www.wjpr.net Vol 7, Issue 8, 2018. 1 Asraret al. World Journal of Pharmaceutical Research
SYNTHESIS AND BIOLOGICAL EVALUATION OF SOME NOVEL
CHALCONE DERIVATIVES AS ANTI-INFLAMMATORY AGENTS
A. Asrar Ahamed1*, M. Mohamed Sihabudeen1, M. Syed Ali2 and M. Syed Abuthakir3
1
PG and Research Department of Chemistry, Jamal Mohamed College (Autonomous),
Affiliated to Bharathidasan University, Tiruchirappalli, Tamil Nadu, India.
2
PG and Research Department of Biotechnology, Mohamed Sathak College of Arts and
Science (Affiliated to University of Madras), Sholinganallur, Chennai, Tamil Nadu, India.
3
Biochematics Laboratary, Department of Bioinformatics, Bharathiar University,
Coimbatore, Tamil Nadu, India.
ABSTRACT
A series of chalcones was synthesized using Claisen- Schmidt
condensation reaction by acetanilide with formyl morphiline. The
resulting chalcones after purification have been converted into
pyrazoline by reaction with hydrazine hydrate and isoniazid. All the
products were tested for purity by TLC and characterized by elemental
analysis (for carbon, hydrogen and nitrogen), IR, 1H-NMR and 13
C-NMR spectral studies. The synthesized compounds have been screened
for their anti-inflammatory activity against both In-vitro and In-silico
studies gives a significant activity as compared to standard.
KEYWORDS: Chalcones, isoniazid, Pyrazolines, anti-inflammatory
activity and docking studies.
INTRODUCTION
The chemistry of chalcones has generated intensive scientific studies throughout the world.
Especially interest has been focused on the synthesis and biodynamic activities of chalcones.
The most convenient methods are the Claisen-Schimdt condensation of equi molar quantities
of an arylmethylketones with aryl aldehyde in the presence of alcoholic alkali. Chalcones are
unsaturated compounds that are major intermediates in the synthesis of several derivatives
like cyanopyridines, pyrazolines isoxazoles, pyrimidines, having different heterocyclic ring
Volume 7, Issue 8, 1-9. Conference Article ISSN 2277–7105
Article Received on 05 March 2018,
Revised on 25 March 2018, Accepted on 15 April 2018
DOI: 10.20959/wjpr20188-10702
*Corresponding Author
A. Asrar Ahamed
PG and Research
Department of Chemistry,
Jamal Mohamed College
(Autonomous),
Affiliated to Bharathidasan
University, Tiruchirappalli,
www.wjpr.net Vol 7, Issue 8, 2018. 2
systems.[1] The chalcones were easily derived from the aldol condensation of aromatic
aldehydes and aromatic ketones.[2]
Chalcones are an important class of natural products and are considered as the precursors of
flavonoids and isoflavonoids. They are widely distributed in fruits, vegetables, tea, spices,
soy based foods and other plant products.[3] Synthesis of these compounds has increased in
the last years because of their use in materials area, because of their interesting biological
activities.[4] Pyrazoles and their reduced forms, pyrazolines, are well known nitrogen
containing heterocyclic compounds and various procedures were developed for their
synthesis. Pyrazole and its derivatives represent one of the most active classes of heterocyclic
compounds possessing a wide spectrum of biological activities.[5, 6] They have been found to
possess oxidant, cancer, HIV, malarial, fungal, microbial,
anti-amoebic and anti-microbial activities.[7-18]
MATERIALS AND METHODS
All reagents and solvents for synthesis were obtained from Alfa Aesar and Sigma-Aldrich
chemicals. All materials used for this experiment are analytical grade. Melting points of the
products were determined in open capillary tube. These are uncorrected. Thin layered
chromatography (TLC) was performed on Merck 60F-254 silica gel plates with ethyl acetate
and n-hexane (3:2) as the solvent system. Spots were visualized using iodine chamber. IR
spectra were recorded on Shimadzu IR spectrophotometer by using KBr pellets technique. 1H
and 13C nuclear magnetic resonance (NMR) spectra were recorded on a BRUKER-AMX 400
MHz (AV 500) instrument using CDCl3 or DMSO-d6 solvent with TMS as an internal
standard. Chemical shifts were recorded in parts per million (ppm).
Procedure for Synthesis of (2E)-3-(morpholin-4-yl)-N-phenylprop-2-enamide (FMAC)
Chalcones of acetanilide (0.01 mole) and formyl morpholine (0.01 mole) was dissolved in 20
ml of ethanol. To this solution added 60% aqueous NaOH solution (10 ml) drop wise with constant stirring on the magnetic stirrer. The reaction temperature was maintained at 0oC
using a cold water bath. After vigorous stirrring for five hours the reaction mixture was
poured into the cold water and neutralized by 2 ml of HCl to obtain dark yellow precipitate
kept at 250C for overnight. The dark yellow colour crystals of chalcones were separated out.
www.wjpr.net Vol 7, Issue 8, 2018. 3
+
NaOHN
H CH3
O
N
O CHO
N H
O
N
O
Scheme I
Procedure for Synthesis of 1-[3-anilino-5-(morpholin-4-yl)-4,5-dihydro-1H
-pyrazol-1-yl]ethan-1-one (FMACH).
A mixture of chalcone (0.02mol) and hydrazine hydrate (0.02 mol) in ethanol (60mL) was
refluxed for 7 hrs by adding glacial acetic acid. The mixture was concentrated under vacuum
and poured into ice water. The precipitate obtained was filtered, washed and recrystalized
from ethanol. The results suggest that the acetic acid acted not only as solvent but also as a
acetylating agent. The reaction was given below Scheme-II.
N H
O
N
O +NH
2-NH2.H2O
Glacial acetic acid
Cyclization
reflux for 800 C HN
N
N
O N
CH3
O
Scheme-II
Procedure for Synthesis of [3-anilino-5-(morpholin-4-yl)-4,5-dihydro-1H
-pyrazol-1-yl](pyridin-4-yl)methanone (FMACIN).
A mixture of chalcone (0.02 mol) and isoniazid (0.02 mol) in ethanol (60 mL) was refluxed
for 9 hrs followed by adding glacial acetic acid. The mixture was concentrated under vacuum
and poured into ice water. The precipitate obtained was filtered, washed and recrystalized
www.wjpr.net Vol 7, Issue 8, 2018. 4 N
H O
N
O
+
Glacial acetic acid
Cyclization reflux for 80 0 C
N H
N
N
O
N N
O
N NH O
NH2
Scheme-III
RESULTS AND DISCUSSION
All the newly synthesized compounds 1a, 2a, 3a have been characterized by their melting
points, CHN analysis and spectroscopic methods such as IR, 1H, 13C, – NMR. The physical characteristics data of the synthesized compound was given in the Table-I.
Table I: Physical Characterization data formyl morpholine cholcones.
Compounds Code
Molecular Formula
Molecular Weight
Mellting Point
% of Yeild
Elemental Analysis (%)
C H N
FMAC C13H16N2O2 232.27 170 85 67.12 6.74 12.46
FMACH C15H20N4O2 288.34 192 80 62.52 6.89 19.23
FMACIN C19H21N5O2 351.40 210 78 64.74 6.09 19.73
(2E)-3-(morpholin-4-yl)-N-phenylprop-2-enamide (FMAC)
IR: 1615 Cm-1 (C=O), 3560 Cm-1 (NH), 1440, 1570, 1688 Cm-1 (C=C), 1320 Cm-1 (C-N),
1
HNMR (300 MHz, DMSO) ppm: 9.8 (s, 1H, -NH-C=O), 7.11-7.84 (m, 15H, aromatic), 3.2
(s, 1H, methine), 13CNMR (300 MHz, DMSO) ppm: 172.3 (C=O), 119.3-128.1 (phenyl
carbons), 138.2 (methine carbon).
1-[3-anilino-5-(morpholin-4-yl)-4,5-dihydro-1H-pyrazol-1-yl]ethan-1-one (FMACH)
IR: 1645 Cm-1 (C=O), 3370 Cm-1 (NH), 1460, 1490, 1578 Cm-1 (C=C), 1390 Cm-1 (C-N),
1
HNMR (300 MHz, DMSO) ppm: 10.1 (s, 1H, C-NH-C), 7.35-8.14 (m, 15H, aromatic), 2.7
(s, 1H, methylene), 13CNMR (300 MHz, DMSO) ppm: 170.3 (C=O), 121.3-128.9 (phenyl
carbons), 136.5 (formyl carbon), 47.2 (methine carbon).
[3-anilino-5-(morpholin-4-yl)-4,5-dihydro-1H-pyrazol-1-yl](pyridin-4-yl)methanone
(FMACIN)
IR: 1665 Cm-1 (C=O), 3680 Cm-1 (NH), 1420, 1474, 1548 Cm-1 (C=C), 1365 Cm-1 (C-N),
1
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1H, indol proton), 2.4 (s, 1H, methylene), 13CNMR (300 MHz, DMSO) ppm: 165.3 (C=O),
112.3-128.1 (phenyl carbons), 147.5, 142.3 (formyl carbons), 41.2 (methine carbon).
Anti-inflammatory activity
The extracts of the different formyl chalcone derivatives were studied for in vitro
anti-inflammatory activity by HRBC membrane stabilization method.[19] The formyl chalcone
derivatives extracts showed significant anti-inflammatory activity in a concentration
dependent manner. Among the three chalcone derivatives tested, FMACIN exhibited
maximum protection of HRBC (71.39±0.52%) in hypotonic solution at a concentration of
600 ppm, whereas the FMAC extracts registered the minimum protection of HRBC
(42.22±0.17%) in hypotonic solution at a concentration of 200 ppm. All the results were
compared with standard hydrocortisone which showed above 90% to 95% protection in Table
II.
Table II: In vitro anti-inflammatory activity of synthesised formyl morpholine
chalcones.
Sample 200ppm 400ppm 600ppm
Standard 90.12±0.24 92.46±0.16 95.24±0.07 FMAC 59.00±0.25 62.21±0.98 63.23±0.47 FMACH 66.41±0.38 67.35±0.49 70.22±0.42 FMACIN 70.32±0.69 75.23±0.31 78.18±0.38
Each average value represents the mean ± SEM (n=3). Hydrocortisone was used as
reference standard and a control was prepared omitting the extracts.
The extracts exhibited membrane stabilization effect by inhibiting hypotonicity induced lyses
of erythrocyte membrane. The erythrocyte membrane is analogous to the lysosomal
membrane and its stabilization implies that the extract may as well stabilize lysosomal
membranes. Stabilization of lysosomal membrane is important in limiting the inflammatory
response by preventing the release of lysosomal constituents of activated neutrophil such as
bactericidal enzymes and proteases, which cause further tissue inflammation and damage
upon extra cellular release. Though the exact mechanism of the membrane stabilization by
the extract is not known yet, hypotonicity-induced hemolysis may arise from shrinkage of the
cells due to osmotic loss of intracellular electrolyte and fluid components. The extract may
inhibit the processes, which may stimulate or enhance the efflux of these intracellular
www.wjpr.net Vol 7, Issue 8, 2018. 6 DOCKING STUDIES
Protein and Ligand preparation:
Crystal co-ordinates of proteins were downloaded (PDB ID: 1DQ8 for HMG CoA Reductase,
1XMU for phosphodiesterase 4b and 3S7S for Aromatase) from the Protein Databank (PDB)
(http://www.pdb.org/). The structure of the compound was drawn by Chemsketch and the
structure was converted from. mol to. pdb by using Openbabel. Drug molecules Roflumilast
was downloaded by Drug Bank. The active site was predicted by the online server Catalytic
site Atlas and CastP finder. The downloaded proteins energy was minimized by Swiss PDB
viewer and docking studies were carried out by AutoDock 4. Hydrogen atoms, charges were
added to protein structure and grid co-ordinates were calculated based upon the active site
and high volume surface area of the protein. Grid co- ordinates were set to generate the grid
box. The docked structure was analysed and visualised by PyMol.
The three newly synthesized different compounds were docked with one targets. Based on the
parameters such as Glide score, no of hydrogen bonds and bond length, it was decided which
one was best for blocking the receptor activity. From this study, the compound FMACIN had
good glide score and more number of hydrogen bonds with active sites of different targets
like HMG CoA reductase, PDE-4B and Aromatase. So, we can use FMACIN as multi
targeting compound and gilde score interaction data is shown in below table – III.
Table III: Interaction Between the Compounds and Pde 4b.
S.NO Compound/Drug
Name
Binding Energy
No.of.H-Bond Interacting Residues Bond Length (Å)
1. Roflumilast -9.78 6 ASP 275, GLU 304, HIS 234, ASP 392 (3)
2.103, 2.208, 2.070, 1.923, 2.127, 2.625.
2.
(2E )-3-(morpholin-4-yl)-N -phenylprop-2-enamide (FMAC)
-6.29 5 ASP 392 (4), HIS 238, HIS 234
1.825, 1.997, 2.031, 2.285, 2.571, 2.001
3.
1-[3-anilino-5- (morpholin-4-yl)-4,5-dihydro-1H -pyrazol-1-yl]ethan-1-one
(FMACH)
-7.37 6
ASP 392 (3), HIS 238, HIS 234, ASP
275
1.848, 2.222, 2.089, 2.427, 2.144, 2.311
4.
[3-anilino-5- (morpholin-4-yl)-4,5-dihydro-1H -pyrazol-1-
yl](pyridin-4-yl)methanone (FMACIN)
-7.11 6 GLN 443, ASP 392 (3), HIS 234
2.038, 1.724, 1.849, 1.720, 2.052.
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FMAC FMACH
FMACIN
CONCLUSION
Novel chalcone derivative has been synthesized and confirmed by chemical analysis, IR, 1H,
and 13C NMR. The in-vitro anti-inflammatory results revealed that most of the target
compounds exhibited anti-inflammatory activity. Especially, compound FMACIN exhibited
high anti-inflammatory activity in-vitro study. Furthermore, docking results indicated that the
anti-inflammatory activities of compounds correlated with their docking results.
ACKNOWLEDGEMENT
The authors thankful to the Management committee and the Principal of Jamal Mohamed
College (Autonomous), Trichirappalli-620020, Tamil Nadu, for their kind support and
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