Synthesis, anti-inflammatory and antioxidant activity of ring-a-monosubstituted chalcone derivatives

Article abstract of DOI:10.1007/s00044-014-1007-z

A library of ring-A-monosubstituted chalcone derivatives (4a-4i, 5a and 5b) was designed and synthesised. The structures as well as the identities of these compounds were established on the basis of spectral (¹H NMR, ¹³C NMR, FT-IR and Mass) and elemental (C, H, N) analyses. All the derivatives were evaluated for their anti-inflammatory and antioxidant activities in vitro using the inhibition of protein denaturation and 2,2-diphenyl-1-picrylhydrazyl radical scavenging assays, respectively. The results indicated a promising anti-inflammatory activity for most of the synthesised compounds with many derivatives showing activities similar to or greater than that of the standard. The sulphonamide-substituted chalcones 4h, 4i, 5a and 5b were found to be the most active derivatives across the concentration range tested. However, all the derivatives exhibited rather mild antioxidant activity compared to the ascorbic acid standard. Interestingly, it was observed that the unsubstituted parent chalcone was one of the optimal compounds with only the trifluoromethyl analogue 4a showing better activity as an antioxidant. The two regioisomeric aminochalcones and 4′-cyanochalcone 4b also seemed to possess decent antioxidant potential.

Full text of DOI:10.1007/s00044-014-1007-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-1007-z
ORIGINAL RESEARCH
Synthesis, anti-inflammatory and antioxidant activity
of ring-A-monosubstituted chalcone derivatives
•
Hiba Iqbal Visakh Prabhakar Atul Sangith
•
•
•
Baby Chandrika Ranganathan Balasubramanian
Received: 3 November 2013 / Accepted: 8 April 2014
Ó Springer Science+Business Media New York 2014
Abstract A library of ring-A-monosubstituted chalcone
derivatives (4a–4i, 5a and 5b) was designed and synthes-
ised. The structures as well as the identities of these
compounds were established on the basis of spectral (1H
NMR, 13C NMR, FT-IR and Mass) and elemental (C, H, N)
analyses. All the derivatives were evaluated for their anti-
inflammatory and antioxidant activities in vitro using the
inhibition of protein denaturation and 2,2-diphenyl-1-
picrylhydrazyl radical scavenging assays, respectively. The
results indicated a promising anti-inflammatory activity for
most of the synthesised compounds with many derivatives
showing activities similar to or greater than that of the
standard. The sulphonamide-substituted chalcones 4h, 4i,
5a and 5b were found to be the most active derivatives
across the concentration range tested. However, all the
derivatives exhibited rather mild antioxidant activity
compared to the ascorbic acid standard. Interestingly, it
was observed that the unsubstituted parent chalcone was
one of the optimal compounds with only the trifluoro-
methyl analogue 4a showing better activity as an antioxi-
dant. The two regioisomeric aminochalcones and 40-
cyanochalcone 4b also seemed to possess decent antioxi-
dant potential.
Keywords Chalcone ꢀ Antioxidant ꢀ Anti-inflammatory ꢀ
Sulphonamide ꢀ Protein denaturation ꢀ DPPH
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-014-1007-z) contains supplementary
material, which is available to authorized users.
Introduction
The chalcone moiety remains a popular scaffold amongst
medicinal chemists owing to its structural simplicity. Not
only are chalcones abundantly found in nature, but also
they are key intermediates for many other medicinally
important natural products like flavonoids, isoflavonoids
and aurones (Detsi et al., 2009; Venkatachalam et al.,
2012). The therapeutic activities of chalcones are attributed
to the presence of the key pharmacophoric a,b-unsaturated
keto group, more specifically a 2-propen-1-one chain (Or-
likova et al., 2011). It is proved that the inherent electro-
philicity of this a,b-unsaturated carbonyl functionality is
involved in the antioxidant and anti-inflammatory proper-
ties of chalcones (Kumar et al., 2011; Maydt et al., 2013).
Both the terminal carbons of the 2-propen-1-one fragment
are attached to a phenyl ring, conventionally referred to as
ring A and ring B on each of which a multitude of func-
tional groups may be appended. Besides such substituted
phenyl groups, the synthetically derived chalcones may
H. Iqbal ꢀ R. Balasubramanian (&)
Department of Pharmaceutical Chemistry, Amrita School of
Pharmacy, Amrita Vishwa Vidyapeetham University, Health
Sciences Campus, Kochi 682041, Kerala, India
e-mail: ranga.balasubramanian@gmail.com
H. Iqbal
e-mail: hiba1442@gmail.com
V. Prabhakar ꢀ A. Sangith
Department of Pharmacy, Birla Institute of Technology
and Sciences, Pilani 333031, Rajasthan, India
e-mail: visakh512@gmail.com
A. Sangith
e-mail: sangith17atul@gmail.com
B. Chandrika
Sophisticated Analytical Instrumentation Facility, Indian
Institute of Technology, Madras, Chennai 600036,
Tamil Nadu, India
e-mail: cbaby@iitm.ac.in
123

Med Chem Res
various mechanisms of inflammatory responses points out
that the presence of antioxidants capable of protecting
oxidative stress-related injury and inflammatory disease
will improve the anti-inflammatory activity (Isa et al.,
2012; Schinella et al., 2002). The notion of this correlation
between antioxidant and anti-inflammatory activity is
strengthened by the findings of research groups that attri-
bute the importance of free radicals in the progression of
oxidative as well as inflammatory damage (Roome et al.,
2008; Saldanha et al., 1990). It is, therefore, felt that the
estimation of antioxidant potential is a logical extension of
studying a compound’s anti-inflammatory activity and
accordingly, the study presented herein deals with the
rational design of a focused library of ring-A-monosub-
stituted chalcones, their synthesis and subsequent in vitro
evaluation for anti-inflammatory and antioxidant activities.
Apart from helping elucidate SAR, estimation of the anti-
oxidant activity of these chalcones also represents our
attempt to probe structural attributes that would confer dual
pharmacological activity to the hits identified.
Fig. 1 Prototypical structure of chalcone
also carry heterocyclic and condensed ring systems as their
aryl substituents (Meng et al., 2007; Solomon and Lee,
2012; Tran et al., 2012). While the presence of a double
bond allows these molecules to exist as cis or trans geo-
metric isomers, the latter configuration has been proven to
be thermodynamically as well as biologically favourable
(Cheng et al., 2000). The structure of the simplest unsub-
stituted chalcone (1) is illustrated in Fig. 1.
The diverse pharmacological applications of chalcones
include their potential utility as antioxidant, antibacterial,
antileishmanial, antiangiogenic, analgesic, antifungal, an-
tiprotozoal, gastric protectant, antimutagenic, antitumori-
genic and anti-inflammatory agents amongst others
(Gacche et al., 2008; Wu et al., 2011; Yadav et al., 2011;
Reddy et al., 2012). Some of the well-known natural anti-
inflammatory chalcones also possessing good antioxidant
potential are butein, xanthohumol, isoliquiritigenin, car-
damonin, licochalcone A, flavokawain A and B, all of
which represent predominantly hydroxylated, methoxylat-
ed and alkylated versions of the basic chalcone scaffold
(Nowakowska, 2007; Srinivasan et al., 2009; Vogel et al.,
2010). Even though many chalcones found in nature are
known to be traditional folk remedies for inflammatory
conditions, the effects of substituting various functional
groups at different positions on the rings can be studied
systematically only if the target molecules are synthesised
chemically. The availability of a simple method to prepare
chalcones via Claisen–Schmidt reaction, involving the
condensation of substituted acetophenone precursors with
substituted benzaldehydes in the presence of a base, has
made it easier to assemble large numbers of derivatives
within a short amount of time (Bandgar et al., 2010; Sing
et al., 2012).
Experimental
Materials and methods
All commercial reagents were used as provided unless
otherwise indicated. All reactions were performed in oven-
dried glassware. TLC was performed on silica gel G 40 lm
particle size-coated glass plates, and spotswere visualizedby
UV and/or I2 chamber. Melting points were determined
using a Roy capillary melting point apparatus and are
1
uncorrected. H and 13C NMR spectra were recorded on a
Bruker Avance 400 or Bruker AV III 500 MHz spectrome-
ter. 19F NMR was recorded on Bruker AV III 500 MHz
spectrometer. Proton, carbon and fluorine chemical shifts are
reported in ppm. The internal standard for proton and carbon
was residual CHCl3 (7.26 and 77.16 ppm, respectively).
Proton chemical data are reported as follows: chemical shift,
multiplicity (ovlp. = overlapping, s = single, d = doublet,
t = triplet, dt = doublet of triplet, tt = triplet of triplet and
m = multiplet), coupling constant and integration. IR
spectra were recorded on a Shimadzu FT-IR spectropho-
tometer or Perkin Elmer spectrum RXI FT-IR spectropho-
tometer. LC–MS chromatograms and mass spectra were
obtained on a Waters e 2695-Waters 3100 instrument with
ESI-PMT arrangement as the mode of ionisation and type of
detector, respectively. Elemental analyses were determined
on an Elementar Vario EL III elemental analyser.
Many of the reports pertaining to synthetic chalcones are
limited in their ability to establish a detailed structure
activity relationship (SAR) footprint with respect to anti-
inflammatory properties of these molecules. The crucial
aspect in our ongoing search for a potent yet safe anti-
inflammatory compound is to bridge such a wide infor-
mation gap that currently exists between structure and anti-
inflammatory potential of this scaffold. A closer look at the
For studying the anti-inflammatory activity, ibuprofen
standard was obtained as a gift from Porus Labs Pvt. Ltd.,
Hyderabad. Protein used in this assay was bovine serum
albumin fraction V (98 %, Nice Chemicals). Analytical
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Med Chem Res
Scheme 1 Synthesis of relevant acetophenone precursors and target chalcones
grade DMF (99.5 %, Otto Chemei, India) was used to
solubilise the test compounds. Absorbance readings were
taken from Schimadzu UV–Vis spectrophotometer. With
regard to the antioxidant assay using DPPH, all chemicals
were purchased from SDFCL, Mumbai unless specified
otherwise. DPPH (90 %) was procured from Sigma Aldrich
(Germany). A Shimadzu UV-1800 Spectrophotometer was
used for measuring the optical density of the samples.
123

Med Chem Res
Design and in silico analysis of target chalcones
40-Trifluoromethylchalcone 4a (Wilhelm et al., 2012)
The design of ring-A-monosubstituted chalcones was pri-
marily based on the electronic properties of the substitu-
ents. The selected functional groups are placed at 20-
(ortho), 30-(meta) or 40-(para) position of ring A of the
scaffold while ring B is left intact. Significant groups like
amino and cyano groups were selected for their mesomeric
(?M/-M) effect; similarly groups like trifluoromethyl and
azido were selected for their inductive (?I/-I) effect. To
test the effect of lipophilicity on the pharmacological
activities of chalcones, sulphonamide functional groups
including methanesulphonamide and benzenesulphona-
mide at ortho and para (5a, 4h, 5b and 4i; Scheme 1)
positions were employed (Balasubramanian and Vijayag-
opal, 2012). The proposed molecules were then subjected
to in silico Lipinski Rule of Five (Ro5) analysis for
assessing drug-likeness using Molsoft (molsoft.com/
mprop/), an online academic software wherein properties
like molecular weight, hydrogen bond donors as well as
acceptors and calculated partition coefficient of the mole-
cules were determined.
Off-white crystals; yield: 77 %; Rf = 0.50 (10 % EtOAc/
1
cyclohexane); mp 94–98 °C; H NMR (CDCl3, 500 MHz)
d 8.10 (d, J = 8 Hz, 2H), 7.83 (d, J = 15.5 Hz, 1H), 7.77
(d, J = 8 Hz, 2H), 7.64–7.66 (m, 2H), 7.49 (d,
J = 15.5 Hz, 1H), 7.42–7.45 (m, 3H); 13C NMR (CDCl3,
125 MHz) d 189.7, 146.2, 141.1, 134.5, 134.2, 133.9,
131.0, 129.1, 128.9, 128.7, 125.8, 125.7, 125.6, 124.8,
122.6, 121.6; 19F NMR (CDCl3, 470 MHz) d -63.00 (s,
3F); IR (KBr) 3060 (arom. –CH str.), 1943 (C–F3 str.),
1666 (a,b-unsaturated keto group, –C=O str.), 1608, 1574
(C=C arom. str., C=C olefinic str.), 1321 (ip arom. C–H
bend.), 985 (oop –CH bend. vibration of alkene), 840 (1,4
disubstitution), 772 (arom. bend.) cm-1; Anal. calcd. for
C16H11F3O: C 69.56, H 4.01. Found: C 69.42, H 3.99; ESI
MS (m/z, relative abundance) 277 [(M?H)?, 100], 298
[(M?Na)?, 44], 320 [(M?2Na)?, 25], [C32H20O22?, 7].
40-Cyanochalcone 4b (Kumar et al., 1985)
White crystals; yield: 23 %; Rf = 0.57 (20 % EtOAc/
1
cyclohexane); mp 92–96 °C; H NMR (CDCl3, 500 MHz)
Synthesis
d 8.08 (d, J = 8.5 Hz, 2H), 7.83 (d, J = 15.5 Hz, 1H),
7.80 (d, J = 8.5 Hz, 2H), 7.64–7.66 (m, 2H), 7.47 (d,
J = 15.5 Hz, 1H), 7.43–7.46 (m, 3H); 13C NMR (CDCl3,
125 MHz) d 189.2, 146.6, 141.5, 134.4, 132.5, 131.2,
129.1, 128.9, 128.7, 121.2, 118.0, 116.0; IR (KBr) 3100
(arom. –CH str.), 2230 (arom. C:N), 1662 (a,b-unsatu-
rated keto group, –C=O str.), 1600, 1574 (C=C arom. str.,
C = C olefinic str.), 984 (oop –CH bend. vibration of
A total of eleven monosubstituted chalcones were sought
out in this study of which ten were successfully pre-
pared. While the synthetic route(s) used to access them
are presented in Scheme 1, experimental procedures and
characterisation data of the respective compounds are
detailed herein. Typically, the classic base-catalysed
aldol-type condensation was employed to assemble the
target compounds 4a, 4b, 4d–4i as well as the congener
4c that acted as a precursor to the two other target
sulphonamides 5a and 5b. Spectral characterisation fol-
lowing the synthesis unambiguously confirmed the
structure of the compounds.
alkene), 833 (1,4 disubstitution), 766 (arom. bend.) cm-1
;
Anal. calcd. for C16H11NO: C 82.38, H 4.75, N 6.00.
Found: C 82.17, H 4.88, N 6.02; ESI MS (m/z, relative
abundance) 234 [(M?H)?, 100].
20-Aminochalcone 4c (Mannich and Dannehl, 1938)
General procedure for synthesis of 4a–4e
Yellow flakes; yield: 52 %; Rf = 0.68 (20 % EtOAc/
cyclohexane); mp 50–54 °C; 1H NMR (CDCl3, 500 MHz) d
7.85 (dd, J = 1.5, 8.5 Hz, 1H), 7.73 (d, J = 15.5 Hz, 1H),
7.62 (dd, J = 1.5, 8.5 Hz, 1H), 7.61 (d, J = 8.5 Hz, 1H),
7.60 (d, J = 15.5 Hz, 1H), 7.41 (ovlp. d, J = 8.5 Hz, 1H),
7.36–7.42 (m, 2H), 7.26–7.30 (m, 1H), 6.66–6.71 (m, 2H),
6.30 (s, 2H); 13C NMR (CDCl3, 100 MHz) d 191.7, 151.0,
142.9, 135.3, 134.3, 131.0, 130.1, 128.9, 128.2, 123.2, 119.1,
117.3, 115.9; IR (KBr) 3444, 3325 (–NH str., primary
amine), 1641 (a,b-unsaturated keto group, –C=O str.), 1605,
1573 (C=C arom. str., C=C olefinic str.), 1336 (arom. amino
group, C–N str.), 738 (1,2 disubstitution) cm-1; ESI MS (m/
z, relative abundance) 224 [(M?H)?, 100].
To a solution of substituted acetophenone (10 mmol) in
rectified spirit (30 mL), benzaldehyde (1.01 mL, 10 mmol)
was added followed by an aqueous solution of 10 % KOH
(10 mL). The mixture was stirred and kept overnight at
room temperature. The contents of the reaction mixture
were poured into crushed ice and acidified with dil. HCl
(0.1–0.2 N). The precipitated chalcone derivative was fil-
tered off and recrystallised from rectified spirit. Column
chromatography (10–20 % EtOAc/cyclohexane) was per-
formed whenever recrystallisation failed to sufficiently
purify the target compound.
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30-Aminochalcone 4d (Karaman et al., 2010)
product 3f. To the solution of this crude 3-azido aceto-
phenone in rectified spirit (30 mL), benzaldehyde
(1.01 mL, 10 mmol) was added followed by an aqueous
solution of 10 % KOH (10 mL). The mixture was stirred
and kept overnight at room temperature. The contents of
the reaction mixture were poured into crushed ice and
acidified with dil. HCl (0.1–0.2 N). The precipitated 30-
azidochalcone was filtered off and recrystallised from
rectified spirit. Further purification by column chromatog-
raphy (20 % EtOAc/cyclohexane) afforded cream-coloured
crystals. Yield: 14 % over two steps; Rf = 0.68 (20 %
EtOAc/cyclohexane); mp 46–50 °C; 1H NMR (CDCl3,
Yellow crystals obtained by column chromatography;
yield: 33 %; Rf = 0.35 (20 % EtOAc/cyclohexane); mp
90–94 °C; 1H NMR (CDCl3, 500 MHz) d 7.79 (d,
J = 16 Hz, 1H), 7.63 (d, J = 5.5 Hz, 1H), 7.62 (d,
J = 5.5 Hz, 1H), 7.48 (d, J = 16 Hz, 1H), 7.41 (ovlp. d,
J = 5.5 Hz, 1H), 7.40 (ovlp. d, J = 5.5 Hz, 1H), 7.38–
7.42 (m, 2H), 7.26–7.32 (m, 2H), 6.88–6.90 (m, 1H), 3.85
(s, 2H); 13C NMR (CDCl3, 100 MHz) d 190.7, 146.9,
144.5, 139.4, 135.0, 130.5, 129.5, 128.9, 128.4, 122.4,
119.4, 118.9, 114.4; IR (KBr) 3367, 3203 (–NH str., pri-
mary amine), 1660 (a,b-unsaturated keto group, –C=O
str.), 1650, 1587 (C=C arom. str., C=C olefinic str.), 1336
(arom. amino group, C–N str.), 759 (1,3 disubstitution)
cm-1; Anal. calcd. for C15H13NO: C 80.69, H 5.87, N 6.27.
Found: C 80.90, H 5.79, N 6.41; ESI MS (m/z, relative
abundance) 224 [(M?H)?,100].
400 MHz)
d 7.75 (d, J = 15.6 Hz, 1H), 7.69 (d,
J = 7.6 Hz, 1H), 7.56–7.59 (m, 3H), 7.41 (d, J = 7.6 Hz,
1H), 7.39 (d, J = 15.6 Hz, 1H), 7.34–7.43 (m, 3H), 7.14–
7.17 (m, 1H); 13C NMR (CDCl3, 100 MHz) d 188.5, 144.6,
139.9, 138.9, 133.7, 129.8, 129.0, 128.0, 127.5, 123.9,
122.1, 120.7, 117.8; IR (thin film) 3062 (arom. –CH str.),
2113 (asym. NNN str.), 1583 (a,b-unsaturated keto group,
–C=O str.), 985 (oop –CH bend. vibration of alkene), 759
(1,3 disubstitution) cm-1; Anal. calcd. for C15H11N3O: C
72.28, H 4.45, N 16.86. Found: C 72.08, H 4.49, N 16.77;
ESI MS (m/z, relative abundance) 222 [(M-N2)?, 100],
250 [(M?H)?, 30].
40-Aminochalcone 4e (Applequist and Gdanski, 1981)
Yellow crystalline needles; yield: 30 %; Rf = 0.42 (20 %
1
EtOAc/cyclohexane); mp 130–136 °C; H NMR (CDCl3,
500 MHz)
d 7.93 (d, J = 8.5 Hz, 2H), 7.78 (d,
J = 15.5 Hz, 1H), 7.62 (d, J = 8.5 Hz, 2H), 7.54 (d,
J = 15.5 Hz, 1H), 7.36–7.41 (m, 3H), 6.69 (d, J = 9 Hz,
2H), 4.22 (s, 2H); 13C NMR (CDCl3, 125 MHz) d 188.1,
151.2, 143.1, 135.3, 131.0, 130.0, 128.8, 128.4, 128.2,
122.0, 113.9; IR (KBr) 3338, 3200 (–NH str., primary
amine), 1628 (a,b-unsaturated keto group, –C=O str.),
1603, 1577 (C=C arom. str., C=C olefinic str.), 1341 (arom.
amino group, C–N str.), 830 (1,4 disubstitution), 766
(arom. bend.) cm-1; Anal. calcd. for C15H13NO: C 80.69,
H 5.87, N 6.27. Found: C 80.81, H 5.95, N 6.22; ESI MS
(m/z, relative abundance) 224 [(M?H)?, 100], 249
[(M?Na)?, 10].
Synthesis of 40-azidochalcone 4g (Zarghi et al., 2006)
Sodium nitrite (1.0 g, 14.7 mmol) was added as a solid to a
30 mL solution of 4-amino acetophenone (1.0 g, 7.4 mmol)
in trifluoroacetic acid at 0 °C. After 45 min of stirring at 0–
5 °C, sodium azide (4.81 g, 74 mmol) was added in small
portions over 10 min, so that the temperature would not
exceed 5 °C. An aliquot of Et2O (20 mL) was added, and the
reaction was allowed to stir in the dark for 2 h. The reaction
mixture was washed with distilled H2O (2 9 15 mL) and
saturated sodium chloride solution (20 mL). The washings
were re-extracted with Et2O (15 mL); the Et2O layers com-
bined, dried over Na2SO4 and evaporated to get a dark brown
residue from which the desired azido acetophenone 3g was
extracted by boiling with hexane (3 9 20 mL). Pure 4-azido
acetophenone (Kym et al., 1993) eventually precipitated as
light yellow crystals after keeping the hexane extract in
refrigerator overnight. Yield: 79 %; Rf = 0.60 (20 % EtOAc/
cyclohexane); mp 40–44 °C; IR (KBr) 3000 (arom. –CH str.),
2128 (asym. NNN str.), 1685 (C=O str.), 1654, 1596 (C=C
Synthesis of 30-azidochalcone 4f
To a solution of 3-amino acetophenone (1.0 g, 7 mmol) in
THF (10 mL), 20 mL of 10 % aqueous HCl was added.
Sodium nitrite (1.0 g, 14.7 mmol) was added as a solid to
this solution at 0 °C. After 45 min of stirring at 0–5 °C,
sodium azide (4.81 g, 74 mmol) was added in small por-
tions over 10 min, so that the temperature would not
exceed 5 °C. The reaction was allowed to stir for a further
1–2 h at room temperature. The reaction mixture was
extracted with Et2O (2 9 15 mL); the organic layers were
washed with distilled water (2 9 20 mL) and saturated
sodium chloride solution (20 mL). The washings were re-
extracted with Et2O (15 mL), the combined Et2O layers
were dried over Na2SO4 and evaporated to get the crude
arom. str.), 831 (1,4 disubstitution), 721 (arom. bend.) cm-1
.
To the solution of 4-azido acetophenone (0.15 g, 0.9
mmol) in rectified spirit (30 mL), benzaldehyde (0.15 mL,
0.9 mmol) was added followed by an aqueous solution of
10 % KOH (5 mL). The mixture was stirred and kept
overnight at room temperature. The contents of the reaction
mixture were poured into crushed ice and acidified with dil.
HCl (0.1–0.2 N). The precipitated 40-azidochalcone was
123

Med Chem Res
filtered off and recrystallised from rectified spirit to afford
(arom. –CH str.), 1653 (a,b-unsaturated keto group, –C=O
str.), 1601, 1500 (C=C arom. str., C=C olefinic str.), 1338
(asym. SO2 str.), 1178 (sym. SO2 str.), 934 (SN str.), 837
(1,4 disubstitution) cm-1; Anal. calcd. for C16H15NO3S: C
63.77, H 5.02, N 4.65, S 10.64. Found: C 63.79, H 4.98, N
4.49, S 10.78; ESI MS (m/z, relative abundance) 300 [(M-
H)?, 100].
a light brown solid. Yield 79 %; Rf = 0.72 (20 % EtOAc/
1
cyclohexane); mp 80–84 °C; H NMR (CDCl3, 500 MHz)
d 8.05 (d, J = 9 Hz, 2H), 7.82 (d, J = 15.5 Hz, 1H), 7.64
(d, J = 9 Hz, 2H), 7.51 (d, J = 15.5 Hz, 1H), 7.42 (ovlp.
d, J = 8.5 Hz, 1H),7.41–7.43 (m, 2H), 7.13 (d,
J = 8.5 Hz, 2H); 13C NMR (CDCl3, 125 MHz) d 188.6,
144.9, 144.7, 134.8, 134.7, 130.6, 130.4, 129.0, 128.4,
121.5, 119.0; IR (KBr) 3050 (arom. –CH str.), 2150 (asym.
NNN str.), 1645 (a,b-unsaturated keto group, –C=O str.),
1600, 1570 (C=C arom. str., C=C olefinic str.), 985 (oop
–CH bend. vibration of alkene), 830 (1,4 disubstitution),
775 (arom. bend.) cm-1; Anal. calcd. for C15H11N3O: C
72.28, H 4.45, N 16.86. Found: C 72.15, H 4.32, N 16.91;
ESI MS (m/z, relative abundance) 222 [(M-N2)?, 100],
250 [(M?H)?, 40].
Synthesis of 40-benzenesulphonamide chalcone 4i
(Moustafa and Ahmad, 2003)
To a solution of 4-aminoacetophenone (0.5 g, 3.7 mmol) in
dry CH2Cl2 (10 mL), Et3N (1.18 mL, 8 mmol) was added
followed by benzenesulphonyl chloride (0.51 mL, 4 mmol)
at 0 °C. After maintaining the reaction mixture between 0
and 5 °C for 3–4 h, the contents were gradually brought up to
room temperature and stirred overnight. After TLC indicated
the completion of the reaction, the mixture was diluted with
CH2Cl2 (20 mL) and washed with water (2 9 15 mL) fol-
lowed by saturated sodium chloride solution (15 mL). Dry-
ing (Na2SO4) and then distillation of the organic layer
afforded the crude acetophenone intermediate 3i as a yellow
solid. This crude 4-benzenesulphonamide acetophenone was
dissolved in rectified spirit (30 mL). Benzaldehyde
(1.01 mL, 10 mmol) was added to it followed by an aqueous
solution of 10 % KOH (10 mL). The mixture was stirred and
kept overnight at room temperature. The contents of the
reaction mixture were poured into crushed ice and acidified
with dilute HCl (0.1–0.2 N). The precipitated product was
filtered off and recrystallised from rectified spirit affording
the title compound as light yellow crystals. Yield: 16 % over
two steps; Rf = 0.44 (20 % EtOAc/cyclohexane); mp 126–
130 °C; 1H NMR (CDCl3, 500 MHz) d 7.99 (d, J = 8.5 Hz,
2H), 7.95 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H),
7.84 (d, J = 16 Hz, 1H) 7.70 (tt, J = 2, 8 Hz, 2H), 7.64–
7.66 (m, 2H), 7.57 (s, 1H), 7.56–7.59 (ovlp. m, 2H), 7.49 (d,
J = 16 Hz, 1H), 7.42–7.45 (m, 2H), 7.17 (dt, J = 2, 8 Hz,
2H); 13C NMR (CDCl3, 125 MHz) d 189.4, 145.8, 139.5,
139.3, 137.9, 134.6, 134.2, 131.9, 130.9, 129.3, 129.2, 129.1,
128.6, 121.5; IR (thin film) 3344 (–NH str.), 3066 (arom.
–CH str.), 1662 (a,b-unsaturated keto group, –C=O str.),
1595, 1489 (C=C arom. str., C=C olefinic str.), 1340 (asym.
SO2 str.), 1166 (sym. SO2 str.), 920 (SN str.), 867 (1,4 di-
substitution) cm-1; Anal. calcd. for C21H17NO3S: C 69.40, H
4.71, N 3.85, S 8.82. Found: C 69.29, H 4.64, N 3.78, S 8.99;
ESI MS (m/z, relative abundance) 362 [(M-H)?, 100].
Synthesis of 40-Methanesulphonamide chalcone 4h
(Zarghi et al., 2006)
To a solution of 4-aminoacetophenone (0.5 g, 3.7 mmol) in
dry CH2Cl2 (10 mL), Et3N (1.18 mL, 8 mmol) was added
followed by methanesulphonyl chloride (0.32 mL,
4 mmol) at 0 °C. After maintaining the temperature
between 0 and 5 °C for 3–4 h, the reaction mixture was
gradually warmed to room temperature and the contents
were stirred overnight. Once TLC indicated the completion
of the reaction, the mixture was diluted with CH2Cl2
(20 mL) and washed with water (2 9 15 mL) followed by
saturated sodium chloride solution (15 mL). Drying
(Na2SO4) and then distillation of the organic extracts
afforded the crude intermediate 3h as a light yellow solid.
To the crude 4-methanesulphonamide acetophenone dis-
solved in rectified spirit (30 mL), benzaldehyde (1.01 mL,
10 mmol) was added followed by an aqueous solution of
10 % KOH (10 mL). The mixture was stirred and kept
overnight at room temperature. The contents of the reaction
mixture were poured into crushed ice and acidified with
dilute HCl (0.1–0.2 N). The precipitated chalcone deriva-
tive was filtered off, recrystallised from rectified spirit and
further purified by column chromatography (20 % EtOAc/
cyclohexane) to get yellow crystals. Yield: 10 % over two
steps; Rf = 0.24 (20 % EtOAc/cyclohexane); mp 112–
116 °C; 1H NMR (CDCl3, 500 MHz)
d 8.06 (d,
J = 7.25 Hz, 1H), 8.05 (d, J = 7.25 Hz, 1H), 7.83 (d,
J = 16 Hz, 1H), 7.65 (d, J = 7.25 Hz, 1H), 7.64 (d,
J = 7.25 Hz, 1H), 7.51 (d, J = 16 Hz, 1H), 7.44 (ovlp. d,
J = 6.25 Hz, 1H), 7.43 (ovlp. d, J = 6.25 Hz, 1H), 7.42–
7.44 (m, 1H), 7.33 (d, J = 6.25 Hz, 1H), 7.32 (d,
J = 6.25 Hz, 1H), 7.15 (s, 1H), 3.12 (s, 3H); 13C NMR
(CDCl3, 125 MHz) d 188.9, 145.1, 141.1, 134.8, 134.4,
131.1, 130.7, 130.6, 129.4, 129.0, 128.5, 128.3, 121.5,
118.4, 118.2, 40.1; IR (thin film) 3248 (–NH str.), 3035
Synthesis of 20-methanesulphonamide chalcone 5a
(Batt et al., 1993)
To a solution of 20-aminochalcone (0.18 g, 0.81 mmol) in
dry CH2Cl2 (5 mL), pyridine (0.66 mL, 0.83 mmol) was
123

Med Chem Res
added followed by methanesulphonyl chloride (0.07 mL,
0.9 mmol) at 0 °C under N2 atmosphere. The contents were
stirred overnight at room temperature by gradually warm-
ing the reaction mixture from 0 °C. After completion of the
reaction as indicated by TLC, the reaction mixture was
diluted with CH2Cl2 (20 mL), washed with water
(2 9 15 mL) and saturated sodium chloride solution
(15 mL), dried over Na2SO4 and distilled off. The crude
compound thus obtained was purified using column chro-
matography (10 % EtOAc/cyclohexane). Bright yellow
crystals; yield: 19 %; Rf = 0.35 (20 % EtOAc/cyclohex-
group, –C=O str.), 1593, 1494 (C=C arom. str., C=C ole-
finic str.), 1336 (asym. SO2 str.), 1163 (sym. SO2 str.), 931
(SN str.), 746 (1,2 disubstitution) cm-1; Anal. calcd. for
C21H17NO3S: C 69.40, H 4.71, N 3.85, S 8.82. Found: C
69.43, H 4.88, N 3.97, S 8.81; ESI MS (m/z, relative
abundance) 362 [(M-H)?, 100].
Determination of biological activity by in vitro methods
Anti-inflammatory activity
1
ane); mp 74–76 °C; H NMR (CDCl3, 500 MHz) d 11.23
A well-established literature protocol that involved inhi-
bition of protein denaturation was followed with minor
modifications for the estimation of in vitro anti-inflamma-
tory activity (Mizushima and Kobayashi, 1968). The
standard drug ibuprofen and test compounds were dis-
solved in minimum amount of DMF and diluted with
phosphate buffer saline (0.2 M, pH 7.4) in such a way that
concentration of DMF in all solutions was less than 2.5 %.
Each of the test solutions was mixed with bovine serum
albumin (BSA) solution (2 mL, 2 mmol) in phosphate
buffer saline and incubated at 27 1 °C in an incubator
for 15 min. Denaturation was induced by keeping the
reaction mixture at 60 1 °C in a water bath for 10 min.
After cooling, the turbidity was measured at 660 nm with a
UV visible spectrophotometer and the percentage of inhi-
bition of denaturation calculated from control where no
drug is added. The percentage of inhibition was calculated
using the formula: percentage inhibition = AC - AS/AC 9
100, where AC = absorbance of control and AS = absor-
bance of test sample.
(s, 1H), 8.04 (dd, J = 1.5, 8.5 Hz, 1H), 7.85 (d,
J = 15.5 Hz, 1H), 7.80 (dd, J = 1.5, 8.5 Hz, 1H), 7.66 (d,
J = 6.75 Hz, 1H), 7.65 (d, J = 6.75 Hz, 1H), 7.59 (d,
J = 15.5 Hz, 1H), 7.58 (t, J = 1 Hz, 1H), 7.43–7.46 (m,
3H), 7.20–7.23 (m, 1H), 3.08 (s, 3H); 13C NMR (CDCl3,
125 MHz) d 192.7, 146.3, 140.7, 134.9, 134.5, 131.1,
131.0, 129.1, 128.7, 123.4, 122.8, 121.8, 118.9, 40.2; IR
(thin film) 3200 (–NH str.), 3053 (arom. –CH str.), 1643
(a,b-unsaturated keto group, –C=O str.), 1593, 1500 (C=C
arom. str., C=C olefinic str.), 1342 (asym. SO2 str.), 1170
(sym. SO2 str.), 981 (SN str.), 767 (1,2 disubstitution)
cm-1; Anal. calcd. for C16H15NO3S: C 63.77, H 5.02, N
4.65, S 10.64. Found: C 63.66, H 5.10, N 4.76, S 10.63;
ESI MS (m/z, relative abundance) 300 [(M-H)?, 100].
Synthesis of 20-benzenesulphonamide chalcone 5b
(Donnelly and Farrell, 1989)
To a solution of 20-aminochalcone (0.4 g, 3 mmol) in dry
CH2Cl2 (10 mL), Et3N (0.7 mL, 5 mmol) was added fol-
lowed by benzenesulphonyl chloride (0.51 mL, 4 mmol) at
0 °C. The contents were stirred overnight at room tem-
perature by gradually warming the reaction mixture from
0 °C. After the completion of the reaction as indicated by
TLC, the reaction mixture was diluted with CH2Cl2
(20 mL) and washed with water (2 9 15 mL) and satu-
rated sodium chloride solution (15 mL). The organic
extracts were then dried over Na2SO4 and evaporated under
reduced pressure to give the crude compound which was
subsequently purified by column chromatography (10 %
EtOAc/cyclohexane) to afford yellow crystals of the title
compound. Yield: 25 %; Rf = 0.47 (20 % EtOAc/cyclo-
hexane); mp 76–78 °C; 1H NMR (CDCl3, 500 MHz) d
11.19 (s, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.79–7.83 (m, 2H),
7.75 (d, J = 8.5 Hz, 1H), 7.66 (d, J = 15.5 Hz, 1H), 7.58–
7.60 (m, 2H), 7.49 (t, J = 7.5 Hz, 1H), 7.38–7.44 (m, 6H),
7.34 (d, J = 15.5 Hz, 1H), 7.14 (t, J = 7.5 Hz, 1H); 13C
NMR (CDCl3, 125 MHz) d 192.8, 146.1, 139.9, 139.4,
134.4, 134.3, 132.9, 131.0, 130.6, 129.1, 129.0, 128.6,
127.3, 124.9, 123.2, 122.1, 120.8; IR (thin film) 3144 (–NH
str.), 3070 (arom. –CH str.), 1641 (a,b-unsaturated keto
Antioxidant activity
The method of Doble and co-workers was adopted with
minor modifications for carrying out the DPPH assay
(Sivakumar et al., 2011). Stock solutions of the com-
pounds were prepared by solubilising 2 mg of the com-
pound in 200 lL of DMF and then making up the volume
to 10 ml with methanol. Corresponding dilutions of 10,
20, 30, 40 and 50 lg mL-1 were prepared with methanol.
0.1 mM DPPH solution and test samples were taken in a
ratio of 1:2, and the mixture was vortexed and kept for
equilibration by incubating in dark at 25 °C for 20 min.
The optical density was measured at 517 nm in a UV
spectrophotometer with ascorbic acid being used as the
positive control. Pure methanol was used as the blank, and
DPPH solution and methanol in a ratio of 1:2 were used
as the control. The actual decrease in the absorption
induced by the test compound was estimated by sub-
tracting the absorbance of the test from that of the control.
The percentage of scavenging of DPPH was calculated
using the formula:
123

Med Chem Res
would make them amenable to oral administration in
humans. Topological polar surface area (TPSA) of the
˚
target compounds and reference standard was below 70 A
Table 1 The Lipinski parameters and TPSA of the selected com-
pounds and ibuprofen
2
Compound Molecular Number of
weight
Number of
hydrogen
bond
Mol TPSA
logP (A )
2
˚
hydrogen
bond
acceptor(s)
implying easy permeability through cell membrane and
ready penetrability across the blood brain barrier (BBB).
(g mol-1
)
donor(s)
Chemistry
4a
4b
4d
4e
4f
276.08
233.08
223.10
223.10
246.09
246.09
301.08
363.09
301.08
363.09
208.09
1
2
1
1
3
3
3
3
3
3
1
2
2
0
2
2
0
0
1
1
1
1
0
1
3.27 17.07
3.71 40.86
3.59 43.09
3.59 43.09
4.07 66.82
3.99 66.82
3.08 55.24
4.66 54.10
3.20 54.48
5.03 53.34
3.95 17.07
3.38 37.30
Out of the ten target compounds obtained, eight were
synthesised by direct Claisen-Schmidt condensation of
substituted acetophenones and benzaldehyde while sulph-
onamides 5a and 5b were prepared from 20-aminochalcone
(4c) as clearly illustrated in Scheme 1. The structures of all
the synthesised derivatives were assigned on the basis of
IR, 1H & 13C NMR, mass spectral data as well as elemental
analysis and comparison with published data. It was
observed that the results are in agreement with the pro-
posed structures.
4g
4h
4i
5a
5b
1
Ibuprofen 206.13
The IR spectra of 4a and 4b showed characteristic peaks
at 1,943 cm-1 corresponding to C–F stretch of the –CF3
group and 2,230 cm-1 that is attributable to C:N
stretching of the –CN group, respectively. The 19F NMR of
30-trifluoromethylchalcone gives a singlet at -63.00 ppm
corresponding to the three equivalent fluorine atoms of the
aromatic –CF3 group. With regard to the amino-substituted
chalcones, it was difficult to purify the meta and para
analogues due to the interference of side products. While
30-aminochalcone (4d) was purified by repeated column
chromatography of the crude product, precipitation of pure
4e occurred upon dissolving it in boiling ethanol and
keeping overnight at room temperature. A broad singlet
around 4 ppm in the 1H NMR represents the two protons of
the –NH2 group. Deuterated water (D2O) exchange spec-
trum of 4d clearly demonstrates the exchange of –NH2
protons with deuterium atom(s). As seen in Fig. 2, the
–NH2 peak disappears and a singlet corresponding to HOD
appears around 4.7 ppm post equilibration of the sample.
The meta and para azidochalcones (4f and 4g, respec-
tively) were synthesised in two steps involving the prepa-
ration of intermediate azido acetophenones and their
subsequent condensation with benzaldehyde to afford the
target compounds. The intermediate 4-azido acetophenone
(3g) was isolated and characterised, whereas 3f could not
be purified and was consequently coupled with benzalde-
hyde as such. The crude 4f was then purified by column
chromatography. It is pertinent to note that 4f gradually
darkens from off-white to brownish colour when kept at
room temperature. The characteristic NNN stretching of
azides was displayed by both the azidochalcones in their IR
spectra at around 2,200–2,100 cm-1. Although the diag-
nostic molecular ion peaks were seen in the mass spectra of
azidochalcones, the characteristic and most intense peak
was at m/z 222 representing the fragment corresponding to
% scavenging of DPPH ¼ ACꢁ AS=AC ꢂ 100;
where AC = absorbance of control and AS = absorbance
of test sample
Statistical analysis
All assays were carried out in triplicate, and results were
expressed as mean SD. All statistical analysis was per-
formed using Graph Pad (Version-6) Prism software.
ANOVA was used to test the differences between the
percentage inhibition values of the different derivatives in
both the anti-inflammatory and antioxidant assays followed
by Tukey’s multiple comparison tests, and p \ 0.05 was
considered statistically significant.
Results and discussion
Drug-likeness assessment
The drug-likeness of compounds was evaluated by calcu-
lating the Lipinski parameters using the high-speed
molecular properties calculator, a free module in the
Molsoft software package. All chalcone derivatives have
less than or equal to two hydrogen bond donors and possess
no more than three hydrogen bond acceptors, the total
maximum permissible count being 10. Besides, none of
their molecular weights exceeded 500 daltons. The octa-
nol–water partition coefficient (logP) is not more than 5 for
any of the ligands except compound 5b. These results that
are shown in Table 1 strongly suggest drug-likeness, and
the target compounds can be said to have properties that
123

Med Chem Res
Fig. 2 D2O exchange observed
in 30-aminochalcone
the loss of nitrogen. The chalcone bearing the azide group
at the 20-(ortho) position was also targeted, but the efforts
for its isolation failed. The formation of this product was
confirmed by the mass spectrum showing an intense peak
at 222 as well as deduced from IR spectrum which dis-
played the characteristic peak of azide group between
2,200 and 2,100 cm-1. The presence of a stubborn impu-
disappearance of amide proton singlet in the range of 11–
11.5 ppm and the concomitant appearance of a singlet
corresponding to HOD in the range of 4.5–5 ppm.
Pharmacological evaluation
In our study, the in vitro anti-inflammatory potential of
chalcone derivatives was evaluated against denaturation of
BSA. The results indicated a concentration-dependent
inhibition of protein denaturation by the entire chalcone
library throughout the concentration range of 10–
30 lg mL-1. Ibuprofen that was used as reference drug also
exhibited a similar concentration-dependent profile of
inhibition. It is clear from the data (Table 2) that the sub-
stitution pattern on the phenyl ring of the acetophenic group
of chalcone moiety plays an important role in modulating
inflammation. Inspection of the tabulated values easily sets
apart the four sulphonamides as the better anti-inflamma-
tory analogues with the 20-benzenesulphonamide chalcone
5b emerging as the best compound within this subset. The
para azido- and aminochalcones also fared better than
ibuprofen, while the rest of the target compounds exhibited
activity similar to that of the reference. Significantly, none
of the library members were inactive in this assay.
1
rity was, however, seen in the compound’s H and 13C
NMR spectra which led to its eventual disqualification
from further in vitro study.
Two benzenesulphonamides and two methanesulph-
onamides, each appended to the 20- or 40- position of the
unsubstituted chalcone, comprise the sulphonamides
designed for the study. The 20-sulphonamide chalcones 5a
and 5b were synthesised by direct sulphonylation of 4c,
whereas the 40-sulphonamides 4h and 4i were accessed
from 4-amino acetophenone (3e) via the intermediary 4-
sulphonamide acetophenones. Pyridine or Et3N was
employed as a base in the sulphonamide synthesis. The
reaction, though slower when carried out using Et3N
compared to pyridine, was, however, driven to completion
under the former conditions. Three out of the four sulph-
onamides were purified by column chromatography which
was difficult to be performed due to the similarity in the Rf
values of the impurities to that of the desired products. The
remaining analogue, 40-benzenesulphonamide chalcone
(4i), was purified by stirring in boiling ethanol for 5–
10 min and filtering the solution. The pure product being
insoluble in hot ethanol is obtained as the filter cake and
the impurities go along with the hot ethanol. Analogous to
the aminochalcones, the presence of an exchangeable
amide proton was tested by recording the D2O exchange
spectra of all the sulphonamides (data not shown). As
expected, the exchange phenomenon resulted in the
From a SAR perspective, electron-withdrawing substitu-
ents in the form of a trifluoromethyl group (via inductive
effect) in 4a and a cyano group (via mesomeric effect) in 4b
had no effect on the anti-inflammatory activity. With respect
to the azide group, the para regioisomer was preferred over
the meta congener. This particular finding is in conformation
with reports published by Knaus and co-workers revealing the
structural as well as functional basis for the incorporation
of an azide as a key bioisosteric pharmacophore in contain-
ing inflammation (Habeeb et al., 2001; Rao et al., 2004).
123

Med Chem Res
Table 2 The anti-inflammatory and antioxidant profiles of the synthesised derivatives (4a–4i, 5a, 5b and 1)
Compound Concentration (lg mL-1
Anti-inflammatory activity (% inhibition)
)
Antioxidant activity (% inhibition)
10
20
30
10
20
30
40
50
4a
4b
4d
4e
4f
80.69 0.33a 84.21 0.55a 85.66 0.12a 15.01 0.30a 17.83 0.18a 20.45 0.26a 22.12 0.26a 24.39 0.18a
81.06 0.33a 84.76 0.16a 85.39 0.16a 14.69 0.63a 16.57 0.59b 17.53 0.52b 18.67 0.19b 19.89 0.25b
80.69 0.33a 84.21 0.55a 85.66 0.12a 8.69 0.30b 10.98 0.32c 12.24 0.32c
13.9 0.37c 15.18 0.24c
84.11 0.36b 84.82 0.41a 85.73 0.62a 9.24 0.24b 10.95 0.14c 13.61 0.37d 15.73 0.20d 17.41 0.15d
81.01 0.51a 82.50 0.36b 82.59 0.48b 1.95 0.35c 3.08 0.33d 4.73 0.14e
83.30 0.54b 85.48 0.73a 86.01 0.33a 2.19 0.31d 3.62 0.23d 5.57 0.28e
6.80 0.35e 9.30 0.23e
7.45 0.10e 9.54 0.33e
4g
4h
4i
85.11 0.18b 87.15 0.18c 88.76 0.09c 7.85 0.36b 8.67 0.21e 9.23 0.23f 10.02 0.17f 11.34 0.44f
84.15 0.43b 85.26 0.04a 86.04 0.20a 8.97 0.55b 9.50 0.23e 10.21 0.28g 11.00 0.12f 11.55 0.14f
g
5a
5b
1
84.94 0.33b 86.69 0.79a 88.65 0.24c 8.06 0.36b 8.87 0.16e 9.76 0.19
10.35 0.14f 11.40 0.18f
5.87 0.18g 7.84 0.28g
85.81 0.48b 87.95 0.24c 91.07 0.09d 2.54 0.18e 3.60 0.22d 4.55 0.39e
81.43 0.27a 81.96 0.16b 85.71 0.15a 14.15 0.39a 16.65 0.36b 17.63 0.29b 19.92 0.40h 22.21 0.45h
80.37 0.51a 80.79 0.41b 84.29 0.72e 93.62 0.17f 94.45 0.30f 95.04 0.18h 95.52 0.23i 96.43 0.21i
Stdi
Data presented as Mean SD of triplicate observations; different letters on the same column show significant differences from each other at
p \ 0.05
i
Reference drugs for anti-inflammatory and antioxidant activity were ibuprofen and ascorbic acid, respectively
A similar and subtler positional effect was mirrored in the case
of the amino substituent as well. Previous studies in our group
have led to the finding that electron-donating substituents,
especially of the mesomeric type like hydroxy- and amino-,
placed at the 20-position potentiate the anti-inflammatory
activity (Balasubramanian et al., 2013). Since free radicals
have been definitively implicated in the progression of
inflammatory damage, a regiochemical preference for the
ortho position can be unambiguously explained by the supe-
rior ability of such a substituent to form an intramolecular
hydrogen bond (iHB) and consequently, the relative ease of
free radical formation. More specifically, the unique hydrogen
bonding ability in 4c allows for the formation of an oxygen-
centred radical rather than a nitrogen-centred radical, the
former being a more favourable process owing to the lower
bond dissociation energy (BDE) of O–H versus N–H bond
(Bendary et al., 2013). Taken together with the results of this
work, a useful activity rank order presents itself in the context
of regioisomeric aminochalcones: ortho [ para [meta.
A marked increase in the percent inhibition of the sul-
phonamides over the parent chalcone underscores the
importance of this functional group as a ring A substituent.
Sulphur-based functional groups have been successfully
employed in blockbuster anti-inflammatory drugs. While
sulphonamides are present in coxibs (celecoxib, valdecoxib
and parecoxib) and oxicams (meloxicam, piroxicam and
tenoxicam), reverse sulphonamides i.e. sulphonanilides are
seen in drugs such as nimesulide and flosulide. The role of
methylsulphonyl group in potentiating anti-inflammatory
activity has been highlighted in depth (Gans et al., 1990;
Talley, 1999). A detailed investigation has provided further
valuable insight into the binding mode and consequently,
the selectivity imparted by a p-NHSO2CH3 substituent in
cyclooxygenase-2 (COX-2) inhibition (Garavito and De-
Witt, 1999; Zarghi et al., 2006). Therefore, it is quite
natural that the sulphonamides have emerged superior in
this library. Even though target-specific in silico analysis
was not a part of our study, it is not difficult to envision the
benzenesulphonamide 5b as the most optimal compound
because the phenyl group therein would likely participate
in hydrophobic interactions with the relevant residues in
the COX-2-binding pocket similar to those established in
the case of a p-NHSO2CH3 group.
Recently, a report published by Menichini and co-
workers (Conforti et al., 2009) as well as findings in our
laboratory (manuscript accepted for publication in Free Rad
and Antiox) have further corroborated the correlation
between antioxidant and anti-inflammatory activity. More-
over, literature suggests that antioxidant and anti-inflam-
matory activities can be expected to co-exist owing to the
involvement of reactive oxygen species (ROS) in cyclo-
oxygenase and lipoxygenase-mediated inflammatory path-
ways from arachidonic acid (Chebil et al., 2007; Melagraki
et al., 2009). The focused library under consideration in this
study was, therefore, felt suitable for carrying out the
antioxidant assay as it would help satisfy the twin objectives
of SAR exploration and an extension of anti-inflamma-
tory:antioxidant activity correlation. However, the results
displayed in Table 2 indicate that the derivatives did not
fare well in their antioxidant activity, with many showing
123

Med Chem Res
Balasubramanian R, Vijayagopal R (2012) Design and in silico
analysis of ring-A monosubstituted chalcones as potential anti-
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negligible percentage inhibition compared to the reference
compound. In fact, identification of the unsubstituted
chalcone as one of the active compounds renders the
members of this focused library ineffective for their ability
to probe antioxidant SAR. Interestingly enough, one of the
suboptimal anti-inflammatory compounds bearing the tri-
fluoromethyl group was the only library member that
exhibited better antioxidant potential than the parent. On a
similar note, the potential anti-inflammatory sulphonamides
had very poor antioxidant ability. These observations pre-
clude the possibility of extending any meaningful correla-
tion between the two activities. The azide-bearing
chalcones 4f and 4g turned out to be the least active ana-
logues in the DPPH radical scavenging assay.
Conclusions
The study reveals the design, synthesis and evaluation of
anti-inflammatory and antioxidant activity of a series of
monosubstituted chalcone derivatives. Most of the target
compounds were found to have significant anti-inflamma-
tory activity with many showing activity profile similar to
that of the standard drug. Some key structural features that
are beneficial for anti-inflammatory activity have been
identified. The utility of sulphonamides in containing
inflammation has been further corroborated in this work.
We have shown that a shift of the azide group from the
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clear regiochemical preference for primary amino groups
being placed in the ortho position of ring A has emerged
reinforcing our earlier observations. 20-Benzenesulphona-
mide derivative (5b) possessed the best anti-inflammatory
potential in this series. These results can act as an impetus
for further research in designing potent anti-inflammatory
synthetic chalcone derivatives. However, we realise that
these compounds are not the right probes for studying
antioxidant activity and consequently, a possible correla-
tion between the two activities could not be extended.
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Acknowledgments This work was partially funded by a grant (No.
051/SPS/2012/CSTE) of Kerala State Council for Science, Technol-
ogy and Environment (KSCSTE). The authors would like to thank the
Department of Organic Chemistry, Cochin University of Science and
Technology (CUSAT) for providing necessary laboratory facilities.
Conflict of interest The authors declare no conflict of interest.
Isa NM, Abdelwahab SI, Mohan S, Abdul AB, Sukari MA, Taha
MME, Syam S, Narrima P, Cheah S, Ahmad S, Mustafa MR
(2012) In vitro anti-inflammatory, cytotoxic and antioxidant
activities of boesenbergin A, a chalcone isolated from Boesen-
bergia rotunda (L.) (fingerroot). Braz J Med Biol Res 45(6):524–
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