Synthesis and Biological Evaluation of Some New Benzimidazole Derivatives For their Anticonvulsant and Anti-inflammatory activity

 

Sanjeev Sharma¹*, P.D. Gokulan¹, P.S. Jadon², Sanjay Singh² and S.S. Shukla³

¹Shri Ram Nath Singh Institute of Pharmaceutical Science and Technology, Sitholi, Gwalior

² Sun Institute of Pharmaceutical Education and Research, Lahar, Gwalior

³Columbia Institute of Pharmacy, Tekari, Raipur, Chhattisgarh

*Corresponding Author E-mail: pharmasanjeev@gmail.com

ABSTRACT:

Benzimidazole is a heterocyclic aromatic organic compound. In step-1, The O-pheylenediamine (13.5 gm and 0.12mol) was placed in a 250 ml round-bottomed flask and 85 percent formic acid (9.2 gm, 8.4 ml and 0.17 mol.) was added to it. The mixture was heated on a water bath for 100^oC for 3 hours. The mixture was cooled, 10 percent sodium hydroxide solution was added slowly, with constant rotation of the flask, until the mixture is just alkaline to litmus. In step-2, the mixture of o-pheylenediamine dihydrochloride (5.43 gm and 0.03 mol.), 20 ml of water, acetic acid (5.4 gm, 5.67ml, and 0.09 mol.) was refluxed for 4 hours. The reaction mixture was cooled and basified with gradual addition of concentrated ammonia solution, the precipitate was filtered, dried and recrystallised from 10 % aqueous ethanol. In step-3, the mixture of O-pheylenediamine dihydrochloride (5.43 gm and 0.03 mol.), 20 ml of water, phenyl acetic acid (12.3 gm, 0.09 mol.) was refluxed for 6 hours. The reaction mixture was cooled and basified with gradual addition of concentrated ammonia solution, the precipitate was filtered, dried and recrystallised from 40% aqueous ethanol.

 

 

INTRODUCTION:

The benzimidazoles contain a phenyl ring fused to an imidazole ring as indicated in the structure. This bicyclic compound consists of the fusion of benzene and imidazole. This important group of substances has found practical applications in a number of fields. Benzimidazole, which is a heterocyclic nucleus, plays an important role in various medicines.¹ Benzimidazole in an extension of the well-elaborated imidazole system has been used as carbon skeletons for N-heterocyclic carbenes. The discovery that 5, 6-dimthylbenzimidazole moiety is part of chemical structure of vitamin B₁₂ has prompted a massive research upon benzimidazoles with particular emphasis on the synthesis of new compounds for pharmacological screening. The most prominent benzimidazole compound in nature is N-ribosyl-dimethylbenzimidazole, which serves as an axial ligand for cobalt in vitamin B12.^2-3 Benzimidazole undergoes a number of reactions such as electrophilic and nucleophilic addition, thermal oxidation and electrocyclic reactions.

 

In early 1950s the vital role of purines in biological systems was established and it was discovered that 5, 6-dimethyl-1-(α-D-ribofuranosyl) benzimidazole is an integral part of the structure of Vitamin B₁₂.⁴ Benzimidazole show the various activities like anti-microbial activity,⁵ antiviral activity,  antitumor activity,⁶ anti-inflammatory activity,⁷ anti-micyobacterial activity⁸ and anti-asthamic activity.⁹

 

EXPERIMENTAL WORK:

The synthesis and analytical studies of the compounds were carried out using laboratory grade and analytical grade reagents as the case may be standard procedure or reported methods were followed with or without modification appropriately as and when required.

 

Step A:  Synthesis of benzimidazole:

O-pheylenediamine (13.5 gm and 0.12mol) was placed in a 250 ml round-bottomed flask and 85 percent formic acid (9.2 gm, 8.4 ml and 0.17mol) was added to it. The mixture was heated on a water bath for 100^oC for 3 hours. The mixture was cooled, 10 percent sodium hydroxide solution was added slowly, with constant rotation of the flask, until the mixture is just alkaline to litmus. The crude product was filtered washed with ice cold water and dried. The product was dissolved in 200 ml of boiling water and about 1 gm decolorizing carbon was added and digested for 15 minutes. Then it was filtered, the filtrate was cooled to about 10^oC, benzimidazole was filtered and washed with water and dry at 100^oC. The completion of reaction was monitored by running TLC.

 

Solvent system: Ethyl acetate, chloroform - (8:2)

Melting Point: 178-180ºC

Yield:  (8.25 gm) 56%

R_{F value}: 0.19

 

 

Step B:  Synthesis of 2-methylbenzimidazole:

The mixture of  O-pheylenediamine dihydrochloride (5.43 gm, 0.03 mol), 20 ml of water, acetic acid  (5.4 gm, 5.67ml, 0.09 mol) was refluxed for 4 hours. The reaction mixture was cooled and basified with gradual addition of concentrated ammonia solution, the precipitate was filtered, dried and recrystallised from 10 % aqueous ethanol. The completion of reaction was monitored by running TLC.

 

Solvent system: Ethyl acetate: chloroform - (8:2)

Melting Point: 184-186ºC.

Yield:  (2.3 gm) 58%

R_{F value}: 0.22

 

 

Step C:  Synthesis of 2-benzylbenzimidazole:

The mixture of O-pheylenediamine dihydrochloride (5.43 gm and 0.03 mol.), 20 ml of water, phenyl acetic acid (12.3 gm and 0.09 mol.) was refluxed for 6 hours. The reaction mixture was cooled and basified with gradual addition of concentrated ammonia solution, the precipitate was filtered, dried and recrystallised from 40% aqueous ethanol. The completion of reaction was monitored by running TLC.

Solvent system: Ethyl acetate, chloroform - (8:2)

Melting Point: 194-196ºC.

Yield:  (3.9 gm) 63%

 R_{f value}: 0.79

 

 

SCHEME OF WORK:

 

LIST OF SYNTHESIZED COMPOUNDS

Table- I

S. No.	Molecular Structure	Mol. Formula	Mol.   Weight	Melting Point (0 C )	% Yield	Colour	Solubility
A (1)	3-(1H-benzimidazol-1-yl)-1-phenylpropan-1-one	C16H14ON2	250.3	164-166	27	White (Amorphous)	Chloroform
A (2)	4-(1H-benzimidazol-1-yl)butan-2-one	C11H12ON2	188.2	192-196	26	White (Amorphous)	Chloroform
A (3)	3-(1H-benzimidazol-1-yl)-1,3-diphenylpropan-1-one	C22H18ON2	326.4	102-104	26	White (Amorphous)	Chloroform
A (4)	4-(1H-benzimidazol-1-yl)-4-phenylbutan-2-one	C17H16ON2	264.3	108-110	29	White (Amorphous)	Chloroform

 

 

 

 

 

 

 

 

Table- II

S. No.	Molecular Structure	Mol. Formula	Mol.  Weight	Melting Point (0 C )	% Yield	Colour	Solubility
B (1)	3-(2-methyl-1H-benzimidazol-1-yl)-1-phenylpropan-1-one	C17H16ON2	264.3	>300	33	Off-white (Amorphous)	Chloroform
B (2)	4-(2-methyl-1H-benzimidazol-1-yl)butan-2-one	C12H14ON2	202.2	98-102	21	White (Amorphous)	Chloroform
B (3)	3-(2-methyl-1H-benzimidazol-1-yl)-1,3-diphenylpropan-1-one	C23H20ON2	340.48	78-82	23	White (Amorphous)	Chloroform
B (4)	4-(2-methyl-1H-benzimidazol-1-yl)-4-phenylbutan-2-one	C18H18ON2	278.36	56-60	29	White (Amorphous)	Chloroform

 

 

 

 

Table- III

S. No.	Molecular Structure	Mol. Formula	Mol.   Weight	Melting Point    (0 C )	% Yield	Colour	Solubility
C (1)	3-(2-benzyl-1H-benzimidazol-1-yl)-1-phenylpropan-1-one	C23H20ON2	340.4	64-66	23	Off-white (Amorphous)	Chloroform
C (2)	4-(2-benzyl-1H-benzimidazol-1-yl)butan-2-one	C18H18ON2	278.3	68-70	15	Off-White (Amorphous)	Chloroform
C (3)	3-(2-benzyl-1H-benzimidazol-1-yl)-1,3-diphenylpropan-1-one	C29H24ON2	416.525	76-78	18	White (Amorphous)	Chloroform
C (4)	4-(2-benzyl-1H-benzimidazol-1-yl)-4-phenylbutan-2-one	C24H22ON2	354.45	66-70	24	White (Amorphous)	Chloroform

BIOLOGICAL ACTIVITY:

ANTICONVULSANT ACTIVITY

Different types of epilepsies, i.e. grand mal, petit mal or psychomotor type, can be studied in laboratory animals. There are the two procedures used to study convulsions, and to test anticonvulsant drugs in laboratory animals. The maximal electro-shock (MES)-induced convulsions in animals represent grand mal type of epilepsy. Similarly, chemo-convulsions due to PTZ which produce clonic–type of convulsions resemble petit mal type of convulsions in man. In MES-convulsion electroshock is applied through the corneal electrodes. Through optic stimulation cortical excitation is produced. The MES-convulsions are divided into five phases such as (a) tonic flexion, (b) tonic extensor, (c) clonic convulsions, (d) stupor and (e) recovery or death.

 

Requirements:

Animals: Mice ( 20-25 gm)

Standard Drug: Diazepam

Method: PTZ (pentylenetetrazole)

 

 

Test Compounds:

(A1):       3-(1H-benzimidazol-1-yl)-1-phenylpropan-1-one

(A2):       4-(1H-benzimidazol-1-yl) butan-2-one

(A3):       3-(1H-benzimidazol-1-yl)-1, 3-diphenylpropan-1-           one

(A4):       4-(1H-benzimidazol-1-yl)-4-phenylbutan-2-one

(B1):       3-(2-methyl-1H-benzimidazol-1-yl)-1-              phenylpropan-1-one

(B2):       4-(2-methyl-1H-benzimidazol-1-yl) butan-2-one

(B3):       3-(2-methyl-1H-benzimidazol-1-yl)-1, 3-          diphenylpropan-1-one

(B4):       4-(2-methyl-1H-benzimidazol-1-yl)-4-              phenylbutan-2-one

(C1):       3-(2-benzyl-1H-benzimidazol-1-yl)-1-               phenylpropan-1-one

(C2):       4-(2-benzyl-1H-benzimidazol-1-yl) butan-2-one

(C3):       3-(2-benzyl-1H-benzimidazol-1-yl)-1, 3-           diphenylpropan-1-one

(C4):       4-(2-benzyl-1H-benzimidazol-1-yl)-4-               phenylbutan-2-one

 

Procedure:-

1.      Animal will be weighed and numbered and divided into two groups each consisting of 4-5 mices. One group will use as control and the other for sample compound treatment.

 

2.      The sample compounds were injected interaperitoneally to a group of 6 mice. After 30 min, PTZ was injecting to the mice. The Onset of clonus and Duration of clonus were noted.¹⁰

 

Table IV- In-vivo anticonvulsant activity

Compound No.	Dose (mg/kg)	Onset of clonus (sec.) Mean ± SEM	Duration of clonus (sec.) Mean ± SEM
Control	-	77.66± 12.19	23.33 ± 3.75
A1	30	222.50± 80.50*	32.50±19.50*
A2	30	214.50± 96.50*	22.50±7.50
A3	30	181.50± 181.50	11.00±11.00
A4	30	69.00± 0.00	16.00±10.00
B1	30	295.00± 189.00*	3.50±1.500
B2	30	81.00± 41.00	27.00±6.00
B3	30	237.00± 159.00*	24.00±10.00
B4	30	279.00± 136.00*	24.00±19.00*
C1	30	240.50± 90.50*	21.00±11.00
C2	30	82.00± 8.00	24.50±5.50
C3	30	237.50± 131.50*	11.50±6.50
C4	30	155.00± 10.00	23.50±0.50
Diazepam (std)	2	136.33± 3.93	13.00 ± 1.73

 

Fig 1.1- Onset of Clonus

 

Fig 1.2 - Duration of Clonus

ANTI-INFLAMMATORY ACTIVITY:

Anti-inflammatory activity was determined in-vivo using the carrageenan-induced paw edema test. Animal of either sex were divide into 14 groups 4 each. A solution of 0.1mL of 1% carrageenan solution in saline was injected subplantarly into the right hind paw of the rats 1 hour after i.p. administration of the compounds.

 

Paw thickness was measured from the ventral to the dorsal surfaces immediately prior to carrageenan injection and then each hour, up to the four hour after the subplantarly injection Edema was calculated as thickness variation between the carrageenan and control treated paws.

 

 

 

Table -V: Anti-inflammatory activity (Paw edema method)

Compounds	Time (h)	Dose (mg/kg)	Volume of edema       (ml)	Inhibition (%)
Control (DMSO)	1 2 3 4	- - - -	0.250±0.0144 0.342± 0.0220 0.400±0.0144 0.383± 0.0167		- - - -
Diclofenac Sodium	1 2 3 4	25 25 25 25	0.142±0.0300 0.108±0.0601* 0.0500±0.0382 0.0167±0.0167	68.42 87.50 95.63	43.20
A1	1 2 3 4	30 30 30 30	0.163±0.0875 0.0750±0.0250 0.238±0.0375 0.0375± 0.0125	78.07   90.20	34.80   40.50
A2	1 2 3 4	30 30 30 30	0.112±0.0625 0.138±0.0375* 0.0750±0.000 0.0500±0.000	59.64	55.2o   81.25 86.94
A3	1 2 3 4	30 30 30 30	0.213±0.0125 0.200± 0.0750 0.163±0.0875* 0.150±0.0750*	59.25 60.83	14.80 41.52
A4	1 2 3 4	30 30 30 30	0.0250±0.000 0.0625±0.0375 0.0750±0.0250 0.1000±0.0250*	81.72 81.25 73.89	90.00
B1	1 2 3 4	30 30 30 30	0.0875±0.0875 0.1000±0.1000* 0.138± 0.0125* 0.138±0.0125*	65.00 70.76 65.50 63.96
B2	1 2 3 4	30 30 30 30	0.225±0.000 0.0625±0.0375 0.1000±0.0500* 0.113±0.0177*	81.72 75.00 70.49	10.00
B3	1 2 3 4	30 30 30 30	0.0750±0.0250 0.0500±0.0500 0.0625± 0.0125 0.0250±0.000	70.00 85.38 84.37	93.47
B4	1 2 3 4	30 30 30 30	0.0750±0.0500 0.163±0.0375 0.125± 0.0500* 0.0500±0.125	70.00   68.75	52.33   86.94
C1	1 2 3 4	30 30 30 30	0.0125±0.0125 0.175±0.000 0.162± 0.0125* 0.0625±0.0625*	95.00   59.50 83.68	48.83
C2	1 2 3 4	30 30 30 30	0.0875±0.0375* 0.0500±0.0500 0.0500± 0.0250 0.0625±0.0375*	65.00 85.38 87.50 83.68
C3	1 2 3 4	30 30 30 30	0.1000±0.000 0.0750± 0.0250 0.0500±0.0250 0.0875±0.0375*	78.07 87.50 77.15	60.00
C4	1 2 3 4	30 30 30 30	0.0250±0.0250 0.1000±0.000* 0.150±0.1000* 0.0500±0.000	90.00 70.76 62.50	86.94

 

 

 

Anti-inflammatory activity was expressed as the percentage of inhibition of the edema when compared with the control group and was calculated by using the formula:

 

% inhibition of Edema =   (V_c-V_t)/ V_c * 100

 

Where, V_t­ and V_c are the mean paw volume of the test and control groups respectively.^11-12

 

Statistics:-

The results were expressed as the Mean ± SEM per group and the data were statistically analyzed by one-way analysis of Variance (ANOVA) followed by Dunnett’s test as post hoc test. P <0.05 was considered to be statistically significant.

THE PHYSICO-CHEMCAL DATA OF INTERMEDIATE COMPOUNDS

 

 

Comp.	Mol. Formula	R	Mol. Wt.	Rf	Color (Appearance)	M. P. (0 C )
A	C7H6N2	-H	118.14	0.19	White (Crystalline)	178-180
B	C8H8N2	-CH3	132.17	0.22	White (Crystalline)	184-186
C	C14H12N2	-CH2C6H5	208.26	0.79	Off-white (Crystalline)	194-196

Solvent System (Ethylacetate : chloroform  8 : 2)

 

THE PHYSICO-CHEMICAL DATA OF TITLE COMPOUNDS

 

 

Comp.	Mol. Formula	R1	R2	R3	Mol. Weight	Rf value	Color	Melting Point (0 C )
A1.	C16H14ON2	-H	-C6H5	-H	250.3	0.18	White (Amorphous)	164-166
A2.	C11H12ON2	-H	-CH3	-H	188.2	0.19	White (Amorphous)	192-196
A3.	C22H18ON2	-H	-C6H5	-C6H5	326.4	0.17	White (Amorphous)	102-104
A4.	C17H16ON2	-H	-CH3	-C6H5	264.3	0.18	White (Amorphous)	108-110
B1.	C17H16ON2	-CH3	-C6H5	-H	264.3	0.21	Off-white (Amorphous)	>300
B2.	C12H14ON2	-CH3	-CH3	-H	202.2	0.20	White (Amorphous)	98-102
B3.	C23H20ON2	-CH3	-C6H5	-C6H5	340.48	0.21	White (Amorphous)	78-82
B4.	C18H18ON2	-CH3	-CH3	-C6H5	278.36	0.23	White (Amorphous)	56-60
C1.	C23H20ON2	-CH2C6H5	-C6H5	-H	340.4	0.81	Off-white (Amorphous)	64-66
C2.	C18H18ON2	-CH2C6H5	-CH3	-H	278.3	0.79	Off-White (Amorphous)	68-70
C3.	C29H24ON2	-CH2C6H5	-C6H5	-C6H5	416.525	0.81	White (Amorphous)	76-78
C4.	C24H22ON2	-CH2C6H5	-CH3	-C6H5	354.45	0.78	White (Amorphous)	66-70

Solvent System (Ethylacetate : Chloroform  8 : 2)

 

 

 

 

Table-VI: SPECTRAL DATA DETAIL

Comp ounds	IR SPECTRAL DATA (Wave Number in cm-1 )	NMR SPECTRAL DATA Chemical Shift (δ, ppm)
A.	3124 (N-H), 3064, 2999(Ar. C-H), 1379(C=N)
B.	3191 (N-H), 3047, 2981(Ar. C-H), 1371(C=N)
C.	3023 (N-H), 2964(Ar. C-H), 2614(-CH2), 1418(C=N)
A1.	2995(Ar. C-H), 1679(C=O), 1447(C=N), 1316(tert. N)	3.38(s, 2H, NCH2CH2), 4.71-5.11(m, 2H, NCH2CH2), 5.94(s, 1H, CH), 7.90 (m, 9H, Ar-H)
A2.	3001(Ar. C-H), 2763(-CH3), 1691(C=O), 1453(C=N), 1362(tert. N)
A3.	3009(Ar. C-H), 2755(-CH3), 1688(C=O), 1465(C=N), 1392(tert. N)
A4.	3017(Ar. C-H), 2726(-CH3), 1629(C=O), 1452(C=N), 1372(tert. N)
B1.	2989(Ar. C-H), 2613(-CH3), 1617(C=O), 1458(C=N), 1393(tert. N)	2.77(s, 3H, CH3), 3.38-3.39(dd, 2H, NCH2CH2), 4.72(m, 2H, NCH2CH2), 7.36-7.70(m, 9H, Ar-H)
B2.	2968(Ar. C-H), 2622(-CH3), 1718(C=O), 1454(C=N), 1309(tert. N)
B3.	3002(Ar. C-H), 2670 (-CH3), 1789(C=O), 1447(C=N), 1335(tert. N)
B4.	3012(Ar. C-H), 2635 (-CH3), 1629C=O), 1455(C=N), 1395(tert. N)
C1.	2931(Ar. C-H), 2624(-CH2), 1709(C=O), 1479(C=N),1384(tert. N)
C2.	2971(Ar. C-H), 2606(-CH2), 1679(C=O), 1461(C=N), 1357(tert. N)
C3.	2968(Ar. C-H), 2613(-CH2), 1619(C=O), 1459(C=N), 1294(tert. N)
C4.	2983(Ar. C-H), 2597(-CH2), 1630(C=O), 1464(C=N), 1292(tert. N)

 

 

 

RESULTS AND DISCUSSION:

Benzimidazole derivatives were synthesized and characterized by spectral analysis. The structures of the synthesized compounds were characterized by its IR, ^HNMR spectral analysis show table-VI.

 

Acute Toxicity Study:

Approximate lethal dose (ALD50) was determine by Karber’s method (>300 mg/kg).

 

Anticonvulsant Study:

All the synthesized title compounds (30mg/kg), showed table-I, II and III were screened for in-vivo anticonvulsant activity by PTZ induced seizures in rats using Diazepam (2 mg/kg) as standard drug. The compounds (C4, A3, A2, A1, C3, B3, C1, B4 and B1) showed table-IV that the onset of clonus (Fig.-1.1) is increased as compare to control group as well as the standard Diazepam. The compounds (C3, A3 and B1) showed potent anticonvulsant activity and decreases duration of clonus (Fig.-1.2) as compare to control as well as the standard Diazepam.

 

Anti inflammatory Study:

Anti-inflammatory activity was determined in-vivo using the carrageenan-induced paw edema test using diclofenac sodium (25 mg/kg) as standard drug. All the compounds showed mild to moderate activity. The compounds (A2, A4, B3, C2 and C3) showed table-V to excellent activity as comparable with standard diclofenac sodium.

 

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Received on 04.02.2011       Modified on 29.03.2011

Accepted on 18.04.2011      © RJPT All right reserved