Synthesis and biological evaluation of novel curcumin analogues as anti-inflammatory, anti-cancer and anti-oxidant agents

Article abstract of DOI:10.1007/s00044-011-9834-7

A series of novel curcumin analogues 5a- m were synthesized by Claisen-Schmidt condensation of various aromatic and heteroaromatic amides of 3-aminoactophenones 4a-m with 3-bromo-2,4,6-trimethoxybenzaldehyde and characterized by IR, ¹H NMR and mass spectroscopic analysis and were evaluated for anti-inflammatory, anti-cancer and anti-oxidant activity. Out of the 13 synthesized compounds, compounds 5f, 5j and 5m were excellent inhibitors of TNF-α and IL-6. Compounds 5c, 5e, 5b and 5d showed potent COX-2 inhibition, compounds 5d and 5f have shown good trypsin inhibition and compounds 5e, 5g and 5c exhibited excellent β-glucuronidase inhibition. Compounds 5l and 5m showed potent anti-cancer activity against selected five human cancer cell lines. All the compounds exhibited moderate free radical scavenging activity, while compounds 5a and 5m were excellent OH radical scavengers. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9834-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3006–3014
DOI 10.1007/s00044-011-9834-7
ORIGINAL RESEARCH
Synthesis and biological evaluation of novel curcumin analogues
as anti-inflammatory, anti-cancer and anti-oxidant agents
•
Babasaheb P. Bandgar Baliram S. Hote
•
•
Shivkumar S. Jalde Rajesh N. Gacche
Received: 2 August 2011 / Accepted: 20 October 2011 / Published online: 6 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A series of novel curcumin analogues 5a–
m were synthesized by Claisen-Schmidt condensation of
various aromatic and heteroaromatic amides of 3-amin-
oactophenones 4a–m with 3-bromo-2,4,6-trimethoxybenz-
aldehyde and characterized by IR, 1H NMR and mass
spectroscopic analysis and were evaluated for anti-inflam-
matory, anti-cancer and anti-oxidant activity. Out of the 13
synthesized compounds, compounds 5f, 5j and 5m were
excellent inhibitors of TNF-a and IL-6. Compounds 5c, 5e,
5b and 5d showed potent COX-2 inhibition, compounds 5d
and 5f have shown good trypsin inhibition and compounds
5e, 5g and 5c exhibited excellent b-glucuronidase inhibi-
tion. Compounds 5l and 5m showed potent anti-cancer
activity against selected five human cancer cell lines. All
the compounds exhibited moderate free radical scavenging
activity, while compounds 5a and 5m were excellent OH
radical scavengers.
Introduction
Curcumin (diferuloylmethane) (Fig. 1) is a b-diketone
constitute of the turmeric obtained from the powdered root
of Curcuma longa Linn. This yellow pigment is the main
constituent of turmeric powder, a widely used spice in
Southeast Asia (Nadkarni, 1976). Curcumin is also used in
traditional medicine to treat wide variety of human ail-
ments such as indigestion, urinary tract infection and liver
diseases (Cooper et al., 1994). Curcumin and its analogues
are also reported to possess anti-inflammatory, anti-oxi-
dant, anti-proliferative, anti-angiogenic and anti-tumori-
genic properties (Ammon and Wahl, 1991; Kawamori
et al., 1999; Huang et al., 1997, 1988).
Amongst the series of newly invented drug targets for
inflammations and related disorders, tumour neurosis fac-
tor-a (TNF-a) has been described as a pro-inflammatory
cytokine produced by monocytes, macrophages, neutro-
phils, T-cells, epithetial cells, osteoblasts and dendritic
cells (Mehta et al.,1997; Tracy and Cerami, 1994). The
overexpression of TNF-a is responsible for number of
pathological disorders like ulcerative colitis (Goldfeld
et al., 1991), diabetes (Newton and Decicco, 1999), mul-
tiple sclerosis (Hotamisligil et al., 1995), atherosclerosis
(Selmaj et al., 1991) and stroke (Rus et al., 1991). More-
over, there exist an evidence to support the fact that TNF-a
plays a key role in the origin and progression of rheumatoid
arthritis (RA) and other immune related disorders (Lover-
ing and Zhang, 2005; Oppenheim and Feldmann, 2001;
Feldmann et al., 1996; Odeh, 1997; Bertolini et al., 1986;
Sakalatvala, 1986; Papadakis and Targan, 2000; Tracey
et al., 2008). After the role of TNF-a was elucidated clearly
in RA, efforts have been made to develop inhibitors of
TNF-a. Its activity may be potentially inhibited at a variety
of sites. There are several strategies to inhibit the
Keywords TNF-a ꢀ IL-6 ꢀ COX-2 ꢀ Anti-cancer activity ꢀ
Anti-oxidant activity
B. P. Bandgar (&)
Medicinal Chemistry Research Laboratory, School of Chemical
Sciences, Solapur University, Solapur 413 255, India
e-mail: bandgar_bp@yahoo.com
B. P. Bandgar ꢀ B. S. Hote ꢀ S. S. Jalde
Organic Chemistry Research Laboratory, School of Chemical
Sciences, Swami Ramanand Teerth Marathwada University,
Nanded 431 606, India
R. N. Gacche
School of Life Sciences, Swami Ramanand Teerth Marathawada
University, Nanded 431 606, India
123

Med Chem Res (2012) 21:3006–3014
3007
production or release of TNF-a from the cell, neutralize
TNF-a on the cell surface or in the soluble phase or block
the TNF-a receptor or its downstream signal transduction
pathway (Elliott and Maini, 1994; Moreland et al., 1997).
Amongst pro-inflammatory cytokines, interleukin-6 (IL-
6) is considered to play a key role of initiation and
extension of the inflammatory process. IL-6 is a multi-
functional cytokine produced by wide range of cells, usu-
ally at site of tissue injury, and it regulated hepatic acute
phase response, the immune response, inflammation and
haematopoiesis. It appears to be the central mediator in a
range of inflammatory diseases, including end-stage renal
disease and rheumatoid arthritis (Dominic, 2009). Inhibi-
tion of IL-6 has received desired attention in drug
discovery.
derivatives as curcumin analogues. Woo et al. (2005) have
synthesized curcumin mimics containing enone and amide
ring (Fig. 1). Herein, we have synthesized the novel cur-
cumin analogues containing enone and amide containing
trimethoxy benzene moiety.
The 1,3,5-trimethoxybenzene on Vilsmeier-Haack formy-
lation gives 2,4,6-trimethoxy benzaldehyde (2), which on
bromination with bromine in glacial acetic acid afford
3-bromo-2,4,6-trimethoxybenzaldehyde (3). Compounds
(4a–m) were prepared by acylation of 3-aminoacetophenone
with different acylchlorides in basic medium. Compounds
(4a–m) on Claisen-Schmidt condensation with 3-bromo-
2,4,6-trimethoxybenzaldehyde (3) under basic media affor-
ded a residue, which on purification by column chromatog-
raphy with 1% ammonia and 0.5–1% methanol in chloroform
as eluting solvent, furnished title compounds (5a–m) in
good yields (Scheme 1). All the synthesized curcumin ana-
logues were characterized by IR, 1H NMR and mass spectral
analysis.
Result and discussion
Although, curcumin is non-toxic and has promising bio-
logical activities, preclinical and clinical studies have
indicated that poor bioavailability and pharmacokinetic
profile are due to its instability under physiological con-
dition which has limited its application in anti-cancer
therapies (Hsu and Cheng, 2007; Pan et al., 1999; Sharma
et al., 2007). Evidences from both in vitro and in vivo
studies show that b-diketone moiety is responsible for
instability and weak pharmacokinetic profiles of curcumin.
During last decade synthetic modifications of curcumin,
which were aimed at enhancing its bioactivities, suggested
that the stability and metabolic profile of curcumin could
be enhanced by deleting b-diketone moiety. To overcome
these barriers, several research groups have synthesized
curcumin analogues. Robinson et al. (2005) have synthe-
sized enone and dienone analogues, Ahn et al. (2004) have
synthesized the bis-alkynyl/alkyl, pyridine and thiophene
All synthesized compounds were evaluated for anti-
inflammatory (TNF-a, IL-6, COX-1 and -2, b-glucuroni-
dase and trypsin), anti-cancer and antioxidant activity.
Anti-inflammatory activity against TNF-a and IL-6 and
results are presented in Table 1. Results revealed that the
synthesized compounds 5a, 5j, 5b, 5c, 5f, 5h, 5g and 5m
have shown excellent inhibiton of TNF-a (91–83%) as
compared with standard dexamethasone (72%). Overall,
compounds 5f, 5j and 5m are the most active among the
series against TNF-a and have least cytotoxicity against
CCK-8 cell line. The structure activity relationship study in
these compounds revealed that compounds containing
electron-withdrawing substituent on amide ring have
higher TNF-a inhibition than their counterpart containing
electron-donating substituent on amide ring with exception
compound 5g. Bioisosteric replacement of aromatic ring by
thiophene there is slight decrease in TNF-a inhibition.
Fig. 1 Curcumin and its
analogues
O
O
O
MeO
HO
OMe
R
R
X
X
X
X
OH
R= Cl, F, CH3, NO2, isopropyl, OBn, phenyl,
OCH3 and OH
X= C, N = benzene, pyridine, furan, pyrrole,
naphthalene, anthracene, biphenyl and
benzo[1,3]dioxole.
1
2
OMe
OMe
OH
O
H
HO
MeO
HO
N
O
N
: alkyl, alkoxyl, halogen substituted
phenyl and heteroaromatic
3
4
123

3008
Med Chem Res (2012) 21:3006–3014
O
O
O
Table 2 Anti-inflammatory activity of novel curcumin analogues at
1 lM conc
CHO
O
CHO
O
ii
i
Compounds
COX-1
(%)
COX-2
(%)
Trypsin
(%)
b-glucuronidase
(%)
O
O
O
O
1
2
Br
3
5a
51.91
39.86
48.89
53.87
41.71
58.92
64.56
44.78
59.76
24.18
31.49
19.55
27.81
–
65.67
87.88
89.25
86.51
88.38
83.28
85.79
79.31
82.54
48.22
43.71
37.95
55.41
36.93
–
58.12
68.41
65.48
78.11
66.45
72.86
69.58
64.75
63.54
51.61
35.52
39.63
41.37
–
49.61
76.86
81.38
78.24
84.68
79.39
82.54
74.24
72.89
66.91
29.72
33.98
44.67
–
O
O
5b
H
N
R
CHO
O
5c
+
O
O
5d
Br
iii
5e
4 a-m
3
5f
5g
O
O
R
H
N
5h
5i
O
O
O
5j
Br
5a = H,
5g = 4 - tert. butyl,
5h = 2 - F,
5 a-m
5b = 2 - CF3,
5c = 3 - CF3,
5d = 3 - Cl,
5e = 3 - Br,
5f = 4 - F,
5k
5i = 3 - F,
5j = 3 - Me,
5l
5k = 2 - Me,
5l = 4 - OMe,
5m = 2 - Thiophene.
5m
ASA
SC 560
SA
Scheme 1 Synthesis of curcumin analogues
36.71
–
–
–
–
84.99
24.77
Table 1 Anti-inflammatory activity against TNF-a and IL-6
The results summarized are the mean values of n = 2
Compounds
% Inhibition at 10 lM
ASA acetyl salicylic acid, SA salicylic acid, SC560 a standard COX-1
TNF-a
IL-6
Toxicity
compound 5g. Compounds with fluorine and trifluoro-
methyl substitution have highest activity than the com-
pounds containing chloro and bromo substitution on amide
ring.
5a
91
85
85
00
04
85
83
84
78
87
72
63
80
72
98
95
97
64
75
97
96
96
93
97
95
94
96
94
65
62
65
25
31
49
56
56
51
56
54
65
53
00
5b
5c
5d
The synthesized compounds are subjected to COX
inhibition study and results are shown in Table 2. Com-
pounds 5c, 5e, 5b, 5d and 5f have shown potent
(89.25–83.28%) COX-2 inhibition. COX-2 is expressed
during inflammation and COX-1 is constitutive, which is
involved in physiological functions. Therefore, compounds
selectively inhibit the COX-2 have good account of anti-
inflammatory activity. Compounds under investigation
have inhibited COX-2 preferentially and are more selective
towards COX-2 rather than COX-1. Structure–activity
relationship study with respect to cyclooxygenase inhibi-
tion revealed that compounds containing electron-with-
drawing substituent on amide ring with exception 5g.
Change in the position of substituent will affect the activ-
ity, changing the position of fluorine from para (5f) to meta
(5i) there is a slight decrease in activity and from meta (5i)
to ortho (5h) further decrease in activity.
5e
5f
5g
5h
5i
5j
5k
5l
5m
DMS (1 lM)
DMS Dexamethasone, The results summarized are the mean values of
n = 2
The synthesized compounds screened for IL-6 inhibition
and results are summarized in Table 1. Results showed that
synthesized compounds are found to be active against IL-6.
Compounds 5a, 5c, 5f, 5j, 5g, 5h, 5m and 5b have shown
excellent inhibition of IL-6 (98–95%) as compared with
standard dexamethasone (94%). Overall, compounds 5f, 5j
and 5m are most active and have lowest cytotoxicity
against CCK-8 cell line. Structure activity relationship
indicates amides containing electron-withdrawing substit-
uents are essential for IL-6 inhibition, with exception
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 target for design of anti-inflammatory drugs
(Bilfinger and George, 2002). From Table 2, compounds
5d (78.11%) and 5f (72.86%) have shown good trypsin
inhibition, while compounds 5g, 5b, 5e, 5c and 5h have
shown moderate trypsin inhibiton (69.58–64.75%) as
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Med Chem Res (2012) 21:3006–3014
3009
compared with standard salicylic acid (84.99%). Electron-
withdrawing groups on amide ring are essential for trypsin
inhibition with exception compound 5g.
Table 4 Summary of the results of DPPH, OH radical scavenging
activity (%) and reducing activity (%) of curcumin analogues at 1 lM
conc
Compounds
DPPH (%)
OH (%)
Reducing activity (%)
The enzyme b-glucuronidase has been considered as one
of the targets in the design of anti-inflammatory agents as it
play the role in the initiation of inflammation. 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. Compounds 5e, 5g, 5c,
5f, 5b and 5h have shown excellent inhibition of b-glucu-
ronidase (84.68–74.24%) as compared with standard salicylic
acid (24.77%). Compounds with electron-withdrawing
groups on amide ring are essential for the b-glucuronidase
inhibition than the compounds containing electron-donating
groups. The aromatic amide (5a) and heteroaromatic amide
(5m) are weak inhibitors of b-glucuronidase.
All the synthesized compounds were screened for anti-
cancer activity against ACHN (renal cell carcinoma),
Panc1 (pancreatic carcinoma), Calu1 (non small cell lung
carcinoma), H460 (non small cell lung carcinoma) and
HCT116 (colon carcinoma) human cancer cell lines
(Table 3). Compounds 5l and 5m have shown potent anti-
cancer activity against selected five cancer cell lines as
compared with standard flavopiridol and gemcitabine.
Compound 5h has good anti-cancer activity against all five
cancer cell lines. Structure–activity relationship study
revealed that compounds containing electron-donating
groups on heteroaromatic amide ring have the highest
cytotoxicity. These compounds showing a broad-spectrum
inhibition against all selected five human cancer cell lines.
5a
49.44
44.65
45.69
45.34
42.56
44.30
43.61
42.91
43.26
36.66
31.11
36.31
35.27
92.36
–
54.14
35.85
NR
46.62
42.87
40.69
38.41
39.68
43.92
36.81
40.55
39.85
29.22
22.31
26.94
32.66
–
5b
5c
5d
NR
5e
23.65
33.41
27.31
43.17
32.19
38.29
34.63
21.21
49.26
–
5f
5g
5h
5i
5j
5k
5l
5m
GA (1 lM)
AA(1 mM)
45.85
78.89
GA gallic acid, AA ascorbic acid, NR no reaction
According to recent studies, free radicals such as reac-
tive oxygen species (ROS) are important mediators that
initiate and propagate inflammatory responses by stimu-
lating release of pro-inflammatory cytokines such as IL-1b
and TNF-a (Geronikaki and Gavalas, 2006). It was also
found that ROS induced by activated neutrophils, eosino-
phils, monocytes and macrophages during the inflamma-
tion process leads to tissue injury by damaging
macromolecules and affecting the lipid peroxidation of
membranes (Gutteridge, 1995). The anti-oxidant therapy is
coming up as an attractive therapeutic approach for variety
of human ailments. The results of anti-oxidant activity of
novel curcumin analogues are summarized in Table 4.
Compounds 5a, 5c, 5d, 5e and 5f exhibited moderate free
radical scavenging activity (49.44–44.30%) as compared
with standard gallic acid (92.30%).
Table 3 Anti-cancer activity of curcumin analogues at 10 lM conc
Compounds
Panel H460 Calu-1 hct116 ACHN
5a
5b
5c
5d
5e
5f
10
03
00
35
00
66
37
68
32
23
03
99
89
14
11
04
17
06
62
51
52
33
32
32
98
72
73
79
06
00
00
26
00
60
32
67
31
22
00
99
85
82
87
00
06
06
28
00
64
10
66
26
30
21
98
98
83
86
21
06
15
26
06
69
35
66
33
28
15
97
82
73
84
Compounds 5a (54.14%) and 5m (49.26%) have shown
potent hydroxyl radical scavenging ability as compared with
ascorbic acid (45.85%), while compounds 5h, 5j, 5b and 5k
have shown good hydroxyl radical scavenging activity.
Aromatic amide (5a) and heteroaromatic nucleus is required
for hydroxyl radical scavenging activity. Compounds 5a, 5f,
5b, 5c, 5h and 5e showed moderate reducing activity.
5g
5h
5i
5j
5k
5l
Experimental
5m
Flavopiridol (700 nM) 68
Gemcitabine (500 nM) 78
General methods
Panc1 human pancreas carcinoa, H460 humann non cell lung carci-
noma, Calu-1 human non cell lung carcinoma, hct 116 human colon
carcinoma, ACHN human renal cell carcinoma
Chemicals were purchased from Aldrich Chemical Co.,
USA. Melting points were determined with a digital
123

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Med Chem Res (2012) 21:3006–3014
thermometer were uncorrected. IR spectra were recorded
1
on FT-IR Shimadzu 8300 spectrophotometer and H NMR
1659, 1616, 1608, 1583, 1548, 1429, 1398, 1015, 910,
806, 758; 1H-NMR (CDCl3, 300 MHz) d; 8.47 (1H, s,
–NH), 8.45 (1H, s, bs), 8.45 (1H, d, J = 1.4 Hz), 8.30
(1H, d, J = 1.6 Hz), 8.29 (1H, t), 7.84 (1H, d, J =
3.4 Hz), 7.97 (2H, d, J = 15.6 Hz), 7.80 (3H, m, Ar–H),
6.35 (1H, s, Ar–H), 3.97 (6H, s, –OCH3 9 2), 3.85 (3H, s,
–OCH3). MS: m/e 566 (M ? 2). Anal. Calcd. For
C26H21BrF3NO5: C 55.33, H 3.75, Br 14.16, F 10.10, N
2.48, O 14.18. Found: C 55.36, H 3.77, Br 14.19, F 10.13,
N 2.51, O 14.21.
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 was per-
formed on pre-coated silica plates (Merck Kiesegel 60F254
,
0.2 mm thickness) sheets. The spots could be visualized
easily under ultraviolet light.
N-(3-((E)-3-(3-bromo-2,4,6-trimethoxyphenyl)acryloyl)
phenyl)-3-chlorobenzamide (5d) (yield 48%); mp 159–
161°C; IR tmax cm-1 (KBr): 3415, 3180, 2984, 2874,
1622, 1609, 1538, 1507, 1438, 1421, 1009, 982, 867, 741;
1H-NMR (CDCl3, 300 MHz) d; 8.30 (1H, s, –NH), 8.17
(1H, s), 8.10 (1H, d, J = 3.4 Hz), 8.02 (2H, d, J =
8.5 Hz), 7.97 (1H, d, J = 15.4 Hz), 7.82 (2H, d, J =
4.3 Hz), 7.67 (1H, d, J = 7.8 Hz), 7.51 (1H, t, Ar–H),
7.36 (1H, t, Ar–H), 6.34 (1H, s, Ar–H), 3.97 (6H, s,
–OCH3 9 2), 3.83 (3H, s, –OCH3). MS: m/e 531 (M ? 1).
Anal. Calcd. For C25H21BrClNO5: C 56.57, H 3.99, Br
15.05, Cl 6.68, N 2.64, O 15.07. Found: C 56.61, H 4.01,
Br 15.07, Cl 6.71, N 2.68, O 15.10.
General procedure for the synthesis
of curcumin analogues (5a–m)
Amides 5a–m (1 mmol) were dissolved in ethanol (15 ml),
NaOH (20%) was added to it and stirred for 5 min.
3-bromo-2,4,6-trimethoxybenzaldehyde 3 (1 mmol) was
added and stirring continued for 24 h at room temperature.
After completion of reaction (TLC), reaction mixture was
poured over crushed ice, extracted with chloroform,
organic extract was washed with water, dried (anhydrous
Na2SO4) and concentrated. Purification was carried using
silica gel column using mixture of 0.5–1% methanol ? 1%
liquor ammonia in chloroform as an eluent to obtain the
title compounds (5a–m).
3-bromo-N-(3-((E)-3-(3-bromo-2,4,6-trimethoxyphenyl)
acryloyl)phenyl)benzamide (5e) (yield 47%); mp 150–
152°C; IR tmax cm-1 (KBr): 3315, 3145, 2989, 2842,
1621, 1614, 1598, 1547, 1420, 1410, 1007, 972, 816, 768;
1H-NMR (CDCl3, 300 MHz) d; 8.30 (1H, s, –NH), 8.18
(1H, s, Ar–H), 8.10 (1H, d, J = 3.6 Hz), 8.05 (2H, d,
J = 8.7 Hz), 7.97 (1H, d, J = 15.6 Hz), 7.82 (2H, d,
J = 4.5 Hz), 7.67 (1H, d, J = 7.8 Hz), 7.51 (1H, t, Ar–H),
7.36 (1H, t, Ar–H), 6.34 (1H, s, Ar–H), 3.97 (6H, s,
–OCH3 9 2), 3.83 (3H, s, –OCH3). MS: m/e 576 (M ? 1).
Anal. Calcd. For C25H21Br2NO5: C 52.20, H 3.68, Br
27.78, N 2.43, O 13.91. Found: C 52.23, H 3.71, Br 27.82,
N 2.46, O 13.94.
(3-((E)-3-(3-bromo-2,4,6-trimethoxyphenyl)acryloyl)
phenyl)benzamide (5a) (yield 58%); mp 141–143°C; IR
tmax cm-1 (KBr): 3470, 3168, 2989, 2857, 1618, 1614,
1
1597, 1567, 1540, 1510, 1410, 1017, 945, 844, 763; H-
NMR (CDCl3, 300 MHz) d; 8.13 (1H, d, J = 2.4 Hz),
8.10 (1H, m), 7.92 (2H, d, J = 8.2 Hz), 7.52 (4H, m,
Ar–H), 7.26 (1H, s, –NH), 6.35 (1H, s, Ar–H), 3.96 (6H,
s, –OCH3 9 2), 3.84 (3H, s, –OCH3). MS: m/e 497
(M ? 1). Anal. Calcd. For C25H22BrNO5: C 60.50, H
4.47, Br 16.10, N 2.82, O 16.12. Found: C 60.53, H
4.50, Br 16.14, N 2.84, O 16.14.
N-(3-((E)-3-(3-bromo-2,4,6-trimethoxyphenyl)acryloyl)
phenyl)-4-fluorobenzamide (5f) (yield 40%); mp 63–65°C;
IR tmax cm-1 (KBr): 3320, 3168, 2989, 2856, 1612, 1618,
1602, 1567, 1410, 1010, 945, 822, 768; 1H-NMR (CDCl3,
300 MHz) d; 8.29 (1H, s, –NH), 8.13 (2H, dd, J =
15.8 Hz), 7.99 (2H, dd, J = 5.86 Hz), 7.77 (2H, d, J =
7.74 Hz), 7.49 (1H, t, Ar–H), 7.26 (1H, s, Ar–H), 7.15
(2H, t), 6.37 (1H, s, Ar–H), 3.70 (6H, s, –OCH3 9 2), 3.49
(3H, s, –OCH3). MS: m/e 515 (M ? 1). Anal. Calcd. For
C25H21BrFNO5: C 58.38, H 4.12, Br 15.54, F 3.69, N 2.72,
O 15.55. Found: C 58.42, H 4.14, Br 15.57, F 3.73, N 2.75,
O 15.58.
N-(3-((E)-3-(3-bromo-2,4,6-trimethoxyphenyl)acryloyl)
phenyl)-2-trifluorobenzamide (5b) (yield 60%); mp 182–
184°C; IR tmax cm-1 (KBr): 3417, 3145, 2968, 2840,
1618, 1608, 1598, 1540, 1510, 1417, 1010, 967, 842, 768;
1H-NMR (CDCl3, 300 MHz) d; 8.45 (1H, s, –NH), 8.02
(4H, m, Ar–H), 7.97 (2H, d, J = 15.6 Hz), 7.90 (1H, d,
J = 12.3 Hz), 7.80 (3H, m, Ar–H), 6.35 (1H, s, Ar–H),
3.97 (6H, s, –OCH3 9 2), 3.85 (3H, s, –OCH3). MS: m/e
566 (M ? 2). Anal. Calcd. For C26H21BrF3NO5: C 55.33,
H 3.75, Br 14.16, F 10.10, N 2.48, O 14.18. Found: C
55.36, H 3.77, Br 14.19, F 10.13, N 2.51, O 14.21.
N-(3-((E)-3-(3-bromo-2,4,6-trimethoxyphenyl)acryloyl)
phenyl)-3-trifluorobenzamide (5c) (yield 53%); mp 131–
133°C; IR tmax cm-1 (KBr): 3537, 3103, 3078, 2970,
4-tertbutyl-N-(3-((E)-3-(3-bromo-2,4,6-trimethoxyphenyl)
acryloyl)phenyl)benzamide (5g) (yield 47%); mp 75–77°C;
IR tmax cm-1 (KBr): 3553, 3061, 2958, 1649, 1618, 1585,
1
1543, 1458, 1410, 1016, 918, 850, 761; H-NMR (CDCl3,
300 MHz) d; 8.14 (1H, s, –NH), 8.02 (4H, m, Ar–H), 7.34
123

Med Chem Res (2012) 21:3006–3014
3011
(2H, d, J = 7.8 Hz), 7.29 (4H, m, Ar–H), 6.36 (1H, s, Ar–H),
3.98 (6H, s, –OCH3), 3.85 (3H, s, –OCH3), 1.35 (9H, s, –CH3
9 3). MS: m/e 554 (M ? 1). Anal. Calcd. For C29H30BrNO5:
C 63.05, H 5.47, Br 14.46, N 2.54, O 14.48. Found: C 63.08, H
5.51, Br 14.49, N 2.58, O 14.52.
N-(3-((E)-3-(3-bromo-2,4,6-trimethoxyphenyl)acryloyl)
phenyl)-4-methoxybenzamide (5l) (yield 53%); mp 105–
107°C; IR tmax cm-1 (KBr): 3415, 3140, 2964, 1620,
1604, 1588, 1465, 1420, 1012, 970, 709; 1H-NMR
(CDCl3, 300 MHz) d; 8.13 (2H, d, J = 7.7 Hz), 8.02 (2H,
d, J = 16.4 Hz), 7.88 (2H, d, J = 5.5 Hz), 7.76 (2H, d,
J = 7.6), 7.26 (1H, s, –NH), 6.96 (2H, d, J = 8.4 Hz),
6.33 (1H, s, Ar–H), 3.96 (6H, s, –OCH3 9 2), 3.90 (3H, s,
–OCH3), 3.87 (3H, s, –OCH3). MS: m/e 527 (M ? 1).
Anal. Calcd. For C26H24BrNO6: C 59.33, H 4.60, Br
15.18, N 2.66, O 18.24. Found: C 59.36, H 4.63, Br 15.21,
N 2.69, O 18.28.
N-(3-((E)-3-(3-bromo-2,4,6-trimethoxyphenyl)acryloyl)
phenyl)-2-fluorobenzamide (5h) (yield 54%); mp 131–
133°C; IR tmax cm-1 (KBr): 3415, 3115, 2984, 1617,
1
1610, 1590, 1538, 1468, 1420, 1016, 982, 828, 760; H-
NMR (CDCl3, 300 MHz) d; 8.61 (1H, d, J = 15.9 Hz),
8.22 (1H, d, J = 8.1 Hz), 8.14 (1H, s, –NH), 8.06 (1H, d,
J = 18.9 Hz), 8.00 (1H, d, J = 15.9 Hz), 7.82 (1H, d,
J = 7.8 Hz), 7.54 (3H, m, Ar–H), 7.35 (1H, d, J =
4.6 Hz), 7.23 (1H, d, J = 4.5 Hz), 6.38 (1H, s, Ar–H),
3.99 (6H, s, –OCH3 9 2), 3.87 (3H, s, –OCH3). MS: m/e
516 (M ? 2). Anal. Calcd. For C25H21BrFNO5: C 58.38,
H 4.12, Br 15.54, F 3.69, N 2.72, O 15.55. Found: C 58.42,
H 4.14, Br 15.57, F 3.73, N 2.75, O 15.58.
N-(3-((E)-3-(3-bromo-2,4,6-trimethoxyphenyl)acryloyl)
phenyl)thiophene-2-carboxamide (5m) (yield 50%); mp
79-81°C; IR tmax cm-1 (KBr): 3317, 3005, 2937, 1651,
1
1610, 1581, 1545, 1462, 1429, 1012, 916, 860, 804; H-
NMR (CDCl3, 300 MHz) d; 8.05 (4H, m, Ar–H), 7.76
(2H, d, J = 7.4 Hz), 7.49 (2H, d, J = 4.89 Hz), 7.38 (1H,
s, –NH), 7.12 (1H, s, Ar–H), 6.33 (1H, s, Ar–H), 3.95 (6H,
s, –OCH3 9 2), 3.82 (3H, s, –OCH3). MS: m/e 504 (M ? 2).
Anal. Calcd. For C23H20BrNO5S: C 54.99, H 4.01, Br
15.91, N 2.79, O 15.92, S 6.38. Found: C 55.01, H 4.03, Br
15.93, N 2.82, O 15.95, S 6.41.
N-(3-((E)-3-(3-bromo-2,4,6-trimethoxyphenyl)acryloyl)
phenyl)-3-fluorobenzamide (5i) (yield 54%); mp 106–
108°C; IR tmax cm-1 (KBr): 3450, 3160, 2965, 1625,
1
1602, 1587, 1507, 1468, 1407, 1009, 925, 816, 720; H-
NMR (CDCl3, 300 MHz) d; 8.30 (1H, s, –NH), 8.17 (1H,
s), 8.10 (1H, d, J = 3.6 Hz), 8.05 (2H, d, J = 8.7 Hz),
7.97 (1H, d, J = 15.6 Hz), 7.82 (2H, d, J = 4.5 Hz), 7.67
(1H, d, J = 7.8 Hz), 7.51 (1H, t, Ar–H), 7.36 (1H, t, Ar–H),
6.34 (1H, s, Ar–H), 3.97 (6H, s, –OCH3 9 2), 3.83 (3H, s,
–OCH3). MS: m/e 516 (M ? 2). Anal. Calcd. For C25H21
BrFNO5: C 58.38, H 4.12, Br 15.54, F 3.69, N 2.72, O 15.55.
Found: C 58.42, H 4.14, Br 15.57, F 3.73, N 2.75, O 15.58.
N-(3-((E)-3-(3-bromo-2,4,6-trimethoxyphenyl)acryloyl)
phenyl)-3-methylbenzamide (5j) (yield 82%); mp 122–
124°C; IR tmax cm-1 (KBr): 3420, 3130, 2974, 1620,
TNF-a and IL-6 inhibition assay
Pro-inflammatory cytokine production by lipopolysaccha-
ride (LPS) in THP-1 cells was measured according to the
method described by Hwang et al. (1933). In brief, THP-1
cells were cultured in RPMI 1640 culture medium (Gibco
BRL, Pasley, UK) containing 100 U/ml penicillin and
100 mg/ml streptomycin (1009 solutions, Sigma Chemical
Co. St. Louis, MO) containing 10% foetal bovine serum
(FBS, JRH). Cells were differentiated with phorbol myr-
istate acetate (PMA, Sigma). Following cell plating, the
test compounds or vehicle (0.5% DMSO) was added to
each well and the plate was incubated for 30 min at 37°C.
Finally, LPS (Escherichia coli 127:B8, Sigma Chemical
Co., St. Louis, MO) was added, at a final concentration of
1 lg/ml. Plates were incubated at 37°C for 24 h, 5% CO2.
Supernatants were harvested and assayed for TNF-a and
IL-6 by ELISA as described by the manufacturer (BD
Biosciences). The cells were simultaneously evaluated for
Cytotoxicity using CCK-8 from Dojindo Laboratories.
Percent inhibition of cytokine release compared with the
control was calculated (Hwang et al., 1993).
1
1609, 1587, 1538, 1478, 1420, 1012, 925, 847, 735; H-
NMR (CDCl3, 300 MHz) d; 9.49 (1H, s, –NH), 8.10 (1H,
s, Ar–H), 7.92 (2H, d, J = 7.8 Hz), 7.74 (3H, m, Ar–H),
7.32 (4H, m, Ar–H), 6.35 (1H, s, Ar–H), 3.96 (6H, s,
–OCH3 9 2), 3.90 (3H, s, –OCH3), 2.33 (3H, s, –CH3).
MS: m/e 514 (M ? 4). Anal. Calcd. For C26H24BrNO5: C
61.19, H 4.47, Br 15.66, N 2.74, O 15.67. Found: C 61.22,
H 4.52, Br 15.69, N 2.77, O 15.70.
N-(3-((E)-3-(3-bromo-2,4,6-trimethoxyphenyl)acryloyl)
phenyl)-2-methylbenzamide (5k) (yield 43%); mp 137–
139°C; IR tmax cm-1 (KBr): 3417, 3140, 2964, 1638,
1
1615, 1590, 1510, 1465, 1415, 1012, 976, 802, 709; H-
NMR (CDCl3, 300 MHz) d; 8.23 (1H, d, J = 1.9 Hz),
8.20 (1H, t, Ar–H), 8.10 (1H, t, Ar–H), 8.10 (1H, s, –NH),
8.08 (1H, d, J = 1.7 Hz), 7.92 (2H, d, J = 7.7 Hz), 7.32
(4H, m, Ar–H), 6.35 (1H, s, Ar–H), 3.97 (6H, s, –OCH3 9
2), 3.90 (3H, s, –OCH3), 2.34 (3H, s, CH3). MS: m/e 514
(M ? 4). Anal. Calcd. For C26H24BrNO5: C 61.19, H
4.47, Br 15.66, N 2.74, O 15.67. Found: C 61.22, H 4.52,
Br 15.69, N 2.774, O 15.70.
COX-1 and 2 inhibition microtitre 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
123

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Med Chem Res (2012) 21:3006–3014
Company, MI, USA. The reaction mixture of 100% initial
activity wells contained 160 ll of assay buffer, 150 ll of
haem 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 haem 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). Aspirin and SC560 (1 mM) were tested
simultaneously as standard COX-1 and -2 inhibitors,
respectively. The COX inhibition activity (%) was calcu-
lated using following formula:
spectrophotometrically at 410 nm. Salicylic acid (1 mM)
was used as a reference compound (Gacche and Dhole,
2006).
Anti-cancer activity using propidium iodide
fluorescence assay
The selected cancer cell lines such as ACHN (human renal
cell carcinoma), Panc 1 (human pancreatic carcinoma),
Calu 1 (human non small cell lung carcinoma), H460
(human non cell lung carcinoma) and HCT 116 (human
colon carcinoma) were used for evaluation of cytotoxic
effect of selected curcumin analogues. The fluorescence
signal intensity of the propidium iodide (PI) is directly
proportional to the amount of DNA in each cell, PI is not
able to penetrate an intact membrane, and so cells must first
be permeabilized. Seed cells of 3,000–7,500 cells/well were
placed in 200 ll of tissue culture grade 96-well plates and
allowed them to recover for 24 h in humidified 5% CO2
incubator at 37°C. After culturing for 24 h, compounds (in
0.1% DMSO) were added into triplicate wells with 10 lM
concentrations. 0.1% DMSO alone was used as control.
After 48 h in humidified 5% CO2 incubator at 37°C con-
dition, the medium was removed and treated with 25 ll of
PI (50 lg/ml in water/medium) per well. The plates were
freeze at -80°C for 24 h then thawed and allowed it to come
at room temperature, and the plate absorbance was read on
Fluorometer (Polar-Star BMG Tech), using 530 nM exci-
tation and 620 nM emission wavelength. Lastly, percent
Cytotoxicity of the compounds was calculated by using
following formula (Dengler et al., 1995):
T
COX inhibition activityð%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
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
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 a method of Salicylic acid (10-6 M) was
used as a reference drug (Tandon et al., 1982; Lowry et al.,
1951).
T
Cytotoxicityð%Þ ¼ 1 ꢁ ꢂ 100
C
where T is the O. D. of treated cells and C is the O. D. of
control.
The results were compared with the standard anti-cancer
drug inhibitors flavopiridol (700 nM) and gemcitabine (500
nM).
DPPH radical scavenging assay
b-glucuronidase inhibition assay
The reaction mixture contained 1 mM concentrations of
individual test sample (in absolute ethanol) and DPPH
radical (10-4 M in absolute ethanol) solution. The contents
of the reaction mixture were observed spectrophotometri-
cally at 517 nm after 20 min. Gallic acid (1 mM) was used
as a reference drug (Bartolome et al., 2004).
The effect of the selected compounds on activity of
b-glucuronidase was studied using a method described by
Demetrios (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-nitro-
phenyl-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
OH radical scavenging activity
OH radicals were generated by using the Ferric ion (Fe3?)/
ascorbic acid reaction system. The detection of OH radicals
123

Med Chem Res (2012) 21:3006–3014
3013
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was carried out by measuring the amount of formaldehyde
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reaction cocktail contained 0.1 mM EDTA, 167 mM Fe3?
,
33 mM DMSO in phosphate buffer of 50 mM pH 7.4. 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 spec-
trophotomertically at 412 nm for the detection of formal-
dehyde. Ascorbic acid (1 mM) was used as a reference
compound (45.85%) for comparative study (Nash, 1953).
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Conclusion
In this study, we have synthesized a series of novel cur-
cumin analogues and evaluated for anti-inflammatory, anti-
cancer and anti-oxidant activity. Compounds 5f, 5j and 5m
were excellent inhibitors of TNF-a and IL-6. Compounds
5c, 5e, 5b, and 5d showed potent COX-2 inhibition,
compounds 5d and 5f have shown good trypsin inhibition
and compounds 5e, 5g, and 5c exhibited excellent b-glu-
curonidase inhibition. Compounds 5l and 5m showed
potent anti-cancer activity against selected five human
cancer cell lines and compounds 5a and 5m were excellent
OH radical scavengers. Compounds 5f, 5j and 5m serve as
a lead for manoeuvering novel TNF-a and IL-6 inhibitors.
Nevertheless, the results of the present investigation also
justify the importance 5l and 5m as leads for the devel-
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Acknowledgements BSH is thankful to the Council of Scientific
and Industrial Research (CSIR), New Delhi for the award of SRF, as
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