INTRODUCTION:

Acetanilide is a versatile substance employed for the synthesis of a large variety of heterocyclic compounds and possess broad spectrum of biological activities like antibacterial, antiviral, antifungal, anti-inflammatory, analgesic. It is also used in the intermediation in rubber accelerator synthesis, dyes and dye intermediate synthesis and camphor synthesis. It is also found as a key intermediate for the manufacture of the sulpha drugs and as a precursor in the synthesis of penicillin and other pharmaceuticals.

From the literature survey^{(1, 2, 6,
8, 9, 10)} it is understood that acetanilide does not have the selected aldehyde derivatives. So, the present work is planned to out the novel synthesis, characterization and microbial activity.

In the present study, the starting material acetanilide can be produced by reacting acetic anhydride with aniline. The obtained acetanilide is allowed to react with some aromatic aldehydes such as 4-dimethyl amino benzaldehyde, 3, 4, 5-trimethoxybenzaldehyde and 2- pyridine carboxyaldehyde. Further the work extended to find acetanilide derivatives in different reaction methods. For that the synthesized acetanilide is allowed to react with chloro sulfonicacid, the result is para-sulphonyl chloride acetanilide is obtained, which is allowed to react with hydrazine and PABA, for getting the new acetanilide derivatives. This study contain different acetanilide derivatives are obtained and their structures are identified by using FTIR, which helps to know the functional group present in the synthesized compounds. These synthesized compounds were evaluated to know the antimicrobial activity based on the presence of different aromatic aldehydes and their structure was compared with standard drug like streptomycin. The aim to perform the present study was to enhance the antimicrobial activity and minimize the dose and side effects that were caused due to the overdose of marketed standard drugs.

MATERIALS AND METHODS:

Chemicals:

Silica gel (Research Lab Fine Chem. Industry), Methanol (Jiangse Hauxi International Trade Co Ltd.), chloroform (Rankem Ltd.), aniline (Finar chemicals Ltd.,), acetic anhydride, glacial acetic acid (Avra synthesis Pvt. Ltd), 3,4-dimethyl amino benzaldehyde (Rolex Chemical Industry), 3, 4, 5 trimethoxy benzaldehyde (Himedia Laboratory Pvt. Ltd.),2-pyridine carbaldehyde (Sigma), sulphonated hydrazine (Finar chemicals Ltd.), sulphonated P-amino benzoic acid (Qualikems Fine Chem Pvt. Ltd), Chlorosulphonic acid (Loba Chem. Pvt. Ltd.,), DMF (Rolex Chemical Industries), Formalin (Finar chemicals Ltd.,).

Apparatus:

Round bottom flask, condenser, conical flask, beaker, volumetric flask, pipette, measuring cylinder, magnetic stirrer, slide, petridishes.

Microorganisms:

All these tested strains are reference and were collected form NCIM (National Collection of Industrial Microorganisms).

Gram –ve:

Escherichia coli ⁽²²⁾, Pseudomonas aeruginosa^(3,
5)

Gram +ve:

Bacillus subtilis⁽¹¹⁾, Bacillus cereus⁽¹²⁾.

Method:

Preparation of acetanilide derivatives using different substituted aromatic aldehydes:

SCHEME -1:

Step-1: Synthesis of Acetanilide

Acetanilide was prepared by taking 0.01 mole of aniline, 0.01 mole of acetic anhydride and glacial acetic acid. The mixture was allowed to reflux for about 1 hour. Then the reaction mixture was cooled and filtered. Then the obtained product was washed with alcohol and distilled water. The resultant product was dried and processed for further step.

Step-2: Acetanilide Substituted with Different Aldehydes.

0.01 mole of above synthesized acetanilide, 0.01 mole of different aromatic aldehydes like 2- pyridine carbaldehyde, 3, 4, 5 – tri methoxy benzaldehyde, 3,4 – dimethyl amino benzaldehyde were added. Then 20 ml of ethanol and catalytic quantity of NaOH were added.

Then the mixture was stirred for about 2-3 hrs at room temperature by using magnetic stirrer. The reaction mixture was monitored with TLC plate and the mixture was cooled in ice bath. It was filtered and three different products were collected.

SCHEME-2:

Step-1: Synthesis of Chloro Sulphonated

Acetanilide.^{(7, 14 21)}

0.01mole of acetanilide was taken in a 500 ml round bottom flask and 32 ml chlorosulfonic acid was added slowly with continuous shaking. Then the reaction mixture was refluxed for about 1hr on water bath. Then the reaction mixture was cooled and poured into 150gm of crushed ice. Then it was filtered and the product was collected and washed with distilled water. The resultant product was dried and processed for further step.

Step-2:⁽¹⁸⁾

0.01 mole of selected amines like PABA, Hydrazine were suspended in 50 ml of water and the pH 9-10 was maintained by adding basic aqueous solution of sodium carbonate (10%).

Then the above synthesized product 4-acetamido benzene sulfonyl chloride of 0.01mole was added slowly over 10-15 minutes. After the completion of addition of compound, the reaction mixture was stirred and monitored with TLC (n-hexane: Ethyl acetate; 7:3) for the completion of the reaction. Then conc. HCl was added slowly to adjust the pH to 2 the reaction mixture was reserved at room temperature for 15 minutes, white solid was filtered, washed with distilled water and dried to obtain the corresponding compound.

Preparation of TLC Plates :^{( 4)}

1. Coating Material:

As stationary phase, especiallyfinely ground matrix silica gel, was used glass plate to form a thin layer (~0.25mm).

2. Preparation of The Plate:

There were many methods for the preparation of TLC plates. The most commonly used method was pouring. Slurry was poured on a plate and then the plate was tipped back and forth to spread uniformly.

3. Activation of Adsorbent:

The plates were dried in air and then dried in oven at 110 – 140 ⁰C for 30 minutes. On heating, the plates were said to be activated.

4. Spotting the TLC Plate:

Few milligrams of sample material were spotted on the TLC plate with the help of capillary tube. The spotting capillary tube must be extremely small. To spot the plate, the end of the capillary tube was gently placed on the coated side of the plate, and care was taken of the sample.

5. Development:

The prepared TLC plates were placed in development chamber. Care was taken to maintain the contact between the solvent and the stationary phase. The development chamber was filled with a small amount of the mobile phase and capped with a lid or cork. The origin of spots should not be below the solvent level in the chamber. If the spots were submerged in the solvent, they get washed off and lost.

6. Solvent System:

Mixture of two solvents was used here for determining R_f value. Chloroform and water in the ratio of 9:1.

7. Visualization:

For some coloured organic compounds there is no necessity of using colouring agents to visual the spots.

Preparation of Nutrient Agar Medium :^{( 15)}

Table-1 Formula:

Ingredients	Quantity taken
Beef extract	0.75 gm.
Peptone	1.25 gm.
Agar	3.75 gm.
NaCl	1.25 gm.
Distilled water	Up to 250 ml.

Weigh all the additives separately by physical balance add all the additives in conical flask. Heat on water bath with stirring till agar completely dissolved. Adjust to pH 8.0-8.4 with 5M NaOH and boil for 10 minutes. If necessary, filter it and adjust to pH 7.2-7.4. Sterilize by autoclave, using 15 lb pressure at 1150C for 30 minutes.

Preparation of agar plate :^{( 16, 19)}

Aseptically transfer the sterile nutrient agar in to the petri plates. This process is performed on the table top of laminar air flow bench. Flame the neck of culture flask. Lift half of the lid of sterile petri-dish and add nutrient agar into it, so that it is equally distributed throughout the plate then replace the closure and allow the plate to solidify.

Screening for anti-microbial activity :^{( 13, 17, 20)}

The synthesized compounds are dissolved in DMSO in the ratio of 1:1 and were used as the sample solutions. Streptomycin injection was used as a standard drug to compare the anti-microbial activity. Microbial inoculum was prepared with the required quantity of test organism. Add prepared microbial suspension in the media and mix it and transfer into petri dish. Prepare the solutions of known concentration of the standard preparation with respect to the concentration of the antibiotics to be examined. Apply the solutions to the filter discs and place them on the prepared agar plate. Leave the plates for 1-4 hours at room temperature. Incubate the plates in inverted position at 20-300C for 18 hours.

Observation:

The zone were observed around the filter disc by antibiotic zone reader or visually.

Results: During the synthesis of acetanilide derivatives the end point for each step was identified by performing TLC. The R_f value was measured for every 30 minutes till two consecutive Rf values were

obtained.

Fig No.1: Rf values during synthesis.

Antimicrobial activity:

Fig No.2: Zone of inhibition of Gram +ve bacteria

Fig No.3: Zone of inhibition of Gram –ve bacteria:

Fig No. 4: Zone of inhibition of standard-Streptomycin:

The antimicrobial activity for synthesized compounds was evaluated and the results were tabulated as follows:

Table No.2: Zone of inhibition of 3, 4, 5- trimethoxy benzaldehyde:

Organism	50 gm.	100 gm.	150 gm.	Standard
E. Coli	8 mm	10 mm	10 mm	19 mm
Pseudomonas Aeruginosa	6 mm	6 mm	7 mm	10 mm
Bacillus Subtilis	8 mm	10 mm	12 mm	12 mm
Bacillus Cereus	5 mm	6 mm	7 mm	15 mm

Table No.3: Zone of Inhibition of 3, 4-Dimetyl Amino Benzaldehyde:

Organism	50 gm.	100 gm.	150 gm.	Standard
E. Coli	15 mm	17 mm	20 mm	19 mm
Pseudomonas Aeruginosa	5 mm	5 mm	6 mm	10 mm
Bacillus Subtilis	7 mm	9 mm	12 mm	12 mm
Bacillus Cereus	8 mm	10 mm	13 mm	15 mm

Table No.4: Zone of Inhibition of 2-Pyridine Carbaldehyde :

Organism	50 gm.	100 gm.	150 gm.	Standard
E. Coli	7 mm	10 mm	8 mm	19 mm
Pseudomonas Aeruginosa	8 mm	10 mm	17 mm	10 mm
Bacillus Subtilis	5 mm	7 mm	9 mm	12 mm
Bacillus Cereus	6 mm	9 mm	10 mm	15 mm

Table No.5: Zone of Inhibition of Para Amino Benzoic Acid:

Organism	50 gm.	100 gm.	150 gm.	Standard
E. Coli	8 mm	10 mm	10 mm	19 mm
Pseudomonas Aeruginosa	6 mm	6 mm	7 mm	10 mm
Bacillus Subtilis	8 mm	10 mm	12 mm	12 mm
Bacillus Cereus	5 mm	6 mm	7 mm	15 mm

Table No.6: Zone of Inhibition of Hydrazine:

Organism	50 gm.	100 gm.	150 gm.	Standard
E. Coli	14 mm	18 mm	21 mm	19 mm
Pseudomonas Aeruginosa	7 mm	5 mm	5 mm	10 mm
Bacillus Subtilis	9 mm	10 mm	12 mm	12 mm
Bacillus Cereus	8 mm	7 mm	7 mm	15 mm

Fig No.5: Compound-1:3, 4, 5-trimethoxy benzaldehyde:

Fig No.6: Compound-2:3, 4-dimethyl amino benzaldehyde:

Fig No.7: Compound-3:2- Pyridine carbaldehyde:

Fig No.8: Compound-4: Sulphonated para amino benzoic acid:

Fig No.9: Compound-5: Sulphonated Hydrazine:

Characterization of Acetanilide Derivatives:

Fig No.10: 3, 4,5trimethoxybenzaldehyde:

IR INTERPRETATION:

Table-7: Acetanilide + 3, 4, 5-tri Methoxy Benzaldehyde:

Functional Group Vibrations	Wave Number
CH (Stretching)	3059.39
CH₂(Bend)	1436.49
C-N	1080.87
CH₃O	3932.76
C=O	1743.70

Fig No.11: 3,4 Dimethyl Amino Benzaldehyde:

Table-8: Acetanilide + 3, 4- dimethyl amino Benzaldehyde:

Functional group vibrations	Wave number
CH (stretching)	3079.33
CH₂(bend)	1487.62
C-N	1065.61
CH₃	2922.23
NH₂	3290.29

Fig No.12: 2-Pyridine Carbaldehyde:

Table-9: Acetanilide + 2-pyridine carbaldehyde:

Functional Group Vibrations	Wave Number
CH (Stretching)	3059.89
CH₂(Bend)	1488.01
C-N	1080.70
C=O	1744.60

Fig No.13: Para amino benzoic acid:

Table-10: Chloro Sulphonated Acetanilide + Para Amino Benzoic Acid:

Functional Group Vibrations	Wave Number
CH (Stretching)	2925.04
CH₂(Bend)	1462.87
C-N	1090.64
COOH	1913.24
Cl	676.91
SO₂	1150.73
NH₂	3311.01

Fig No.14: Hydrazine:

Table-11: Chloro Sulphonated Acetanilide + Hydrazine:

Functional Group Vibrations	Wave Number
CH (stretching)	3071.19
CH₂(bend)	1469.56
C-N	1157.41
Cl	692.02
SO₂	1312.61

DISCUSSION:

The R_f values were used to determine the end point of each step involved in the synthesis of acetanilide derivative. Two consecutive constant R_f values represent that the reaction is complete.

The antimicrobial activity was performed by using synthesized acetanilide derivatives (2-PCA, 3, 4, 5-TBA, 3,4-DMBA, PABASO2A, HySO2A) and streptomycin as standard on both gram negative (Escherichia coli, Pseudomonas aeruginosa) and gram positive(Bacillus subtilus and Bacillus cereus) organism. The zone of inhibition was observed around the paper discs placed on the nutrient agar in petridishes. This zone was measured by antibiotic zone reader. These can be easily interpreted using histograms. Two histograms were plotted that represent, the effect of synthesized compounds (2-PCA, 3, 4, 5-TBA, 3,4-DMBA, PABASO2A, HySO2A) on different microorganisms.

Among all the synthesized compounds acetanilide with sulphonated Para amino benzoic acid, acetanilide with sulphonated Hydrazine, 3,4- dimethyl amino benzaldehyde shows more antimicrobial activity when compared with the standard streptomycin as the zone of inhibition of compound acetanilide with 3,4dimethyl amino benzaldehyde, acetanilide with sulphonated Para amino benzoic acid, acetanilide with sulphonated hydrazine is more than all the synthesized compounds and also the standard. Among all the synthesized compounds compound 2-pyridine carbaldehyde shows more antimicrobial activity when compared with the standard streptomycin, as the zone of inhibition of compound 2-pyridine carbaldehyde is more than all the synthesized compound and also the standard.

Among all the synthesized compounds compound acetanilide with sulphonated Para amino benzoic acid and acetanilide with sulphonated hydrazine shows more antimicrobial activity when compared with the standard streptomycin as the zone of inhibition of compound acetanilide with sulphonated Para amino benzoic acid and acetanilide with sulphonated hydrazine is more than all the synthesized compounds and also the standard.

All the synthesized compounds show very significant activity like streptomycin. The functional groups were analyzed by FTIR spectroscopy elemental analysis.

CONCLUSION:

The functional groups that were present in the synthesized compounds were identified by using FTIR spectroscopy. The antimicrobial study was positive for gram negative (Escherichia coli, Pseudomonas aeruginosa) and gram positive (Bacillus subtilus, Bacillus cereus) microorganisms.The novel synthesis of acetanilide derivatives showed significant difference from standard drugs in zone of inhibition especially it was found more in the acetanilide derivatives of 3, 4- dimethyl benzaldehyde, 2-pyridine carbaldehyde, and sulphonated acetanilide with Para amino benzoic acid and sulphonated acetanilide with hydrazine compounds.

The antimicrobial effect of synthesized compounds was significant when compared with the standard drug streptomycin.

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Received on 20.06.2016 Modified on 27.06.2016

Research J. Pharm. and Tech 2016; 9(11): 1846-1854

DOI: 10.5958/0974-360X.2016.00377.2