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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,

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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.

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www.wjpr.net Vol 7, Issue 8, 2018. 3

+

NaOH

N

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

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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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www.wjpr.net Vol 7, Issue 8, 2018. 5

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

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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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www.wjpr.net Vol 7, Issue 8, 2018. 7

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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