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Rasayan J. Chem., 17(1), 281-287(2024)
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SPECTRAL ELUCIDATION, ANTIMICROBIAL AND
ANTIOXIDANT STUDY OF NEWLY SYNTHESIZED
PYRAZOLINE DERIVATIVES
V. Kanchana1, L. Mayavan2, A. Kistan3, and S. Mohan4
1Department of Chemistry, Sree Sastha Institute of Engineering and Technology,
Chennai, (Tamil Nadu) India
2Department of English, Panimalar Engineering College (Autonomous),
Chennai-600123, (Tamil Nadu) India
3Department of Chemistry, Panimalar Engineering College (Autonomous),
Chennai-600123, (Tamil Nadu) India
4Department of Chemistry, Rajalakshmi Engineering College (Autonomous),
Chennai-602105,(Tamil Nadu) India
Corresponding Author: drakistan14@panimalar.ac.in
ABSTRACT
The preparation of Chalcones and Pyrazoline derivatives from different aromatic ketones and substituted aldehydes
is detailed. Chalcones were produced by reacting aldehydes with substituted aromatic ketones, such as
acetophenones, in the Claisen-Schmidt condensation reaction. Cyclohexenone derivatives are produced when ethyl
aceto aromatic ketones undergo base-catalyzed cyclo-condensation to Chalcones under microwave radiation. The
synthesized compounds are characterized using spectrum techniques from 1H NMR, 13C NMR, and IR. These are
examined for their antioxidant qualities as well as their antibacterial action against Staphylococcus aureus,
Escherichia coli, Aspergillus niger, and Aspergillus flavus.
Keywords: Chalcones, Pyrazoline, Antibacterial Activity, Antifungal Activity, Spectral Elucidation.
RASĀYAN J. Chem., Vol. 17, No.1, 2024
INTRODUCTION
Because of their numerous potential uses in scientific fields like synthetic and medicinal chemistry,
natural Chalcones and their synthetic derivatives have garnered a lot of interest.1,2 These are useful
intermediates in organic synthesis from a chemical perspective, and when basic catalysis is present, they
can function as activated unsaturated systems in the conjugated addition reaction of carbanions.3,4
Utilizing this kind of reaction could lead to the production of derivatives of cyclohexenone that are highly
functionalized5,6 Chalcones have a remarkable range of biological activities linked to them, such as blood
platelet anti-aggregating activity and antitubercular, antiparkinson, antitubercular, anti-inflammatory,
anticancer, antiasthmatic, antibacterial, and antihypertensive properties. Chalcones and their derivatives
are important biologically, but they also have a wide range of uses in cosmetic compositions and dyes.7-18
Another class of cyclic compounds with intriguing biological activities are cyclohexenone derivatives of
chalcones; among these, its antimicrobial activity is well-documented18,19 Cyclohexenone are therefore
highly desirable target molecules in synthetic organic chemistry. Chalcones are used more frequently in
the synthesis of cyclohexenone, which is an effective precursor for the synthesis of isoxazolones,
benzisoxazoles, carbazole derivatives, and indazoline pyrazoline. Derivatives of cyclohexenone are well-
known lead compounds for the management of autoimmune disorders and inflammation.20-24
Under microwave radiation, chemical reactions can be sped up and the resulting products' selectivity can
be achieved.25 Compared to traditional heating, this method has a number of benefits, including fast and
instantaneous heating, high temperature homogeneity, and selective heating.26 Because microwave-
assisted synthesis delivers on its promise of being a quick synthesis technique, synthetic organic
chemistry has made extensive use of the microwave heating technique. Given the significance of
Chalcones' cyclohexenone derivatives both chemically and biologically, a report has been made on the
high yield synthesis of these derivatives using the microwave technique.
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PYRAZOLINE DERIVATIVES A. Kistan et al.
1- Pyrazoline 2 - Pyrazoline 3- Pyrazoline
Fig.-1
In vitro antimicrobial activity against Staphylococcus aureus, Escherichia coli, Aspergillus Niger, and
Aspergillus flavus is assessed after the synthesized compounds undergo structural elucidation. Because of
the encouraging outcomes, we decided to further test them for antioxidant activity.27
EXPERIMENTAL
Chemicals
All chemical reagents were procured from Sigma Aldrich and the reputable Merck pharmaceutical
company located in Mumbai, India. Glacial acetic acid and solvents were provided by S.D. Fine
(Mumbai, India) and were of LR mark. Spectrochem, India, supplied the ethanol solvent. The following
were obtained from Sigma-Aldrich and CDH (New Delhi, India): Cyanuric chloride, 4-amino acetanilide,
1,3-benzodioxole-5-carbaldehyde(piperonal), 4-fluorobenzaldehyde, 4-chlorbenzaldehyde, thiophene-2-
carbaldehyde, malononitril, and hydrazine hydrate. The entire set of reagents and solvents used in this
investigation's heterocyclic derivative synthesis were purified using conventional methods. As developing
solvents, benzene, ethyl acetate, and chloroform were employed.28
Thin Layer Chromatography (TLC)
A vision apparatus was used to record the melting points. Using silica gel G (Merck) as the immobile
phase and an iodine chamber and UV lamp to visualize the TLC points, reaction improvement was
monitored on TLC. The exposure to iodine gas, heating the plates dipped in diluted KMnO4 stain, and
ultraviolet lighting all contributed to the visualization of points on TLC plates. Highly pure silica gel for
the Column Chromatography (CC) technique was acquired from Sigma Aldrich & Co. Absolute ethanol
was used to recrystallize every compound that was synthesized.
IR Spectra
Using a KBr pellet and a JASCO spectrophotometer, the solid-state IR spectra were recorded using
Perkin-Elmer Spectrum IR version 10.6.0. FT-IR data were also obtained with a Jasco-400 spectrometer
for the synthetic organic mixes.
NMR Spectra
Every NMR spectrum recorded from the instruments was recorded using a Bruker AMX 400 MHz
instrument and five-millimetre PABBO/BB-1H pipes. Proton (1H) NMR spectra were recorded at
frequencies between 300 and 400 MHz using 0.03 M solutions in CDCl3 or d6-DMSO, with
Tetramethylsilane serving as the internal reference chemical shift. Using Tetra-methylsilane as the inner
reference chemical shift, 13C NMR spectra were recorded at 75 MHz or 100 MHz using approximately
0.05 M solutions in CDCl3 or d6-DMSO.
Mass Spectra
The electron spray ionization-MS electrode WATERS-Q-TOF premier-HAB213 was used to perform the
mass spectrum.
Elemental Analysis
A thermo fanning Flash EA1112 CHN (STIUJA) elemental analyser was used to obtain the elemental
analysis. The yield that is reported is the isolated yield following compound purification.
General Procedure for Pyrazoline Derivative Synthesis(a-e)
Synthesis of Derivatives of Pyrazoline Standard protocol for N1-(4, 6-dichloro-1,3,5-triazin-2-yl)
synthesis -N4-benzene-1,4-diamine (a-f) (4,5-dihydro-5-substituted-1H-pyrazol 3yl)
Equimolar amounts of diluted substituted hydrazine hydrate and substituted benzaldehydes (0.01 M) in 40
millilitres of ethanol. For eight hours, this mixture was refluxed. After that, it was added to crushed ice
and vented. After being separated, strained, cleaned with distilled water, and recrystallized from Power
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PYRAZOLINE DERIVATIVES A. Kistan et al.
alcohol, the resulting products were deemed to be (a-f). TLC was used to track the reaction's progress
while n-hexane (1:3) and ethyl acetate served as the solvents. After ethanol was used to recrystallize the
resultant product, it was dried. Six final compounds and all six intermediate compounds were synthesized.
Melting point values, IR spectral data, H1 NMR data, C13 NMR data, mass spectra data, and elemental
analysis were used to describe the structures of the synthesized compounds.
Fig.-2: Synthesis of Derivatives of Pyrazoline (1a – 1f)
Table-1: Antibacterial Activity of Pyrazoline Derivatives
S.No. Compounds Bacillus
subtilis
(mm)
Staphylococcus
aureus
(mm)
Salmonella
typhi
(mm)
Escherichia
coli
(mm)
1
1
-
a
5
8
9
8
2
1
-
b
7
10
11
10
3
1
-
c
3
9
5
8
4
1
-
d
6
9
14
9
5
1
-
e
5
11
8
10
6 1-f 6 8 9 7
Standard
Ciprofloxacin
(mm)
8
12
20
15
Control
DMSO
-
-
-
-
Table-2: Antifungal Activity of Pyrazoline Derivatives
S.
No
.
Compounds
Aspergillus niger
(mm)
Candida Albicans
(mm)
1
1
-
a
8
2
2
1
-
b
4
2
3
1
-
c
2
3
4
1
-
d
8
7
5
1
-
e
6
3
6 1-f 5 2
Standard Amphotericin-B(mm) 12 10
Control
DMSO
-
-
Larvicidal Activity
Larvae were separated into five collections of 20 and placed in 249 millilitres of distilled water and 1.0
millilitres of the intended chemical extract concentration for the bioassay tests. After a 24-hour exposure,
the number of dead larvae was counted, and the percentage of mortality was calculated using the mean of
five replicates (Table-3).28-30
Mosquito Larvae Consumed
Larvae of Anopheles subpictus were acquired from Pondicherry's Vector Control Research Centre. The
larvicidal action was measured using the World Health Organization's (WHO) methodology with minor
modifications.31, 32
Table-3: Larvicidal Activity of Pyrazoline Derivatives
S.No. Compounds designation % of Inhibition
1
1
-
a
70%
2
1
-
b
68%
3
1
-
c
79%
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PYRAZOLINE DERIVATIVES A. Kistan et al.
Antioxidant Activity
Hi Media Laboratory Pvt. Ltd. provided ascorbic acid and DPPH (1, 1-diphenyl-2-picrylhydrazyl), and all
other reagents, chemicals, and solvents used in the study were of analytical grade.
In-vitro Anti-Oxidant Activity
DPPH-Scavenging Activity
To evaluate the 1, 3-diphenyl-2-propene-1-one derivatives' ability to scavenge free radicals, the most
stable radical, 2-diphenyl-picrylhydrazyl (DPPH), was used. 1 ml of 1, 3 diphenyl 2-propene-1-one
derivatives (0.1 ml & 0.2 ml) prepared in DMSO was added to 1 ml of DPPH (1x10-4 ml) solution made
in DMSO, and the resultant compound was allowed to complete suspension with vigorous agitation. The
DPPH solution served as the reference sample. The aluminium test tubes were used to seal the sample test
tubes before adding the DPPH radical. The amount of organic matter was measured using a UV-visible
spectrometer at a wavelength of 517 nm using DMSO as the blank solution. The following formula was
used to evaluate the 1, 3-diphenyl-2-propene-1-one derivatives' 2, 2-diphenylpicrylhydrazyl (DPPH)
scavenging abilities.
DPPH-scavenging effect = [(A Control – A Sample)/A Sample] x 100
Table-4 lists the anti-oxidant activity of the standard and 1, 3-diphenyl-2-propene-1-one derivatives at
different concentrations. Figure-6 and 7 show the plot of the percentage of inhibition against the different
concentrations of the solutions and standard Ascorbic acid. Ascorbic acid was used as the standard.174
Table-4: Pyrazoline Derivatives' Antioxidant Activity as Measured by DPPH Scavenging
S. No.
Compound
% of DPPH scavenging activity
100µgml
-1
200µgml
-1
300µgml
-1
1.
6
-
a
69.13
70.62
77.20
2.
6
-
b
64.43
76.56
78.96
3.
6
-
c
40.32
56.07
67.45
4.
6
-
d
36.95
48.87
67.04
5.
6
-
e
42.37
56.06
66.34
6.
6
-
f
40.19
54.10
63.07
Standard
Ascorbic
Acid
53.97
54.42
55.42
Fig-3: Anti-Oxidant Activity of Pyrazoline Derivatives by DPPH Scavenging Method
RESULTS AND DISCUSSION
Compounds 1-a, 1-b, 1-d, and 1-e exhibit good to moderate potency, while compounds 6-c and 6-f exhibit
moderate to low inhibition against all tested strains. Trials were conducted on compounds with low
activity for enhanced concentration. The synthesized compounds exhibited moderate activity at higher
0
20
40
60
80
100
6a 6b 6c 6d 6e 6f Ascarbic
Acid
% of DPPH scavenging
activity
Compound
100µgml-1
200µgml-1
300µgml-1
4
1
-
d
64%
5
1
-
e
55%
6
1
-
f
59%
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PYRAZOLINE DERIVATIVES A. Kistan et al.
concentrations. Verified compounds' antimicrobial activity can be linked to structural variations and
divergences of the corresponding compounds. Tables-1 and 2 provide statistics on antibacterial activity.
Among these compounds, 1-b (-4-CH3), 1-d (-4-F), and 1-f (pipernol) demonstrated exceptional activity
against B. subtilis. Compounds 1-d (-4-F) showed the greatest activity against S. typhi. Good activity
against E. coli was demonstrated by compounds 1-b (-4-CH3), and 1-e (thiophene). C. albicans is
demonstrated to be moderately active against 1-d (-4-F). Compounds 1-b, 1-d, 1-e, and 1-f) had moderate
activity, while compounds 1-a, and 1-c demonstrated virtuous larvicidal activity. The findings of these
investigations are presented in Table-4; remarkably, the majority of them demonstrated strong larvicidal
activity. Compounds (1c-1f)) showed moderate activity. The screening results for the antimicrobial,
larvicidal, and antioxidant properties suggest that all of the recently manufactured mixtures be moderately
exposed to worthy activity against the indicated organisms (Figure 4).
Anti-Bacterial Plate
Aspergillus Niger
Standard –Amphotericin-B
A1 - Compound 1-a
B1 –Compound 1-c
C1 - Compound 1-e
Anti-fungal Activity Plate
F Candida albicans
Standard – Amphotericin-B
A1 - Compound 1-a
B1 - Compound 1-c
C1 - Compound 1-e
Fig - 4: Anti-Bacterial & Antifungal activity Plates
CONCLUSION
In the current work, various Chalcones types with therapeutic requests are made, and their extensive
biological demands are further exploited through further modification. We have synthesized and
evaluated the antioxidant, larvicidal, and antimicrobial properties of a few novel Chalcones with the core
s-triazine moiety. Chalcones compounds, which are primarily pharmacologically active substances, are
identified chemically as 1- 3, diphenylprop 2-en-1-one byproducts. For this research project, we prepared
eighteen bioactive s-triazine derivatives. Each synthetic derivative was successfully made in two or three
steps. We discovered in this study that derivatives of Pyrazoline and cyanopyridine were useful
substances. The newly prepared compounds' 1H NMR, 13C NMR, MS (ESI), IR spectral data, and melting
point analysis all contributed to the explanation of their structure. Finally, we have successfully
synthesized a new series of derivatives containing Pyrazoline and cyanopyridine, and some of these
compounds also contain a bioactive heterocyclic moiety. Surprisingly, the majority of them demonstrated
superior antibacterial and antifungal properties. Compounds 1c and 1f showed moderate and little
inhibition against all of the strains tested, while compounds 1a, 1b, 1d, and 1e showed better and
moderate activity. Low-activity compounds were confirmed to have higher concentrations. The fluoro
substituent compound (1-d) exhibits good activity against the fungi under experimentation. The
synthesized compounds showed reasonable activity at higher concentrations Compounds (1-a to 1-f)
demonstrated good and moderate inhibition towards the compounds, according to their larvicidal activity.
The most active compounds, however, were compounds [1-a & 1-b]. There was good and moderate
inhibition towards the compounds (1-a to 1-f). It is recommended by the screening results for antioxidant,
larvicidal, and antimicrobial activities that all newly synthesized compounds exhibit reasonable to good
activity against the verified organisms.
ACKNOWLEDGEMENTS
The Panimalar Engineering College (Autonomous) in Chennai, India, provided the authors with the
support they needed as well as the essential research facilities, for which they are grateful.
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PYRAZOLINE DERIVATIVES A. Kistan et al.
CONFLICT OF INTERESTS
The authors declare that there is no conflict of interest.
AUTHOR CONTRIBUTIONS
All the authors contributed significantly to this manuscript, participated in reviewing/editing, and
approved the final draft for publication. The research profile of the authors can be verified from their
ORCID IDs, given below:
A. Kistan https://orcid.org/0000-0003-1334-4331
L. Mayavan https://orcid.org/0000-0001-9934-5436
V. Kanchana https://orcid.org/0000-0003-3528-5491
S. Mohan https://orcid.org/0000-0001-6538-3055
Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s)
and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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