Nitric oxide inhibitory activity and antioxidant evaluations of 2-benzoyl-6-benzylidenecyclohexanone analogs, a novel series of curcuminoid and diarylpentanoid derivatives

Article abstract of DOI:10.1016/j.bmcl.2015.05.056

Abstract A series of twenty-four 2-benzoyl-6-benzylidenecyclohexanone analogs were synthesized and evaluated for their nitric oxide inhibition and antioxidant activity. Six compounds (3, 8, 10, 17, 18 and 19) were found to exhibit significant NO inhibitory activity in LPS/IFN-induced RAW 264.7 macrophages, of which compound 10 demonstrated the highest activity with the IC₅₀ value of 4.2 ± 0.2 μM. Furthermore, two compounds (10 and 17) displayed antioxidant activity upon both the DPPH scavenging and FRAP analyses. However, none of the 2-benzoyl-6-benzylidenecyclohexanone analogs significantly scavenged NO radical. Structure-activity comparison suggested that 3,4-dihydroxylphenyl ring is crucial for bioactivities of the 2-benzoyl-6-benzylidenecyclohexanone analogs. The results from this study and the reports from previous studies indicated that compound 10 could be a candidate for further investigation on its potential as a new anti-inflammatory agent.

Full text of DOI:10.1016/j.bmcl.2015.05.056

Accepted Manuscript
Nitric oxide inhibitory activity and antioxidant evaluations of 2-benzoyl-6-ben-
zylidenecyclohexanone analogues, a novel series of curcuminoid and diary-
lpentanoid derivatives
Sze Wei Leong, Siti Munirah Mohd Faudzi, Faridah Abas, Mohd Fadhlizil
Fasihi Mohd Aluwi, Kamal Rullah, Lam Kok Wai, Mohd Nazri Abdul Bahari,
Syahida Ahmad, Chau Ling Tham, Khozirah Shaari, Nordin H. Lajis
PII:
S0960-894X(15)00530-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.05.056
BMCL 22746
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 January 2015
11 May 2015
22 May 2015
Please cite this article as: Leong, S.W., Faudzi, S.M.M., Abas, F., Aluwi, M.F.F., Rullah, K., Wai, L.K., Bahari,
M.N.A., Ahmad, S., Tham, C.L., Shaari, K., Lajis, N.H., Nitric oxide inhibitory activity and antioxidant evaluations
of 2-benzoyl-6-benzylidenecyclohexanone analogues, a novel series of curcuminoid and diarylpentanoid
derivatives, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.05.056
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Nitric oxide inhibitory activity and antioxidant
evaluations of 2-benzoyl-6-benzylidenecyclohexanone
analogues, a novel series of curcuminoid and
Leave this area blank for abstract info.
diarylpentanoid derivatives
Sze Wei Leonga, Siti Munirah Mohd Faudzia,f, Faridah Abasa,b*, Mohd Fadhlizil Fasihi Mohd Aluwic, Kamal
Rullahc, Lam Kok Waic, Mohd Nazri Abdul Baharid, Syahida Ahmadd, Chau Ling Thame, Khozirah Shaaria,f,
Nordin H. Lajisa,*

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Nitric oxide inhibitory activity and antioxidant evaluations of 2-benzoyl-6-
benzylidenecyclohexanone analogues, a novel series of curcuminoid and
diarylpentanoid derivatives
Sze Wei Leonga, Siti Munirah Mohd Faudzia,f, Faridah Abasa,b*, Mohd Fadhlizil Fasihi Mohd Aluwic, Kamal Rullahc,
Lam Kok Waic, Mohd Nazri Abdul Baharid, Syahida Ahmadd, Chau Ling Thame, Khozirah Shaaria,f, Nordin H.
Lajisa,*
aLaboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
bDepartment of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
cDrug and Herbal Research Centre Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abd. Aziz, 50300 Kuala Lumpur, Malaysia.
dDepartment of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
eDepartment of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
fDepartment of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
ARTICLE INFO
ABSTRACT
Article history:
Received
A series of twenty-four 2-benzoyl-6-benzylidenecyclohexanone analogues were synthesized and
evaluated for their nitric oxide inhibition and antioxidant activity. Six compounds (3, 8, 10, 17,
18 and 19) were found to exhibit significant NO inhibitory activity in LPS/IFN-induced RAW
264.7 macrophages, of which compound 10 demonstrated the highest activity with the IC50
value of 4.2 0.2 µM. Furthermore, two compounds (10 and 17) displayed antioxidant activity
upon both the DPPH scavenging and FRAP analyses. However, none of the 2-benzoyl-6-
benzylidenecyclohexanone analogues significantly scavenged NO radical. Structure-activity
comparison suggested that 3,4-dihydroxylphenyl ring is crucial for bioactivities of the 2-
benzoyl-6-benzylidenecyclohexanone analogues. The results from this study and the reports
from previous studies indicated that compound 10 could be a candidate for further investigation
on its potential as a new anti-inflammatory agent.
Received in revised form
Accepted
Available online
Keywords:
Anti-inflammatory
Antioxidant
2-benzoyl-6-benzylidenecyclohexanone
RAW 264.7
Stability
2014 Elsevier Ltd. All rights reserved.
Curcumin is a well-known chemical constituent abundantly
found in turmeric (Curcuma longa), which has received
enormous attention by numerous researchers for its broad range
of pharmacological properties with extremely mild side effects.1-7
Curcumin has been reported to exhibit incredible antioxidant,
anti-inflammatory and anti-cancer properties upon their excellent
superoxide and free radical inhibition, pro-inflammatory
cytokines and enzymes suppression, and cancer cells apoptosis
induction.8-11 Curcumin has also been shown to acts as
antibacterial and anti-malarial agents based on its cytotoxic effect
on several bacteria and parasites, such as Helicobacter pylori and
Plasmodium falciparum.12,13 However, the practical use of
curcumin is limited by its poor absorbability and stability,
resulting in low oral bioavailability, which therefore diminishes
its usefulness in clinical trials.14 On this account, synthetic
modifications of curcumin have been widely employed and
intensively studied in many laboratories in order to develop a
molecule with improved bioactivities and stability.
Diarylpentanoids, a 5-carbon spacer series resulted from the
synthetic modification of curcumin which has gained increasing
attention due to its curcumin-like medicinal properties.
Diarylpentanoids have been shown to exhibit remarkable anti-
inflammatory activity based on their positive inhibitions on a
wide range of pro-inflammatory cytokines, enzymes and
mediators such as nitric oxide (NO), prostaglandins (PGs), tumor
necrosis factor alpha [TNF-α, interleukins, lipoxygenase (LOX)
and cyclooxygenases].15-18 They are also found to display great
anti-cancer properties as they significantly inhibited the growth
of various cancer cell lines including breast, lung, colon,
pancreas and prostate cancer cells, through their anti-proliferative
and
anti-angiogenic
activities.19-23
Unlike
curcumin,
diarylpentanoids are chemically stable at physiological pH and
metabolically stable in rat liver microsomes, which make them
the most impactful candidates among curcuminoids deserving
further intensive investigations towards developing novel
bioactive molecules with good stability and bioavailability
Previously, we have shown that α,β-unsaturated β-diketone and
*Corresponding authors. Tel.: +603 8947 1491
E-mail addresses: faridah_abas@upm.edu.my (Faridah Abas);
nordinlajis@gmail.com (N.H.Lajis)

cyclohexanone fragments are crucial for diarylpentanoids’
bioactivities in different studies.15,26 In continuing our effort on
the search of new anti-inflammatory agent, we have now
synthesized a series of 2-benzoyl-6-benzylidenecyclohexanone
analogues, a novel family of diarylpentanoid which incorporated
the integration of α,β-unsaturated β-diketone and cyclohexanone
moieties.
basic condition. The synthesized compounds were purified
through flash chromatography and characterized by H-NMR,
1
13C-NMR,
and
high-resolution
electron
impact-mass
spectrometry. The 1H-NMR spectra of all 2-benzoyl-6-
benzylidenecyclohexanone analogues exhibited an intense and
sharp singlet at 16-17 ppm, indicative of the chelated hydroxyl
groups and that these compounds are more stable in the keto-enol
rather than in their diketo form. All purified compounds used for
the NO inhibition and antioxidant evaluations were of 95 to 99%
purity based on their respective HPLC profiles.
Scheme 1. General synthetic steps for compounds 1-24x
The synthesized compounds were evaluated for their nitric
oxide (NO) inhibitory activity in IFN-γ/LPS-stimulated RAW
264.7 macrophages.29 Nitric oxide is an important mediator
involved in the inflammatory process. It is biosynthesized
endogenously from L-arginine, oxygen and NADPH, and
catalyzed by various nitric oxide synthase (NOS) enzymes.
Appropriate level of NO produced is responsible for body’s
defense mechanism against abnormalities.30 However, the
overproduction of NO may promote tissue injuries through cell
lipids peroxidation and deoxyribonucleic acid (DNA) mutation,
which may eventually lead to several chronic degenerative
diseases including arthritis, multiple sclerosis, ulcerative colitis,
neurodegenerative diseases, cardiovascular disorder, and
cancers.31-35 Therefore, pharmacological intervention of NO
production is a rapid and promising strategy in the search for new
potent drugs against inflammatory related degenarative diseases.
xReagents and conditions: (a) p-toluene-sulphonic acid, toluene, reflux (2h);
(b) benzoic anhydride, RT (24h); (c) H2O, reflux (0.5h); (d) B2O3, tributyl
borate, n-butylamine, ethyl acetate, 70°C (3 h); (e) HCl, reflux (0.5 h); (f)
benzaldehyde, acetic acid, H2SO4, RT (overnight).
As represented in Scheme 1, compounds 1-24 (III) were
successfully synthesized through a series of reactions, which
involved benzoylation of cyclohexanone and aldol condensation
with aryl aldehydes. The benzoylation of cyclohexanone was
accomplished by Stork enamine acylation as prescribed by Kaiho
et.al. in 1989.27 Accordingly, cyclohexanone was first reacted
with pyrrolidine to afford N-(1-cyclohexenyl)pyrrolidine (I), an
essential enamine intermediate for benzoylation. The reaction
was performed in Dean-Stark apparatus in order to eliminate the
water side-product, which would otherwise hydrolyze the N-(1-
cyclohexenyl)pyrrolidine back to its precursors. The N-(1-
cyclohexenyl)pyrrolidine was then reacted with benzoic
anhydride to obtain the corresponding crude product of 2-
benzoylcyclohexanones (II). Subsequent purification with flash
The preliminary evaluation for NO inhibitory activity was
conducted on LPS/IFN induced RAW 264.7 cells at 50 µM test
concentration. The results showed that six compounds were
significantly suppressed the NO production with the percentage
of inhibition at greater than 50%. The six bioactive compounds
were then subjected to IC50 determination and the values obtained
were compared to that of the positive control, curcumin. MTT
assay was subsequently carried out to confirm that NO inhibition
is not due to cytotoxicity. The NO inhibitory effect of 2-benzoyl-
6-benzylidenecyclohexanone analogues are presented in Table 1.
chromatography
yielded
2-benzoylcyclohexanone
with
conversion rate of 30-40%. The purified 2-benzoylcyclohexanone
was subsequently reacted with a range of different aryl aldehydes
to achieve the desired compounds III through aldol
condensation.28 In this step, acidic condition was the method of
choice since it can accomplish higher yields of the desired
products, and in a shorter time as compared to that conducted in
Table 1. NO Suppression activity and cytotoxicity of compounds 1-24 on RAW 264.7 Cells.
NO inhibition at 50 µM
(%) S.E.M
NO inhibition IC50
(µM) S.E.M
Cytotoxicity IC50
(µM) S.E.M
Compounds
Ar
Curcumin
-
phenyl
2-methoxyphenyl
3-methoxyphenyl
4-methoxyphenyl
2-chlorophenyl
99.3 0.2
5.3 2.7
6.9 5.7
14.7 0.2
ND
ND
28.8 0.8
ND
ND
1
2
3
71.6 0.9
30.8 5.1
21.8 2.8
34.5 5.4
2.0 1.7
32.1 1.0
ND
ND
>100
ND
ND
4
5
6
3-chlorophenyl
4-chlorophenyl
ND
ND
ND
ND
7
8
3-hydroxyphenyl
4-hydroxyphenyl
3,4-dihydroxyphenyl
naphthalen-1-yl
80.0 1.5
25.6 2.9
86.0 2.0
15.0 2.2
9.3 4.9
23.3 0.6
ND
87.8 1.9
ND
9
10
11
12
13
14
15
16
17
18
19
4.2 0.2
ND
ND
53.9 2.9
ND
ND
naphthalen-2-yl
2,3-dimethoxyphenyl
2,5-dimethoxyphenyl
3,4-dimethoxyphenyl
3,4,5-trimethoxyphenyl
4-hydroxy-3-methoxyphenyl
3-chloro-4-hydroxyphenyl
3-bromo-4-hydroxyphenyl
29.8 7.4
3.7 1.8
8.2 2.2
ND
ND
ND
ND
ND
ND
ND
ND
15.8 2.4
69.0 2.6
73.4 4.9
83.5 3.0
35.3 1.4
22.7 0.5
15.2 0.8
98.5 0.4
61.6 3.3
56.4 1.6

20
21
5-methylfuran-2-yl
thiophen-2-yl
15.6 4.7
8.2 3.5
7.6 4.0
8.8 5.0
9.1 4.0
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
22
23
5-methylthiophen-2-yl
3-fluorophenyl
2,3,4-trimethoxyphenyl
24
ND = Not determine
Two compounds (10 and 19) were found to exhibit strong NO
inhibitory activity, giving IC50 values of 4.2 and 15.2 µM,
respectively. Meanwhile, four other compounds (3, 8, 17 and 18)
displayed moderate NO inhibitory activity with the IC50 values of
22 to 47 µM. Based on these results, it appears that phenolic
group is essential for NO suppression activity as five out of six
active compounds bearing at least one hydroxyl group on their
phenyl ring. Besides, it has also been found that the substitution
pattern of functional groups does affect the NO inhibitory
activity. The comparison of meta- and para- hydroxylated
compounds (compounds 8 and 9, respectively) shows that
substitution at meta-position is preferable as indicated by a
relatively stronger NO inhibitory activity of 8 over compound 9.
A similar trend was also noted in the comparison of compounds
2-4, of which compound 3, a meta-methoxylated analog
exhibited much stronger activity than the other two analogs.
These results are consistent with the results in previous studies,
which found that meta-oxygenated phenyl ring is favored for
anti-inflammatory and anti-cancer properties’ enhancement.36,37
Surprisingly, apart from oxygenated functionalities, the activity
enhancement effect of meta-substitution was also been observed
in meta-halogenated analogs. This can be seen in the comparison
of compounds 9, 18 and 19, of which compounds 18 and 19, the
meta-halogenated analogs displayed much stronger NO
inhibitory effect than its meta-unsubstituted analog 9. All these
observations suggested that the presence of functionalities at
meta-position of phenyl ring could improve the anti-
inflammatory potential.
Fig. 1B. NO secretion of LPS/IFN induced RAW 264.7 cells upon treatment
with compound 10 at different concentration.
Fig. 1C. Cell viability of LPS/IFN-induced RAW 264.7 macrophage cells
upon treatment with compound 10 at different concentration using MTT
assay.
More importantly, 3,4-dihydroxylphenyl moiety appeared to
be the most significant contributing factor as compound 10, a
Similar observation has also been made in other bioactive
scaffolds such as flavonoids, diarylheptanoids, and aurones,
which further support the anti-inflammatory potential of catechol
containing compound.38-40 Interestingly, a previous study by
Chiang and co-workers has revealed that the activity enhancing
effect of catechol moiety could be boosted by a neighboring α,β-
unsaturated carbonyl moiety.41 On account of these and the
results from this study, it is thus reasonable to suggest that
compound 10 could be a potent drug candidate that deserve a
further in-depth in vitro and in vivo analyses as it contains both
catechol and α,β-unsaturated carbonyl moieties. Unlike our
previous study on 1,5-diphenyl-1,3-pentenediones, the electron
density does not significantly affects the activity of the present
compound series as all high (13-16) and low (5-7) electron
density compounds are poor in NO inhibitory activity. Therefore,
it is conceivable to conclude that hydrogen bonding capacity is
the only responsible factor for anti-inflammatory activity of 2-
benzoyl-6-benzylidenecyclohexanone analogues.
3,4-dihydroxylphenyl
containing
2-benzoyl-6-
benzylidenecyclohexanone, which exhibited the strongest NO
inhibitory activity in a dose-dependent manner (see Fig. 1A and
Fig. 1B). It displayed the IC50 value of 4.2 µM, which is 4-fold
stronger than curcumin (14.7 µM). Subsequent MTT assay has
proven that the strong activity of compound 10 was not due to its
cytotoxic effect (see Fig. 1C). This finding is consistent with our
previous finding that a catechol moiety is important for anti-
inflammatory activity.26
All synthesized compounds were also evaluated for their
antioxidant properties through NO scavenging, 2,2-diphenyl-1-
picrylhydrazyl (DPPH) scavenging and ferric reducing
antioxidant power (FRAP) assays.42 The results obtained were
analyzed and presented in Table 2.
Fig. 1A. Dose-response curve of compound 10 on NO inhibition in LPS/IFN-
induced RAW 264.7 macrophages.

Table 2. Direct NO scavenging, DPPH scavenging and FRAP activity of compounds 1-24.
DPPH radical
scavenging activity
Direct NO
Scavenging activity
at 500 µM
(%)) S.E.M
2.6 1.2
FRAP
(mmole AAE/L)
Compounds
at 200 µM
(%)) S.E.M
-
IC50
(µM) S.E.M
Ascorbic acid
BHT
-
-
-
6.5 2.6
-
0.798
0.395
0.803
0.015
0.024
0.018
0.032
0.011
0.008
0.001
0.042
0.080
0.756
0.015
0.073
0.034
0.178
0.237
0.038
0.379
0.079
0.086
0.177
0.177
0.140
0.018
0.131
Trolox
Curcumin
12.4 1.1
88.3 2.1
17.9 2.1
27.3 3.3
28.3 1.1
31.7 2.3
17.8 3.0
15.8 1.0
30.9 1.0
6.5 2.9
-
-
84.2 1.6
14.0 2.4
13.6 2.3
21.8 1.5
20.1 1.7
17.1 3.2
25.0 1.2
14.0 1.7
17.3 1.8
15.5 3.2
85.1 1.3
17.8 1.8
11.7 1.4
9.9 1.2
5.1 4.2
7.7 1.2
8.3 2.9
67.5 1.1
15.4 2.3
18.0 2.0
27.0 1.9
21.8 2.1
18.4 2.0
23.4 2.3
8.0 3.4
23.6 1.1
ND
ND
1
2
3
ND
ND
4
5
ND
ND
6
7
ND
ND
8
9
10
3.5 2.0
ND
30.5 4.2
14.8 2.6
21.0 3.0
9.9 3.1
11.5 0.4
ND
ND
11
12
13
14
ND
ND
13.8 1.5
10.9 1.3
3.4 1.8
15
16
ND
ND
17
18
19
20
7.4 2.4
47.6 2.1
ND
ND
27.6 1.2
31.7 2.0
4.9 2.1
ND
ND
ND
ND
21
22
3.3 2.1
1.6 0.6
23
24
1.9 0.9
9.3 3.7
ND
ND = Not determine
Two compounds, 10 and 17 were found to display significant
activity in both DPPH radical scavenging and FRAP assays, of
which compound 10 demonstrated the highest antioxidant
potential. It exhibited a 2-fold higher activity than curcumin in
DPPH scavenging assay and comparable activity to butylated
hydroxytoluene (BHT) in FRAP analysis. The dose-dependent
response of these compounds and the respective positive controls
in DPPH and FRAP assays are shown in Fig. 2 and Fig. 3,
respectively. On the other hand, all 2-benzoyl-6-
benzylidenecyclohexanones were found to be inactive towards
the direct NO scavenging as none of the compounds significantly
inhibited NO radical even at high concentration of 500 µM. This
finding is in agreement with the results in our previous study,
which found that all mono-carbonyl diarylpentanoids failed to
perform NO scavenging effect.
Fig. 2b.DPPH scavenging activity of compound 17 at different
concentrations.
Fig. 2a. DPPH scavenging activity of compounds 10 at different
concentration.
Fig. 2c. DPPH scavenging activity of curcumin at different concentrations.

Fig. 3. Ferric-reducing antioxidant power of compounds 10, 17, curcumin,
and the reference antioxidants (ascorbic acid, BHT and trolox) at different
concentrations
Fig. 4A. Ultraviolet-visible absorption spectra of curcumin.
The water solubility of Curcumin and compound10 were
further evaluated according to the method described by
Colclough et al. with some modifications.43 Generally, the water
solubility test was carried out by introducing DMSO stock
solution of test compounds into phosphate buffer (pH 7.4) in the
ratio of 1 to 99. After 24 hours of stirring, the concentrations of
respective compounds in phosphate buffer were determined by
quantitative HPLC analysis. The results from this experiment are
presented in Table 3. Compound 10 was found to possess 3-fold
better water solubility than curcumin with the concentration of
3.3 µM or 1.1 µg/mL. Further modification or formulation of 10
may be necessary to improve its water solubility since 1.1 µg/mL
is categorized as slightly soluble according to the General
Notices and Requirements in USP 38 NF 33.44
Fig. 4B. Ultraviolet-visible absorption spectra of compound 10.
To further investigate the potential of compound 10, in vitro
metabolic stability analysis was also performed based on the
procedure described by Stout et.al.46 The test was carried out by
observing the changes in the concentration of the compound due
to the metabolic action of human liver microsome, which are
measured at different time points. Fig. 5 depicted the metabolic
stability of curcumin and compound 10 in human liver
microsome.
Table 3. Water solubility of curcumin and compound 10.
Solubility
Compounds
µM
µg/mL
Curcumin
1.1
0.4
10
3.3
1.1
Curcumin and compound 10 were also subjected to chemical
stability test. The stability of compound 10 at physiological pH
was compared with curcumin based on the procedure described
by Zhang et al.45 The test was carried out by observing the
ultraviolet spectra changes of targeted compounds for 30 minutes
with 5-minutes interval in phosphate buffer (pH 7.4). Fig. 4A and
Fig. 4B showed the ultraviolet changes of curcumin and
compound 10, respectively. As shown in Fig. 4, the maximum
absorption peaks of curcumin gradually decreased over time.
However, no significant changes was observed in the case of
compound 10 which indicated that compound 10 is chemically
more stable than curcumin in vitro. This finding is consistent
with the previous results of Zhang et.al, in which showed that
mono-carbonyl diarylpentanoid derivatives are chemically more
stable than curcumin.
Fig. 5. Metabolic stability of curcumin and compound 10 in human liver
microsome.
As shown in Fig. 5, concentrations of both curcumin and
compound 10 gradually decreased over the time of the
experiment. However, the concentration of curcumin was found
to be reduced in much faster rate than that of compound 10, as
less than 20% of curcumin was detected after 120 minutes of
metabolism, while more than 60% of compound 10 was still
detected upon the same treatment. These observations indicated
that compound 10 exhibited much better metabolic stability than
curcumin. Taken both chemical and metabolic stability analyses
together, we concluded that compound 10 exhibits better in vitro
stability in comparison with curcumin.
.

In summary, our previous and current findings suggested that
3,4-dihydroxylphenyl is a crucial moiety for bioactivity of
diarylpentanoids and might have promising therapeutic potential
as anti-inflammatory agents. Based upon this, we concluded that
compound 10 could be used as a lead to be further developed into
a new anti-inflammatory agent as it exhibited remarkable NO
inhibitory and antioxidant activities, and possess excellent
stability. However, further mechanistic studies such as iNOS
protein and RNA expressions, release and expression of pro-
inflammatory mediators, regulatory protein expression in
inflammation-related pathways are required to understand NO
inhibition mechanism of compound 10 in RAW 264.7 cells.
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Acknowledgments
The authors thank the Ministry of Education (MOE) of Malaysia
and Universiti Putra Malaysia and for financial support under the
project ERGS/1/11/STG/UPM/01/24. NHL also thanks the
Scientific Chair Unit, Taibah University for its support. The first
author acknowledges the support from the Malaysian Ministry of
Science, Technology and Innovation (MOSTI) for scholarship
under the National Science Foundation (NSF).
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Liu, Z.; Liang, G.; Yang, S. Drug Des. Devel. Ther. 2014,
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Rullah, K.; Wai, L. K.; Bahari, M. N.; Ahmad, S.; Tham, C.
L.; Shaari, K.; Lajis, N. H. Molecules 2014, 19, 16058.
27. Kaiho, T.; San-Nohe, K.; Kajiya, S.; Suzuki, T.; Otsuka, K.;
Ito, T.; Kamiya, J.; Maruyama, M. J. Med. Chem. 1989, 32,
351.
References and notes
1. Naik, S. R.; Thakare, V. N.; Patil, S. R. Exp. Toxicol.
Pathol. 2011, 63, 419.
2. Gupta, S. C.; Sung, B.; Kim, J. H.; Prasad, S.; Li, S.;
Aggarwal, B. B. Mol. Nutr. Food Res. 2013, 57, 1510.
3. Wang, Y.; Lu, Z.; Wu, H.; Lv, F. Int. J. Food Microbiol.
2009, 136, 71.
4. Reddy, R. C.; Vatsala, P. G.; Keshamouni, V. G.;
Padmanaban, G.; Rangarajan, P. N. Bioche.m Biophys. Res.
Commun. 2005, 326, 472.
5. Limtrakul, P.; Lipigorngoson, S.; Namwong, O.;
Apisariyakul, A.; Dunn, F. W. Cancer Lett. 1997, 116, 197.
6. Gururaj, A. E.; Belakavadi, M.; Venkatesh, D. A.; Marme,
D.; Salimath, B. P. Biochem. Biophys. Res. Commun. 2002,
297, 934.
7. Bisht, K.; Wagner, K. H.; Bulmer, A. C. Toxicology 2010,
278, 88.
8. Rao, C. V. Adv. Exp. Med. Biol. 2007, 595, 213.
9. Ben, P.; Liu, J.; Lu, C.; Xu, Y.; Xin, Y.; Fu, J.; Huang, H.;
Zhang, Z.; Gao, Y.; Luo, L.; Yin, Z. Int.
Immunopharmacol. 2011, 11, 179.
General procedure for synthesis of I and II. A catalytic
amount of p-toluenesulphonic acid was added into a
mixture of cyclohexanone (20 mmol) and pyrrolidine (20
mmol) in 30 mL of toluene kept in 100 mL SNRB at room
temperature. The mixture was then refluxed on a Dean &
Stark apparatus for 2 hours to prepare I. Upon completion,
20 mmol of benzoic anhydride in 20 mL of toluene was
added dropwise into the reaction solution (I) and stirred for
24 hours. Distilled water (10 ml) was then added and
further refluxed for 30 min. The resulting reaction mixture
was extracted thrice with 3M HCl and once with 20 mL
water. The toluene layer was dried over anhydrous
magnesium sulphate and concentrated in vacuo to give
crude II. The crude of II was purified by gravitational
column chromatography
28. Lim, G. S.; Jung, B. M.; Lee, S. J.; Song, H. H.; Kim, C.;
Chang, J. Y. Chem. Mater. 2007, 19, 460.
10. Ak, T.; Gulcin, I. Chem. Biol. Interact. 2008, 174, 27.
11. Wu, S. H.; Hang, L. W.; Yang, J. S.; Chen, H. Y.; Lin, H.
Y.; Chiang, J. H.; Lu, C. C.; Yang, J. L.; Lai, T. Y.; Ko, Y.
C.; Chung, J. G. Anticancer Res. 2010, 30, 2125.
12. De, R.; Kundu, P.; Swarnakar, S.; Ramamurthy, T.;
Chowdhury, A.; Nair, G. B.; Mukhopadhyay, A. K.
Antimicrob. Agents Chemother. 2009, 53, 1592.
13. Cui, L.; Miao, J. Antimicrob. Agents Chemother. 2007, 51,
488.
General procedure for the synthesis of III (pathway d &
e). Boron trioxide (1.5 g) was added into a 50 mL SNRB
flask containing 10 mL of ethyl acetate in the presence of 2-
0
benzoylcyclohexanone, and stirred at 70 C for 3 hours.
Appropriate benzaldehyde (20 mmol) and tributyl borate
(20 mmol) were then added into the solution and the
mixture was stirred for 30 minutes. Catalytic amount of n-
butylamine was added dropwise and stirred for overnight
followed by reflux with 10 mL of 1 M HCl for 30 minutes.
The reaction mixture was extracted with EA and dried over
anhydrous magnesium sulphate. The desired product was
purified by gravitational column chromatography.
14. Anand, P.; Kunnumakkara, A. B.; Newman, R. A.;
Aggarwal, B. B. Mol. Pharm. 2007, 4, 807.
15. Lee, K. H.; Ab Aziz, F. H.; Syahida, A.; Abas, F.; Shaari,
K.; Israf, D. A.; Lajis, N. H. Eur. J. Med. Chem. 2009, 44,
3195.
16. Liang, G.; Li, X.; Chen, L.; Yang, S.; Wu, X.; Studer, E.;
Gurley, E.; Hylemon, P. B.; Ye, F.; Li, Y.; Zhou, H.
Bioorg. Med. Chem. Lett. 2008, 18, 1525.
General procedure for the synthesis of III (pathway f).
In a 50 mL SNRB, 2-benzoylcyclohexanone (5 mmol) and
appropriate aromatic aldehyde (5 mmol) were dissolved in
30 mL of acetic acid. Catalytic amount of concentrated
sulfuric acid was added and reaction mixture was stirred for
overnight. The resulting mixture was extracted with EA and
washed with 10% sodium bicarbonate solution. The organic
layer was then dried over anhydrous magnesium sulphate
and evaporated using rotatory evaporator. The target
17. Zhao, C.; Yang, J.; Wang, Y.; Liang, D.; Yang, X.; Li, X.;
Wu, J.; Wu, X.; Yang, S.; Liang, G. Bioorg. Med. Chem.
2010, 18, 2388.
18. Ahmad, W.; Kumolosasi, E.; Jantan, I.; Bukhari, S. N.;
Jasamai, M. Chem. Biol. Drug Des. 2014, 83, 670.

compound was purified by gravitational column
chromatography.
- 2.57 (m, 2 H) 2.70 - 2.76 (m, 2 H) 7.37 (s, 4 H) 7.42 - 7.50
(m, 3 H) 7.58 (dd, J=8.0, 1.3 Hz, 2 H) 7.69 (s, 1 H) 16.69
(s, 1 H). 13C NMR (126 MHz, CDCl3) δ: 23.5, 27.2, 27.5,
108.6, 127.6, 128.1, 128.6, 130.7, 131.3, 131.9, 133.1,
134.0, 134.8, 138.2, 175.6, 195.8
2-benzoyl-6-(3-hydroxybenzylidene)cyclohexen-1-ol (8).
Yellow amorphous powder; 45.14%; m.p.: 120-121ºC;
Mass calculated: 306.1256; Mass found: 306.1258. 1H
NMR (500 MHz, CDCl3) δ: 1.66 (quin, J=6.0 Hz, 2 H) 2.53
(t, J=6.0 Hz, 2 H) 2.76 (t, J=5.5 Hz, 2 H) 5.39 (br. s., 1 H)
6.80 (dd, J=8.0, 2.2 Hz, 1 H) 6.91 (s, 1 H) 7.02 (d, J=7.9
Hz, 1 H) 7.22 - 7.29 (m, 1 H) 7.40 - 7.50 (m, 3 H) 7.58 (d,
J=7.3 Hz, 2 H) 7.70 (s, 1 H) 16.71 (s, 1 H). 13C NMR (126
MHz, CDCl3) δ: 23.5, 27.2, 27.6, 108.6, 115.4, 116.7,
122.7, 127.6, 128.2, 129.5, 130.7, 132.8, 133.1, 137.8,
138.2, 155.5, 176.2, 195.8
2-benzoyl-6-(4-hydroxybenzylidene)cyclohexen-1-ol (9).
Yellow amorphous powder; 43.74%; m.p.: 158-160ºC;
Mass calculated: 306.1256; Mass found: 306.1268. 1H
NMR (500 MHz, CDCl3) δ: 1.68 (quin, J=6.0 Hz, 2 H) 2.53
(t, J=5.8 Hz, 2 H) 2.77 (t, J=5.5 Hz, 2 H) 6.87 (d, J=8.5 Hz,
2 H) 7.38 (d, J=8.2 Hz, 2 H) 7.41 - 7.49 (m, 3 H) 7.57 (d,
J=7.3 Hz, 2 H) 7.71 (s, 1 H) 16.87 (s, 1 H). 13C NMR (126
MHz, CDCl3) δ: 23.6, 27.1, 27.7, 108.1, 115.4, 127.6,
128.1, 129.1, 130.5, 130.6, 132.1, 133.4, 138.2, 155.8,
177.5, 194.4
2-benzoyl-6-(3,4-dihydroxybenzylidene)cyclohexen-1-ol
(10). Yellow amorphous powder; 36.45%; m.p.: 212-213ºC;
Mass calculated: 322.1205; Mass found: 322.1215. 1H
NMR (500 MHz, acetone) δ ppm 1.67 (quin, J=5.6 Hz, 2 H)
2.54 (t, J=5.2 Hz, 2 H) 2.79 (t, J=6.0 Hz, 2 H) 6.87 - 7.00
(m, 2 H) 7.09 (s, 1 H) 7.44 - 7.56 (m, 3 H) 7.58 - 7.68 (m, 3
H) 8.22 (br. s., 2 H) 17.12 (s, 1 H). 13C NMR (126 MHz,
acetone) δ: 23.4, 26.7, 27.5, 107.8, 115.4, 117.2, 123.4,
127.6, 128.1, 128.3, 129.9, 130.5, 133.7, 138.2, 144.9,
146.1, 177.8, 193.8
2-benzoyl-6-benzylidenecyclohexen-1-ol (1). Yellow
amorphous powder; 58.33%; m.p.: 126-127ºC; Mass
calculated: 290.1307; Mass found: 290.1324. 1H NMR (500
MHz, CDCl3) δ: 1.68 (quin, J=6.0 Hz, 2 H) 2.50 - 2.57 (m,
2 H) 2.75 - 2.82 (m, 2 H) 7.29 - 7.35 (m, 1 H) 7.38 - 7.49
(m, 7 H) 7.59 (dd, J=7.9, 1.5 Hz, 2 H) 7.78 (s, 1 H) 16.78
(s, 1 H).13C NMR (126 MHz, CDCl3) δ: 23.6, 27.2, 27.6,
108.4, 127.6, 128.1, 128.2, 128.4, 130.1, 130.6, 132.6,
133.4, 136.3, 138.3, 176.3, 195.0
2-benzoyl-6-(2-methoxybenzylidene)cyclohexen-1-ol (2).
Yellow amorphous powder; 54.12%; m.p.: 127-128ºC;
Mass calculated: 320.1412; Mass found: 320.1422. 1H
NMR (500 MHz, CDCl3) δ: 1.66 (dt, J=11.4, 5.8 Hz, 2 H)
2.53 (t, J=5.7 Hz, 2 H) 2.70 (t, J=5.8 Hz, 2 H) 3.88 (s, 3 H)
6.93 (d, J=8.2 Hz, 1 H) 6.98 (t, J=7.4 Hz, 1 H) 7.29 - 7.37
(m, 2 H) 7.41 - 7.48 (m, 3 H) 7.54 - 7.61 (m, 2 H) 7.96 (s, 1
H) 16.77 (s, 1 H). 13C NMR (126 MHz, CDCl3) δ: 23.7,
27.4, 27.6, 55.5, 108.1, 110.6, 119.9, 125.3, 127.6, 128.1,
129.2, 129.7, 130.2, 130.1, 132.4, 138.5, 158.0, 176.4,
195.5
2-benzoyl-6-(3-methoxybenzylidene)cyclohexen-1-ol (3) .
Yellow amorphous powder; 58.75%; m.p.: 95-97ºC; Mass
calculated: 320.1412; Mass found: 320.1424. 1H NMR (500
MHz, CDCl3) δ: 1.67 (quin, J=6.1 Hz, 2 H) 2.51 - 2.56 (m,
2 H) 2.75 - 2.81 (m, 2 H) 3.84 (s, 3 H) 6.88 (dd, J=8.2, 2.0
Hz, 1 H) 6.99 (s, 1 H) 7.05 (d, J=7.6 Hz, 1 H) 7.32 (t, J=7.9
Hz, 1 H) 7.41 - 7.50 (m, 3 H) 7.58 (dd, J=8.0, 1.6 Hz, 2 H)
7.74 (s, 1 H) 16.75 (s, 1 H). 13C NMR (126 MHz, CDCl3) δ:
23.6, 27.2, 27.6, 55.3, 108.5, 113.8, 115.5, 122.6, 127.6,
128.1, 129.3, 130.6, 132.8, 133.2, 137.7, 138.3, 159.5,
176.2, 195.5
2-benzoyl-6-(4-methoxybenzylidene)cyclohexen-1-ol (4).
Yellow amorphous powder; 59.01%; m.p.: 120-121ºC;
Mass calculated: 320.1412; Mass found: 320.1421. 1H
NMR (500 MHz, CDCl3) δ: 1.68 (quin, J=6.0 Hz, 2 H) 2.54
(t, J=6.0 Hz, 2 H) 2.78 (t, J=5.4 Hz, 2 H) 3.85 (s, 3 H) 6.94
(d, J=8.7 Hz, 2 H) 7.40 - 7.47 (m, 5 H) 7.58 (dd, J=7.7, 1.6
Hz, 2 H) 7.74 (s, 1 H) 16.93 (s, 1 H). 13C NMR (126 MHz,
CHLOROFORM-d) δ ppm 23.6, 27.1, 27.7, 55.4, 108.0,
113.9, 127.7, 128.1, 129.0, 130.5, 130.6, 131.9, 133.4,
138.3, 159.7, 177.5, 194.3
2-benzoyl-6-(1-naphthalenylmethylene)cyclohexen-1-ol
(11). Yellow amorphous powder; 68.12%; m.p.: 109-110ºC;
Mass calculated: 340.1463; Mass found: 340.147. H NMR
1
(500 MHz, CDCl3) δ: 1.64 (quin, J=6.0 Hz, 2 H) 2.56 (t,
J=5.8 Hz, 2 H) 2.63 (t, J=5.4 Hz, 2 H) 7.43 - 7.56 (m, 7 H)
7.59 - 7.64 (m, 2 H) 7.84 (d, J=8.2 Hz, 1 H) 7.87 - 7.91 (m,
1 H) 8.03 - 8.08 (m, 1 H) 8.31 (s, 1 H) 16.74 (s, 1 H). 13C
NMR (126 MHz, CDCl3) δ: 23.7, 27.5, 27.6, 108.4, 124.9,
125.1, 126.1, 126.4, 126.9, 127.6, 128.2, 128.5, 128.5,
130.6, 131.4, 131.9, 133.5, 133.5, 134.4, 138.5, 175.5,
196.4
2-benzoyl-6-(2-naphthalenylmethylene)cyclohexen-1-ol
(12). Yellow amorphous powder; 63.68%; m.p.: 114-115ºC;
Mass calculated: 340.1463; Mass found: 340.1488. 1H
NMR (500 MHz, CDCl3) δ:1.71 (quin, J=5.8 Hz, 2 H) 2.57
(t, J=5.7 Hz, 2 H) 2.89 (t, J=5.5 Hz, 2 H) 7.42 - 7.53 (m, 5
H) 7.55 - 7.64 (m, 3 H) 7.81 - 7.89 (m, 3 H) 7.93 (br. s., 2
H) 16.80 (s, 1 H). 13C NMR (126 MHz, CDCl3) δ: 23.6,
27.2, 27.7, 108.5, 126.4, 126.7, 127.6, 127.6, 127.9, 128.1,
128.3, 129.7, 130.6, 132.8, 132.9, 133.2, 133.4, 133.9,
138.3, 176.2, 195.5
2-benzoyl-6-(2,3-dimethoxybenzylidene)cyclohexen-1-ol
(13). Orange amorphous powder; 53.72%; m.p.: 69-71 ºC;
Mass calculated: 350.1518; Mass found: 350.1535. 1H
NMR (500 MHz, CDCl3) δ: 1.65 (dt, J=11.9, 6.2 Hz, 2 H)
2.53 (t, J=5.8 Hz, 2 H) 2.66 (t, J=5.4 Hz, 2 H) 3.84 (s, 3 H)
3.89 (s, 3 H) 6.93 (dd, J=16.0, 7.8 Hz, 2 H) 7.04 - 7.09 (m,
1 H) 7.41 - 7.49 (m, 3 H) 7.56 - 7.59 (m, 2 H) 7.89 (s, 1 H)
16.69 (s, 1 H). 13C NMR (126 MHz, CDCl3) δ: 23.6, 27.3,
27.6, 55.9, 61.1, 108.2, 112.4, 122.1, 123.4, 127.5, 128.1,
2-benzoyl-6-(2-chlorobenzylidene)cyclohexen-1-ol
(5).
Yellow amorphous powder; 66.21%; m.p.: 92-94ºC; Mass
calculated: 324.0917; Mass found: 324.0932. 1H NMR (500
MHz CDCl3) δ: 1.67 (quin, J=6.1 Hz, 2 H) 2.54 (t, J=6.0
Hz, 2 H) 2.59 - 2.64 (m, 2 H) 7.23 - 7.30 (m, 2 H) 7.35 (dd,
J=7.0, 2.0 Hz, 1 H) 7.41 - 7.50 (m, 4 H) 7.58 (dd, J=8.2, 1.5
Hz, 2 H) 7.85 (s, 1 H) 16.56 (s, 1 H). 13C NMR (126 MHz,
CDCl3) δ: 23.6, 27.3, 27.4, 108.7, 126.2, 127.5, 128.1,
129.2, 129.7, 130.1, 130.5, 130.7, 134.2, 134.7, 134.8,
138.4, 174.7, 196.7
2-benzoyl-6-(3-chlorobenzylidene)cyclohexen-1-ol
(6).
Yellow amorphous powder; 64.93%; m.p.: 97-99ºC; Mass
calculated: 324.0917; Mass found: 324.0928. 1H NMR (500
MHz, CDCl3) δ: 1.68 (quin, J=6.0 Hz, 2 H) 2.54 (t, J=6.0
Hz, 2 H) 2.71 - 2.78 (m, 2 H) 7.27 - 7.35 (m, 3 H) 7.40 -
7.51 (m, 4 H) 7.58 (dd, J=8.0, 1.3 Hz, 2 H) 7.67 (s, 1 H)
16.62 (s, 1 H). 13C NMR (126 MHz, CDCl3 CDCl3) δ: 23.5,
27.2, 27.5, 108.7, 127.6, 128.0, 128.2, 128.2, 129.6, 129.6,
130.7, 131.5, 133.8, 134.3, 138.14, 138.2, 175.2, 196.2
2-benzoyl-6-(4-chlorobenzylidene)cyclohexen-1-ol
(7).
Yellow amorphous powder; 68.32%; m.p.: 129-130ºC;
Mass calculated: 324.0917; Mass found: 324.0925. 1H
NMR (500 MHz, CDCl3) δ: 1.68 (quin, J=6.1 Hz, 2 H) 2.50

128.9, 130.5, 130.7, 133.5, 138.4, 148.0, 152.8, 175.9,
195.9
2-benzoyl-6-(5-methyl-2-furanylmethylene)cyclohexen-
1-ol (20). Red amorphous powder; 44.94%; m.p.: 104-
106ºC; Mass calculated: 294.1256; Mass found: 294.1265.
1H NMR (500 MHz, CDCl3) δ: 1.72 (quin, J=6.1 Hz, 2 H)
2.38 (s, 3 H) 2.50 - 2.55 (m, 2 H) 2.84 (t, J=5.5 Hz, 2 H)
6.13 (d, J=2.3 Hz, 1 H) 6.54 (d, J=3.2 Hz, 1 H) 7.40 - 7.48
(m, 4 H) 7.57 (dd, J=7.7, 1.6 Hz, 2 H) 16.85 (s, 1 H). 13C
NMR (126 MHz, CDCl3) δ: 14.0, 23.0, 27.0, 27.4, 108.2,
108.9, 116.6, 120.5, 127.7, 127.9, 128.1, 130.4, 138.3,
151.6, 154.8, 177.4, 193.6
2-benzoyl-6-(2,5-dimethoxybenzylidene)cyclohexen-1-ol
(14). Yellow amorphous powder; 43.89%; m.p.: 128-129ºC;
Mass calculated: 350.1518; Mass found: 350.1545. 1H
NMR (500 MHz, CDCl3) δ: 1.66 (quin, J=6.0 Hz, 2 H) 2.53
(t, J=5.8 Hz, 2 H) 2.71 (t, J=5.4 Hz, 2 H) 3.80 (s, 3 H) 3.84
(s, 3 H) 6.85 (d, J=1.5 Hz, 2 H) 6.91 (s, 1 H) 7.40 - 7.50 (m,
3 H) 7.55 - 7.59 (m, 2 H) 7.91 (s, 1 H) 16.71 (s, 1 H). 13C
NMR (126 MHz, CDCl3) δ: 23.6, 27.3, 27.6, 55.8, 56.1,
108.2, 111.5, 114.0, 116.4, 126.1, 127.6, 128.1, 128.9,
130.5, 132.7, 138.5, 152.4, 152.9, 176.1, 195.6
2-benzoyl-6-(3,4-dimethoxybenzylidene)cyclohexen-1-ol
(15). Yellow amorphous powder; 42.21%; m.p.: 152-153ºC;
Mass calculated: 350.1518; Mass found: 350.1542 1H NMR
(500 MHz, CDCl3) δ: 1.69 (quin, J=6.0 Hz, 2 H) 2.52 - 2.57
(m, 2 H) 2.76 - 2.84 (m, 2 H) 3.92 (s, 3 H) 3.92 (s, 3 H)
6.91 (d, J=8.5 Hz, 1 H) 7.00 (d, J=1.8 Hz, 1 H) 7.09 (dd,
J=8.3, 1.6 Hz, 1 H) 7.41 - 7.49 (m, 3 H) 7.58 (dd, J=7.7, 1.6
Hz, 2 H) 7.72 (s, 1 H) 16.89 (s, 1 H). 13C NMR (126 MHz,
CDCl3) δ: 23.6, 27.1, 27.7, 55.9, 56.0, 108.1, 110.9, 113.5,
123.5, 127.7, 128.1, 129.3, 130.5, 130.8, 133.5, 138.3,
148.7, 149.3, 177.2, 194.4
2-benzoyl-6-(thien-2-ylmethylene)cyclohexen-1-ol (21).
Orange amorphous powder; 54.28%; m.p.: 110-111ºC;
Mass calculated: 296.0871; Mass found: 296.0896. 1H
NMR (500 MHz, CDCl3) δ: 1.77 (dt, J=12.1, 6.2 Hz, 2 H)
2.52 - 2.58 (m, 2 H) 2.75 - 2.82 (m, 2 H) 7.14 (dd, J=5.0,
3.8 Hz, 1 H) 7.33 (d, J=3.5 Hz, 1 H) 7.43 - 7.48 (m, 3 H)
7.51 (d, J=5.0 Hz, 1 H) 7.58 (dd, J=7.9, 1.5 Hz, 2 H) 7.95
(s, 1 H) 16.87 (s, 1 H). 13C NMR (126 MHz, CDCl3) δ:
23.0, 26.8, 27.7, 108.4, 126.5, 127.6, 127.7, 128.1, 129.3,
129.4, 130.5, 131.9, 138.1, 139.9, 177.3, 193.8
2-benzoyl-6-(5-methyl-2-thienylmethylene)cyclohexen-1-
ol (22). Yellow amorphous powder; 58.16%; m.p.: 100-
102ºC; Mass calculated: 310.1028; Mass found: 310.1035.
1H NMR (500 MHz, CDCl3) δ: 1.75 (quin, J=6.2 Hz, 2 H)
2.49 - 2.58 (m, 5 H) 2.75 (t, J=5.4 Hz, 2 H) 6.79 - 6.82 (m,
1 H) 7.14 (d, J=3.5 Hz, 1 H) 7.39 - 7.48 (m, 3 H) 7.57 (dd,
J=7.9, 1.5 Hz, 2 H) 7.86 (s, 1 H) 16.96 (s, 1 H). 13C NMR
(126 MHz, CDCl3) δ: 15.7, 23.0, 26.8, 27.6, 108.1, 126.1,
127.2, 127.8, 128.0, 128.1, 130.4, 132.5, 138.0, 138.1,
144.9, 178.0, 193.0
2-benzoyl-6-(3-fluorobenzylidene)cyclohexen-1-ol (23).
Yellow; 68.57%; m.p.: 121-122ºC; Mass calculated:
308.1213; Mass found: 308.1228. 1H NMR (500 MHz,
CDCl3) δ: 1.68 (quin, J=6.1 Hz, 2 H) 2.51 - 2.58 (m, 2 H)
2.73 - 2.79 (m, 2 H) 7.02 (td, J=8.5, 1.8 Hz, 1 H) 7.14 (d,
J=10.2 Hz, 1 H) 7.21 (d, J=7.6 Hz, 1 H) 7.36 (td, J=8.0, 6.1
Hz, 1 H) 7.41 - 7.51 (m, 3 H) 7.55 - 7.61 (m, 2 H) 7.70 (s, 1
H) 16.64 (s, 1 H). 13C NMR (126 MHz, CDCl3 CDCl3) δ:
23.5, 27.2, 27.5, 108.7, 115.0, 116.5, 125.9, 127.6, 128.2,
129.9, 130.7, 131.8, 133.6, 138.2, 138.5, 163.6, 175.3,
196.1
2-benzoyl-6-(3,4,5-trimethoxybenzylidene)cyclohexen-1-
ol (16). Yellow amorphous powder; 27.85%; m.p.: 150-
151ºC; Mass calculated: 380.1624; Mass found: 380.1631.
1H NMR (500 MHz, CDCl3) δ: 1.69 (t, J=5.7 Hz, 2 H) 2.54
(t, J=6.0 Hz, 2 H) 2.80 (t, J=5.2 Hz, 2 H) 3.89 (s, 6 H) 3.89
(s, 3 H) 6.69 (s, 2 H) 7.42 - 7.48 (m, 3 H) 7.58 (dd, J=7.9,
1.5 Hz, 2 H) 7.69 (s, 1 H) 16.79 (s, 1 H). 13C NMR (126
MHz, CDCl3) δ: 23.6, 27.1, 27.7, 56.2, 56.2, 61.0, 107.6,
108.3, 127.6, 128.1, 130.6, 131.8, 131.9, 131.9, 133.5,
138.2, 138.4, 153.0, 176.4, 195.2
2-benzoyl-6-(4-hydroxyl-3-
methoxybenzylidene)cyclohexen-1-ol
(17).
Yellow;
51.66%; m.p.: 153-154ºC; Mass calculated: 336.1362; Mass
found: 336.13822. 1H NMR (500 MHz, CDCl3) δ: 1.67
(quin, J=6.0 Hz, 2 H) 2.53 (t, J=5.8 Hz, 2 H) 2.78 (t, J=5.4
Hz, 2 H) 3.93 (s, 3 H) 5.67 (s, 1 H) 6.89 (d, J=8.4 Hz, 1 H)
7.00 (d, J=8.5 Hz, 1 H) 7.10 (s, 1 H) 7.39 - 7.49 (m, 3 H)
7.58 (d, J=6.7 Hz, 2 H) 7.68 (s, 1 H) 16.88 (s, 1 H). 13C
NMR (126 MHz, CDCl3) δ: 23.5, 27.1, 27.7, 56.0, 108.1,
110.4, 115.9, 123.4, 127.7, 128.1, 129.8, 130.5, 131.0,
133.4, 138.3, 145.3, 146.8, 177.3, 194.4
2-benzoyl-6-(2,3,4,-trimethoxybenzylidene)cyclohexen-
1-ol (24). Yellow amorphous powder; 41.21%; m.p.: 140-
141ºC; Mass calculated: 380.1624; Mass found: 380.1643.
1H NMR (500 MHz, CDCl3) δ: 1.63 - 1.69 (m, 2 H) 2.53 (t,
J=5.8 Hz, 2 H) 2.69 (t, J=5.2 Hz, 2 H) 3.90 (s, 6 H) 3.91 (s,
3 H) 6.69 (d, J=8.7 Hz, 1 H) 7.09 (d, J=8.7 Hz, 1 H) 7.40 -
7.48 (m, 3 H) 7.55 - 7.59 (m, 2 H) 7.88 (s, 1 H) 16.80 (s, 1
H). 13C NMR (126 MHz, CDCl3) δ: 23.7, 27.3, 27.7, 56.1,
61.0, 61.5, 106.7, 107.9, 123.3, 125.0, 127.6, 128.1, 128.7,
130.4, 131.8, 138.5, 142.3, 153.1, 154.1, 176.8, 195.1
29. Cell-based NO Inhibitory Assay. Cell culture. ATCC
RAW264.7 murine macrophages were grown in Dulbecco’s
Modified Eagle’s Medium (DMEM) containing 10% FBS
and 1% penicillin/streptomycin in a 95% air and 5% CO2
atmosphere at 37 °C. Nitrite determination. RAW 264.7
cells at 90-95% confluency were detached and seeded
(50000 cells/well) into a 96-well culture plate with 50 µL of
DMEM and incubated for 24 h. The cells were then
stimulated in 5 mg/mL of LPS (Escherichia coli, serotype
0111:B4) and 1 ng/ml of interferon-gamma (IFN-γ) in the
presence or absence of test compounds for 17 hours. Nitrite
concentration was then determined by Griess assay by
reacting 50 µL of cell culture supernatant with 50 µL of
Griess reagent (1% sulfanilamide and 0.1% N-(1-
naphthyl)ethylenediamine dihydrochloride in 2.5%
2-benzoyl-6-(3-chloro-4-
hydroxybenzylidene)cyclohexen-1-ol
(18).
Yellow
amorphous powder; 62.13%; m.p.: 164-165ºC; Mass
calculated: 340.0866; Mass found: 340.0878. 1H NMR (500
MHz, CDCl3) δ: 1.69 (quin, J=6.0 Hz, 2 H) 2.51 - 2.56 (m,
2 H) 2.69 - 2.78 (m, 2 H) 5.74 (s, 1 H) 7.05 (d, J=8.4 Hz, 1
H) 7.30 (dd, J=8.6, 1.9 Hz, 1 H) 7.42 - 7.50 (m, 4 H) 7.56 -
7.60 (m, 2 H) 7.63 (s, 1 H) 16.75 (s, 1 H). 13C NMR (126
MHz, CDCl3) δ: 23.5, 27.1, 27.6, 108.4, 116.2, 120.0,
127.6, 128.1, 130.1, 130.5, 130.6, 130.7, 131.7, 131.8,
138.2, 151.3, 176.3, 195.2
2-benzoyl-6-(3-bromo-4-
hydroxybenzylidene)cyclohexen-1-ol
(19).
Yellow
amorphous powder; 65.19%; m.p.: 172-173ºC; Mass
calculated: 384.0361; Mass found: 384.0379. 1H NMR (500
MHz, CDCl3) δ: 1.69 (quin, J=6.0 Hz, 2 H) 2.50 - 2.57 (m,
2 H) 2.71 - 2.78 (m, 2 H) 5.70 (br. s., 1 H) 7.05 (d, J=8.5
Hz, 1 H) 7.34 (dd, J=8.5, 2.0 Hz, 1 H) 7.41 - 7.50 (m, 3 H)
7.55 - 7.61 (m, 3 H) 7.63 (s, 1 H) 16.74 (s, 1 H). 13C NMR
(126 MHz, CDCl3) δ: 23.5, 27.1, 27.6, 108.3, 110.3, 116.0,
127.6, 128.1, 130.5, 130.6, 131.4, 131.5, 131.9, 133.6,
138.2, 152.2, 176.2, 195.2

phosphoric acid) at room temperature. The optical density
was measured at 550 nm after 5 minutes of incubation at
dihydrochloride in 2.5% phosphoric acid). The absorbance
was measure at 550 nm.
room temperature with
a
microplate reader. Cell
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cytotoxicity determination (MTT assay). Supernatant in
each well was removed followed by addition of 100 µL
DMEM. Following this, 20 µL of 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml) was
then added and the plate was incubated in a 95% air and 5%
CO2 atmosphere at 37 °C for 4 hours. The mixture of
culture media and MTT in all wells were removed and the
purplish formazan crystals formed were dissolved in
dimethyl sulfoxide (DMSO) and further incubated for 15
minutess at room temperature. Color intensity was then
measured at 570 nm at room temperature.
Solubility Test. A DMSO stock solution of 10 mM
curcumin or active compound 10 were prepared and diluted
with phosphate buffer (pH 7.4) in the ratio of 1 to 99. The
respective mixtures were stirred for 24 hours at room
temperature to achieve equilibrium. The mixtures were then
filtered through a 0.45 µm syringe filter. The filtrates were
further diluted with HPLC grade methanol as co-solvent in
the ratio of 1 to 2 and the content was measured by the
quantitative reverse phase HPLC analysis on a 4.3 x 150
mm XBridgeTM C18 column (5 µm, Part no: 186003116).
The mobile phase was composed of 0.1% formic acid and
methanol for both curcumin (40:60) and compound 10
(30:70). A flow rate of 1.0 ml/min was used. Curcumin and
compound 10 were detected at 360 nm.
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Chemical Stability Test. A stock solution of 50 mM
curcumin or active compound 10 were prepared and diluted
by phosphate buffer (pH 7.4), containing 1% dimethyl
sulfoxide (DMSO), to a final concentration of 20 µM. The
ultraviolet absorption spectra were collected for over 30
min at 5-min intervals at 25 °C. All spectral measurements
were carried out in a 1 cm path-length quartz cuvette.
Absorbance readings were taken from 300–600 nm.
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Microsomal Stability Test. A mixture of pH 7.4 phosphate
buffer (100 mM, 183 µL), human liver microsome solution
(20 mg/mL, 5 µL) and test sample (1 mM, 2 µL) were pre-
incubated at 37 °C for 5 minutes. The reaction was
commenced upon the addition of NAPDH solution (40mM,
10 µL) and further incubated at 37 °C for different time
points (0, 15, 30, 45, 60, 90 and 120 minutes). The reaction
was quenched by the addition of 200 µL HPLC grade
methanol followed by vortex homogenization and
centrifugation for 5 minutes. The concentration of the
remaining test sample in supernatant was determined by
quantitative HPLC analysis as described above.
42. Antioxidant evaluations. DPPH Free Radical
Scavenging Assay. Generally 100 µL of DPPH solution
(100 µM) and 100 µL of sample dissolved in DMSO (100
µM) were added to a 96-well microplate. The mixture was
then shaken gently and incubated in the dark for 30 minutes
followed by absorbance measurement at 517 nm. Active
compounds were further analyzed through serial dilution
technique to obtain their IC50 value. FRAP Assay.
Generally 20 µL of sample or standard dissolved in 3%
DMSO in buffer was added to a 96-well microplate in
triplicate, followed by the addition of potassium phosphate
buffer (40 µL, 0.2 M, pH 7.2) and potassium ferricyanide
(40 µL, 1% w/v). The reaction mixtures were incubated at
50 ◦C for 20 min followed by adding 40 µL of 10% w/v
trichloroacetic acid, 150 µL of distilled water and 30 µL of
0.1% w/v FeCl3. The reaction mixtures were further
incubated at room temperature for 30 min in the dark.
Absorbance readings were measured at 630 nm. Active
compounds were further analyzed through serial dilution
technique to obtain a dose-dependent graph. Nitrite
Scavenging Assay. Briefly, 75 µL of SNP (10 mM)
solution were incubated alone or in combination with 75 µL
of 500 µM test compounds for 60 min with light exposure
at room temperature. The nitrite levels of the mixture were
then determined by adding Griess reagent (1%
sulfanilamide and 0.1% N-(1-naphthyl)ethylenediamine