Synthesis, spectral characterization and antibacterial activity of functionalized hydrazones

Article abstract of DOI:10.13005/ojc/350208

New hydrazones have been synthesized by condensing 2-acetylthiophene with acetic hydrazide and benzhydrazide. The synthesized hydrazones viz. 2-acetylthiophene acetoylhydrazone (ATAH), 2-acetylthiophene benzoylhydrazone (ATBH) are characterized in the lig

Full text of DOI:10.13005/ojc/350208

ISSN: 0970-020 X
CODEN: OJCHEG
2019, Vol. 35, No.(2):
Pg. 557-562
ORIENTAL JOURNAL OF CHEMISTRY
An International Open Access, Peer Reviewed Research Journal
www.orientjchem.org
Synthesis, Spectral Characterization and Antibacterial activity
of Functionalized Hydrazones
D. KASIMBI1, K. HUSSAIN REDDY1* and N. DEVANNA2
1Department of Chemistry, Sri Krishnadevaraya University, Ananthapuramu–515 003, India.
2Department of Chemistry, Jawaharlal Nehru Technological University, Ananthapur–515 002. India
*Corresponding author E-mail: khussainreddy@yahoo.co.in
http://dx.doi.org/10.13005/ojc/350208
(Received: January 04, 2019; Accepted: March 31, 2019)
ABSTRACT
New hydrazones have been synthesized by condensing 2-acetylthiophene with acetic
hydrazide and benzhydrazide.The synthesized hydrazones viz.2-acetylthiophene acetoylhydrazone
(ATAH), 2-acetylthiophene benzoylhydrazone (ATBH) are characterized in the light of physicochemical
and analytical data.Structures of ATAH and ATBH are confirmed by FT-IR, 1H-NMR and Mass spectral
data. The hydrazones are screened for their anti-bacterial activities against E coli, Bacillus cereus,
Staphylococcus aureus and Pseudomonas aureoginos.Acetoyl hydrazones are found to show more
antibacterial activity than the corresponding benzoyl hydrazones.
Keywords: Synthesis, Characterization, Functionlized Hydrazones, Antibacterial action.
INTRODUCTION
applications15,16. Survey of literature1,17,18 revealed
that hydrazones possessing heterocyclic group
show interesting biological and pharmacological
properties.
Hydrazoneshavinganazomethine (>C=N-NH-)
aggregate establish an imperative class of ligands for
new advancements in medication1.In this way, it is of
interest to prepare new hydrazones and to evaluate
theirbiologicalactivities.Hydrazonespossessdiverse
biological and pharmacological properties2,3 such
as antimicrobial4, anti-tubercular5 anticonvulsant6,
anti-inflammatory7, analgesic, antifungal, vasodilator8,
antiviral, anticancer9.10, antiplatelet, antimalarial,
cardio protective, antihelmintic, antiprotozoal, anti-
trypanosomal, cytotoxic11 and antischistosomiasis
activities. The chemical properties of hydrazones
have been widely investigated due to their chelating
capability12, pharmacologicalactivity13,14 andanalytical
Thiophenes are important class of
heterocyclic compounds. Thiophene derivatives
are widely used as building blocks in many
pharmaceuticals19. Thiophene derivatives are well
known for their therapeutic applications. Thiophene
nucleus is one of the most important heterocycles
exhibiting remarkable pharmacological activities20.
For example, 2-butylthiophene has been used as a
raw material in the synthesis of anticancer agents.
In the light of the above literature and in
This is an
Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC- BY).
Published by Oriental Scientific Publishing Company © 2018

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REDDY et al., Orient. J. Chem., Vol. 35(2), 557-562 (2019)
continuation of our progressing research work, here
in we report synthesis, spectral characterization
and antibacterial activity of 2-acetylthiophene
acetoylhydrazone(ATAH), 2-acetylthiophene
benzoylhydrazone (ATBH).
in 20 ml methanol was added to a hot methanolic
solution(20 ml) of 2-Acetylthiophene (0.03 mol,
2.7 ml) in a 100ml round bottom flask. Glacial acetic
acid (3-4 drops) was added to the reaction mixture.
The contents were refluxed over water bath for 2
h and cooled to room temperature. The crystalline
compound formed was collected by filtration,
washed several times with hot water, recrystallized
from methanol and dried in vacuo. Yield, 85%;
M.P., 198-200oC. Preparation of 2-acetylthiophene
benzoylhydrozone (ATBH) is shown in Scheme 2.
EXPERIMENTAL
2-acetylthiophene (AR grade) was procured
from Sigma-Aldrich Chemicals Pvt.Ltd.India.Acetic
hydrazide and benzhydrazide were purchased from
Merck chemicals. N,N- dimethyl formamide(DMF),
ethanol were of AR quality and utilized as provided.
Elemental data were obtained using a Perkin-
Elmer 2400 CHNS/O analyzer. Mass spectra
(in EI+ ionization mode)of compounds were recorded
using JEOLGCMATEIIGC-Mass spectrometer. IR
spectra of hydazones in solid state were recorded in
4000-400 cm-1 range on a Perkin-Elmer 100 FT-IR
spectrophotometer. 1H-NMR spectra were recorded
using Avanc-400 Brucker, NMR spectrometer.
Scheme 2. Synthesis of 2-acetylthiophene
benzoylhydrozone (ATBH)
Preparationof2-acetylthiopheneacetoylhydrozone
(ATAH)
The antibacterial activity of hydrazones
were screened against the Bacillus subtilis and
Staphylococcus aureus which are gram positive
and Pseudomonas aureoginosa and Escherichia
coli which are gram negative organisms
A 3.0 g (0.03) of acetic hydrazide dissolved
in 20 ml methanol was added to a hot methanolic
solution (20 ml) of 2-acetylthiophene (0.03 mol, 5.03
ml)in a 100 ml round bottom flask. Glacial acetic
acid (3-4drops) was added to the reaction mixture.
The contents were refluxed over water bath for
2 h and cooled to room temperature.The crystalline
compound formed was obtained by filtration . It was
washed a few times with hot water, recrystallized
from methanol and dried in vacuo. Yield: 85%
M.P:176-178oC. Preparation of 2-acetylthiophene
acetoylhydrozone (ATAH) is shown in Scheme 1.
Antibacterial Activity
Procedure for the Development Media:
Bacillus subtilis and Staphylococcus aureus which
are gram positive and Pseudomonas aureoginosa
and Escherichia coli which are gram negative
organisms were chosen in light of their clinical and
pharmacological significance. The bacterial strains
were got from Division of Microbiology, Osmania
College, Hyderabad.Activities of present hydrazones
are investigated on these microorganisms. The
bacterial stock societies were hatched for one
day at 37°C on supplement agar. The microscopic
organisms were developed on Mueller-Hinton agar
plates at 37°C. The stock cultures were kept up
at 4°C for the development of organisms potato
dextrose agar was utilized. Antibacterial activity
of hydrazones was determined by using zone of
hindrance technique
Scheme 1. Synthesis of 2-acetylthiophene
acetoylhydrozone (ATAH)
Preparationof2-acetylthiopheneBenzoylhydrozone
(ATBH)
Preparation of Discs
Whatman No.1 channel paper plates of
5mm width were autoclaved by keeping in a spotless
A 3 g (0.02mol) of benzhydrazide dissolved

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REDDY et al., Orient. J. Chem., Vol. 35(2), 557-562 (2019)
and dry Petri plate. The plates were absorbed
compound answers for 5 h were taken as test
material. Following 5 h the circles were dried in
shade. The groupings of compound arrangements
per plate are represented 0.1 g /1 ml. In this manner
they were cautiously exchanged to spread on refined
Petri plates. Channel paper plates inundated in
ethanol are arranged and utilized as positive control
and streptomycin as negative control.
the plates independently containing set agar media.
The prepared sterile channel paper circles were
impregnated with the sample solution and shaken
completely and these test plates hatched for 2 days
at 37oC for the improvement of inhibitory zones and
the average of two readings were recorded.
Measuring the diameter of inhibition zone
The inhibition zones were measured after
1 day at 37oC. The diameter of the inhibition zone
was measured and recorded with the aid of plastic
ruler. Five paper discs placed in one Petri plate.
Testing of antibacterial activity
LB agar medium was prepared and it
was sanitized at 121°C for 30 minutes. The agar
plates were prepared21 by pouring about 10 ml
of the medium into 10 cm Petri dishes under
aseptic condition and left undisturbed for 2 h to
harden the medium. 1 ml of inoculum (containing
suspension) of microorganisms culture (Bacillus
subtilis, Staphylococcus aureus, Pseudomonas
aureoginosa and Escherichia coli) was poured on to
RESULTS AND DISCUSSION
The hydrazones are partially soluble in water,
less soluble methanol more soluble in ethanol and
readily soluble in acetonitrile (CH3CN), DMF and DMSO.
The colours, formula weight, yields and elemental
analysis of hydrazones are summarized in Table 1
Table 1: Physicochemical and analytical data of hydrazones
S.No
Hydrazone
Mol.Wt.
Colour
Yield% %C Found (Calc.) %H Found (Calc.)
%N Found (Calc.)
01
02
ATAH
ATBH
182
244
Colourless
Pale White
85
83
53.25(52.74)
64.50(63.93)
5.525.49
4.85(4.91)
15.30(15.38)
11.60(11.47)
The hydrazones are further characterized
using (i) Fourier-Transform Infrared spectroscopy, (ii)
NMR spectroscopy and (iii) Mass spectrometry (i)
FT-IR spectroscopy: IR spectra of ATAH and ATBH
are shown in Fig.1 and 2 respectively.IR spectral data
values and assignment of peaks are given in Table 2.
Fig. 1. IR spectrum of ATAH in KBr medium
Fig. 2. IR spectrum of ATBH in KBr medium
(ii) 1H-NMR spectroscopy
Fig. 2. IR spectrum of ATBH in KBr medium
1H-NMR spectra of ATAH and ATBH are
shown in Fig.3 and 4 respectively.Peak assignments
are given in Table 3.
ATAH
ATBH
Assignment
3312
3072
2928
1666
1606
1017
3327
3073
-
1651
1576
1020
υNH (stretching)
υCH (Aromatic stretching)
υCH ( Aliphatic stretching)
υC=O (stretching)
υC=N (stretching)
υN-N (stretching)
(iii) Mass spectra
Mass spectra of ATAH and ATBH are shown

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REDDY et al., Orient. J. Chem., Vol. 35(2), 557-562 (2019)
Table 3: Assignment of peaks observed in NMR
spectra of ATAH and ATBH
in Fig.5 and 6 respectively.Fragmentation Schemes
are given in Figs. 7 and 8.
Hydazone Chemical
Multiplicity No. of
protons
Assignment
Shift(ppm)
ATAH
ATBH
2.28
2.35
7.00-7.30
9.5
2.34
7.03-8.00
Singlet
Singlet
Multiplet
Singlet
Singlet
Multiplet
3
3
3
1
3
8
Acetoyl CH
Acetoyl CH33
Thiophene H
Imine H i.e.,>NH
Acetyl CH3
Thiphene H+
Benzoyl H
8.885
Singlet
1
Imine H i.e.,>Nh
Mass spectra of ATAH and ATBH show
molecular ion peak at m/z values 182 and 244
respectively corresponding to their molecular
weights. According to nitrogen rule even mass
numbers of ATAH and ATBH indicate even number
of nitrogen atoms. Fragmentation Scheme of ATAH
is shown in Figure 7.
Fig. 3. 1H-NMR spectrum of ATAH in CDCl3
CH3
N
NH
C
O
CH3
C
S
(-15)
m/z = 167
CH3
NH
(-)CH3
(-15)
C
C
N
S
S
(-)CH3CO
(-43)
N
NH2
m/z = 124
m/z = 139
O
C
(-105)
CH3
CH3
NH NH
CH3
C
H
S
C
S
N
O
NH
CH3
m/z = 111
C
m/z = 182
O
C CH3
CH2
C
H
N
NH
Fig. 4. 1H-NMR spectrum of ATBH in CDCl3
(-99)
S
m/z = 83
O
C4H3S CH3
C
N
N
C CH3
m/z = 99
Fig. 7. Mass fragmentation Scheme of ATAH
Mass fragmentation pattern scheme of
ATBH is given in Figure 8
PhC
O
CH3
N
NH
C
O
C
N
(-105)
C
H
S
S
(-15)
NH2
m/z = 229
m/z = 113
O
NH NH
C
C6H5
NH
C
S
(-134)
Fig. 5. Mass spectrum of ATAH
m/z = 110
CH3
C
PhC
(-105)
O
CH2
CH3
N
2H
C
S
S
C
S
(-29)
N
NH
N
O
NH
C
m/z = 110
m/z = 139
m/z = 244
C6H5C
O
CO
(C6H7N2S)
(-139)
C6H9N2OS
(-139)
C6H5
m/z = 105
m/z = 77
C
O
m/z = 105
Ph
O
NH
C
N
H3C
C
(-161)
S
Fig. 8. Mass fragmentation Scheme of ATBH
Fig. 6. Mass spectrum of ATBH

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REDDY et al., Orient. J. Chem., Vol. 35(2), 557-562 (2019)
The diameters of the zones of complete
inhibition were measured in millimeters(mm) and
data are given in the Table 4.
gram negative bacterial species but at maximum
concentrations the activity is not as expected due
to less diffusion rate in agar medium.
In our present study we are investigating
our compounds which are more antibacterial in
nature and it makes platform for preparation of
effective chemotherapeutics agents for therapy of
human pathogenic diseases.
Antibacterial assay
The antibacterial activity of hydrazones
were screened against the Bacillus subtilis and
Staphylococcus aureus which are Gram positive
and Pseudomonas aureoginosa and Escherichia
coli which are Gram negative organisms. Typical
photographs are shown Figure 9.
The antibacterial activities of our hydrazone
compounds are comparable to with standard drug
Streptomycin. Acetoyl hydrazones (APAH and ATAH)
compounds show more activity when compared with
benzoyl hydrazone(APBH and ATBH) compounds,
All the compounds show gradually increasing activity
with concentrations towards both gram positive and
Bacillus
E.Coli
Staphylococcus
Fig. 9. Typical photographs of agar plates showing
antibacterial activity of 1)APAH, 2) APBH, 3)ATAH, 4) ATBH,
5) Etanol blank(Control) and 6) Streptomycin(Reference)
Table 4: Antibacterial activity of a group of closely ATAH and ATBH and closely related
Hydrazones (Zone of inhibition is given in mm)
Compound
E Coli
Bacillus
Staphylococcus aureus
P.aureoginosa
A
B
C
A
B
C
A
B
C
A
B
C
APAH1
4.2
2.0
4.0
5.5
5.5
5.6
4.0
6.0
4.5
6.6
6.6
6.0
6.5
5.5
7.5
5.0
5.0
5.0
4.0
6.6
6.0
5.5
6.0
5.0
7.5
6.5
6.0
7.0
6.0
9.5
7.0
4.0
8.0
6.0
9.0
8.0
7.0
6.0
5.0
5.0
6.0
8.0
7.0
5.0
5.0
8.0
8.5
7.5
APBH2
ATAH3
6.0
8.0
9.0
6.0
ATBH4
4.0
6.0
7.0
6.0
Streptomycin
10.0
12.0
13.0
10.0
1APAH = 2-Acetylpyridine acetoylhydrazone : 2APBH = 2-Acetylpyridine benzoylhydrazone
3ATAH = 2-Acetylthiophene acetoylhydrazone: 4ATBH = 2-Acetyl thiophene benzoylhydrazone
Compound Taken: A= 200mg; B = 300mg; C = 500mg
CONCLUSION
assistance provided by UGC, New Delhi in the form
of One-time Grant Scheme [ Lr. No. F. 19-106/2013
(BSR)]. The authors also thank UGC and DST, New
Delhi for giving equipment facility under SAP and FIST
programs respectively.Further, the authors thank SAIF,
IIT-M for providing 1H-NMR and Mass spectral data.
We have synthesized and characterized
two closely related functionalized hydrazones
bearing thiophenemoiety and evaluated their
anti-bacterial activity. Acetoyl hydrazones are
found to show more antibacterial activity than the
corresponding benzoyl hydrazones.
CONFLICT OF INTEREST
The authors declare that there is no
conflict of interests regarding the publication of
this article.
ACKNOWLEDGEMENT
KHR gratefully acknowledges the financial
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