenediamine (5.4 g, 0.25 mol) and Formic acid 90% (3.2 g, 0.34 mol) was refluxed on water bath for about 2h at 100̊ C. Then slowly cooled and resultant mixture was basified with aqueous sodium hydroxide solution. The solid obtained was filtered, decolorized from boiling water and recrystallized with ethanol.14
Synthesis of 2-phenyl-1H-benzoimidazole (1b):
A mixture of (6 g, 0.05 mol) o-phenylenediamine, (6 g, 0.05 mol) of benzoic acid and 25 mL of 4N dilute HCl was refluxed on water bath for 2h at 180-185 ̊C. The reaction mixture was cooled and poured on to the crushed ice. The product was recrystallised in boiled water using charcoal.14
Synthesis of 2-benzylbenzimidazole (1c):
A mixture (5.43 g, 0.03 mol) of o-phenylenediamine, 20ml of H2O, (12.3 g, 0.09 mol) Phenyl acetic acid were refluxed for 45 min. The reaction mixture was cooled and basified with conc. ammonia solution. Further the precipitated solid was filtered and recrystallized from 40% aqueous ethanol solution.14
Synthesis of 2-(4-Aminophenyl) benzimidazole (1d): A mixture of (1.2 g, 0.01 mol) o-phenylenediamine and p-amino benzoic acid (1.37 g, 0.1 mol) was refluxed on a water bath for 2 h. It was cooled and add 10% sodium hydroxide solution slowly with constant stirring until just alkaline. The crude product was filtered, washed with ice-cold water, decolorized and recrystallized from ethanol.15
General Procedure for synthesis of 1, 2 disubstituted Benzimidazole derivatives (2a-2d):
In a conical flask 10% NaOH solution (0.01 mol) was taken. To this add (0.01 mol) of 2-substituted benzimidazole derivatives 1a-1d with continuous stirring. The reaction mixture was cooled on ice bath and 1.5 mL of chloroacetyl chloride was added drop wise by dropping funnel in fuming hood. The addition of chloroacetyl chloride was stopped till the fumes stops completely. After complete addition of chloroacetyl chloride the reaction mixture was cooled for some time. The product is separated out by filtration, washed with water, filtered and dried. The yields of the products varied from 70-90%. The Schematic representation has been given in Scheme 1. The physical data of the synthesized compounds were given in Table 1.
1-benzoimidazol-1yl-2-chloro ethanone (2a):
IR ( cm-1 , KBr) : 1335.07 (C-N), 1664.75 (C=O), 2851.97 (CH2), 770.77 (C-Cl ) ; 1H NMR : 7.26-7.70 (m, 4H, Ar.H), 4.49 (s,2H,CH2), 8.08 (dd,1H,CH).
2-Chloro-1-(2-phenyl-benzoimidazol-1-yl)-ethanone (2b):
IR ( cm-1 , KBr) : 1328.35 (C-N), 1658.19 (C=O ), 2852.39 (CH2), 691.15 (C-Cl ) ; 1H NMR : 7.26-7.70 (m, 4H, Ar.H), 7.22-7.48 (m, 5H, Ar.H), 4.49(s,2H,CH2)
1-(2-benzyl-benzoimidazol-1-yl)-2-chloro ethanone (2c):
IR ( cm-1 , KBr) : 1170.69 (C-N), 1654.56 (C=O ), 2854.64, 2797.96 (CH2), 685.27(C-Cl) ; 1H NMR : 7.26-7.70 (m, 4H, Ar.H), 7.06-7.14 (m, 5H, Ar.H) , 4.49(s,2H,CH2), 3.81 (s,2H,CH2)
1-[2-(4-amino-phenyl)-benzoimidazol-1-yl]-2-chloro ethanone (2d):
IR ( cm-1 , KBr) : 1168.33 (C-N), 1660.44 (C=O), 2841.86 (CH2), 762.81(C-Cl) ; 1H NMR : 7.26-7.70 (m, 4H, Ar.H), 6.52-7.23 (m, 4H, Ar.H), 4.49 (s,2H,CH2), 6.27 (s,1H,NH)
Scheme 1: Synthesis of 1, 2 - disubstituted benzimidazole derivatives (2a-2b)
1a-1b 2a-2b
1a: R= H 2a: R= H
1b: R= Ph 2b: R= Ph
1c: R= CH2C6H4 2c: R= CH2C6H4
1d: R=p-NH2C6H4 2d: R=p-NH2C6H4
Table 1. Physical data of synthesized compounds: (2a-2d)
|
Compd code |
Physical state |
Structure |
Mol.Formula |
Mol.Wt. |
m.p. (0C) |
Yield (%) |
|
2a |
Pale cream solid |
|
C9H7ClN2O |
194.62 |
190-192 |
88 |
|
2b |
Pale pink solid |
|
C15H11ClN2O |
270.71 |
200-210 |
72 |
|
2c |
Brown solid
|
|
C16H13ClN2O |
284.74 |
185-190 |
89 |
|
2d |
Dark brown solid |
|
C15H12ClN3O |
285.73 |
290-292 |
75 |
Invitro Anti Inflammatory Activity
HRBC Membrane Stabilization Method
The principle concerned in this method is stabilization of human red blood cell membrane by hypo tonicity induced membrane lysis.16 Blood was collected (2 mL) from healthy volunteers and was mixed with equal volume of sterilized Alsevers solution ( 2% dextrose, 0.8% sodium citrate, 0.5 % citric acid, and 0.42 % NaCl in distilled water) and centrifuged at 3000 rpm .The packed cells were washed with isosaline solution and a 10 % v/v suspension was prepared with normal saline. Different concentrations of synthesized 1, 2-disubstituted benzimidazole derivatives 2a-2d (25 μg/mL, 50 μg/mL, 100 μg/mL), Diclofenac sodium (25 μg/mL, 50 μg/mL, 100 μg/mL) as standard and control (distilled water instead of hypo saline to produce 100% haemolysis) were separately mixed with 1 mL of phosphate buffer, 2 mL hyposaline solution and 0.5mL of 10% HRBC suspension was added to prepared reaction mixture. All the assay mixtures were incubated at 37 oC for 30 min and centrifuged at 3000 rpm for 20 min and hemoglobin content of the supernatant solution was estimated spectrophotometrically at 560 nm. The percentage of HRBC membrane stabilization or protection was calculated by using the formula.
% Membrane Stabilization= {(Absorbance of control-Absorbance of test)/Absorbance of control} × 100
Table 2-- Results of anti-inflammatory activity
|
Conc. (µg/mL) |
2a |
2b |
2c |
2d |
Standard (Diclofenac) |
|
25 |
0.09±0.0002
|
0.166±0.015 |
0.15 ±0.003 |
0.053 ±0.001 |
0.163 ±0.0005 |
|
50 |
0.032± 0.0001 |
0.027 ±0 |
0.021 ±0 |
0.037± 0 |
0.054 ±0.012 |
|
100 |
0.017 ±0 |
0.022±0.006 |
0.002 ±0.018 |
0.017 ±0.0005 |
0.013 ±0.0005 |
Each value represents the mean ± SEM. N=3, Experimental group were compared with control p<0.01, considered extremely significant.
Figure: 1. Effect of di substituted benzimidazoles on HRBC membrane stabilization
RESULTS AND DISCUSSION:
Heterocyclic compound previously reported substituted benzimidazole were synthesized by treating o-phenylenediamine with various carboxylic acids in presence of suitable solvent undergoes condensation to form required 2-substituted bezimidazole derivatives which were coded as 1a-1d. Later compounds 1a-1d were treated with acid chloride i.e., chloro acetyl chloride in presence of base 10% NaOH to yield the respective title compounds 1, 2 disubstituted benzimidazole derivatives 2a-2d in good yields. All synthesized compounds were purified by means of recrystalisation method and characterized by physical data prescribed in Table 1.
The molecular formula of the compounds was confirmed by elemental analysis and their structures were determined from IR and 1H NMR and melting point, which were consistent with the literature data. The 1H NMR of the synthesized compounds gave characteristic peaks in the expected region.
The title compounds were also screened for their biological activity i.e., invitro anti-inflammatory by using HRBC membrane stabilization method. Since, HRBC membrane is similar to lysosomal membrane components, the prevention of hypotonicity induced HRBC membrane lysis was taken as a measure of anti-inflammatory activity. The title compounds showed highly significant activity when compared to that of standard Diclofenac sodium. The results of anti-inflammatory activity were shown in Table 2 and Figure 1.
CONCLUSION:
In this present study, the four derived compounds have been successively synthesized and characterized by using various spectroscopic methods. All these compounds showed potent anti-inflammatory activity invitro with potent HRBC membrane stabilization using 3 different concentrations (25 μg/mL, 50 μg/mL, 100 μg/mL) tested. The results obtained revealed that the four mentioned compounds of 1, 2-disubstituted benzimidazole derivatives showed significant invitro anti-inflammatory activity. Basing on the results further work may be carried on using animal models.
ACKNOWLEDGEMENTS:
The authors gratefully acknowledge Sri Vasavi Institute of Pharmaceutical Sciences, Pedatadepalli, Tadepalligudem for providing laboratory facilities and thanking Mr. B. Mohan Gandhi, Asst. Professor, Sri Vasavi Institute of Pharmaceutical Sciences for his suggestions and assistance.
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Received on 18.08.2016 Modified on 15.09.2016
Accepted on 06.10.2016 © AJRC All right reserved
Asian J. Research Chem. 2016; 9(10):462-468.
DOI: 10.5958/0974-4150.2016.00069.9