Research J. Pharm. and Tech.

9(11): November 2016

ISSN 0974-3618 (Print) www.rjptonline.org

0974-360X (Online)

RESEARCH ARTICLE

Synthesis, Characterization and Antimicrobial Activity of Acetanilide

Derivatives by using Aromatic Aldehydes and Sulphonamide Derivatives.
G. Muthu Bhupathi1*, K. Padmalatha, Akkiraju Anusha, Abdul Rameeza,
Makina Geethika Sravanthi, Sunnam Praneetha.
Department of Medicinal Chemistry, Vijaya Institute of Pharmaceutical Sciences for Women.
NH-5, Enikepadu, Vijayawada - 521 108.
*Corresponding Author E-mail: [email protected]

ABSTRACT:
Acetanilide was the first aniline derivative serendipitously found to possess analgesic as well as antipyretic
property. The literature review shows that the synthesis and characterization of acetanilide derivatives do not
have the selected aldehyde derivatives. The present work was planned to synthesis acetanilide derivatives using
different aromatic aldehyde derivatives like 4-dimethyl amino benzaldehyde; 3, 4, 5-trimethoxy benzaldehyde;
2-pyridine carbaldehyde; sulphonated hydrazine and PABA. Acetanilide was prepared by reacting aniline, acetic
anhydride and glacial acetic acid. The produced acetanilide is the substituted with different aromatic aldehydes.
Then the antimicrobial activity of synthesized acetanilide derivatives and streptomycin as standard was
performed on both gram negative (E. coli, Pseudomonas aeruginosa) and gram positive (Bacillus subtilus and
Bacillus cereus) organisms. The acetanilide derivatives of 3, 4- dimethyl benzaldehyde; 2- pyridine
carbaldehyde; sulphonated acetanilide with PABA and sulphonated acetanilide with hydrazine compounds
shows more zone of inhibition when compared streptomycin as standard. The antimicrobial effect of synthesized
compounds was significant when compared with the standard drug streptomycin.

KEYWORDS: Acetanilide, Aldehyde Derivatives, Streptomycin, Gram Positive and Gram Negative
Organisms.

INTRODUCTION:
Acetanilide is a versatile substance employed for the From the literature survey (1, 2, 6, 8, 9, 10) it is understood
synthesis of a large variety of heterocyclic compounds that acetanilide does not have the selected aldehyde
and possess broad spectrum of biological activities like derivatives. So, the present work is planned to out the
antibacterial, antiviral, antifungal, anti-inflammatory, novel synthesis, characterization and microbial activity.
analgesic. It is also used in the intermediation in rubber In the present study, the starting material acetanilide can
accelerator synthesis, dyes and dye intermediate be produced by reacting acetic anhydride with aniline.
synthesis and camphor synthesis. It is also found as a The obtained acetanilide is allowed to react with some
key intermediate for the manufacture of the sulpha drugs aromatic aldehydes such as 4-dimethyl amino
and as a precursor in the synthesis of penicillin and other benzaldehyde, 3, 4, 5-trimethoxybenzaldehyde and 2-
pharmaceuticals. pyridine carboxyaldehyde. Further the work extended to
find acetanilide derivatives in different reaction
methods. For that the synthesized acetanilide is allowed
Received on 20.06.2016 Modified on 27.06.2016 to react with chloro sulfonicacid, the result is para-
Accepted on 02.07.2016 © RJPT All right reserved sulphonyl chloride acetanilide is obtained, which is
Research J. Pharm. and Tech 2016; 9(11): 1846-1854 allowed to react with hydrazine and PABA, for getting
DOI: 10.5958/0974-360X.2016.00377.2 the new acetanilide derivatives. This study contain
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different acetanilide derivatives are obtained and their The mixture was allowed to reflux for about 1 hour.
structures are identified by using FTIR, which helps to Then the reaction mixture was cooled and filtered. Then
know the functional group present in the synthesized the obtained product was washed with alcohol and
compounds. These synthesized compounds were distilled water. The resultant product was dried and
evaluated to know the antimicrobial activity based on processed for further step.
the presence of different aromatic aldehydes and their
structure was compared with standard drug like Step-2: Acetanilide Substituted with Different
streptomycin. The aim to perform the present study was Aldehydes.
to enhance the antimicrobial activity and minimize the 0.01 mole of above synthesized acetanilide, 0.01 mole of
dose and side effects that were caused due to the different aromatic aldehydes like 2- pyridine
overdose of marketed standard drugs. carbaldehyde, 3, 4, 5 – tri methoxy benzaldehyde, 3,4 –
dimethyl amino benzaldehyde were added. Then 20 ml
MATERIALS AND METHODS: of ethanol and catalytic quantity of NaOH were added.
Chemicals: Then the mixture was stirred for about 2-3 hrs at room
Silica gel (Research Lab Fine Chem. Industry), temperature by using magnetic stirrer. The reaction
Methanol (Jiangse Hauxi International Trade Co Ltd.), mixture was monitored with TLC plate and the mixture
chloroform (Rankem Ltd.), aniline (Finar chemicals was cooled in ice bath. It was filtered and three different
Ltd.,), acetic anhydride, glacial acetic acid (Avra products were collected.
synthesis Pvt. Ltd), 3,4-dimethyl amino benzaldehyde
(Rolex Chemical Industry), 3, 4, 5 trimethoxy SCHEME-2:
benzaldehyde (Himedia Laboratory Pvt. Ltd.),2-pyridine Step-1: Synthesis of Chloro Sulphonated
carbaldehyde (Sigma), sulphonated hydrazine (Finar Acetanilide. (7, 14 21)
chemicals Ltd.), sulphonated P-amino benzoic acid 0.01mole of acetanilide was taken in a 500 ml round
(Qualikems Fine Chem Pvt. Ltd), Chlorosulphonic acid bottom flask and 32 ml chlorosulfonic acid was added
(Loba Chem. Pvt. Ltd.,), DMF (Rolex Chemical slowly with continuous shaking. Then the reaction
Industries), Formalin (Finar chemicals Ltd.,). mixture was refluxed for about 1hr on water bath. Then
the reaction mixture was cooled and poured into 150gm
Apparatus: of crushed ice. Then it was filtered and the product was
Round bottom flask, condenser, conical flask, beaker, collected and washed with distilled water. The resultant
volumetric flask, pipette, measuring cylinder, magnetic product was dried and processed for further step.
stirrer, slide, petridishes.
Step-2:(18)
Microorganisms: 0.01 mole of selected amines like PABA, Hydrazine
All these tested strains are reference and were collected were suspended in 50 ml of water and the pH 9-10 was
form NCIM (National Collection of Industrial maintained by adding basic aqueous solution of sodium
Microorganisms). carbonate (10%).
Gram –ve:
Escherichia coli (22), Pseudomonas aeruginosa(3, 5) Then the above synthesized product 4-acetamido
Gram +ve: benzene sulfonyl chloride of 0.01mole was added slowly
Bacillus subtilis(11), Bacillus cereus(12). over 10-15 minutes. After the completion of addition of
Method: compound, the reaction mixture was stirred and
Preparation of acetanilide derivatives using different monitored with TLC (n-hexane: Ethyl acetate; 7:3) for
substituted aromatic aldehydes: the completion of the reaction. Then conc. HCl was
SCHEME -1: added slowly to adjust the pH to 2 the reaction mixture
Step-1: Synthesis of Acetanilide was reserved at room temperature for 15 minutes, white
Acetanilide was prepared by taking 0.01 mole of aniline, solid was filtered, washed with distilled water and dried
0.01 mole of acetic anhydride and glacial acetic acid. to obtain the corresponding compound.

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Scheme -I

Scheme -II

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Preparation of TLC Plates :( 4) between the solvent and the stationary phase. The
1. Coating Material: development chamber was filled with a small amount of
As stationary phase, especiallyfinely ground matrix the mobile phase and capped with a lid or cork. The
silica gel, was used glass plate to form a thin layer origin of spots should not be below the solvent level in
(~0.25mm). the chamber. If the spots were submerged in the solvent,
2. Preparation of The Plate: they get washed off and lost.
There were many methods for the preparation of TLC 6. Solvent System:
plates. The most commonly used method was pouring. Mixture of two solvents was used here for determining
Slurry was poured on a plate and then the plate was Rf value. Chloroform and water in the ratio of 9:1.
tipped back and forth to spread uniformly. 7. Visualization:
3. Activation of Adsorbent: For some coloured organic compounds there is no
The plates were dried in air and then dried in oven at necessity of using colouring agents to visual the spots.
110 – 140 0C for 30 minutes. On heating, the plates were
said to be activated. Preparation of Nutrient Agar Medium :( 15)
4. Spotting the TLC Plate: Table-1 Formula:
Few milligrams of sample material were spotted on the Ingredients Quantity taken
Beef extract 0.75 gm.
TLC plate with the help of capillary tube. The spotting Peptone 1.25 gm.
capillary tube must be extremely small. To spot the Agar 3.75 gm.
plate, the end of the capillary tube was gently placed on NaCl 1.25 gm.
the coated side of the plate, and care was taken of the Distilled water Up to 250 ml.
sample.
5. Development: Weigh all the additives separately by physical balance
The prepared TLC plates were placed in development add all the additives in conical flask. Heat on water bath
chamber. Care was taken to maintain the contact with stirring till agar completely dissolved. Adjust to pH
8.0-8.4 with 5M NaOH and boil for 10 minutes. If
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necessary, filter it and adjust to pH 7.2-7.4. Sterilize by organism. Add prepared microbial suspension in the
autoclave, using 15 lb pressure at 1150C for 30 minutes. media and mix it and transfer into petri dish. Prepare the
solutions of known concentration of the standard
Preparation of agar plate :( 16, 19) preparation with respect to the concentration of the
Aseptically transfer the sterile nutrient agar in to the antibiotics to be examined. Apply the solutions to the
petri plates. This process is performed on the table top of filter discs and place them on the prepared agar plate.
laminar air flow bench. Flame the neck of culture flask. Leave the plates for 1-4 hours at room temperature.
Lift half of the lid of sterile petri-dish and add nutrient Incubate the plates in inverted position at 20-300C for
agar into it, so that it is equally distributed throughout 18 hours.
the plate then replace the closure and allow the plate to
solidify. Observation:
The zone were observed around the filter disc by
Screening for anti-microbial activity :( 13, 17, 20) antibiotic zone reader or visually.
The synthesized compounds are dissolved in DMSO in Results: During the synthesis of acetanilide derivatives
the ratio of 1:1 and were used as the sample solutions. the end point for each step was identified by performing
Streptomycin injection was used as a standard drug to TLC. The Rf value was measured for every 30 minutes
compare the anti-microbial activity. Microbial inoculum till two consecutive Rf values were
was prepared with the required quantity of test obtained.

Fig No.1: Rf values during synthesis.

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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
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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.8: Compound-4: Sulphonated para amino benzoic acid:

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

Fig No.6: Compound-2:3, 4-dimethyl amino benzaldehyde: Characterization of Acetanilide Derivatives:

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

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

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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.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
Fig No.11: 3,4 Dimethyl Amino Benzaldehyde: Cl 676.91
SO2 1150.73
Table-8: Acetanilide + 3, 4- dimethyl amino Benzaldehyde: NH2 3311.01
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.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

Fig No.12: 2-Pyridine Carbaldehyde: DISCUSSION:

The Rf values were used to determine the end point of
Table-9: Acetanilide + 2-pyridine carbaldehyde:
Functional Group Vibrations Wave Number each step involved in the synthesis of acetanilide
CH (Stretching) 3059.89 derivative. Two consecutive constant Rf values represent
CH2(Bend) 1488.01 that the reaction is complete.
C-N 1080.70
C=O 1744.60 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
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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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