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: muthuboopathi@rediffmail.com

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
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.
Received on 20.06.2016 Modified on 27.06.2016
Accepted on 02.07.2016 © RJPT All right reserved
Research J. Pharm. and Tech 2016; 9(11): 1846-1854
DOI: 10.5958/0974-360X.2016.00377.2
1846
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
 Research J. Pharm. and Tech. 9(11): November 2016
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.
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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.
 Research J. Pharm. and Tech. 9(11): November 2016

Scheme -I

Scheme -II

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 Research J. Pharm. and Tech. 9(11): November 2016

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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 Research J. Pharm. and Tech. 9(11): November 2016
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
Fig No.1: Rf values during synthesis.
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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 Rf value was measured for every 30 minutes
till two consecutive Rf values were
obtained.
 Research J. Pharm. and Tech. 9(11): November 2016

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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 Research J. Pharm. and Tech. 9(11): November 2016

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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 Research J. Pharm. and Tech. 9(11): November 2016

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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 Research J. Pharm. and Tech. 9(11): November 2016
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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