Synthesis and Anti-inflammatory Screening of Various Benzimidazole Derivatives

 

John Joseph*, P. Perumal, Preethimol Francis

Nirmala College of Pharmacy, Muvattupuzha, Kerala-686661

 

ABSTRACT:

The compounds having benzimidazole heterocyclic ring system have been proven to elicit a variety of biological activities like anti-inflammatory, antispasmodic, antihistaminic, antimicrobial, antitumor analgesic and antitubercular. The presence of electron donating and electron withdrawing groups have shown different degree of magnitude in their biological activities. The present study involves the synthesis of mannich bases of 2-substituted benzimidazoles with various electron donating and electron withdrawing groups in the side chains and were screened for their potency in reducing inflammatory response. The compounds were screened by both invitro and invivo anti-inflammatory experimental models. The study revealed that the benzimidazole derivatives having the presence of both electron donating and electron withdrawing groups in their side chain showed potent anti-inflammatory effects.

 

 

 

INTRODUCTION:

Benzimidazole is a bicyclic  heterocyclic  aromatic organic compound formed by the fusion of benzene and imidazole rings. Benzimidazole nucleus is a vital structural component in many of the biologically active molecules and some are very effective and potent drugs. They are extensively used for their anti-inflammatory¹, antispasmodic², antihistaminic³, antimicrobial⁴, antitumour⁵, analgesic⁶ and anti tubercular⁴ activities. Although a number of benzimidazole derivatives are available as therapeutic agents, the synthesis of newer compounds may be beneficial in improving the potency or reducing the side effects for better therapeutic benefits.

 

Inflammation is a protective mechanism of the body against injury to tissues. However, when inflammation persists for longer periods, it can lead to other complications including permanent disability. The major local mediators in inflammation and associated pain are eicosanoids, prostaglandins, kinins, histamine and 5-HT⁷etc. Most of the anti-inflammatory agents act by blocking such local mediators. The present study aims to synthesize various benzimidazole derivatives with electron withdrawing and donating groups in their side chain and to evaluate their potency in anti-inflammatory activity.

 

MATERIALS AND METHODS:

A). Synthesis of Benzimidazole Derivatives

a)   Synthesis of Benzimidazole: Equimolar proportions of o-phenylenediamine and the substituted aromatic aldehyde were mixed with ethanol in the presence of NH₄Cl as catalyst. Stirred well and heated for 2-4 hours at 80 °C. The mixture was poured into ice cold water to precipitate the product, filtered, dried and were recrystallized⁸.

 

B). Synthesis of Mannich bases of Benzimidazoles: The prepared benzimidazole derivatives (J₁-J₇), formaldehyde and the respective substituted aromatic amine, in equimolar proportions were mixed in ethanol and refluxed for 20 hours⁹. The completion of reaction was checked using TLC with Ethyl acetate:Hexane (1:2) as eluting solvent system. The mixture was poured into ice cold water to precipitate the product, filtered, dried and recrystallized.

                               

Figure 1. Scheme of Synthesis of Mannich bases of Benzimidazole Derivatives

 

Table.1: The Substitutions in synthesized Mannich bases of Benzimidazole Derivatives

 

In vitro Anti-inflammatory activity screening by HRBC membrane stabilization method^{10, 11}

 

i) Preparation of Human Red Blood Cells (HRBC) Suspension:

Fresh whole human blood (from healthy volunteers who had not taken NSAIDs for 2 weeks) was collected and mixed with equal volume of sterilized Alsever solution (2 % dextrose, 0.8 % sodium citrate, 0.05 % citric acid and 0.42 % sodium chloride in water). It was then centrifuged at 3000 rpm for 10 min, the supernatant was decanted and packed cells were washed three times with isosaline, (pH 7.2). The volume of the blood was measured and reconstituted as 10 % v/v suspension with isosaline.

 

ii)  Hypotonicity Induced Hemolysis:

The principle involved here is stabilization of human red blood cell membrane by hypo tonicity induced membrane lysis. The assay mixture contains 1ml phosphate buffer [pH 7.4, 0.15 M], 2 ml hyposaline [0.36 % NaCl],  0.5 ml HRBC suspension [10 % v/v] with 0.5 ml of solution of the synthesized drugs of concentration 100 μg/ml, and standard drug diclofenac sodium of concentration 50 μg/ml and control (distilled water instead of hypo saline) were incubated at 37°C for 30 min and centrifuged. The intensity of colour of hemoglobin content in the supernatent was estimated using spectrophotometer at 560 nm.

 

The percentage of hemolysis of HRBC membrane was calculated as follows:

Percentage of Hemolysis = (Optical density of Test sample / Optical density of Control) X 100

 

The percentage of HRBC membrane stabilization was calculated as follows:

 

 

Protection =  100 -   Optical density of drug treated sample X 100

Optical density of control

 

 

4. In vivo Anti-inflammatory activity screening by Carrageenan induced rat-paw Edema Method^12,13

Experimental animals:

Male wistar rats (6-8 weeks old) were used. Animals were placed in polypropylene cages at a controlled room temperature of 22±3° C and relative humidity of 30–70 %, in animal house. They were maintained with standard pellet diet and water ad libitum. Animals were acclimatized to laboratory conditions for three days before the commencement of experiment. Ethical clearance was obtained from Institutional Animal Ethical Committee.

 

Experiment methodology:

The selected male wistar rats were fasted overnight before the commencement of experiment. The animals were segregated into Group I to Group V and each group contained 6 animals and received test/standards as given below.

Group-I               : Served as a negative control (0.1ml of 1% carrageenan)

Group-II: Served as standard which received Diclofenac sodium (10mg/kg, i.p) +(0.1ml of 1% carrageenan)

Group-III: Received J₁ M₄ (100mg /kg) + (0.1ml of 1% carrageenan)

Group IV: Received J₁ M₈ (100 mg/kg) + (0.1ml of 1% carrageenan)

Group V: Received J₇M₈  (100 mg/kg) + (0.1ml of 1% carrageenan)

 

The samples were prepared with distilled water. Just before carrageenan injection, Group-II received the standard diclofenac sodium 10 mg/kg through i.p, Group III, IV and V received the test compounds J₁M₄, J₁M₈ and J₇M₈ at a dose of 100 mg/kg through oral route. Inflammatory pain was induced in animals belonging to all the groups by injection of carrageenan (0.1ml of 1% suspension) into the intraplantar region of the right hind paw using a 27-gauge needle attached to a Hamilton syringe.

 

The change in paw volume was measured at 0 min, 30 min, 1hr, 2 hr, 3 hr, 4 hr, 5 hr and 6 hr after carrageenan injection, using a digital plethysmograph. The percentage change in paw volume was calculated at each time interval and compared with that of the standard.

 

Statistical analysis:

Results of biochemical estimation were reported as mean ± SEM. The total variation present in a data was analyzed by one way analysis of variance (ANOVA). Difference among the mean and the comparison of the remaining data before and after drug administration, was done by using the Post hoc Dunnett’s test by using the software SPSS Demo Version.

 

RESULTS AND DISCUSSION:

With a view to synthesize  some novel benzimidazole derivatives with various biological activities, few  Benzimidazole derivatives were prepared which were then condensed with a variety of aromatic amines which had both electron withdrawing groups like -NO₂, halide and electron donating groups like –CH₃, -OCH₃ etc.

 

In vitro Anti-inflammatory Activity by HRBC membrane Stabilization method

The inhibition of hypotonicity induced HRBC membrane lysis i.e, stabilization of HRBC membrane was taken as a measure of the anti-inflammatory activity. The test compounds labelled as J₁M_4,  J₁M_7, J₁M_8, J₂M₅, J₃M₃ and J₇M₈  have shown  significant  reduction in hemolysis as compared with standard. From these compounds, J₁M_4,  J₁M₈  and J₇M₈  which showed better activity were taken for further  in vivo anti-inflammatory screening procedure.

 

Table.2: Results of In vitro anti-inflammatory screening by HRBC method

Sl. No.	Sample	Concentration in μg/ml	% Hemolysis	% Stabilization
1.	J1M2	100μg/ml	67.95	32.05
2.	J1M4	100μg/ml	16.05	83.92
3.	J1M6	100μg/ml	35.73	64.29
4.	J1M7	100μg/ml	21.42	78.57
5.	J1M8	100μg/ml	22.32	80.20
6.	J2M3	100μg/ml	58.68	41.32
7.	J2M5	100μg/ml	21.42	78.57
8.	J2M6	100μg/ml	65.26	34.82
9.	J2M8	100μg/ml	35.73	64.29
10.	J3M3	100μg/ml	26.79	73.21
11.	J3M8	100μg/ml	57.21	42.79
12.	J4M2	100μg/ml	86.16	13.84
13.	J4M3	100μg/ml	67.95	32.05
14.	J4M4	100μg/ml	88.37	11.61
15.	J5M4	100μg/ml	36.63	63.29
16.	J5M5	100μg/ml	86.63	13.40
17.	J5M8	100μg/ml	76.16	23.84
18.	J6M1	100μg/ml	60.95	39.05
19.	J6M4	100μg/ml	51.84	48.16
20.	J6M8	100μg/ml	35.79	66.96
21.	J7M8	100μg/ml	33.95	66.07
22.	Control	--	100
23.	Standard	50 μg/ml	28.95	71.05

 

In vivo Anti-inflammatory Activity by Carrageenan induced Rat-paw edema method

The results of anti-inflammatory activity of test samples by carrageenan induced paw edema is depicted in table 3 and 4.

 

 

 

Table 3: Percentage inhibition of paw edema by the test samples at 0 min,1 and 2 h

Group	Mean paw volume before carrageenan injection	Paw Volume after injection with Carrageenan Increase in paw volume (ml) after carrageenan injection (mean ± SEM)			(% )Percent  inhibition of edema
	0 min	30 min	1h	2h	30 min	1h	2h
Control	3.985±0.1638	5.788±0.2053	6.56±2.08	7.563±0.107	45.24%	64.61%	89.78%
Standard	4.19±0.2259	4.85±0.103*	6.66±2.10	6.095±0.1468***	15.75%	58.94%	45.46%
J1 M4	3.76±0.2599	5.845±0.3294ns	6.08±1.93	6.848±0.2027ns	55.45%	61.70%	82.12%
J1 M8	3.97±0.4392	5.39±0.3122ns	6.14±1.95	7.18±0.2806ns	35.76%	54.65%	80.85%
J7 M8	3.838±0.2504	5.788±0.1591ns	6.04±1.91	7.113±0.1596ns	50.80%	57.37%	85.33%

Values are expressed as Mean ±SEM; ns -not significant; *p<0.01;**p<0.05;***p<0.001

Table 4: Percentage inhibition of paw edema by the test samples at 3,4,5and 6 h.

Group	Mean paw volume before carrageenan injection	Paw Volume after injection with carrageenan  Increase in paw volume (ml) after  carrageenan  injection (mean ± SEM)/			Percent  (%) inhibition of edema
	3h	4h	5h	6h	3h	4h	5h	6h
Control	7.548± 0.1463	7.375± 0.3544	6.425± 0.2175	5.118± 0.2723	89.41%	85.06%	61.22%	28.43%
Standard	6.42± 0.1857***	5.475± 0.1315***	5.62± 0.1158ns	6.745± 0.1506**	53.22%	30.66%	34.12%	60.97%
J1 M4	6.735± 0.1148**	6.125± 0.1031**	5.75± 0.119ns	6.135± 0.1209ns	79.12%	62.89%	47.34%	63.16%
J1 M8	7.145± 0.1762ns	5.35± 0.3775***	5.75± 0.2958ns	6.215± 0.3839*	79.97%	34.76%	44.83%	54.28%
J7 M8	6.865± 0.1099*	6.275± 0.04787*	5.7± 0.3082ns	5.795± 0.3185ns	81.47%	63.49%	48.51%	50.99%

Values are expressed as Mean ±SEM; ns-not significant; *p<0.01;**p<0.05;***p

 

The inflammation induced by carrageenan injection into the rat paw is an acute, non-immune, well researched and highly reproducible method¹⁴. Oral doses of 100mg/Kg of J₁M₄, J₁M₈, J₇M₈ 100mg/kg and diclofenac sodium at a dose of 10 mg/kg significantly inhibited carrageenan induced paw edema at all of the recorded incidents. Compounds J₁M₄, J₁M₈, J₇M₈ at a dose of 100mg/Kg significantly inhibited paw edema formation up to 5^th hr after carrageenan injection (Table 4). Edema induced by carrageenan consists of three phases. The first phase, during the first 1.5 hr, is mediated by histamine and 5-HT; the second phase (1.5-2.5 h) is mediated by Bradykinins; and the third phase is attributed to local production of Prostaglandins from 2.5 to 6 h after carrageenan injection¹⁵. The results show that J₁M₄, J₁M₈, J₇M₈   and diclofenac sodium exhibited edema inhibition at all assessment times. Compounds J₁M₄, J₁M₈, J₇M₈ exhibit anti-inflammatory effect and it is suggested that it may inhibit all the mediators which are associated with inflammation and pain.

 

The potent anti-inflammatory activity of   J₁M₄, J₁M₈, J₇M₈   may be due to the presence of either electron donating or withdrawing groups in their side chain, as the compounds with either one of the groups had not shown a significant variation in the anti-inflammatory activity. These new synthetic compounds may be having inhibitory effects on the pain mediators like Prostaglandins, kinins and 5-HT. However, to understand a clear mechanism, further studies are required.

 

CONCLUSION:

The Benzimidazole and their derivative compounds are widely used in a number of dosage regimens available in the market for various ailments. In this study, different electron withdrawing groups and electron donating groups were substituted on the benzimidazole ring. These compounds were then screened for their anti-inflammatory potential. The study revealed that the Benzimidazole derivatives with both electron withdrawing and donating groups have shown a significant anti-inflammatory activity as compared to that produced by the standard drug. Further studies may explore the synthesis of more potent anti-inflammatory agents.

 

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interests regarding the publication of this paper

 

ACKNOWLEDGEMENT:

The authors are thankful to KMCH College of Pharmacy, Coimbatore for helping in the in vivo studies

 

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Accepted on 02.07.2016        © RJPT All right reserved