Synthesis and Evaluation of Some New Benzofuran Derivative for Antimicrobial Activity

 

Ramesh V.¹*, S.M. Shantakumar² and M. Bhagavanraju²

¹Department of Pharmaceutical Chemistry, K.L.E. College of Pharmacy, K.L.E University, Belgaum-590010

²Department of Pharmaceutical chemistry, V.L. College of Pharmacy, Raichur-584101

*Corresponding Author E-mail: rameshkinhal@yahoo.co.in

ABSTRACT:

Most of the diseases in the current scenario are found to be due to infections caused by microbes like bacteria, fungi, rickettsia, etc. Many antibiotics have been discovered till date for treatment of such diseases but most of them are associated with superinfection, development of resistance, destruction of normal non pathogenic bacterial flora and selective toxicity like aplastic anemia, kidney damage etc. The present study is based upon preparation of antibiotics with high potency, specificity and less toxicity. Benzofuran derivatives are widely distributed in nature and they have been shown to have very interesting pharmacological activities like, antibacterial, anti-inflammatory and anti fungal. When one biologically active molecule is linked to another, the resultant molecule generally has increased potency. Hence in the present study the two pharmacophores, benzofuran and Mercaptotriazoles are fused to obtain highly potent, more specific and less toxic antibiotics.

 

 

 

INTRODUCTION:

Benzofuran compounds occur in nature in a variety of structural forms, which ranges from a simple molecule such as 5-methoxybenzofuran to a highly complicated molecule like morphine. Due to wide scope for synthetic investigation leading to more potent synthetic leads, voluminous synthetic work has been done.  Several monographs^1-5 devoted to the study of such natural and synthetic benzofurans⁶ have appeared in literature from time to time.

 

Several benzofuran compounds are reported to  posses anti-microbiall^7-10,  anti-inflammatory, anti-depressant, analgesic activity, hypoglycemic activity. Substitutes triazoles have been reported for antimicrobial activity.  Hence we plan by coupling the benzofuran nucleus with Mercapto triazoles¹¹ derivatives and screen for Antimicrobial activity.

 

As far as triazoles are concerned, the first studies of triazole were concerned with structural isomerism. Modern instrumental and theoretical methods achieved much success in dealing with tautomeric problems, the complexity of which is one of the enduring charms of the chemistry of triazoles.

 

However, some structural and many tautomeric problems require further study, kinetic and other quantitative mechanistic studies are scarce, the stereochemistry and photochemistry of triazole are virtually unexplored.

Triazoles can be considered as cyclic hydrazines with Hydrogen or some other substituent on either a hydrazide nitrogen or an amide nitrogen.

 

3-Amino-1,2,4-triazole (Amitrole, Amizol, ATA, Cytrol, ENT 25445, Weedazol) was the first 1,2,4-triazole to be manufactured on a large scale from aminoguanidine format for use as a herbicide and defoliant of cotton. 4-Butyltriazole is fungicide but injures seedlings. Such damage is avoided if used as a complex. Newer herbicide include triazolidinonethiones with aromatic and lower aliphatic substituents. Compounds of a similar nature are insecticides, ascaricides and nematociees. Many alkyl and aryl derivatives of mercaptotriazole are active against fungi and other bacteria¹². Relatively simple 1,2,4-triazoles display biological activity such as inhibition of cholinesterase, interference with mitosis and reversible denaturation of serum proteins. But the pharmacological significance of 1,2,4-triazoles is slight in comparison with some other azoles.

 

Scheme – 1

 

MATERIALS AND METHODS: -

Diethylbromomalonate: -

Diethylmalonate (160, 1 mol) and carbon tetra chloride (150 ml) were placed in three necked flask fitted with a reflux condenser and a dropping funnel. Condeser may be fixed with a cork inserted with a glass tube which may be connected to rubber tubing, the end of which may be dipped in water for absorption of hydrogenbromide. In the separating funnel 165 gm (53 ml ; 1.03 mol) of dry bromine was added slowly, the large electric bulb was held under flask until the reaction starts. Then the rest of the bromine was added gradually at such a rate so as to keep the reaction mixture boiling gently. After the addition, the reaction mixture was refluxed until no more hydrobromic acid gas evolved (about 1 hour).  The reaction mixture was cooled and washed five times with 50 ml portions of anhydrous sodium carbonate solution and solvent was removed under reduced pressure. The pure diethylbromomalonate which distilled at 132-133^oC under 33 mm pressure was collected (175 gr).

 

2-Carboxyethoxy benzofuran:-

A solution of salicylaldehyde (10 gr, 0.082 mol) and diethylbromomalonate (20gr, 0.083 mol) in ethyl methyl ketone (40 ml) was treated with anhydrous potassium carbonate (10 gr).  The reaction mixture was heated under reflux for 8 hours on a steam bath. Solvent was distilled off under reduced pressure and the residual salts were dissolved in about 200 ml of water and cooled in an ice-bath and carefully acidified with dilute sulphuric acid. The product was extracted with ether and ethereal extract was washed with saturated sodium bicarbonate solution and dried over calcium chloride. Removal of solvent gave the product as a semisolid (11 gr, 90%) which was sufficiently pure for use in the next stage.

 

Benzofuran-2-carboxy hydrazide (compound II):-

To a solution of 2-carbethoxy benzofuran (10gr, 0.053 mol) in ethanol (30 ml), hydrazine hydrate (5 ml, 99%) was added and the mixture was magnetically stirred at room temperature for 2 hours. The colourless crystalline carboxy hydrazide started separating with in a few minutes and was complete in about 2 hours. The reaction mixture was cooled thoroughly in ice bath and carboxy hydrazide was collected by filtration. On crystallization from ethanol, it was obtained as colourless needles (5.8 gr, 62%), Melting point 158^oC.

 

1)      General method of synthesis of Mercapto Triazole Benzofuran

Benzofuran –2- carboxyhydrazide (0.01 mole) was taken in a round bottom flask, to it different  isothiocyantes in 10 ml of ethanol are added and refluxed for 5 hours on a water bath. The product was cooled and diluted with water and   the precipitate filtered, 50ml of 5% NaoH was added and refluxed for 1 hours on a water bath. Then acidified with dil HCl white precipitate separates out which was filtered and recrytallised from ethanol.

 

 

 

Table No 1 Analytical data

Sl No	Prod Code	R	RI	RII	RIII	Molecular Formula	Melting Point	Rf * Value	Yield%
1	OK1	Phenyl	Benzyl	H	H	C23H17ON3S	175	0.70	78
2	OK2	Ethyl	Benzyl	H	H	C18H19ON3S	156	0.67	75
3	OK3	Phenyl	Methyl	H	H	C19H15ON3S	180	0.71	70
4	OK4	Ethyl	Methyl	H	H	C13H15ON3S	181	0.65	72
5	OK5	Phenyl	Ethyl	H	H	C18H17ON3S	160	0.71	76
6	OK6	Ethyl	Ethyl	H	H	C14H15ON3S	171	0.62	88
7	OK7	Phenyl	Benzyl	Br	Br	C23H15ON3SBr2	192	0.61	66
8	OK8	Phenyl	Methyl	Br	Br	C19H13ON3SBr2	194	0.64	65
9	OK9	Phenyl	Ethyl	Br	Br	C18H15ON3SBr2	198	0.75	80
10	OK10	Phenyl	Benzyl	Br	H	C23H16ON3SBr	181	0.67	80

 

Table No 2 Microanalysis

Sl. No.	Prod Code	C%		H%		N%
		Required	Found	Required	Found	Required	Found
1.	OK1	72.06	71.06	4.43	4.41	10.96	10.92
2.	OK2	65.59	65.29	5.46	5.06	13.50	13.46
3.	OK3	70.10	69.7	4.46	4.06	14.43	14.03
4.	OK4	60.23	59.83	5.01	4.61	16.21	15.81
5.	OK5	67.28	66.88	4.67	4.27	13.08	9.9
6.	OK6	61.53	61.13	5.49	5.09	15.38	14.98
7.	OK7	51.01	50.61	2.77	2.37	7.76	7.36
8.	OK8	43.87	43.47	2.36	1.96	9.03	8.63
9.	OK9	45.09	44.69	2.71	2.31	8.76	8.36
10.	OK10	65.59	65.19	5.46	5.06	3.60	2.66

 

 

Subtituted Mercapto triazole  Benofuran

Mercapto triazole (0.01) was dissolved in alcoholic NaoH to this substituted iodides  was added dropwise at room temperature. The reaction mixture was stirred for 2hours. The solid separated is filtered and washed with water and recrystalised from ethanol. Purity was checked by TLC plates using Benzene:Chloroform (1:2) as solvent system.

 

Pharmacological Screening:

The compounds synthesized during the present investigation were screened for their antibacterial activity. The antibacterial tests were conducted on five common microorganisms such as stabphylococcus aureus, Bacillus subtilis, Escherichia coli, Bacillus pumiills and Pseudomonas aeruginosa which are the representative types of gram positive and gram negative or organisms respectively. The antibacterial activity of the compounds was assessed by cup-plate method.

 

1.      Preparation of sub-cultures: Nutrient broth is prepared by dissolving Peptone (Bacteriological) 20 g, Beef extract (Bacteriological) 5 g, Sodium Chloride 5 g and distilled water up to 1000 ml. Stream for about 2 hour and adjust the reaction mixture P^H to about 7.2 and autoclave at 15 lbs/in2 pressure for 20 minutes. One day prior to the testing, the organisms obtained from the laboratory stock were sub-cultured into sterile nutrient broth and incubated at 37^oC for 18-24 hours. The culture growth thus obtained was used as inoculum for the antibacterial testing.

 

2.      Preparation of nutrient agar media: Weighed quantities of peptone (20g), beef extract (5g) were dissolved in distilled water by gentle warming, then the specified amount of agar (20g) was dissolved by heating on boiling bath. Then the P^H of the above solution is adjusted by adding sodium chloride (5g) and the volume of final solution is made upto 1000 ml with distilled water. Then the above prepared nutrient agar media is sterilized by autoclave at 121^oC for 20 minutes at 15 lbs/in2 pressure.

 

3.      Preparation of solutions of test compounds: 10mg of the test compound was dissolved in 10 ml. of Chloroform. From this 10 ml of solution was taken and diluted to 100 ml with Chloroform. Now the concentration of the test compound is 100 μg/1 ml.

 

4. Preparation of standard solution: The standard drug used in this testing is Penicillin –G, Streptomycin. Since both are water soluble, the concentration of this drug is adjusted so as to contain 100μg/1 ml.

 

Spectral studies.

 

Table No 3 I R Benzofuran-2-Carboxyhydrazide

Types of  vibrations	Group frequency   in wave  number (cm-1)
C-H C-O St C=C C=O st CONH	827 1234 1602 1656 3319

 

Table No-4

Types of vibrations	Group frequency in wave number (cm-1)
C-H st (Aromatic) S-C C=N C-N Monosubstituted benzene C-S	2923 2350 1597 1496 755 693

 

 

Table No- 5 Nuclear magnetic Resonance spectra (OK₁ )

Value (d)	Nature of segment	Type
6.0 – 8.0   2.5	Multiplet   Singlet	Aromatic and Hetro aromatic CH2 of S-CH2

Mass spectra.

 

Table No-6

Types of ionic peak	fragmentation
m/z OK1 fragment ions m/z OK2 fragment ions	275, 152, 135, 120, 92, 77,65 199, 171, 153, 140, 127, 111, 99, 77, 65.

 

 

Method of testing:

The above prepared nutrient agar media is cooled to 45^oC with gentle shaking to bring about uniform cooling.To this 0.5 – 0.6 ml of 18-24 hours old culture was injected aseptically and mixed well by gentle shaking. This was poured onto the petridishes (20-25 ml in each petridish of big size) and was allowed to set (1 hour).

 

Thereafter the cups were made by punching into the set agar with a sterile cork borer and scooping out the punched part of the agar. The diameter of each cup was 6mm. To these cups 0.1 ml of the test compound was put, which was prepared in chloroform. After adding the drug solution, it was allowed to diffuse for about 45 minutes, at room temperature. Then the plates were incubated at 37^oC hours in an incubator.The extent of diameter of inhibition after 24 hour was measured as the zone of inhibition in millimeters.

 

The antibacterial results are summarized in the following manner.

Antimicrobial Activity of New Subtituted Mercapto Triazole Benzofuran

 

 

Zone of inhibition in mm

Table No 7:

Sample Code	S. aureus	B. substilis	B. pumilus	E. coli	P. aeruginosa
OK1	10	14.6	12	-	8.6
OK2	-	-	-	-	-
OK3	-	-	-	-	-
OK4	10.6	10	10	7.3	10.6
OK5	12.6	14	14	12	-
OK6	23	23	22	21	21
OK7	09	-	13	18	-
OK8	09	-	09	18	10
OK9	11	16	14	08	-
OK10	09	10	11	10	-
Penicillin ( 10 u / ml )	14	18	18	-	-
Streptomycin (10 mcg/ml)	14	15	21	21	21
Chloroform	-	-	-	-	-

Note: ‘-‘ denotes no activity

Concentration of standard is 100mg/ml

 

Antifungal activity:

From the observation based on the literature survey, some of the compounds synthesized were screened for antifungal activity against a fungus Asperigillus niger.

 

The procedure used for testing antifungal activity is same as the procedure used for testing antibacterial activity, except, that the medium used for growing the fungi is different. Here for growing the fungal culture, potato dextrose medium is used.

 

Antifungal activity of New Subtituted Mercapto Triazole Benzofuran

Zone of inhibition in mm

Table No 8

Sample Code	A.niger	C.utillis
OK1	-	10
OK2	-	-
OK3	-	-
OK4	-	-
OK5	11	8
OK6	19	18
OK7	-	-
OK8	18	8
OK9	-	-
OK10	9.5	7
Grisofluvin ( 100m g / ml )	20	18
Chloroform	-	-

NOTE: Concentration of test compound is 100 mg/ml

 

RESULTS AND DISCUSSION:

The resulted 10 compounds were screened for antibacterial activity studies at a concentration of 100mg/ml using chloroform as a control against Escherchia coli, Staphylococcus aureus, B. pumillus B. substilis, and P.aeruginosa by cup plate method on nutrient agar Himedia, penicillin 10U/ml and Streptomycin 10 mg/ml are used as standard against Gram positive and Gram negative bacteria respectively. Table No 7 indicates Antimicrobial and Table No 8 indicates Antifungal activity.

 

The data in the Table 1 indicate that OK6 compound was found to possess a broad spectrum activity. While compounds OK1,OK5,and OK9 were found  to exhibit moderate activities. Among the various compound OK7 and OK8 were showed good activities against E.Coli. The tested other compounds showed good to moderate activity.

 

The antifungal activity was tested on potato dextrose agar Himedia against A.niger and C.utilis. The results showed that OK6 showed siginificant activity against both A.niger and C.utilis, but OK8 showed good activity. The other tested compounds OK5 OK10 showed good activity, but OK1 showed little antiyeast The activities were compared with  Grisofulvin (100mg/ml) as standard.

 

Table No 2 indicates Microanalysis of Synthesised compounds.Table No 3and Table No 4  indicates I R Data.Table No 5 indicates NMR data and Table No 6 Mass spectra.

 

It can be concluded that substituted mercaptotriazole as a part of synthesized molecules is responsible for antimicrobial activity. As the Carbon chain length increased at the thiol group it increases the antimicrobial activity. Bromination at benzofuran increases the Antifungal activity.The above results establish the fact that substituted benzofuran with the substituted mercaptotriazole can be studied further to search for new antimicrobial compounds.

 

REFERENCES: -

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Received on 22.03.2011        Modified on 13.04.2011

Accepted on 11.05.2011        © AJRC All right reserved