Synthesis and biological evaluation of novel series of chalcone derivatives as inhibitors of cyclooxygenase and LPS-induced TNF-α with potent antioxidant properties

Article abstract of DOI:10.1007/s00044-011-9746-6

Novel series of chalcones were synthesized and were evaluated as possible anti-inflammatory agents targeting the cyclooxygenase-1 and 2 (COX-1 and 2), β-glucuronidase, trypsin, and TNF-α. Amongst the tested chalcones the compound 4k was found to be most effective inhibitor of TNF-α exhibiting 85% inhibition activity (IC₅₀ = 0.1 μM). The compounds 4a, 4f, 4l, and 4m were found to inhibit the COX-1 activity in as a range of 79.95-68.47% and COX-2 inhibition ranging 84.45-74.77%. The compounds 4l (81.71%) and 4f (72.10%) were found to be excellent inhibitors of trypsin and β-glucuronidase, respectively. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9746-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2292–2299
DOI 10.1007/s00044-011-9746-6
ORIGINAL RESEARCH
Synthesis and biological evaluation of novel series of chalcone
derivatives as inhibitors of cyclooxygenase and LPS-induced
TNF-a with potent antioxidant properties
•
Babasaheb P. Bandgar Baliram S. Hote
•
•
Nagesh A. Dhole Rajesh N. Gacche
Received: 26 January 2011 / Accepted: 8 July 2011 / Published online: 28 July 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Novel series of chalcones were synthesized and
were evaluated as possible anti-inflammatory agents target-
ing the cyclooxygenase-1 and 2 (COX-1 and 2), b-glucu-
ronidase, trypsin, and TNF-a. Amongst the tested chalcones
the compound 4k was found to be most effective inhibitor of
TNF-a exhibiting 85% inhibition activity (IC50 = 0.1 lM).
The compounds 4a, 4f, 4l, and 4m were found to inhibit the
COX-1 activity in as a range of 79.95–68.47% and COX-2
inhibition ranging 84.45–74.77%. The compounds 4l
(81.71%) and 4f (72.10%) were found to be excellent
inhibitors of trypsin and b-glucuronidase, respectively.
diaryl-2-propen-1-ones, belong to the flavonoid family.
Chemically they consist of open chain flavonoids wherein
the two aromatic rings are joined by a three-carbon a,
b-unsaturated carbonyl system. Varieties of naturally
occurring chalcones are polyhydroxylated in the aryl rings.
The radical quenching properties of the phenolic groups
present in many chalcones have raised interest in using the
compounds or chalcones rich plant extract as drugs or food
preservatives (Dhar, 1981). A vast body of literature has
accumulated in the recent past linking the importance of
chalcones as anti-inflammatory agents involved in inhibi-
tion of cell migration and inhibition of TNF-a synthesis in
mouse (Herencia et al., 2001). Many citations describe the
diverse biological activities of chalcones and related
derivatives as anti-cancer (Xia et al., 2000; Bois et al.,
1998), anti-inflammatory (Hsieh et al., 1998, 2000; Her-
encia et al.,1998), antimitotic (Ducki et al., 1998), antitu-
bercular (Linn et al., 2002), cardiovascular (Furman et al.,
2001), and hyperglycemic agents (Satyanarayana et al.,
2004).
Keywords WBCs ꢀ TNF-a ꢀ COX-1 ꢀ COX-2 ꢀ
Antioxidant ꢀ Enzyme inhibition
Introduction
Chalcone is one of the major classes of natural product
with wide spread distribution in fruit, vegetables, spices,
tea, and soya-based foodstuff and it has been recently
subjects of great interest for their interesting pharmaco-
logical activities (Di Carlo et al., 1999). Chalcones, or 1,3-
Cyclooxygenase (COX) is the rate-limiting enzyme of
the prostanoid biosynthetic pathway and it catalyzes the
conversion of arachidonic acid to important inflammatory
mediators such as prostaglandins (PGs), prostacyclins, and
thromboxanes (Vane and Botting, 1996). The existence of
enzyme cyclooxygenase in its two distinct isoforms and
thus nonselective action of classical non-steroidal anti-
inflammatory drugs (NSAIDs) results in certain mecha-
nism-based side effects including dyspepsia, gastrointesti-
nal ulcerations, bleeding, and nephrotoxicity (Pairet and
Engelhardt, 1996). Both the isoforms differ in their regu-
lation and expression. The constitutive COX-1 is respon-
sible for the biosynthesis of PGs, which involves the
cytoprotection of gastrointestinal tract and platelet aggre-
gation (Kujubu et al., 1991). COX-2 is induced by pro-
B. P. Bandgar (&)
Medicinal Chemistry Research Laboratory, School of Chemical
Sciences, Solapur University, Solapur, Maharashtra, India
e-mail: bandgar_bp@yahoo.com
B. P. Bandgar ꢀ B. S. Hote
Organic Chemistry Research Laboratory, School of Chemical
Sciences, SRTM University, Nanded, Maharashtra, India
N. A. Dhole ꢀ R. N. Gacche
Biochemistry Research Laboratory, School of Life Sciences,
SRTM University, Nanded, Maharashtra, India
123

Med Chem Res (2012) 21:2292–2299
2293
inflammatory molecules such as interleukin-1 (IL-1), tumor
necrosis factor-a (TNF-a), lipopolysaccharide (LPS), car-
rageenan, etc. that leads to inflammation (Marnett, 2000).
COX-2 levels are undetectable in most tissues under normal
physiological conditions, but are significantly up regulated
in acute and chronic inflammations. Inhibition of both iso-
forms by classical NSAIDs with preferential binding
affinity for enzyme COX-1 causes serious side effects. The
association of COX-2 with induced inflammation has led to
the hypothesis that selective inhibition of COX-2 over
COX-1 might provide good anti-inflammatory agents with
reduced side effects than classical NSAIDs. Searching the
alternatives to currently used NSAIDs has remained a major
thrust area of the anti-inflammatory research.
inflammation process leads to tissue injury by damaging
macromolecules and effecting the lipid peroxidation of
membranes (Gutteridge, 1995). This indicates that free
radical scavengers might be a useful means of attenuating
inflammatory effects.
In this study, series of novel chalcones were synthesized
and evaluated as possible inhibitors of cyclooxygenase,
LPS-stimulated TNF-a and pro-inflammatory enzymes
such as trypsin and b-glucuronidase (Bandgar et al., 2010).
Free radical scavenging assays were also performed so as
determine the antioxidant potential.
Result and discussion
Tumor necrosis factor-a, an important cytokine pro-
duced by activated monocytes/macrophages, has been
identified as an endotoxin-induced serum factor that causes
hemorrhagic necrosis of transplanted solid tumors (Miy-
achi et al., 1996). TNF-a plays a critical role in several
physiological immune systems, and can cause severe
damage when it is produced in excess. Therefore, TNF-a
can be regarded as possessing both favorable and unfa-
vorable effects. The favorable effects include direct tumor
killing action, stimulation of the host’s immune system,
and acting as a growth factor for normal B cells. The
unfavorable effects include induction of tissue inflamma-
tion, a tumor-promoting action, stimulation of human
immunodeficiency virus (HIV) replication, and induction
of insulin resistance. These pleiotropic effects associated
with TNF-a clearly demonstrate that the cytokine produc-
tion enhancers and inhibitors could be of use as biological
response modifiers under various circumstances (Shibata
et al., 1994; Miyachi et al., 1997).
The novel chalcone derivatives (4a–p) were synthesized
(Scheme 1). In this study, 1-(2-hydroxyl-4,6-dimethoxy-
phenyl)ethanone (2) was prepared by reacting commercially
available 1,3,5-trimethoxy benzene (1) with acetyl chloride
in dry ether using anhydrous AlCl3 (Ahluwalia, 2005). The
1-(2-hydroxyl-4,6-dimethoxyphenyl)ethanone (2) on bro-
mination with Br2 in glacial acetic acid with constant stirring
at 20°C for 1 h obtained 1-(3-bromo-2-hydroxyl-4,6-dime-
thoxyphenyl)ethanone (3) (Cechinel-Filho et al., 1996).
Finally the different substituted benzaldehydes treated with
1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)ethanone (3)
by Claisen–Schmidt condensation (Cechinel-Filho et al.,
1996). The desired derivatives were obtained in 46–83%
yield after purification. All synthesized compounds were
characterized by using IR, 1H NMR, mass spectra, and ele-
mental analysis.
Series of chalcone derivatives synthesized (Table 1)
were biologically evaluated as antioxidant and possible
inhibitors of pro-inflammatory cytokine TNF-a and
enzymes such as COX, trypsin, and b-glucuronidase. The
results of the TNF-a inhibition are shown in (Table 2). It
was observed that the compounds 4k (85.0%), 4o (78.0%),
4g (72.0%), and 4d (63.0%) were found to inhibit the LPS-
induced release of TNF-a in WBCs significantly as com-
pared to other compounds which showed the TNF-a inhi-
bition in a range of (37–57%) at 10 lM. Nevertheless, the
minimum IC50 values were calculated for 4k (0.1 lM) and
4o (0.6 lM). From the results, it can be stated that the
chalcones (4k and 4o) containing heterocyclic B-rings
seems to be an important configuration for the demon-
stration of effective TNF-a inhibition. Moreover, the
chalcones containing electron withdrawing substituents
(4e, 4d, and 4i) were also observed to be effective inhibi-
tors of TNF-a.
Trypsin is a member of the serine proteases family.
These proteases are involved in initiation of inflammation;
moreover, serine protease inhibition has been considered as
one of the targets for design of anti-inflammatory drugs
(Bilfinger and George, 2002). The enzyme b-glucuronidase
has been considered as one of the target in the design of
anti-inflammatory agents as it plays pro-inflammatory role
in the initiation of inflammation reaction. The lysosomes of
the polymorphonuclear neutrophils are rich in b-glucu-
ronidase. This enzyme is attributed as one of the mediators
for initiating the process of inflammation. The enzyme
inhibition assay was performed as per the method descri-
bed (Nicolaides et al., 1998).
According to recent studies, reactive oxygen species
(ROS) are important mediators that initiate and propagate
inflammatory responses by stimulating release of pro-
inflammatory cytokines such as interleukin-1b (IL-1b) and
tumor necrosis factor-a (Geronikaki and Gavalas, 2006). It
was also found that ROS induced by activated neutrophils,
eosinophils, monocytes, and macrophages during the
The results of the COX-1 inhibition summarized in
(Table 3) clearly indicated that the compounds 4a
(79.95%), 4f (71.39%), 4l (69.47%), and 4b (62.61%) were
effective, however, the compounds 4a (84.45%), 4f
123

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Med Chem Res (2012) 21:2292–2299
O
O
O
O
O
O
b
a
H
R
+
O
O
O
OH
O
OH
Br
2
3
1
c
O
O
4 a = 4 - OMe
4 i = 4 - Cl
4 b = 2, 5 - (OMe)2
4 c = 2, 4 - (OMe)2
4 d = 3 - F
4 j = 3 - CF3
R
4 k = 3 - Pyridine
4 l = 2 - F
O
OH
Br
4 e = 3 - CN
4 m = 3 - NO2
4 f = 3 - Br
4 g = 2 - Cl
4 a - p
4 n = 3, 5 - (OMe)2
4 o = 2 - Pyrrole
4 h = 2, 4 - Cl2
4 p = 4 - Cl, 3 - NO2
Scheme 1 Synthesis of novel series of chalcones
(78.41%), 4l (74.77%), and 4b (68.19%) were found to be
most active COX-2 inhibitors. It is interesting to note that
all the tested compounds are more selective toward COX-2
as compared to COX-1. The results obtained were com-
pared with standard COX-1 and 2 inhibitors such as aspirin
(34.90%) and SC 560 (38.54%). The results of the trypsin
and b-glucuronidase inhibition are summarized in
(Table 3). The compounds 4l (81.71%), 4j (71.84%), and
4i (67.81%) were observed to be effective inhibitors of
trypsin, while all other compounds demonstrated the
trypsin inhibition in a range of (33.67–65.92%) as com-
pared to salicylic acid (86.64%). It was found that the
compounds 4f (72.10%), 4l (68.46%), and 4j (62.11%)
inhibited b-glucuronidase significantly as compared to
other compounds having inhibition activity in a range of
(38.45–61.32%). The results of the enzyme inhibition
studies shows that the majority of compounds possessing
electron withdrawing substituted chalcones (4f, 4l, and 4m)
showed considerable pro-inflammatory enzyme inhibition
activity. However, 4a was exceptionally found to be more
active as compared to other compounds.
scavenging, and reducing activity, respectively. All other
compounds were found to possess moderate to good anti-
oxidant activities. The free radicals are closely implicated
in the initiation and progression of inflammatory disorders.
It has been also reported that the compounds possessing
significant reducing abilities can be considered as a
potential candidate for the design and development of
novel COX inhibitors (Gacche et al., 2008).
Experimental
General methods
Chemicals were purchased from Aldrich Chemical Co.,
USA. Melting points were determined with a digital ther-
mometer were uncorrected. IR spectra were recorded on
1
FT–IR Shimadzu 8300 spectrophotometer and H NMR
spectra were recorded on a Bruker 300 MHz spectrometer
in CDCl3 using tetramethylsilane as an internal standard
and chemical shifts are reported in d units and the coupling
constants (J) are reported in hertz. Mass spectra were
obtained with a Shimadzu LCMS-2010 EV. Chromato-
graphic purification was performed with silica gel
(100–200 mesh). Thin layer chromatography (TLC) was
performed on pre-coated silica plates (Merck Kiesegel
60F254, 0.2 mm thickness) sheets. The spots could be
visualized easily under ultraviolet light.
The results of the free radical scavenging activities are
depicted in (Table 4). With few exceptions, all the syn-
thesized chalcones exhibited moderate to good free radical
scavenging and reducing activities. Amongst the tested
chalcones, the compound 4a was found to possess con-
siderable antioxidant potential which demonstrated
(38.37%), (49.41%), and (34.32%) DPPH, OH radical
123

Med Chem Res (2012) 21:2292–2299
2295
Table 1 Analytical data of chalcone derivatives
Table 2 Profile of the inhibition of LPS-induced TNP-a production
by selected chalcones
O
Entry
Inhibition of TNF-a (%)
(IC50 lM)
1
1'
1 lM
10 lM
2
3
2'
3'
B
A
4
4a
4b
4c
38
22
21
36
26
13
40
46
43
27
45
24
31
25
42
34
72
53
57
59
63
57
57
72
53
51
52
85
38
50
37
78
49
87
7.4
5
5'
10.9
10.2
1.2
4'
4d
4e
0.7
Entry A ring
1
B ring
10 20
Yield
(%)
4f
8.2
2
3
4
5
30
H
40
50
4g
4h
4i
15.9
8.9
4a
4b
4c
4d
4e
4f
OMe H OMe Br OH H H
OMe H
40
1.4
OMe H OMe Br OH H OMe H
OMe H OMe Br OH H OMe H
H
OMe 38
4j
3.2
OMe H
47
55
55
60
83
62
80
70
52
62
60
4k
4l
0.1
OMe H OMe Br OH H H
OMe H OMe Br OH H Cl
OMe H OMe Br OH H H
OMe H OMe Br OH H Cl
OMe H OMe Br OH H Cl
OMe H OMe Br OH H H
OMe H OMe Br OH H H
OMe H OMe Br OH H H
OMe H OMe Br OH H F
OMe H OMe Br OH H H
OMe H OMe Br OH H H
OMe H OMe Br OH H H
OMe H OMe Br OH H H
F
H
H
H
H
Cl
Cl
H
N
H
H
H
H
H
H
H
II
II
H
H
H
N.A.
10.1
11.4
0.6
CN
Br
H
4m
4n
4o
4p
DMS
4g
4h
4i
H
14.2
1
H
4j
CF3
H
N.A. not active, DMS betamethasone
4k
4l
H
4m
4n
4o
4p
NO2
OMe H
d, J = 11.2 Hz), 7.78 (2H, d, J = 1.7 Hz), 7.52 (2H, d,
J = 1.9 Hz), 6.12 (1H, s, Ar-H), 4.04 (3H, s, –OCH3), 3.94
(6H, s, –OCH3 X 2). MS: m/e 394 (M ? 1). Anal.:
C18H17BrO5/393.
OMe 67
H
N
–
43
36
NO2 CI
H
(E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(2,5-
dimethoxyphenyl)prop-2-en-1-one (4b)
General Procedure for the synthesis of (4a–p)
To a mixture of 1-(3-bromo-2-hydroxy-4,6-dimethoxy-
phenyl)ethanone 3 (1 mmol) in ethanol (15 ml) was added
NaOH (2.5 mmol, 2–3 drops of water) and stirred for
5 min. Then, added substituted benzaldehyde (1 mmol)
and stirred the reaction mixture at room temperature. The
progress of reaction was checked by TLC and the reaction
mixture was poured over crushed ice and acidified with
acetic acid. The precipitated solid was filtered, washed with
water, and oven dried. It was purified by column chroma-
tography using silica gel mesh size (100–200 mesh) and
elution with petroleum ether and ethyl acetate.
Recrystallized from ethanol; (yield 38%); mp 134–136°C;
IR tmax cm-1 (KBr): 3430, 3140, 3008, 2964, 1678, 1638,
1560, 1465, 1420, 1021, 962, 765; H-NMR (CDCl3) d;
14.52 (1H, s, –OH), 7.83 (2H, d, J = 1.2 Hz), 7.82 (2H, d,
J = 12.2 Hz), 7.59 (1H, d, J = 11.4 Hz), 6.06 (1H, s, Ar-
H), 4.02 (3H, s, –OCH3), 3.94 (9H, s, –OCH3 X 3). MS: m/
e 424 (M ? 1). Anal.: C19H19BrO6/423.
1
(E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(2,4-
dimethoxyphenyl)prop-2-en-1-one (4c)
Recrystallized from ethanol; (yield 47%); mp 152–154°C;
IR tmax cm-1 (KBr): 3620, 3110, 2984, 1686, 1635, 1590,
1438, 1420, 1009, 865, 728; H-NMR (CDCl3) d; 14.79
(E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(4-
methoxyphenyl)prop-2-en-1-one (4a)
1
(1H, s, –OH), 8.04 (1H, d, J = 4.3 Hz), 7.36 (1H, d,
J = 1.4 Hz), 7.21 (1H, d, J = 9.2 Hz), 7.94 (2H, d,
J = 12.3 Hz), 7.72 (1H, d, J = 13.2 Hz), 6.09 (1H, s, Ar-
H), 4.06 (6H, s, –OCH3 X 2), 3.93 (6H, s, –OCH3 X 2).
MS: m/e 424 (M ? 1). Anal.: C19H19BrO6/423.
Recrystallized from ethanol; (yield 40%); mp 154–156°C;
IR tmax cm-1 (KBr): 3420, 3130, 2974, 1685, 1615, 1587,
1538, 1478, 1420, 1011, 920, 748; H-NMR (CDCl3) d;
14.59 (1H, s, –OH), 7.89 (1H, d, J = 11.2 Hz), 7.62 (1H,
1
123

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Med Chem Res (2012) 21:2292–2299
Table 3 Profile of COX, trypsin, and b-glucuronidase inhibitory
activity of (4a–p) chale one derivatives at 1 mM concentration
Table 4 Summary of the results of DPPH, OH radical scavenging
activity (%), and reducing activity (%) of (4a–p) chalcone derivatives
at concentration (1 mM)
Entry COX-1
(%)
COX-2
(%)
Trypsin
(%)
b-glucluronidase
(%)
Entry
DPPH (%)
OH (%)
Reducing activity (%)
4a
4b
4c
79.95
62.61
52.42
21.84
62.42
71.39
32.78
37.47
44.00
11.78
21.72
69.47
68.47
57.23
35.18
18.62
–
84.45
68.19
57.82
33.54
58.21
78.41
34.74
39.57
51.80
12.62
35.57
74.77
76.41
78.42
61.38
38.76
38.54
–
65.14
42.85
39.47
55.42
48.20
51.90
57.61
54.24
67.81
71.84
48.00
81.71
65.92
65.92
33.67
49.73
–
52.91
48.45
41.29
61.32
47.29
72.10
48.21
44.72
54.59
62.11
44.27
68.46
61.85
61.85
38.45
38.95
–
4a
38.37
22.98
20.24
12.00
27.42
24.12
15.75
17.29
18.41
13.67
21.11
29.95
23.67
NR
49.41
31.58
28.76
NR
34.32
22.90
18.40
12.20
17.27
19.60
14.29
17.38
15.70
10.00
16.56
29.70
26.22
18.40
26.00
26.00
–
4b
4c
4d
4e
4d
4e
25.62
38.23
32.27
30.18
38.23
47.05
44.11
16.76
18.82
23.52
NR
4f
4f
4g
4h
4i
4g
4h
4i
4j
4j
4k
4l
4k
4l
4m
4n
4o
4p
ASA
4m
4n
4o
26.69
14.27
91.02
–
4p
NR
GA (1 mM)
AA (1 mM)
–
SC 560 34.90
SA
–
–
41.17
76.60
–
–
86.64
26.48
1 mM is the stock concentration, from which only 10 lM was
pipetted. Therefore, the actual concentration in the reaction mixture is
lM only
The results summarized are the mean values of n = 2
ASA acetyl salicylic acid, SA salicylic acid, SC560 a standard COX-1
inhibitor
GA gallic acid, AA ascorbic acid, NR no reaction
(E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(3-
bromophenyl)prop-2-en-1-one (4f)
(E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(3-
fluorophenyl)prop-2-en-1-one (4d)
Recrystallized from ethanol; (yield 60%); mp 151–153°C;
IR tmax cm-1 (KBr): 3320, 3110, 3008, 2987, 1678, 1624,
1510, 1468, 1410, 1010, 968, 818, 726; H-NMR (CDCl3)
Recrystallized from ethanol; (yield 55%); mp 165–167°C;
IR tmax cm-1 (KBr): 3450, 3131, 3010, 2986, 1667, 1623,
1530, 1454, 1423, 1012, 945, 815, 724; H-NMR (CDCl3)
1
1
d; 14.61 (,1H, s, –OH), 7.80 (1H, d, J = 16 Hz), 7.72 (1H,
d, J = 16 Hz), 7.71 (2H, d, J = 1.7 Hz), 7.55 (2H, d,
J = 11.4 Hz), 7.28 (1H, d, J = 8 Hz), 6.05 (1H, s, Ar-H),
4.01 (3H, s, –OCH3), 3.95 (3H, s, –OCH3). MS: m/e 443
(M ? 2). Anal.: C17H14Br2O4/441.
d; 14.52 (1H, s, –OH), 7.85 (1H, d, J = 8.6 Hz), 7.82 (1H,
d, J = 1.6 Hz), 7.60 (1H, d, J = 1.6 Hz), 7.52 (1H, d,
J = 8.7 Hz), 7.11 (2H, d, J = 8.8 Hz), 6.05 (1H, s, Ar-H),
4.02 (3H, s, –OCH3), 3.92 (3H, s, –OCH3). MS: m/e 382
(M ? 1). Anal.: C17H14BrFO4/381.
(E)-3-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(2-
chlorophenyl)prop-2-en-1-one (4g)
3-((E)-3-(3-bromo-2-hydroxy-4,6-dimethoxyphenyl)-3-
oxoprop-1-enyl)benzonitrile (4e)
Recrystallized from ethanol; (yield 83%); mp 154–156°C;
IR tmax cm-1 (KBr): 3310, 3080, 3010, 2981, 1682, 1622,
1525, 1422, 1403, 1010, 936, 810, 712; H-NMR (CDCl3)
Recrystallized from ethanol; (yield 55%); mp 170–172°C;
IR tmax cm-1 (KBr): 3456, 3110, 3009, 2991, 1678, 1633,
1561, 1464, 1410, 1019, 933, 817, 720; H-NMR (CDCl3)
1
1
d; 14.61 (1H, s, OH), 7.89 (1H, d, J = 9.6 Hz), 7.82 (1H,
d, J = 9.6 Hz), 7.80 (1H, d, J = 0.4 Hz), 7.72 (1H, t, Ar-
H), 7.67 (1H, t, Ar-H), 7.53 (1H, d, J = 0.3 Hz), 6.12 (1H,
s, Ar-H), 4.01 (3H, s, –OCH3), 3.92 (3H, s, –OCH3). MS:
m/e 398 (M ? 1). Anal.: C17H14BrClO4/397.
d; 14.61 (1H, s, –OH), 7.86 (2H, d, J = 12.3 Hz), 7.51
(2H, d, J = 0.4 Hz), 7.35 (1H, d, J = 0.6 Hz), 7.13 (1H, d,
J = 3.5 Hz), 6.10 (1H, s, Ar-H), 3.52 (6H, s, –OCH3 X 2).
MS: m/e 389 (M ? 1). Anal.: C18H14BrNO4/388.
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2297
(E)-3-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(2,4-
dichlorophenyl)prop-2-en-1-one (4h)
d; 14.82 (1H, s, –OH), 7.95 (1H, d, J = 14.6 Hz), 7.88
(1H, d, J = 14.6 Hz), 7.53 (1H, d, J = 2 Hz), 7.34 (1H, d,
J = 3 Hz), 7.15(2H, m, Ar-H), 6.11 (1H, s, Ar-H), 3.98
(6H, s, –OCH3 X 2). MS: m/e 383 (M ? 2). Anal.:
C17H14BrFO4/381.
Recrystallized from ethanol; (yield 62%); mp 152–154°C;
IR tmax cm-1 (KBr): 3430, 3129, 3010, 2964, 1678, 1635,
1590, 1520, 1470, 1410, 1010, 928, 825, 728; H-NMR
1
(CDCl3) d; 14.60 (1H, s, –OH), 8.24 (1H, d, J = 12.1 Hz),
7.82 (1H, d, J = 12.1 Hz), 7.74 (1H, d, J = 0.3 Hz), 7.62
(1H, d, J = 0.3 Hz), 6.14 (1H, s, Ar-H), 4.03 (3H, s, –
OCH3), 3.92 (3H, s, –OCH3). MS: m/e 433 (M ? 1). Anal.:
C17H13BrCl2O4/432.
(E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(3-
nitrophenyl)prop-2-en-1-one (4m)
Recrystallized from ethanol; (yield 60%); mp 141–143°C; IR
t
max cm-1 (KBr): 3410, 3122, 3124, 2987, 1684, 1662, 1575,
1468, 1425, 1012, 931, 825, 748; 1H-NMR (CDCl3) d; 14.59
(1H, s, -OH), 8.47 (1H, d, J = 2 Hz), 8.25 (1H, d, J = 3 Hz),
7.94 (2H, d, J = 15.5 Hz), 7.62 (1H, d, J = 15.5 Hz), 7.24
(1H, d, J = 3 Hz), 7.15 (1H, d, J = 2.1 Hz), 6.02 (1H, s, Ar-
H), 3.97 (3H, s, –OCH3), 3.94 (3H, s,–OCH3). MS: m/e 410
(M ? 2). Anal.: C17H14BrNO6/408.
(E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(4-
chlorophenyl)prop-2-en-1-one (4i)
Recrystallized from ethanol; (yield 80%); mp 217–219°C;
IR tmax cm-1 (KBr): 3343, 3164, 3110, 2995, 1681, 1612,
1577, 1494, 1425, 1010, 943, 813, 736; H-NMR (CDCl3)
1
d; 14.24 (1H, s, –OH), 7.90 (1H, d, J = 15.6 Hz), 7.81
(1H, d, J = 19.6 Hz), 7.66 (2H, d, J = 8.6 Hz), 7.45 (2H,
d, J = 8.5 Hz), 6.21 (1H, s, Ar-H), 3.95 (3H, s, –OCH3),
3.92 (3H, s, –OCH3). MS: m/e 398 (M ? 1). Anal.:
C17H14BrClO4/397.
(E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(3,5-
dimethoxyphenyl)prop-2-en-1-one (4n)
Recrystallized from ethanol; (yield 67%); mp 170–172°C;
IR tmax cm-1 (KBr): 3470, 3168, 3008, 2989, 2858, 1678,
1618, 1567, 1590, 1410, 1021, 945, 822; 1H-NMR (CDCl3)
d; 14.64 (1H, s, –OH), 7.82 (1H, d, J = 2.4 Hz), 7.64 (1H,
d, J = 2.4 Hz), 7.62 (1H, d, J = 2.4 Hz), 7.61 (2H, d,
J = 12 Hz), 6.04 (1H, s, Ar-H), 4.03 (6H, s, –OCH3 X 2),
3.92 (6H, s, –OCH3 X 2). MS: m/e 424 (M ? 1). Anal.:
C19H19BrO6/423.
(E)-3-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(3-
(trifluoromethyl)phenyl)prop-2-en-1-one(4j)
Recrystallized from ethanol; (yield 70%); mp 139–141°C;
IR tmax cm-1 (KBr): 3420, 3120, 3024, 2978, 1678, 1656,
1516, 1458, 1413, 1009, 928, 824, 721; H-NMR (CDCl3)
1
d; 14.62 (1H, s, –OH), 7.88 (1H, d, J = 7.6 Hz), 7.81 (1H,
d, J = 9.5 Hz), 7.73 (2H, t, Ar-H), 7.62 (1H, d,
J = 1.7 Hz), 7.53 (1H, d, J = 6.1 Hz), 6.13 (1H, s, Ar-H),
4.01 (3H, s, –OCH3), 3.92 (3H, s, –OCH3). MS: m/e 432
(M ? 1). Anal.: C18H14BrF3O4/431.
(E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(1H-
pyrrol-3-yl)prop-2-en-1-one (4o)
Recrystallized from ethanol; (yield 43%); mp 156–158°C;
IR tmax cm-1 (KBr): 3410, 3109, 3144, 2968, 1681, 1638,
1510, 1456, 1420, 1012, 933, 822, 751; H-NMR (CDCl3)
1
(E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-
(pyridine-3-yl)prop-2-en-1-one (4k)
d; 14.21 (1H, s, –OH), 7.90 (1H, s, Ar-H), 7.82 (1H, d,
J = 10.2 Hz), 7.60 (2H, d, J = 10.2 Hz), 7.44 (1H, d,
J = 0.5 Hz), 7.42 (1H, s), 6.02 (1H, s, Ar-H), 4.02 (3H, s,
–OCH3), 3.94 (3H, s, –OCH3). MS: m/e 352 (M ? 1).
Anal.: C15H14BrNO4/351.
Recrystallized from ethanol; (yield 52%); mp 166–168°C;
IR tmax cm-1 (KBr): 3310, 3144, 3110, 2987, 1681, 1632,
1570, 1456, 1415, 1014, 934, 813, 751; H-NMR (CDCl3)
1
d; 14.72 (1H, s, –OH), 8.84 (1H, d, J = 2 Hz), 8.61 (1H, d,
J = 3 Hz), 7.43 (2H, d, J = 11 Hz), 7.35 (1H, d,
J = 12 Hz), 6.11 (1H, s, Ar-H), 3.97 (6H, s, –OCH3 X 2).
MS: m/e 366 (M ? 2). Anal.: C16H14BrNO4/364.
(E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(4-
chloro-3-nitrophenyl)prop-2-en-1-one (4p)
Recrystallized from ethanol; (yield 36%); mp 176–178°C;
IR tmax cm-1 (KBr): 3420, 3145, 3109, 2987, 2842, 1685,
1616, 1598, 1547, 1422, 1012, 972, 815, 725; H-NMR
1
(E)-3-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(2-
fluorophenyl)prop-2-en-1-one (4l)
(CDCl3) d; 14.21 (1H, s, –OH), 7.92 (2H, d, J = 14.6 Hz),
7.72 (1H, s, Ar-H), 7.69 (1H, d, J = 12.2 Hz), 7.62 (1H, d,
J = 1.7 Hz), 6.04 (1H, s, Ar-H), 4.09 (3H, s, –OCH3),
3.96 (3H,–s, OCH3). MS: m/e 443 (M ? 1). Anal.:
C17H13BrClNO6/442.
Recrystallized from ethanol; (yield 62%); mp 152–154°C;
IR tmax cm-1 (KBr): 3400, 3165, 3021, 2974, 1678, 1665,
1545, 1526, 1470, 1010, 978, 824, 765; H-NMR (CDCl3)
1
123

2298
Med Chem Res (2012) 21:2292–2299
Whole blood cell culture (WBCs)
phosphate buffer, pH 7.6) was added after 20 min. The
reaction mixture was incubated for 25 min at 37°C. The
reaction was terminated by the addition 3 ml of
CCl3COOH (5%, w/v). The acid soluble fractions were
obtained by centrifuging the contents at 5,000 RPM for
15 min. The amount of protein in the acid soluble fractions
was estimated by this method (Tandon et al., 1982; Lowry
et al., 1951).
TNF-a production by lipopolysaccharide (LPS) in whole
blood was measured according to the method described by
Wilson et. al. In brief, K3EDTA blood from healthy donors
were diluted 1:5 in RPMI 1640 culture medium containing
100 U/ml penicillin and 100 mg/ml streptomycin with no
added serum. 100 ll/well of diluted blood was transferred
into 96 well culture plates. Following cell plating, 80 ll of
medium and 1 ll of the test compounds or vehicle (0.5%
DMSO) were added to each well and the plate was incu-
bated at 37°C for 30 min. Finally 20 ll (10 lg/ml) per well
of LPS (Escherichia coli 0127:B8) was added, for a final
concentration of 1 lg/ml. Plates were incubated at 37°C up
to 5 h in 5% carbon dioxide humidifier. Supernatants were
harvested and assayed for TNF-a release compared to the
control (Wilson et al., 1997).
b-glucuronidase inhibition assay
The effect of the selected compounds on activity of b-
glucuronidase was studied using a method described by
(Nicolaides et al., 1998). One millimolar concentration of
test sample (0.1 ml) in 0.1 M acetate buffer pH 7.4 for
5 min at 37°C were preincubated with 0.8 mL of 2.5 mM
p-nitrophenyl-b-D-glucopyranosiduronic acid and 0.1 ml of
b-glucuronidase was added. The mixture was incubated for
30 min. Reaction was terminated by addition of 2 ml of
0.5 N NaOH. The reaction mixtures were observed spec-
trophotometrically at 410 nm (Gacche and Dhole, 2006).
COX-1 and 2 inhibition micro-titer assay
The COX-1 and 2 inhibition assay was performed as per
the assay protocol instructions of ‘‘Colorimetric COX
(ovine) inhibitor Screening Assay Kit,’’ Cayman Chemical
Company, MI, USA. The reaction mixture of 100% initial
activity wells contained 160 ll of assay buffer, 150 ll of
heme, and 10 ll of either COX-1 or 2 enzyme solutions.
While the reaction mixture of inhibitor wells was com-
prised of 150 ll of assay buffer, 10 ll of heme, and 10 ll
of either enzyme COX-1 or 2, 10 ll of the test samples
(1 mM). The plates were carefully shaken for 5 s and were
incubated for 5 min at 25°C. After 5 min incubation, 20 ll
of the colorimetric substrate solution was added to all the
wells, followed by the addition of 20 lL of arachidonic
acid to all the wells. The plates were shaken gently for few
seconds and again incubated for 5 min at 25°C. The
absorbance of all the wells was read at 590 nm using
Thermo make Automatic Ex-Microplate Reader (M
51118170). The COX inhibition activity (%) was calcu-
lated using following formula.
DPPH radical scavenging assay
The DPPH radical scavenging assay was performed as
described by Bartolome et al. (2004). The reaction mixture
contained 1 mM concentrations of individual test sample
(in absolute ethanol) and DPPH radical (10-4 M in abso-
lute ethanol) solution. The contents of the reaction mixture
were observed spectrophotometrically at 517 nm after
20 min (Bartolome et al., 2004).
OH radical scavenging activity
OH radicals were generated by using the Ferric ion (Fe3?)/
ascorbic acid reaction system. The detection of OH radicals
was carried out by measuring the amount of formaldehyde
generated from the oxidation of dimethyl sulfoxide. The
reaction cocktail contained 0.1 mM EDTA, 167 mM Fe3-
,
33 mM DMSO in phosphate buffer of 50 mM pH 7.4, and
0.1 ml individual compound (1 mM) solution. Ascorbic
acid (150 ll, 10 mM in phosphate buffer) was added
finally to initiate the reaction. Trichloroacetic acid (17%,
w/v) was used to terminate the reaction. The contents were
observed spectrophotomertically at 412 nm for the detec-
tion of formaldehyde (Nash, 1953).
T
COX inhibition activity ð%Þ ¼ 1 ꢁ ꢂ 100
C
where T is the absorbance of the inhibitor well at 590 nm,
C is the absorbance of the 100% initial activity without
inhibitor well at 590 nm (Bandgar et al., 2010).
Trypsin inhibition assay
Reducing activity assay
The method is based on the measurement of inhibition of
trypsin induced hydrolysis of bovine serum albumin
(BSA). Trypsin (0.075 mg/ml) was initially incubated with
1 mM individual concentrations of test sample of 0.1 ml
for 20 min. The substrate BSA (6 g/100 ml, in 0.1 M
Reduction of Fe3? of K3Fe(CN)6 to Fe2? by antioxidants is
the underlying mechanism of the reducing activity assay.
The reducing potential of selected compounds was calcu-
lated by the fall in extinction of K3Fe(CN)6 at 420 nm
123

Med Chem Res (2012) 21:2292–2299
2299
activity of chalcone derivatives. Bioorg Med Chem Lett
8:1169–1174
against appropriate blank. The reaction mixture contained
500 ml solution of individual sample (1 mM in 0.5% v/v
dimethyl sulfoxide) in 3 ml of 1 mM potassium ferricynide
solution and the absorbance was recorded at 420 nm after
10 min reaction time (Sasaki et al., 1991).
´
´
Herencia F, Ferrandiz ML, Ubeda A, Guillen I, Dominguez JN,
Charris JE, Lobo GM, Alcaraz MJ (2001) 4-dimethylamino-3,4-
dimethoxy chalcones down regulates inos expression and exerts
anti-inflammatory effects. Free Radic Biol Med 30:43–50
Hsieh H-K, Lee T-H, Wang J-P, Wang J-J, Lin C-N (1998) Synthesis
and anti-inflammatory effect of chalcones and related com-
pounds. Pharm Res 15:39–46
Acknowledgment Authors are thankful to the Council of Scientific
and Industrial Research (CSIR), New Delhi for the award of SRF to
Mr. B.S.Hote, as well as to Mr. Mahesh Nambiar and Mrs. Asha
Almeida, Piramal life Sciences Ltd., Mumbai for TNF-screening of
the compounds.
Hsieh H-K, Tsao L-T, Wang J-P (2000) Synthesis and anti-inflamma-
tory effect of chalcones. J Pharm Pharmacol 52:163–171
Kujubu DA, Fletcher BS, Varnum BC, Lim RW, Herschman HR
(1991) TIS10, a phorbol ester tumor promoter-inducible mRNA
from Swiss 3T3 cells, encodes a novel prostaglandin synthase/
cyclooxygenase homologue. J Biol Chem 266:12866–12872
Linn Y-M, Zhou Y, Flavin MT, Zhou L-M, Nie W, Chen F-C (2002)
Chalcones and flavonoids as anti-tuberculosis agents. Bioorg
Med Chem 10:2795–2802
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measurement with the folin phenol reagent. Biol Chem 193:
265–275
Marnett LJ (2000) Cyclooxygenase mechanisms. Curr Opin Chem
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