Synthesis and evaluation of DPPH and anti-inflammatory activities of 2,6-bisbenzylidenecyclohexanone and pyrazoline derivatives

Article abstract of DOI:10.1007/s00044-010-9521-0

A series of thirty three 2,6-bisbenzylidenecyclohexanone and pyrazoline derivatives were synthesized and evaluated for inhibitory activities on IFN-γ/LPS-activated RAW 264.7 cells and DPPH radical scavenging activity. Compounds 8, 9, and 11a demonstrated significant NO inhibitory activity as compared to L-NAME and curcumin with IC₅₀ values of 6.68 ± 0.16, 6.09 ± 0.46, and 6.84 ± 0.12 μM, respectively. Apparently the suppression upon NO secretion was not due to cell death since the active compounds did not reduce the cell viability in close proximity to the IC ₅₀ of NO inhibition. Overall, incorporation of pyrazoline ring as part of the linker chain improved cell viability compared to the 2,6-bisbenzylidenecyclohexanone derivatives. Meanwhile, compound 11 (IC ₅₀ = 13.27 ± 1.78 μM) bearing ortho hydroxyls on the aromatic rings recorded the highest radical scavenging activity as compared with quercetin (IC₅₀ = 21.46 ± 0.85 μM). Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9521-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:333–344
DOI 10.1007/s00044-010-9521-0
ORIGINAL RESEARCH
Synthesis and evaluation of DPPH and anti-inflammatory
activities of 2,6-bisbenzylidenecyclohexanone and pyrazoline
derivatives
•
Kok Wai Lam Chau Ling Tham Choi Yi Liew
•
•
•
Ahmad Syahida Mohd. Basyaruddin Abdul Rahman
•
•
Daud A. Israf Nordin H. Lajis
Received: 5 April 2010 / Accepted: 18 November 2010 / Published online: 7 January 2011
Ó Springer Science+Business Media, LLC 2010
Abstract A series of thirty three 2,6-bisbenzylidenecy-
clohexanone and pyrazoline derivatives were synthesized
and evaluated for inhibitory activities on IFN-c/LPS-acti-
vated RAW 264.7 cells and DPPH radical scavenging
activity. Compounds 8, 9, and 11a demonstrated significant
NO inhibitory activity as compared to L-NAME and cur-
cumin with IC50 values of 6.68 0.16, 6.09 0.46, and
6.84 0.12 lM, respectively. Apparently the suppression
upon NO secretion was not due to cell death since the
active compounds did not reduce the cell viability in close
proximity to the IC50 of NO inhibition. Overall, incorpo-
ration of pyrazoline ring as part of the linker chain
improved cell viability compared to the 2,6-bisbenzylide-
necyclohexanone derivatives. Meanwhile, compound 11
(IC50 = 13.27 1.78 lM) bearing ortho hydroxyls on the
aromatic rings recorded the highest radical scavenging
activity as compared with quercetin (IC50 = 21.46
0.85 lM).
Keywords 2,6-Bisbenzylidenecyclohexanone Á
DPPH Á RAW 264.7 Á Pyrazoline Á Curcumin
Introduction
Nitric oxide (NO) plays a pivotal role in complex biolog-
ical systems as a physiological signaling molecule. Evi-
dence of the role of NO in intravascular endocrine NO
transport, hypoxic vasodilatation, cellular signaling path-
ways, and cytoprotection further demonstrate the impor-
tance of this simple yet essential molecule (Anggard,
1994). Excessive production of NO, however, poses major
health risks through the induction of both acute and chronic
inflammatory diseases including cancer, rheumatoid
arthritis, diabetes, liver cirrhosis, and septic shock (Gonz-
alez et al., 2008; Lechner et al., 2005). Studies have shown
that NO biosynthesis is regulated by different signaling
pathways, including the transcriptional and post-transcrip-
tional levels in activated macrophages (Aktan, 2004). Thus,
finding new chemical entity capable of inhibiting either the
iNOS expression or in the transcriptional level in upper
stream still remains an important target in drug discovery.
The choice of treatment for inflammation currently in
use is that of non-steroidal anti-inflammatory drugs
(NSAIDs) (Singh and Triadafilopoulos, 1999). However,
the effectiveness of NSAIDs is limited due to its potential
side effects including nausea, stroke, heartburn, and heart
attacks if consumed over long period. The diversity of
natural compounds provides a vast pool of alternative drug
leads that do not only possess enhanced potency but also
afford more specific targeting, hence providing safer usage
(Harvey, 2008). Recently, curcumin has gained renewed
attention in the area of drug discovery because of its low
K. W. Lam Á N. H. Lajis (&)
Laboratory of Natural Products, Institute of Bioscience,
University Putra Malaysia, 43400 UPM Serdang,
Selangor Darul Ehsan, Malaysia
e-mail: nhlajis@ibs.upm.edu.my
C. L. Tham Á C. Y. Liew Á D. A. Israf
Department of Biomedical Science, Faculty of Medicine
and Health Sciences, University Putra Malaysia,
43400 UPM Serdang, Selangor Darul Ehsan, Malaysia
A. Syahida
Department of Biochemistry, Faculty of Biotechnology
and Molecular Sciences, University Putra Malaysia,
43400 UPM Serdang, Selangor Darul Ehsan, Malaysia
Mohd. B. Abdul Rahman Á N. H. Lajis
Department of Chemistry, Faculty of Science, University Putra
Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia
123

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Med Chem Res (2012) 21:333–344
toxicity and wide range of therapeutic properties (Hatcher
et al., 2008). Compounds containing a divinylketone
moiety such as curcumin and zerumbone are also known to
exhibit potent anti-inflammatory activity (Hatcher et al.,
2008; Murakami et al., 2004; Takada et al., 2005).
Curcumin is the major constituent isolated from the
plant Curcuma longa (Shishodia et al., 2005; Tomita et al.,
2005). It has potent anti-inflammatory properties and is
therapeutically active in the treatment of bowel inflam-
mation and rheumatoid arthritis (Hsu and Cheng, 2007).
However, preclinical and clinical studies on curcumin
showed that this compound has a relatively poor bio-
availability and weak pharmacokinetic profiles, making it a
poor drug candidate (Pan et al., 2000). The major cause for
the poor bioavailability is the rapid metabolism by
sequential reduction and glucuronidation resulting in rapid
elimination by the body (Pandeya, 1997). Various curcu-
min analogs have been synthesized to overcome the poor
bioavailability. These analogs have demonstrated potent
pharmacological effects, such as anti-proliferation (Wei
et al., 2005), anti-angiogenesis (Chandru et al., 2007),
antioxidant (Shishodia et al., 2005), and anti-tumorous
(Duvoix et al., 2005) properties.
or propionic anhydrides in the presence of 4-dimethyl-
aminopyridine (DMAP). Benzoylation of pyrazoline deriv-
atives was accomplished using benzoyl chloride in the
presence of pyridine to afford high yields of the respective
products.
Discussion
DPPH radical scavenging activity
Among the 2,6-bisbenzylidenecyclohexanone derivatives,
compounds 1, 4, 6, 7, 8, and 11 exhibited DPPH radical
scavenging activity at 100 lM as compared to quercetin
(positive control). Further tests showed that compounds 11,
4, and 7 demonstrated the highest antioxidant activity with
IC50 values of 13.27 1.78, 14.81 2.09, and 20.71
1.11 lM, respectively. These compounds exhibited radical
scavenging activity similar to that of quercetin (IC50
=
21.46 0.85 lM) possibly because of the presence of an
ortho hydroxyl group in the phenyl rings. Srinivasan et al.
(2007) showed that one of the critical factors contributing
to radical scavenging properties was the presence of a
hydroxyl group in the phenyl ring. The radical scavenging
activity was less likely to be influenced by the size of the
middle ring since scavenging by both 11 and 4 were of a
similar magnitude. Non-adjacent hydroxyl phenolic com-
pounds recorded lower radical scavenging activity ranging
from 50 to 75 lM.
Pyrazole, pyrazoline, and pyrazolidine belong to a
class of five-membered ring compounds having two
nitrogen atoms located adjacent to each other. This class
of compounds has been known to exhibit potent anti-
inflammatory activities (Bansal et al., 2001; Barsoum
et al., 2006; Rani et al., 2004). In this study, a series of
active NO inhibitors of 2,6-bisbenzylidenecyclohexanone
derivatives merging with the active pyrazoline moiety
were synthesized and evaluated for their anti-inflamma-
tory properties.
Nitric oxide inhibitory activity
The results for NO inhibitory activity are summarized in
Table 1. Overall, 2,6-bisbenzylidenecyclohexanone deriv-
atives demonstrated higher NO inhibitory activity com-
pared to the pyrazoline derivatives. Compounds 8, 9, and
11a suppressed the production of (NO) significantly with
IC50 values of 6.68 0.16, 6.09 0.46, and 6.84
0.12 lM, respectively. The positive controls, L-NAME and
curcumin, were less active with IC50 values of 26.21
1.64 and 21.58 2.06 lM, respectively. These com-
pounds demonstrated almost a threefold improvement in
NO inhibition relative to the positive controls which may
possibly be related to the stability in the conformation and
improved pharmacokinetics compared to the structure of
b-diketone moiety in curcumin (Liang et al., 2009a, b).
Unlike curcumin, which possesses a flexible conformational
structure, the presence of a cyclohexanone ring in these
compounds contributed to the compound’s rigidity and sta-
bility. Figure 1 shows the dose–response of compounds 8, 1,
9, and 8c toward the secretion of NO. In this experiment,
IFN-c/LPS-induced RAW 264.7 cells produced large
amounts of nitrite (&65 lM). NO inhibitory activity
Results
Synthesis
The syntheses of 2,6-bisbenzylidenecyclohexanone- and
pyrazoline-derived compounds are outlined in Scheme 1.
2,6-Bisbenzylidenecyclohexanone derivatives were obtained
via an effective Claisen–Schmidt condensation reaction.
Catalytic amounts of sodium hydroxide (base) were used in
the reaction. Acid-catalyzed reactions are highly favored
when aromatic aldehydes bearing hydroxyl groups are
involved. Pyrazoline derivatives were synthesized by
reacting hydrazinium hydroxide with selected 2,6-bisben-
zylidenecyclohexanone derivatives in the presence of a
catalytic amount of acetic acid in ethanol. Product yields
were increased when the electron-donating group at
para-carbon was replaced with an electron-withdrawing
group. Acylation was generally accomplished using acetic
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Med Chem Res (2012) 21:333–344
335
2,6-Bisbenzylidenecyclopentanone derivatives
O
O
R1
O
R1
R1
O
R1
R
R2
R3
R2
R3
R2
R3
R2
R3
(a)
(b)
2
1a. R1 = H, R2 = OMe, R3 = OAc
4a. R1 = H, R2 = OAc, R3 = OAc
1. R1 = H, R2 = OMe, R3 = OH
2. R1 = H, R2 = OH, R3 = H
3. R1 = H, R2 = H, R3 = OH
4. R1 = H, R2 = OH, R3 = OH
5. R1 = H, R2 = H, R3 = F
6. R1 = OH, R2 = H, R3 = OH
7. R1 = OH, R2 = OH, R3 = H
2,6-Bisbenzylidenecyclohexanone derivatives
R1
O
R1
O
O
R1
O
R1
R
R2
R3
R2
R3
R2
R3
R2
R3
(a)
(b)
2
8a. R1 = H, R2 = OMe, R3 = OAc
8b. R1 = H, R2 = OMe, R3 = Oprop
11a. R1 = H, R2 = OAc, R3 = OAc
8. R1 = H, R2 = OMe, R3 = OH
9. R1 = H, R2 = OH, R3 = H
10. R1 = H, R2 = H, R3 = OH
11. R1 = H, R2 = OH, R3 = OH
12. R1 = H, R2 = OMe, R3 = OMe
13. R1 = H, R2 = H, R3 = NO2
14. R1 = H, R2 =H, R3 = F
H
R3
N N
R1
N N
R1
R2
R1
R2
R3 = Ac
R1
R2
(c)
(d) / (e)
R2
8c. R1 = OMe, R2 = OH
10c. R1 = H, R2 = OH
12c. R1 = OMe, R2 = OMe
13c. R1 = H, R2 = NO2
14c. R1 =H, R2 = F
R3 = Benzoyl
8d. R1 = OMe, R2 = OAc 8e. R1 = OMe, R2 = OH
10d. R1 = OMe, R2 = OMe 10e. R1 = OMe, R2 = OMe
13d. R1 = H, R2 = NO2
14d. R1 =H, R2 = F
13e. R1 = H, R2 = NO2
14e. R1 =H, R2 = F
Scheme 1 Synthesis of 2,6-bisbenzylidenecyclopentanone, 2,6-bis-
benzylidenecyclohexanone and their pyrazoline derivatives. Reagents
and conditions: a NaOH and ethanol, reflux, 2 h; or HCl, and ethanol,
reflux, 1 h; b Acetic anhydride or propionic anhydride, CHCl3 and
DMAP, rt, 3 h; c NH2NH2.H2O, ethanol, and few drops acetic acid,
reflux, 4 h; d Acetic anhydride, CH2Cl2, DMAP, rt, 3 h; e Benzoyl
chloride, CH2Cl2, pyridine, and KHCO3, 0–10°C
appeared to be in the order of 9 [ 8 [ 8c [ 1. The reduced
NO inhibitory activity shown by compound 1 compared with
8 could be due to the contraction of the middle ring which
affected the conformation while reduction in the inhibitory
effect of 8c as compared with 9 could be rationalized by the
absence of ketone and double bond moieties.
1998; Tetko et al., 2005; Veber et al., 2002). All the active
compounds recorded the log P values of lower than 5,
which fulfilled the Lipinski, by the statement that most
‘‘drug-like’’ molecules should not exceed this value.
Compounds 7, 8, 9, 11, 8b, and 11a, which have molecular
volumes and weights below 500 recorded a more signifi-
cant NO inhibition. These results are consistent with the
fact that larger molecular size hinders the interaction
between the compound and the active site. In addition, the
Lipinski-calculated profiles as presented in Table 1 also
indicated that an increase in both the numbers of hydrogen
bond donor and acceptor in pyrazoline as compared with
2,6-bisbenzylidenecyclohexanone derivatives did not dis-
play improvement on the NO inhibitory activities.
The NO inhibitory effects were further analyzed based
on the compounds’ different theoretical parameters
including log P, molecular weight, and volume, and the
number of hydrogen bond donor and acceptor groups,
according to the Lipinski profiles (Lipinski et al., 2001).
The lack of NO inhibitory activity demonstrated by com-
pounds 5 and 14 could be ascribed to the high log P values
due to the presence of hydrophobic fluorine groups.
According to Lipinski rule, log P is an important criteria
for good drug absorption and permeation (Balanco et al.,
The presence of an electron-withdrawing group such as
the fluorine atom did not play a significant role in
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Med Chem Res (2012) 21:333–344
Table 1 The IC50 values of anti-inflammatory activity, DPPH radical scavenging activity and Lipinski’s Rule of 5 values of 2,6-bisbenzyli-
denecyclohexanone derivatives
Compound
Anti-inflammatory
activity (lM)
DPPH radical scavenging
activity (lM)
Lipinski Rule of 5d
(Mean SEMa)
(Mean SEMa)
miLog P
Volume
MW
HBA
HBD
1
13.94 0.24
C50
74.96 2.53
C100
C100
14.81 2.09
C100
50.64 1.18
20.71 1.11
51.85 2.47
C100
C100
13.27 1.78
C100
C100
C100
C100
C100
C100
C100
C100
NT
3.141
3.456
3.504
2.526
4.790
2.978
2.577
3.646
3.961
4.009
3.031
4.261
4.886
5.295
2.746
1.737
3.252
4.589
2.242
3.697
4.060
4.312
4.937
5.346
2.909
3.918
4.543
4.952
4.975
5.338
5.590
6.215
6.624
2.303
ND
319.158
268.067
268.067
284.102
261.894
284.102
284.102
335.960
284.868
284.868
300.904
371.016
315.501
278.695
278.695
430.148
408.983
442.586
446.950
348.036
296.945
383.092
327.577
290.771
456.985
419.018
363.503
326.698
438.810
387.718
473.865
418.351
381.545
332.182
ND
352.386
292.334
292.334
324.332
296.316
324.332
324.332
366.413
306.361
306.361
338.359
394.467
364.357
310.343
310.343
492.480
450.487
478.541
506.507
380.444
320.392
408.498
378.388
324.374
506.555
450.535
420.425
366.411
484.552
424.500
512.606
482.496
428.482
368.385
ND
5
2
2
3
2
3
C50
3
2
4
20.49 3.40
C50
5
4
5
1
0
6
C50
5
4
7
7.94 0.46
6.68 0.16
6.09 0.46
41.59 2.55
9.43 0.79
19.77 1.16
29.36 3.36
C50
5
4
8
5
2
9
3
2
10
3
2
11
5
4
12
5
0
13
7
0
14
1
0
1a
C50
1
0
4a
C50
9
0
8a
13.27 0.80
8.02 0.01
6.84 0.12
12.22 0.65
26.87 0.15
38.73 2.77
C50
7
0
8b
7
0
11a
8c
9
0
6
3
10c
12c
13c
14c
8d
NT
4
3
NT
6
1
NT
8
1
C50
NT
2
1
C50
NT
9
0
10d
13d
14d
8e
C50
NT
7
0
C50
NT
9
0
C50
NT
3
0
47.08 0.63
25.10 2.73
C50
NT
7
2
10e
12e
13e
14e
Curcumin
L-NAMEb
Quercetinc
NT
5
2
NT
7
0
C50
NT
9
0
C50
NT
3
0
21.58 2.06
26.21 1.64
NT
NT
6
2
NT
ND
ND
ND
ND
21.46 0.85
ND
ND
ND
NT not tested, ND not determined
a
Standard error of the mean
b
Positive control used for anti-inflammatory assay
c
Positive control used for DPPH radical scavenging assay
d
Calculation was generated online on www.molinspiration.com website (Molinspiration Property Engine v2009.01). Values colored in red
showed that it contravened with the Lipinski Rule of 5
improving the NO inhibition. However, replacement of the
fluorine with a hydroxyl group increased the NO inhibition
significantly. This could be best explained by the fact that
hydroxyl group could act as both the hydrogen bond
donor and acceptor simultaneously, while fluorine could
only serve as a hydrogen bond acceptor. Interestingly, the
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Med Chem Res (2012) 21:333–344
337
100
100
80
60
40
20
0
80
***
***
60
40
20
0
*
**
***
***
***
***
***
***
***
C
0
0.78 1.56 3.13 6.25 12.5 25
50 250
C
0
0.78 1.56 3.13 6.25 12.5 25
50 250 μM
-20
L-NAME
L-NAME
compound 1
compound 8
IFN-γ/LPS
IFN-γ/LPS
100
80
60
40
20
0
100
80
60
40
20
0
***
***
*
**
***
**
***
***
***
***
***
0.78 1.56 3.13 6.25 12.5 25
***
***
μM
50 250
C
0
0.78 1.56 3.13 6.25 12.5 25
C
0
50 250 μM
-20
L-NAME
compound 8c
L-NAME
compound 9
IFN-γ/LPS
IFN-γ/LPS
Fig. 1 Nitric oxide (NO) production level of RAW 264.7 in the
presence of compounds 8, 1, 9, and 8c. C: basal level of nitrite concen-
tration without IFN-c/LPS-treatment. All values are mean SEM of
three different experiments. *P \ 0.05, **P \ 0.01, ***P \ 0.001
significantly different from the IFN-c/LPS-treated control group
presence of meta methoxyl group in compound 8 increased
the activity by sixfolds as compared with compound 10
suggesting the importance of different electronic effects
and molecular volume in this region. This is further sup-
ported by the fact that replacement of methoxyl with a
hydroxyl group at the meta-position decreased the NO
activity in compound 11 (IC50 = 9.43 0.79 lM). The
authors suggest that both the electron-donating hydroxyl
groups at meta- and para-positions might contribute to
higher electron density in the phenyl ring, which is legible
for a p interaction to take place in the active site. In
addition, both the hydroxyl groups on the phenyl rings are
also capable of participating in a certain hydrogen bonding
interaction in the active site, which is important in sus-
taining the NO inhibitory activity. When methylation and
acylation are carried out on the para hydroxyl group, the
NO inhibitory activity was surprisingly decreased as indi-
cated by the inhibitory activity of compounds, 12 and 8a,
which exhibited IC50 values of 19.77 1.16 lM and
13.27 0.80 lM, respectively. Therefore, the presence of
a para hydroxyl group remains an important factor for NO
inhibition. However, the authors could not explain further
on the activity of compound 9 which recorded the most
potent activity with IC50 value of 6.09 0.46 lM. The
authors believe it might be due to its participation in a
different NO inhibitory mechanism as compared to the
previous two compounds, 8 and 10. This is further sup-
ported by the fact (Fig. 2) that compound 9 exhibited
higher cellular toxicity than that of 8 at a concentration of
25 lM.
Pyrazoline derivatives were synthesized to mimic the
diketone linker found in curcumin to establish the effect of
electronic effects of this section of the molecule upon NO
inhibitory activity. The synthesis was performed on selected
2,6-bisbenzylidenecyclohexanone derivatives as they dis-
played higher NO inhibitory activity compared to the 2,
6-bisbenzylidenecyclopentanone derivatives. Compound 8c
was found to be twofold more active than curcumin but
failed to match the activity of the respective ketone, 8.
However, electronic density induced by the introduction of
pyrazoline moiety, which increased the hydrogen bonding
capability as compared with the parent compound 8, failed
to improve NO inhibition. Both acylation and benzoylation
were carried out on the pyrazoline moiety but failed to
improve NO inhibition. This could possibly be explained by
the violation in one of the Lipinski’s rules including log P,
volume, and molecular weight criteria. As an example,
compounds from series e exhibited high log P values (more
than 5). Both compounds 8d and 12e recorded molecular
weight parameter exceeding the permitted value of 500 in
Lipinski profiles. Based on the results obtained by the
authors’ group on dose–response NO inhibitory studies of
compound 8, the authors have suggested that its action was
due to the suppression of both iNOS gene and enzyme
expression, and not due to effects upon scavenging of nitrite
(Tham et al., 2010).
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Med Chem Res (2012) 21:333–344
150
100
50
150
100
50
*
**
***
***
***
50 250
0
0
C
0
0.78 1.56 3.13 6.25 12.5 25
μM
C
0
0.78 1.56 3.13 6.25 12.5 25
50 250
μM
L-NAME
L-NAME
compound 8
compound 1
IFN-γ/LPS
IFN-γ/LPS
150
100
50
150
100
50
***
***
***
***
***
***
0
0
C
0
0.78 1.56 3.13 6.25 12.5 25
50 250
μM
C
0
0.78 1.56 3.13 6.25 12.5 25
50 250
μM
L-NAME
compound 9
L-NAME
compound 8c
IFN-γ/LPS
IFN-γ/LPS
Fig. 2 RAW 264.7 cell viability in the presence of compounds 8, 1,
9, and 8c. C: basal level of nitrite concentration without IFN-c/
LPS-treatment. All values are mean SEM of three different
experiments. *P \ 0.05, **P \ 0.01, ***P \ 0.001 significantly
different from the IFN-c/LPS-treated control group
The effect upon cell viability of RAW 264.7 cells of the
most active compounds at different concentrations are
represented in Fig. 2. It is evident that compound 8, 1, 9,
and 8c are toxic to cells at concentrations above 50 lM.
However, the inhibitory effect on NO secretion was
apparently not due to cell death as these compounds 8, 1, 9,
and 8c did not suppress the cell viability in close proximity
to the IC50 of NO inhibition. A slight improvement in cell
viability demonstrated by compounds 1 and 8c could be
related to the removal of one double bond moiety as well as
the size of the ketone linker ring and the replacement of
ketone by pyrazoline moiety in the linker, respectively. The
presence of the meta hydroxyl groups in compound 9
showed that it is highly toxic to cell even at 25 lM. In
general, low cell toxicity recorded by the pyrazoline
compounds highlighted its future potential by replacement
of the b-diketone in curcumin or divinyl ketones moieties
in the future drug designing work.
mediators, gene and protein expression, cytokines, and
cell-signaling pathways are currently being carried out.
Assessment on miLog P and molecular weight of the
active compounds provided us with supplementary data in
solving the differences in structural and functional char-
acteristics compared to curcumin for future design of
potent anti-inflammatory drug leads.
Experimental
Chemistry
All the chemicals are purchased in research grade form and
used without further purification from Sigma–Aldrich,
Merck, and Acros Organics. All the solvents used in the
experiments were either of HPLC-grade or dried and dis-
tilled before use. The typical work-up included washing
with brine and followed by drying the organic layer with
magnesium sulfate (anhydrous) before allowing it to con-
centrate in vacuo. Analytic TLC was carried out on silica
gel F254 precoated (0.2-mm thickness, Merck) aluminum
TLC sheets. The TLC plates were spotted with samples
using a fine glass capillary tube and developed in a chro-
matographic tank saturated with solvent vapor at room
In conclusion, altering the backbone of the 2,6-bisben-
zylidenecyclohexanone derivatives by incorporation of a
pyrazoline ring resulted in decreased NO inhibitory activity
but improved the overall cell viability. The mechanism of
NO inhibitory action of compounds 7, 8, 9, 8b, 11a, and 8c
remains to be explained. Further dissection of the mecha-
nism including the effects on various inflammatory
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339
temperature. IR Spectra was recorded on a Perkin Elmer
RXI FT-IR spectrometer as KBr disk or thin film. Mass
spectra were measured on Thermo Finnigan POLARISQ
spectrometer, with ionization induced by electron impact at
70 eV. Nuclear Magnetic Resonance Spectra were recor-
ded in CDCl3, CD3OD, or DMSO-d6 using Varian
500 MHz NMR Spectrometer.
(125 MHz, CD3OD): d 26.2, 115.7, 127.5, 122.4, 132.9,
134.2, 157.3, 188.4; EIMS m/z (rel. int.) calcd for C19H16O3
(M?, %): 292 (M?, 60), 291 (100), 275 (24), 131 (21).
2,5-bis(3,4-dihydroxybenzylidene)cyclopentanone (4);
Yield: 38%; yellow crystals; IR(KBr) (cm-1) mmax: 3397
(–OH), 2941 (C–H stretch), 1591 (C=O), 1443 (C=C),
1252 (C–O aromatic), 1160, 875; 1H NMR (500 MHz,
CD3OD): d 2.83 (s, 4H, C–H), 6.96 (d, J = 8.0 Hz, 2H,
Ar–H), 7.12 (d, J = 8.0 Hz, 2H, Ar–H), 7.22 (s, 2H,
Ar–H), 7.34 (s, 2H, C–H). 13C NMR (125 MHz, CD3OD):
d 26.4, 115.9, 117.4, 124.4, 128.6, 132.8, 135.3, 145.4,
147.2, 194.8; EIMS m/z (rel. int.) calcd for C19H16O5 (M?,
%): 324 (M?, 100), 307 (49).
General procedures for the synthesis of a series com-
pounds (Refer to Scheme 1). (Base Catalyzed) Four drops
of 40% NaOH in ethanol was added dropwise to a vigor-
ously stirred mixture of appropriate aromatic aldehyde
(20 mmol, 2 equiv) and ketone (10 mmol, 1 equiv) dis-
solved in 20 ml of absolute ethanol. The mixture was
stirred and refluxed for 2 h. Then, 100 ml of distilled water
was added and then neutralized with dilute HCl. The solid
was filtered off, washed with distilled water, and subjected
to further purification by recrystallization method or col-
umn chromatography. (Acid Catalyzed) A mixture of
appropriate hydroxylated aromatic aldehyde (20 mmol, 2
equiv) and ketone (10 mmol, 1 equiv) was dissolved in
20 ml of absolute ethanol. To this solution, concentrated
HCl (2 ml) was then added dropwise to the stirred mixture.
The mixture was further refluxed for 1 h, and 100 ml of
distilled water was then poured into the dark viscous
solution and extracted with ethyl acetate. This layer was
collected and subjected to further purification using
recrystallization method or column chromatography.
2,5-bis(4-hydroxy-3-methoxybenzylidene)cyclopentanone
(1); Yield: 46%; yellow solid; IR(KBr) (cm-1) mmax: 3361
(–OH), 2915 (C–H), 1590 (C=O), 1523 (C=C), 1260 (C–O
2,5-bis(4-fluorobenzylidene)cyclopentanone (5); Yield:
73%; yellow powder; IR(KBr) (cm-1) mmax: 1584 (C=O),
1
1507 (C=C), 1104, 825; H NMR (500 MHz, CDCl3): d
3.11 (s, 4H, C–H), 7.15 (d, J = 8.0 Hz, 4H, Ar–H), 7.60
(d, J = 8.0 Hz, 4H, Ar–H), 7.61 (s, 2H, C–H). 13C NMR
(125 MHz, CDCl3): d 26.7, 102.2, 107.4, 115.5, 128.8,
133.7, 160.8, 197.8; EIMS m/z (rel. int.) calcd for
C19H14F2O (M?, %): 296 (M?, 45), 295 (100), 133 (30).
2,5-bis(2,4-dihydroxybenzylidene)cyclopentanone (6);
Yield: 63%; red crystals; IR(KBr) (cm-1) mmax: 3393
(–OH), 1601 (C=O), 1444 (C=C), 1040; 1H NMR
(500 MHz, CD3OD): d 3.03 (s, 4H, C–H), 6.36 (s, 2H,
Ar–H), 6.39 (d, J = 8.5 Hz, 2H, Ar–H), 7.47 (d,
J = 8.5 Hz, 2H, Ar–H), 8.00 (s, 2H, C–H). 13C NMR
(125 MHz, CD3OD): d 26.7, 102.2, 107.4, 115.5, 128.8,
131.1, 133.7, 159.8, 160.8, 197.8; EIMS m/z (rel. int.) calcd
for C19H16O5 (M?, %): 324 (M?, 11), 306 (58), 186 (100).
2,5-bis(2,3-dihydroxybenzylidene)cyclopentanone (7);
Yield: 42%; yellow solid; IR(KBr) (cm-1) mmax: 3430
(–OH), 1591 (C=O), 1441 (C=C), 1199; 1H NMR (500 MHz,
CD3OD): d 3.06 (s, 4H, C–H), 6.73 (t, J = 8.0 Hz, 2H,
Ar–H), 6.85 (d, J = 8.0 Hz, 2H, Ar–H), 7.10 (d, J = 8.0 Hz,
2H, Ar–H), 8.02 (s, 2H, C–H). 13C NMR (125 MHz,
CD3OD): d 26.5, 116.2, 119.0, 120.8, 123.3, 128.9, 136.8,
145.4, 146.3, 197.7; EIMS m/z (rel. int.) calcd for C19H16O5
(M?, %): 324 (M?, 10), 306 (47), 184 (100).
1
aromatic), 1177; H NMR (500 MHz, CD3OD): d 3.13 (s,
4H, C–H), 3.92 (s, 6H, OCH3), 6.90 (d, J = 8.5 Hz, 2H,
Ar–H), 7.17 (d, J = 8.5 Hz, 2H, Ar–H), 7.21 (s, 2H, Ar–H),
7.46 (s, 2H, C–H). 13C NMR (125 MHz, CD3OD): d 26.3,
55.3, 114.1, 115.6, 125.3, 128.1, 134.5, 135.0, 148.0, 149.0,
197.2; EIMS m/z (rel. int.) calcd for C21H20O5 (M?, %): 352
(M?, 100), 335 (32), 321 (16), 309 (11), 137 (15).
2,5-bis(3-hydroxybenzylidene)cyclopentanone (2); Yield:
78%; yellow powder; IR(KBr) (cm-1) mmax: 3392 (–OH),
2920 (C–H stretch), 1584 (C=O), 1448 (C=C), 1213; H
1
2,6-bis(4-hydroxy-3-methoxybenzylidene)cyclohexanone
(8); Yield: 52%; yellow solid; IR(KBr) (cm-1) mmax: 3392
(–OH), 2926 (C–H), 1585 (C=O), 1512 (C=C), 1259 (C–O
NMR (500 MHz, CD3OD): d 3.15 (s, 4H, C–H), 6.85 (d,
J = 8.0 Hz, 2H, Ar–H), 7.09 (s, 2H, Ar–H), 7.14 (d,
J = 8.0 Hz, 2H, Ar–H), 7.29 (t, J = 8.0 Hz, 2H, Ar–H),
7.49 (s, 2H, C–H). 13C NMR (125 MHz, CD3OD): d 26.4,
116.8, 117.0, 122.4, 129.7, 134.2, 137.1, 137.7, 157.8, 197.3;
EIMS m/z (rel. int.) calcd for C19H16O3 (M?, %): 292 (M?,
83), 275 (100).
1
aromatic), 1161; H NMR (500 MHz, acetone-d): d 1.81
(p, J = 6.0 Hz, 2H, C–H), 2.98 (t, J = 6.0 Hz, 4H, C–H),
3.92 (s, 6H, OCH3), 6.93 (d, J = 8.5 Hz, 2H, Ar–H), 7.10
(d, J = 8.5 Hz, 2H, Ar–H), 7.16 (s, 2H, Ar–H), 7.85 (s, 2H,
C–H). 13C NMR (125 MHz, acetone-d): d 23.2, 28.6, 55.9,
114.3, 115.3, 124.6, 128.3, 134.3, 136.3, 147.6, 147.8,
188.5; EIMS m/z (rel. int.) calcd for C22H22O5 (M?, %):
366 (M?, 100), 349 (47), 335 (25), 321 (17), 309 (16).
2,6-bis(3-hydroxybenzylidene)cyclohexanone (9); Yield:
53%; yellow powder; IR(KBr) (cm-1) mmax: 3339 (–OH),
1586 (C=O), 1446 (C=C), 1235 (C–O aromatic), 1163,
2,5-bis(4-hydroxybenzylidene)cyclopentanone (3); Yield:
61%; yellow solid; IR(KBr) (cm-1) mmax: 3401 (–OH), 2941
(C–H stretch), 1598 (C=O), 1440 (C=C), 1262 (C–O aro-
1
matic), 1165, 1053; H NMR (500 MHz, CD3OD): d 3.11
(s, 4H, C–H), 6.70 (d, J = 8.0 Hz, 4H, Ar–H), 7.53
(d, J = 8.0 Hz, 4H, Ar–H), 7.47 (s, 2H, C–H). 13C NMR
123

340
Med Chem Res (2012) 21:333–344
787; 1H NMR (500 MHz, CD3OD): d 1.78 (p, J = 6.5 Hz,
2H, C–H), 2.93 (t, J = 6.5 Hz, 4H, C–H), 6.81 (d,
J = 8.0 Hz, 2H, Ar–H), 6.93 (s, 2H, Ar–H), 6.95 (d,
J = 8.0 Hz, 2H, Ar–H), 7.24 (t, J = 8.0 Hz, 2H, Ar–H),
7.65 (s, 2H, C–H). 13C NMR (125 MHz, CD3OD): d 22.9,
26.3, 115.8, 116.7, 121.8, 129.4, 136.4, 137.1, 137.2,
157.4, 191.0; EIMS m/z (rel. int.) calcd for C20H18O3 (M?,
%): 306 (M?, 78), 289 (100).
J = 5.5 Hz, 2H, C–H), 7.10 (d, J = 8.5 Hz, 4H, Ar–H),
7.46 (d, J = 8.5 Hz, 4H, Ar–H), 7.76 (s, 2H, C–H). 13C
NMR (125 MHz, CDCl3): d 23.1, 28.6, 115.7, 115.8,
132.3, 132.5, 136.1, 163.9, 190.2; EIMS m/z (rel. int.) calcd
for C20H16 F2O (M?, %): 310 (M?, 100), 309 (100), 282
(14), 253 (48), 233 (24).
General procedures for the synthesis of b series com-
pounds. Purified intermediate from respective a series
(5 mmol) was dissolved in chloroform followed by addi-
tion of acetic anhydride or propionic anhydride (5.5 mmol)
and stirred at room temperature for 3 h in the presence of
DMAP. The mixture was subsequently concentrated and
purified through silica gel column.
2,6-bis(4-hydroxybenzylidene)cyclohexanone (10); Yield:
58%; yellow powder; IR(KBr) (cm-1) mmax: 3348 (–OH),
1594 (C=O), 1508 (C=C), 1279 (C–O aromatic), 1164, 838;
1H NMR (500 MHz, CD3OD): d 1.80 (p, J = 6.0 Hz, 2H,
C–H), 2.93 (t, J = 6.0 Hz, 4H, C–H), 6.86 (d, J = 8.5 Hz,
4H, Ar–H), 7.41 (d, J = 8.5 Hz, 4H, Ar–H), 7.67 (s, 2H,
C–H). 13C NMR (125 MHz, CD3OD): d 23.0, 28.3, 115.3,
127.4, 132.5, 133.8, 137.3, 158.7, 191.2; EIMS m/z (rel. int.)
calcd for C20H18O3 (M?, %): 306 (M?, 68), 305 (100), 289
(40), 278 (33), 249 (54).
2,5-bis(4-acetoxy-3-methoxybenzylidene)cyclopentanone
(1a); Yield: 86%; pale yellow powder; IR(KBr) (cm-1
)
m
max: 2916 (C–H stretch), 1765, 1626 (C=O), 1511 (C=C),
1
1231 (C–O aromatic), 1030; H NMR (500 MHz, CDCl3):
d 2.35 (s, 6H, OCOCH3), 3.12 (s, 4H, C–H), 3.89 (s, 6H,
OMe), 7.11 (d, J = 8.0 Hz, 2H, Ar–H), 7.18 (s, 2H, Ar–H),
7.22 (d, J = 8.0 Hz, 2H, Ar–H), 7.57 (s, 2H, C–H). 13C
NMR (125 MHz, CDCl3): d 20.9, 26.6, 56.2, 114.9, 123.5,
125.7, 133.6, 135.0, 137.5, 140.9, 151.4, 169.1, 198.2;
EIMS m/z (rel. int.) calcd for C27H24O7 (M?, %): 436 (M?,
5), 393 (43), 352 (100), 335 (20).
2,6-bis(3,4-dihydroxybenzylidene)cyclohexanone (11);
Yield: 52%; yellow solid; IR(KBr) (cm-1) mmax: 3342
(–OH), 1586 (C=O), 1441, 1116; 1H NMR (500 MHz,
CD3OD): d 1.80 (p, J = 5.5 Hz, 2H, C–H), 2.93 (t,
J = 5.5 Hz, 2H, C–H), 6.83 (d, J = 8.0 Hz, 2H, Ar–H),
6.93 (d, J = 8.0 Hz, 2H, Ar–H), 7.01 (s, 2H, Ar–H), 7.61
(s, 2H, C–H). 13C NMR (125 MHz, CD3OD): d 23.0, 28.4,
115.2, 117.5, 123.9, 128.0, 133.7, 137.7, 145.1, 146.9,
191.3; EIMS m/z (rel. int.) calcd for C20H18O5 (M?, %):
338 (M?, 99), 337 (100), 327 (77), 200 (26).
2,5-bis(3,4-diacetoxybenzylidene)cyclopentanone (4a);
Yield: 79%; yellow powder; IR(KBr) (cm-1) mmax: 1765,
1628 (C=O), 1502 (C=C), 1195 (C–O aromatic), 755; H
1
NMR (500 MHz, CDCl3): d 2.32 (s, 6H, OCOCH3), 2.33
(s, 6H, OCOCH3), 3.08 (s, 4H, C–H), 7.27 (d, J = 8.0 Hz,
2H, Ar–H), 7.42 (s, 2H, Ar–H), 7.45 (d, J = 8.0 Hz, 2H,
Ar–H), 7.52 (s, 2H, C–H). 13C NMR (125 MHz, CDCl3): d
20.9, 21.0, 26.5, 124.0, 125.3, 129.3, 132.5, 134.8, 138.2,
142.5, 143.0, 168.3, 168.4, 196.0; EIMS m/z (rel. int.) calcd
for C27H24O9 (M?, %): 492 (M?, 6), 449 (17), 408 (27),
366 (55), 349 (36), 324 (100).
2,6-bis(3,4-dimethoxybenzylidene)cyclohexanone (12);
Yield: 88%, yellow powder; IR(KBr) (cm-1) mmax: 2934
(C–H stretching), 1597 (C=O), 1513 (C=C), 1252 (C–O
aromatic), 1024, 730; 1H NMR (500 MHz, CDCl3): d 1.85
(p, J = 5.5 Hz, 2H, C–H), 2.97 (t, J = 5.5 Hz, 4H, C–H),
3.94 (s, 12H, OCH3), 6.94 (d, J = 7.5 Hz, 2H, Ar–H), 7.05
(s, 2H, Ar–H), 7.14 (d, J = 7.5 Hz, 2H, Ar–H), 7.78 (s, 2H,
C–H). 13C NMR (125 MHz, CDCl3): d 23.3, 28.8, 56.1,
56.2, 111.2, 113.9, 124.2, 129.2, 134.7, 137.0, 148.9,
149.9, 190.3; EIMS m/z (rel. int.) calcd for C24H26O5 (M?,
%): 394 (M?, 83), 379 (100), 363 (97), 335 (33), 320 (11).
2,6-bis(4-nitrobenzylidene)cyclohexanone (13); Yield:
89%; yellow powder; IR(KBr) (cm-1) mmax: 1669, 1593
2,6-bis(4-acetoxy-3-methoxybenzylidene)cyclohexanone
(8a); Yield: 88%; yellow powder; IR(KBr) (cm-1) mmax
:
1
2930 (C–H), 1765 (ester), 1600 (C=O), 1197, 1031; H
NMR (500 MHz, CDCl3): d 1.81 (p, J = 6.0 Hz, 2H, C–
H), 2.34 (s, 6H, OCOCH3), 2.94 (t, J = 6.0 Hz, 4H, C–H),
3.87 (s, 6H, OMe), 7.06 (s, 2H, Ar–H), 7.08 (overlapped,
4H, Ar–H), 7.76 (s, 2H, C–H). 13C NMR (125 MHz,
CDCl3): d 20.9, 23.1, 28.6, 56.2, 114.8, 123.0, 123.1,
135.1, 136.5, 136.6, 140.2, 151.2, 169.2, 190.3; EIMS m/
z (rel. int.) calcd for C26H26O7 (M?, %): 450 (M?, 10), 408
(38), 391 (49), 365 (73), 349 (100).
1
(C=O), 1515 (C=C), 1266 (C–O aromatic), 853; H NMR
(500 MHz, CDCl3): d 1.90 (p, J = 6.0 Hz, 2H, C–H), 2.98
(t, J = 6.0 Hz, 4H, C–H), 7.63 (d, J = 8.5 Hz, 4H, Ar–H),
7.82 (s, 2H, C–H), 8.30 (d, J = 8.5 Hz, 4H, Ar–H). 13C
NMR (125 MHz, CDCl3): d 22.8, 28.6, 123.9, 131.1,
135.1, 138.9, 142.4, 147.6, 189.5; EIMS m/z (rel. int.) calcd
for C20H16N2O5 (M?, %): 364 (M?, 5), 347 (100), 317
(47), 215 (14).
2,6-bis(4-propionate-3-methoxybenzylidene)cyclohexanone
(8b); Yield: 89%; yellow crystal; IR(KBr) (cm-1) mmax
:
2941 (C–H), 1763 (ester), 1601 (C=O), 1508 (C=C), 1247
(C–O aromatic), 1130 (ester); 1H NMR (500 MHz,
CDCl3): d 1.31 (t, J = 7.5 Hz, 6H, C–H), 1.83 (p,
J = 5.5 Hz, 2H, C–H), 2.67 (q, J = 7.5 Hz, 4H), 2.95 (t,
J = 5.5 Hz, 4H, C–H), 3.86 (s, 6H, OMe), 7.05 (s, 2H,
2,6-bis(4-fluorobenzylidene)cyclohexanone (14); Yield:
76%; yellow crystals; IR(KBr) (cm-1) mmax: 2954(C–H),
1609 (C=O), 1507 (C=C), 1222, 840; 1H NMR (500 MHz,
CDCl3): d 1.83 (p, J = 5.5 Hz, 1H, C–H), 2.90 (t,
123

Med Chem Res (2012) 21:333–344
341
Ar–H), 7.08 (overlapped, 4H, Ar–H), 7.76 (s, 2H, C–H).
13C NMR (125 MHz, CDCl3): d 9.4, 23.1, 27.5, 28.6, 56.2,
114.7, 123.0, 123.1, 134.9, 136.4, 136.7, 140.4, 151.2,
172.7, 190.3; EIMS m/z (rel. int.) calcd for C28H30O7 (M?,
%): 478.51 (M?, 12), 349 (100).
154.8, 157.2, 157.3; EIMS m/z (rel. int.) calcd for
C20H20N2O2 (M?, %): 320 (M?, 40), 319 (100), 198 (50).
7-(3,4-dimethoxybenzylidene)-3-(3,4-dimethoxyphenyl)-
3,3a,4,5,6,7-hexahydro-2H-indazole (12c); Yield :74%;
yellow powder; IR(KBr) (cm-1) mmax: 3340 (N–H), 2930
(C–H stretching), 1592 (C=N), 1513 (C=C), 1260 (C–O
2,6-bis(3,4-diacetoxybenzylidene)cyclohexanone (11a);
Yield: 91%; pale; yellow crystals; IR(KBr) (cm-1) mmax
1772, 1591 (C=O), 1502 (C=C), 1206 (C–O aromatic),
1013; 1H NMR (500 MHz, CDCl3): d 1.78 (p, J = 5.5 Hz,
2H, C–H), 2.29 (s, 6H, OCOCH3), 2.30 (s, 6H, OCOCH3),
2.90 (t, J = 5.5 Hz, 4H, C–H), 7.23 (d, J = 8.0 Hz, 2H,
Ar–H), 7.28 (s, 2H, Ar–H), 7.32 (d, J = 8.0 Hz, 2H, Ar–
H), 7.70 (s, 2H, C–H). 13C NMR (125 MHz, CDCl3): d
20.8, 20.9, 22.9, 28.4, 123.7, 125.3, 129.0, 134.8, 135.4,
137.0, 142.1, 142.4, 168.3, 168.4, 189.9; EIMS m/z (rel.
int.) calcd for C28H26O9 (M?, %): 506 (M?, 8), 463 (78),
379 (63), 363 (100), 337 (51), 321 (51).
1
aromatic), 1025; H NMR (500 MHz, CDCl3): d 1.57 (m,
2H, C–H), 1.98 (m, 2H, C–H), 2.46 (m, 2H, C–H), 3.08 (m,
1H, C–H), 3.94 (overlapped –OMe, 12H, OMe), 4.49 (dd,
J = 14.0/4.0 Hz, 1H, C–H), 6.87 (overlapped, 4H, Ar–H),
6.96 (overlapped, 2H, Ar–H), 7.16 (s, 1H, C–H), 7.20 (d,
J = 4.0 Hz, 1H, N–H). 13C NMR (125 MHz, CDCl3): d
24.7, 28.8, 28.9, 54.0, 56.2 (2 –OMe), 73.4, 109.9, 111.1,
111.3, 113.2, 119.7, 122.8, 126.3, 129.9, 130.1, 133.1,
148.6, 148.8, 148.9, 149.4, 157.3; EIMS m/z (rel. int.) calcd
for C24H28N2O4 (M?, %): 408 (M?, 35), 407 (100), 242
(18).
General procedures for the synthesis of c series com-
pounds. A mixture of purified intermediate from respective
a series (5 mmol) and hydrazinium hydroxide (6 mmol) in
ethanol was stirred and refluxed for 4 h in the presence of a
few drops of acetic acid as catalyst. Later, the mixture was
concentrated and slowly poured into ice-cooled distilled
water. The white precipitate was filtered and subjected to
further purification using recrystallization method or col-
umn chromatography.
7-(4-nitrobenzylidene)-3-(4-nitrophenyl)-3,3a,4,5,6,7-
hexahydro-2H-indazole (13c); Yield: 62%; orange pow-
der; IR(KBr) (cm-1) mmax: 1593 (C=N), 1514 (N–O),
1
1340 (N–O), 1108, 1033; H NMR (500 MHz, DMSO-
d6): d 1.59 (m, 2H, C–H), 1.97 (m, 2H, C–H), 2.86 (m,
2H, C–H), 3.37 (overlapped with solvent peak), 4.61 (dd,
J = 14.5 Hz/3.5 Hz, 1H, C–H), 7.12 (s, 1H, C–H), 7.64
(d, J = 8.5 Hz, 2H, Ar–H), 7.71 (d, J = 8.5 Hz, 2H,
Ar–H), 7.74 (d, J = 3.5 Hz, 1H, N–H) 8.21 (d,
J = 8.5 Hz, 2H, Ar–H), 8.24 (d, J = 8.5 Hz, 2H, Ar–H).
13C NMR (125 MHz, DMSO-d6): d 24.4, 28.4, 28.9,
53.6, 72.2, 122.9, 124.2, 124.3, 128.9, 131.0, 136.1,
144.1, 146.3, 147.5, 150.1, 153.6; EIMS m/z (rel. int.)
calcd for C20H18N2O2 (M?, %): 378 (M?, 35), 377 (100),
331 (18), 228 (14), 181 (17).
4-(7-(4-hydroxy-3-methoxybenzylidene)-3,3a,4,5,6,7-hexa-
hydro-2H-indazol-3-yl)-2-methoxyphenol (8c); Yield: 74%;
white powder; IR(KBr) (cm-1) mmax: 3335 (O–H), 2925 (C–H
stretch), 1595 (C=N), 1514 (C=C), 1269 (C–O aromatic), 1032;
1H NMR (500 MHz, DMSO-d6): d 1.41 (m, 2H, C–H), 1.88
(m, 2H, C–H), 2.36 (m, 2H, C–H), 2.88 (m, 1H, C–H), 3.77
(overlapped, 6H, OMe), 4.24 (dd, J = 16.0 Hz/4.0 Hz, 1H,
C–H), 6.76 (m, 4H, Ar–H), 6.91 (overlapped, 2H, Ar–H), 7.00
(s, 1H, C–H), 7.19 (d, J = 4.0 Hz, 1H, N–H). 13C NMR
(125 MHz, DMSO-d6): d 24.7, 28.7, 28.8, 53.4, 56.3, 56.5,
73.0, 111.54, 114.4, 115.9, 116.0, 120.1, 123.2, 125.0, 128.7,
129.7, 132.6, 146.5, 146.7, 148.0, 148.2, 154.8; EIMS m/z (rel.
int.) calcd for C22H24N2O4 (M?, %): 380 (39), 379 (100), 228
(40).
7-(4-fluorobenzylidene)-3-(4-fluorophenyl)-3,3a,4,5,6,7-
hexahydro-2H-indazole (14c); Yield: 72%; pale yellow
powder; IR(KBr) (cm-1) mmax: 3298 (N–H), 2935 (C–H
stretching), 1602 (C=N), 1506 (C=C), 1224 (C–O aro-
matic), 1157, 835; 1H NMR (500 MHz, DMSO-d6): d 1.49
(m, 2H, C–H), 1.87 (m, 2H, C–H), 2.36 (m, 2H, C–H), 2.80
(m, 1H, C–H), 4.39 (dd, J = 16.0 Hz/4.0 Hz, 1H, C–H),
6.98 (s, 1H, C–H), 7.14 (d, J = 7.5 Hz, 2H, Ar–H), 7.18
(d, J = 7.5 Hz, 2H, Ar–H), 7.37 (d, J = 4.0 Hz, 1H, N–
H), 7.40 (d, J = 7.5 Hz, 2H, Ar–H), 7.47 (d, J = 7.5 Hz,
2H, Ar–H). 13C NMR (125 MHz, DMSO-d6): d 24.5, 28.5,
28.7, 53.5, 72.2, 115.8, 115.9, 123.6, 129.6, 132.0, 132.1,
133.6, 137.9, 154.5, 160.7, 162.7; EIMS m/z (rel. int.) calcd
for C20H18F2N2 (M?, %): 324 (M?, 39), 323 (100), 200
(52).
4-(7-(4-hydroxybenzylidene)-3,3a,4,5,6,7-hexahydro-2H-
indazol-3-yl)phenol (10c); Yield: 68%; yellow powder;
IR(KBr) (cm-1) mmax: 3397 (N–H), 1603 (C=N), 1512
(C=C), 1441, 1259 (C–O aromatic), 1047; 1H NMR
(500 MHz, DMSO-d6): d 1.34 (m, 2H, C–H), 1.84 (m, 2H,
C–H), 2.33 (m, 2H, C–H), 2.56 (m, 1H, C–H), 3.96 (dd,
J = 15.5 Hz/4.0 Hz, 1H, C–H), 6.74 (d, J = 8.5 Hz, 2H,
Ar–H), 6.77 (d, J = 8.5 Hz, 2H, Ar–H), 6.90 (s, 1H, C–H),
7.15 (d, J = 4.0 Hz, 1H, N–H), 7.18 (d, J = 8.5 Hz, 2H,
Ar–H), 7.22 (d, J = 8.5 Hz, 2H, Ar–H). 13C NMR
(125 MHz, DMSO-d6): d 24.7, 28.6, 28.8, 53.4, 72.7,
115.7, 115.9, 124.6, 128.2, 128.8, 129.5, 131.5, 131.8,
General procedures for the synthesis of d and e series
compounds. (d) Purified intermediate from c series
(5 mmol) was dissolved in chloroform followed by addi-
tion of acetic anhydride or propionic anhydride (6 mmol)
and was stirred in room temperature for 3 h in the presence
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Med Chem Res (2012) 21:333–344
of DMAP. The mixture was subsequently concentrated and
purified through silica gel column to obtain the respective
pure d series compounds. (e) Purified c series compounds
(5 mmol) were dissolved in 10 ml of pyridine and stirred
while keeping it immersed in ice bath 0°C. Benzoyl chlo-
ride (6 mmol) was added dropwise into the mixture. The
reaction was monitored with TLC with variable ratios of
ethyl acetate and chloroform depending on the ascending
rate, and when the reaction was completed, the mixture was
poured into crushed ice, and the white precipitate was fil-
tered and collected.
J = 9.0 Hz, 2H, Ar–H), 8.22 (d, J = 9.0 Hz, 2H, Ar–H),
8.26 (d, J = 9.0 Hz, 2H, Ar–H). 13C NMR (125 MHz,
CDCl3): d 22.3, 24.4, 29.3, 29.9, 56.9, 67.8, 123.9, 124.5,
126.6, 126.8, 130.5, 134.1, 142.6, 147.1, 147.6, 148.9,
157.8, 170.9; EIMS m/z (rel. int.) calcd for C22H20N4O5
(M?, %): 420 (M?, 6), 377 (97), 228 (100).
1-(7-(4-fluorobenzylidene)-3-(4-fluorophenyl)-3,3a,4,5,
6,7-hexahydro-2H-indazol-2-yl)ethanone (14d); Yield:
93%; white powder; IR(KBr) (cm-1) mmax: 2942 (C–H
stretch), 1671 (C=N), 1598 (C=O), 1511 (C=C), 1217
1
(C–O aromatic), 1068; H NMR (500 MHz, CDCl3): d
4-(7-(4-acetoxy-3-methoxybenzylidene)-2-acetyl-3,3a,4,
5,6,7-hexahydro-2H-indazol-3-yl)-2-methoxyphenyl ace-
1.71 (m, 2H, C–H), 1.95 (m, 2H, C–H), 2.38 (s, 3H,
OCOCH3), 2.41 (m, 2H, C–H), 3.00 (m, 1H, C–H), 4.93
(d, J = 9.5 Hz, 1H, C–H), 7.06 (overlapped, 4H, Ar–H),
7.15 (s, 1H, C–H), 7.26 (d, J = 8.5 Hz, 2H, Ar–H), 7.35
(d, J = 8.5 Hz, 2H, Ar–H). 13C NMR (125 MHz, CDCl3):
d 22.5, 24.5, 29.2, 30.2, 57.4, 67.5, 115.5, 115.8, 127.2,
127.5, 130.6, 131.6, 132.3, 137.9, 158.7, 161.3, 163.3,
170.6; EIMS m/z (rel. int.) calcd for C22H20F2N2O (M?,
%): 366 (M?, 9), 323 (96), 201 (100).
tate (8d); Yield: 79%; white powder; IR(KBr) (cm-1
)
m
max:: 2938 (C–H stretch), 1765 (C=O), 1666 (C=O), 1603
1
(C=N), 1509 (C=C), 1197 (C–O aromatic), 1032, 753; H
NMR (500 MHz, CDCl3): d 1.50 (m, 2H, C–H), 1.96 (m,
2H, C–H), 2.31 (s, 3H, OCOCH3), 2.33 (s, 3H, OCOCH3),
2.41 (s, 3H, OCOCH3), 2.43 (m, 2H, C–H), 3.09 (m, 1H,
C–H), 3.8 (overlapped, 6H, OCH3), 4.91 (d, J = 9.0 Hz,
1H, C–H), 6.84 (overlapped, 2H, Ar–H), 6.94 (overlapped,
2H, Ar–H), 7.02 (d, J = 8.0 Hz, 1H, Ar–H), 7.05 (d,
J = 8.0 Hz, 1H, Ar–H), 7.15 (s, 1H). 13C NMR
(125 MHz, CDCl3): d 20.9, 22.5, 24.5, 24.6, 29.3, 30.3,
56.2, 56.3, 57.3, 68.0, 110.1, 114.0, 117.8, 122.3, 122.9,
123.3, 127.7, 131.1, 135.2, 139.2, 139.5, 140.9, 151.1,
151.5, 158.8, 169.2, 169.4, 170.7; EIMS m/z (rel. int.)
calcd for C28H30N2O7 (M?, %): 506 (M?, 12), 421 (27),
271 (77), 229 (100).
(7-(4-hydroxy-3-methoxybenzylidene)-3-(4-hydroxy-3-
methoxyphenyl)-3,3a,4,5,6,7-hexahydro-2H-indazol-2 yl)
(phenyl)methanone (8e); Yield: 91%; white powder;
IR(KBr) (cm-1) mmax: 3370 (-OH), 2936 (C–H stretching),
1738 (C=O), 1622 (C=N), 1514 (C=C), 1266 (C–O aro-
matic), 1031; 1H NMR (500 MHz, DMSO-d6): d 1.48 (m,
2H, C–H), 2.08 (m, 2H, C–H), 2.42 (m, 2H, C–H), 2.99 (m,
1H, C–H), 3.76 (s, 3H, OMe), 3.77 (s, 3H, OMe), 5.01 (d,
J = 10.0 Hz, 1H, C–H), 6.81 (overlapped, 4H, Ar–H),
6.95 (overlapped, 3H, Ar–H), 7.45 (overlapped, 3H), 7.82
(d, J = 7.5 Hz, 2H, Ar–H). 13C NMR (125 MHz, DMSO-
d6): d 24.3, 29.2, 29.7, 40.8, 56.2, 56.4, 68.6, 110.8, 114.5,
116.0, 116.2, 118.8, 123.5, 127.7, 128.3, 128.4, 128.5,
128.7, 130.0, 131.6, 133.5, 146.4, 147.3, 147.9, 148.3,
159.9, 168.2; EIMS m/z (rel. int.) calcd for C29H28N2O5
(M?, %): 483 (M?, 6), 362 (25), 229 (100).
1-(7-(3,4-dimethoxybenzylidene)-3-(3,4-dimethoxyphenyl)-
3,3a,4,5,6,7-hexahydro-2H-indazol-2-yl)ethanone (10d);
Yield: 85%; white powder; IR(KBr) (cm-1) mmax: 2935
(C–H stretch), 1666 (C=N), 1602 (C=O), 1507 (C=C),
1224 (C–O aromatic), 1063, 833; 1H NMR (500 MHz,
CDCl3): d 1.53 (m, 2H, C–H), 1.97 (m, 2H, C–H), 2.40 (s,
3H, OCOCH3), 2.47 (m, 2H, C–H), 3.11 (m, 1H, C–H),
3.90 (overlapped –OMe, 12H), 4.88 (d, J = 9.0 Hz, 1H,
C–H), 6.79 (s, 1H, Ar–H), 6.86 (overlapped, 2H, Ar–H),
6.92 (overlapped, 2H, Ar–H), 6.99 (d, J = 8.5 Hz, 1H,
Ar–H), 7.19 (s, 1H, C–H). 13C NMR (125 MHz, CDCl3): d
22.5, 24.6, 29.3, 30.3, 55.9, 56.1, 56.2, 56.4, 57.5, 67.9,
109.2, 111.2, 111.8, 113.2, 118.2, 122.9, 128.1, 128.2,
129.3, 134.8, 148.8, 148.6, 149.0, 149.5, 159.0, 170.7;
EIMS m/z (rel. int.) calcd for C26H30N2O5 (M?, %): 450
(M?, 3), 390 (11), 243 (100).
(7-(3,4-dimethoxybenzylidene)-3-(3,4-dimethoxyphenyl)-
3,3a,4,5,6,7-hexahydro-2H-indazol-2-yl)(phenyl)methanone
(10e); Yield: 87%; yellow powder; IR(KBr) (cm-1) mmax
:
2934 (C–H stretching), 1597 (C=N), 1513 (C=C), 1252
(C–O aromatic), 1139, 1024; 1H NMR (500 MHz, CDCl3):
d 1.57 (m, 2H, C–H), 1.98 (m, 2H, C–H), 2.45 (m, 2H,
C–H), 3.10 (m, 1H, C–H), 3.94 (overlapped, 12H, OMe),
5.16 (d, J = 10.0 Hz, 1H, C–H), 6.88 (overlapped, 7H,
Ar–H), 7.16 (overlapped, 2H), 7.43 (overlapped, 2H, Ar–H),
8.06 (d, J = 7.0 Hz, 1H, Ar–H). 13C NMR (125 MHz,
CDCl3): d 24.6, 28.8, 29.3, 53.9, 56.2 (overlapped –OMe),
73.4, 109.5, 110.0, 111.2, 111.9, 113.2, 118.4, 119.7,
122.9, 127.9, 128.6, 129.9, 130.4, 131.5, 133.1, 134.5,
148.9, 149.6, 157.3, 159.8, 168.7; EIMS m/z (rel. int.) calcd
for C31H32N2O5 (M?, %): 511 (M?, 7), 390 (17), 245 (11).
(7-(4-nitrobenzylidene)-3-(4-nitrophenyl)-3,3a,4,5,6,7-
hexahydro-2H-indazol-2-yl)(phenyl)methanone (13e); Yield:
1-(7-(4-nitrobenzylidene)-3-(4-nitrophenyl)-3,3a,4,5,6,7-
hexahydro-2H-indazol-2-yl)ethanone (13d); Yield: 92%;
yellow powder; IR(KBr) (cm-1) mmax: 2939 (C–H stretch),
1667 (C=O), 1594 (C=N), 1517 (N–O), 1343 (N–O), 1109;
1H NMR (500 MHz, CDCl3): d 1.76 (m, 2H, C–H), 2.04
(m, 2H, C–H), 2.40 (s, 3H, OCOCH3), 2.52 (m, 2H, C–H),
3.02 (m, 1H, C–H), 5.03 (d, J = 9.0 Hz, 1H, C–H), 7.27 (s,
1H, C–H), 7.46 (d, J = 9.0 Hz, 2H, Ar–H), 7.52 (d,
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Med Chem Res (2012) 21:333–344
343
75%; yellow powder; IR(KBr) (cm-1) mmax: 2939 (C–H
stretching), 1648 (C=O), 1516 (N–O), 1342 (N–O), 1109,
749; 1H NMR (500 MHz, DMSO-d6): d 1.85 (m, 2H,
C–H), 2.14 (m, 2H, C–H), 2.89 (m, 2H, C–H), 3.10 (m, 1H,
C–H), 5.38 (d, J = 10.0 Hz, 1H, C–H), 7.18 (s, 1H), 7.53
(overlapped, 3H, Ar–H), 7.67 (d, J = 9.0 Hz, 2H, Ar–H),
7.70 (d, J = 8.5 Hz, 2H, Ar–H), 7.89 (d, J = 8.5 Hz, 2H,
Ar–H), 8.24 (d, J = 9.0 Hz, 2H, Ar–H), 8.26 (d,
J = 8.5 Hz, 2H, Ar–H). 13C NMR (125 MHz, DMSO-d6):
d 24.2, 29.2, 40.8, 55.6, 68.4, 124.3, 124.7, 126.2, 127.9,
128.6, 130.3, 131.3, 131.9, 134.6, 135.1, 142.9, 146.9,
147.4, 149.8, 159.3, 168.2; EIMS m/z (rel. int.) calcd for
C27H22N4O5 (M?, %): 482 (M?, 3), 228 (100), 105 (53).
(7-(4-fluorobenzylidene)-3-(4-fluorophenyl)-3,3a,4,5,6,
7-hexahydro-2H-indazol-2-yl)(phenyl)methanone (14e);
Yield: 78%; white powder; IR(KBr) (cm-1) mmax: 2936
induced with 100 U/ml of recombinant mouse interferon-
gamma (IFN-c) and 5 lg/ml of LPS (Escherichia coli,
serotype 0111:B4) in the presence or absence of test
compounds for 17–20 h. The NO production was then
determined by Griess assay, and the percentage of activity
was calculated.
Griess assay
The NO scavenging(Mirkov et al., 2004) and NO produc-
tion in cell culture were determined by measuring the
nitrite (NO2-) formation in the SNP solution mixtures and
supernatants of spent cell culture media, respectively using
Griess assay (Tsikas, 2007). In brief, 50 ll of Griess
Reagent (1% sulfanilamide and 0.1% naphtylethyenedi-
amine dihydrochloride in 2.5% H3PO4) was added to 50 ll
of SNP solution mixtures and/or cell culture supernatant.
The color density was measured at 550 nm after 10-min
incubation at room temperature.
1
(C–H stretch), 1647 (C=O); H NMR (500 MHz, DMSO-
d6): d 1.46 (m, 2H, C–H), 1.71 (m, 2H, C–H), 2.37 (m, 2H,
C–H), 2.83 (m, 1H, C–H), 5.16 (d, J = 10.5 Hz, 1H, C–H),
7.03 (s, 1H, C–H), 7.21 (overlapped, 4H, Ar–H), 7.51
(overlapped, 7H, Ar–H), 7.85 (d, J = 7.5 Hz, 2H, Ar–H).
13C NMR (125 MHz, DMSO-d6): d 24.2, 29.1, 49.3, 56.1,
68.1, 115.9, 116.1, 126.8, 128.5, 129.2, 130.2, 131.3,
131.7, 132.3, 132.7, 135.1, 138.6, 159.6, 161.1, 162.9,
168.2; EIMS m/z (rel. int.) calcd for C26H30N2O5 (M?, %):
428 (M?, 3), 201 (100), 105 (36).
Cell viability determination (MTT assay)
After removal of culture media, all wells were topped-up
with 100 ll of DMEM, followed by the addition of 20 ll
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT, 5 mg/ml). The cells were incubated under
5% CO2 at 37 °C for 4 h. The formazan crystals formed
were dissolved in DMSO and the absorbance was read at
570 nm and the percentage of cell viability was calculated.
Bioassay procedures
DPPH radical scavenging assay
Statistical analysis
The free radical scavenging activity assay was carried out
according to Tagashira and Ohtake (1998) with some
modification. In brief, stock solution of test compound was
prepared at 50 lM concentration in 100% DMSO. The
solutions were serially diluted to different concentrations in
a 96-well microtiter plate, from 7.81 to 100 lM. Then, 5 ll
of diphenylpicrylhydrazine radical (DPPH) solution (1 mg/
ml dissolved in methanol) was then added to each well.
The plate was shaken gently and incubated in the dark for
30 min. The absorbance was measured at 517 nm, and the
percentage of total radical scavenging activity was
calculated.
The IC50 values were calculated using GraphPad Prism
software with one parameter model [y = 100/(1 ? a/x)].
Differences between groups were determined by one-way
analysis of variance (ANOVA) followed by Dunnett test
method. Statistical significance of differences between
groups was accepted at P \ 0.05.
Acknowledgments This study was financially supported by a grant
from the Ministry of Higher Education, Malaysia under the Funda-
mental Research Grant Scheme (FRGS) (01-11-08-651FR). Kok-Wai
Lam was a recipient of Graduate Research Fellowship (GRF) scheme
supported by UPM.
Cell cultures and treatment
References
The RAW 264.7 cells (ATCC) were cultured in a plastic
culture flask in Dulbecco’s Modified Eagle’s Medium
(DMEM) supplemented with 10% FBS and 1% penicillin/
streptomycin under 5% CO2 at 37 °C. Cells at confluency
of 80–90% were scrapped out and seeded into 96-well plate
at 5 9 104 cells/50 ll well. Attached cells were then
Aktan F (2004) iNOS-mediated nitric oxide production and its
regulation. Life Sci 75(6):639–653
Anggard E (1994) Nitric oxide: mediator, murderer, and medicine.
Lancet 343(8907):1199–1206
Balanco JM, Pral EM, da Silva S, Bijovsky AT, Mortara RA, Alfieri
SC (1998) Axenic cultivation and partial characterization of
123

344
Med Chem Res (2012) 21:333–344
Leishmania braziliensis amastigote-like stages. Parasitology
116(Pt 2):103–113
proinflammatory cytokine genes in human colon adenocarci-
noma cell lines. BioFactors 21(1–4):95–101
Bansal E, Srivastava VK, Kumar A (2001) Synthesis and anti-
inflammatory activity of 1-acetyl-5-substituted aryl-3-(beta-am-
inonaphthyl)-2-pyrazolines and beta-(substituted aminoethyl)
amidonaphthalenes. Eur J Med Chem 36(1):81–92
Barsoum FF, Hosni HM, Girgis AS (2006) Novel bis(1-acyl-2-
pyrazolines) of potential anti-inflammatory and molluscicidal
properties. Bioorg Med Chem 14(11):3929–3937
Pan MH, Lin-Shiau SY, Lin JK (2000) Comparative studies on the
suppression of nitric oxide synthase by curcumin and its
hydrogenated metabolites through down-regulation of IkappaB
kinase and NFkappaB activation in macrophages. Biochem
Pharmacol 60(11):1665–1676
Pandeya SN (1997) An introduction to drug design. New Age
International Publishers Limited, New Delhi
Chandru H, Sharada AC, Bettadaiah BK, Kumar CS, Rangappa KS,
Sunila, Jayashree K (2007) In vivo growth inhibitory and anti-
angiogenic effects of synthetic novel dienone cyclopropoxy
curcumin analogs on mouse Ehrlich ascites tumor. Bioorg Med
Chem 15(24):7696–7703
Rani P, Srivastava VK, Kumar A (2004) Synthesis and antiinflam-
matory activity of heterocyclic indole derivatives. Eur J Med
Chem 39(5):449–452
Shishodia S, Sethi G, Aggarwal BB (2005) Curcumin: getting back to
the roots. Ann N Y Acad Sci 1056:206–217
Duvoix A, Blasius R, Delhalle S, Schnekenburger M, Morceau F,
Henry E, Dicato M, Diederich M (2005) Chemopreventive and
therapeutic effects of curcumin. Cancer Lett 223(2):181–190
Gonzalez FM, Shiva S, Vincent PS, Ringwood LA, Hsu LY, Hon YY,
Aletras AH, Cannon RO III, Gladwin MT, Arai AE (2008)
Nitrite anion provides potent cytoprotective and antiapoptotic
effects as adjunctive therapy to reperfusion for acute myocardial
infarction. Circulation 117(23):2986–2994
Singh G, Triadafilopoulos G (1999) Epidemiology of NSAID induced
gastrointestinal complications. J Rheumatol 26(SUPPL. 56):18–24
Srinivasan M, Sudheer AR, Menon VP (2007) Ferulic Acid:
therapeutic potential through its antioxidant property. J Clin
Biochem Nutr 40(2):92–100
Tagashira M, Ohtake Y (1998) A new antioxidative 1,3-benzodioxole
from Melissa officinalis. Planta Med 64(6):555–558
Takada Y, Murakami A, Aggarwal BB (2005) Zerumbone abolishes
ˆ
ˆ
ˆ
Harvey AL (2008) Natural products in drug discovery. Drug Discov
Today 13(19–20):894–901
Hatcher H, Planalp R, Cho J, Torti FM, Torti SV (2008) Curcumin:
from ancient medicine to current clinical trials. Cell Mol Life Sci
65(11):1631–1652
Hsu CH, Cheng AL (2007) Clinical studies with curcumin. Adv Exp
Med Biol 595:471–480
Lechner M, Lirk P, Rieder J (2005) Inducible nitric oxide synthase
(iNOS) in tumor biology: the two sides of the same coin. Semin
Cancer Biol 15(4):277–289
Liang G, Shao L, Wang Y, Zhao C, Chu Y, Xiao J, Zhao Y, Li X,
Yang S (2009a) Exploration and synthesis of curcumin ana-
logues with improved structural stability both in vitro and in vivo
as cytotoxic agents. Bioorg Med Chem 17(6):2623–2631
Liang G, Yang S, Zhou H, Shao L, Huang K, Xiao J, Huang Z, Li X
(2009b) Synthesis, crystal structure and anti-inflammatory
properties of curcumin analogues. Eur J Med Chem 44(2):
915–919
Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (2001) Exper-
imental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv
Drug Deliv Rev 46(1–3):3–26
Mirkov SM, Djordjevic AN, Andric NL, Andric SA, Kostic TS,
Bogdanovic GM, Vojinovic-Miloradov MB, Kovacevic RZ
(2004) Nitric oxide-scavenging activity of polyhydroxylated
fullerenol, C60(OH)24. Nitric Oxide 11(2):201–207
Murakami A, Miyamoto M, Ohigashi H (2004) Zerumbone, an
anti-inflammatory phytochemical, induces expression of
NF-I8B and II8BI kinase activation leading to suppression of
antiapoptotic and metastatic gene expression, upregulation of
apoptosis, and downregulation of invasion. Oncogene 24(46):
6957–6969
Tetko IV, Gasteiger J, Todeschini R, Mauri A, Livingstone D, Ertl P,
Palyulin VA, Radchenko EV, Zefirov NS, Makarenko AS,
Tanchuk VY, Prokopenko VV (2005) Virtual computational
chemistry laboratory—design and description. J Comput Aided
Mol Des 19(6):453–463
Tham CL, Liew CY, Lam KW, Mohamad AS, Kim MK, Cheah YK,
Zakaria ZA, Sulaiman MR, Lajis NH, Israf DA (2010) A synthetic
curcuminoid derivative inhibits nitric oxide and proinflammatory
cytokine synthesis. Eur J Pharmacol 628(1–3):247–254
Tomita M, Holman BJ, Santoro CP, Santoro TJ (2005) Astrocyte
production of the chemokine macrophage inflammatory protein-
2 is inhibited by the spice principle curcumin at the level of gene
transcription. J Neuroinflammation 2(1):8
Tsikas D (2007) Analysis of nitrite and nitrate in biological fluids by
assays based on the Griess reaction: appraisal of the Griess
reaction in the L-arginine/nitric oxide area of research. J Chro-
matogr B Analyt Technol Biomed Life Sci 851(1–2):51–70
Veber DF, Johnson SR, Cheng HY, Smith BR, Ward KW, Kopple KD
(2002) Molecular properties that influence the oral bioavailabil-
ity of drug candidates. J Med Chem 45(12):2615–2623
Wei QY, Ma JP, Cai YJ, Yang L, Liu ZL (2005) Cytotoxic and
apoptotic activities of diarylheptanoids and gingerol-related
compounds from the rhizome of Chinese ginger. J Ethnophar-
macol 102(2):177–184
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