Synthesis of unsymmetrical monocarbonyl curcumin analogues with potent inhibition on prostaglandin E2 production in LPS-induced murine and human macrophages cell lines

Article abstract of DOI:10.1016/j.bmcl.2016.03.092

The syntheses and bioactivities of symmetrical curcumin and its analogues have been the subject of interest by many medicinal chemists and pharmacologists over the years. To improve our understanding, we have synthesized a series of unsymmetrical monocarbonyl curcumin analogues and evaluated their effects on prostaglandin E₂ production in lipopolysaccharide-induced RAW264.7 and U937 cells. Initially, compounds 8b and 8c exhibited strong inhibition on the production of PGE₂ in both LPS-stimulated RAW264.7 (8b, IC₅₀ = 12.01 M and 8c, IC₅₀ = 4.86 μM) and U937 (8b, IC₅₀ = 3.44 μM and 8c, IC₅₀ = 1.65 μM) cells. Placing vanillin at position Ar₂ further improved the potency when both compounds 15a and 15b significantly lowered the PGE₂ secretion level (RAW264.7: 15a, IC₅₀ = 0.78 μM and 15b, IC₅₀ = 1.9 μM while U937: 15a, IC₅₀ = 0.95 μM and 15b, IC₅₀ = 0.92 μM). Further experiment showed that compounds 8b, 8c, 15a and 15b did not target the activity of downstream inflammatory COX-2 mediator. Finally, docking simulation on protein targets COX-2, IKK-β, ERK, JNK2, p38α and p38β were performed using the conformation of 15a determined by single-crystal XRD.

Full text of DOI:10.1016/j.bmcl.2016.03.092

Accepted Manuscript
Synthesis of unsymmetrical monocarbonyl curcumin analogues with potent in-
hibition on prostaglandin E2 production in LPS-induced murine and human
macrophages cell lines
Mohd Fadhlizil Fasihi Mohd Aluwi, Kamal Rullah, Bohari M. Yamin, Leong
Sze Wei, Mohd Nazri Abdul Bahari, Lim Sock Jin, Siti Munirah Mohd Faudzi,
Juriyati Jalil, Faridah Abas, Norsyahida Mohd Fauzi, Nor Hadiani Ismail,
Ibrahim Jantan, Lam Kok Wai
PII:
S0960-894X(16)30324-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.03.092
BMCL 23735
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 December 2015
21 January 2016
25 March 2016
Please cite this article as: Aluwi, M.F.F., Rullah, K., Yamin, B.M., Wei, L.S., Bahari, M.N.A., Jin, L.S., Faudzi,
S.M.M., Jalil, J., Abas, F., Fauzi, N.M., Ismail, N.H., Jantan, I., Wai, L.K., Synthesis of unsymmetrical
monocarbonyl curcumin analogues with potent inhibition on prostaglandin E2 production in LPS-induced murine
and human macrophages cell lines, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/
10.1016/j.bmcl.2016.03.092
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Graphical Abstract
Synthesis of unsymmetrical monocarbonyl
curcumin analogues with potent inhibition
on prostaglandin E2 production in LPS-
induced murine and human macrophages
cell lines
Mohd Fadhlizil Fasihi Mohd Aluwia , Kamal Rullaha , Bohari M. Yamind , Leong Sze Weib , Mohd
Nazri Abdul Baharib , Lim Sock Jina , Siti Munirah Mohd Faudzib, e , Juriyati Jalila , Faridah Abasb ,
Norsyahida Mohd Fauzia , Nor Hadiani Ismailc , Ibrahim Jantana , Lam Kok Waia, 
RAW 264.7
100
8c
80
15a
curcumin
60
40
20
0
-7
-6
-5
-4
Log [inhibitor], M
U937
100
80
60
40
20
0
8c
15a
curcumin
-6.0
-5.5
-5.0
-4.5
-4.0
Log [inhibitor], M

Bioorganic & Medicinal Chemistry Letters
Synthesis of unsymmetrical monocarbonyl curcumin analogues with potent
inhibition on prostaglandin E2 production in LPS-induced murine and human
macrophages cell lines
Mohd Fadhlizil Fasihi Mohd Aluwia, Kamal Rullaha, Bohari M. Yamind, Leong Sze Weib, Mohd Nazri
Abdul Baharib, Lim Sock Jina, Siti Munirah Mohd Faudzib,e, Juriyati Jalila, Faridah Abasb, Norsyahida
Mohd Fauzia, Nor Hadiani Ismailc, Ibrahim Jantana, Lam Kok Waia, 
aDrug and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300, Kuala Lumpur, Malaysia
bLaboratory of Natural Product, Institute of Bioscience, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia
cAtta-Ur- Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA Kampus Puncak Alam, 42300, Puncak Alam, Selangor, Malaysia
dSchool of Chemical Sciences and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia
eDepartment of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The syntheses and bioactivities of symmetrical curcumin and its analogues have been the subject
of interest by many medicinal chemists and pharmacologists over the years. To improve our
understanding, we have synthesized a series of unsymmetrical monocarbonyl curcumin
analogues and evaluated their effects on prostaglandin E2 production in lipopolysaccharide-
induced RAW264.7 and U937 cells. Initially, compounds 8b and 8c exhibited strong inhibition
on the production of PGE2 in both LPS-stimulated RAW264.7 (8b, IC50 = 12.01 µM and 8c, IC50
= 4.86 µM) and U937 (8b, IC50 = 3.44 µM and 8c, IC50 = 1.65 µM) cells. Placing vanillin at
position Ar2 further improved the potency when both compounds 15a and 15b significantly
lowered the PGE2 secretion level (RAW264.7: 15a, IC50 = 0.78 µM and 15b, IC50 = 1.9 µM
while U937: 15a, IC50 = 0.95 µM and 15b, IC50 = 0.92 µM). Further experiment showed that
compounds 8b, 8c, 15a and 15b did not target the activity of downstream inflammatory COX-2
mediator. Finally, docking simulation on protein targets COX-2, IKK-β, ERK, JNK2, p38α and
p38β were performed using the conformation of 15a determined by single-crystal XRD.
Keywords:
Unsymmetrical curcumin analogues
Prostaglandin E2
RAW264.7
U937
Single-crystal XRD
Cyclooxygenase-2
2009 Elsevier Ltd. All rights reserved.
studies showed that consumption of curcumin is extremely safe
The production of prostaglandin E2 (PGE2) can be triggered
by the event of infection or inflammation. It is known that
accumulation of excessive PGE2 in the body could lead to
various diseases such as cancer, rheumatoid arthritis,
atherosclerosis and pain. The biosynthesis of PGE2 is initiated
from the stimulus-induced liberation of arachidonic acid (AA)
from membrane phospholipids by the phospholipase A2 (PLA2)
enzyme. AA is then sequentially metabolized into prostaglandin
G2 and further converted to prostaglandin H2 (PGH2) by either
cyclooxygenase-1 (COX-1) or cyclooxygenase-2 (COX-2).
Finally, the unstable intermediate PGH2 can be catalyzed by any
of the three forms of prostaglandin synthase (PGES), namely
mPGES-1, mPGES-2, and cPGES, [1-3] to form the bioactive
lipid prostaglandin E2 (PGE2) (Figure 1).
even at very high doses in various animal models [4] or human
studies [5]. Nevertheless, curcumin has been shown to exhibit
great chemical and pharmacological potential as antioxidant,
anti-proliferation, anti-angiogenesis, anti-tumour and anti-
inflammation [6-10]. It has also been investigated for COX
inhibitory activity using the bovine seminal vesicles, microsomes
and cytosol from homogenates of mouse epidermis with reported
IC50 values of 2 µM, 52 µM and 5–10 µM, respectively [11-13].
Despite that, accumulating evidence suggests that curcumin is
highly unstable and has a poor bioavailability when tested in
vitro and in vivo. Some of the efforts to improve the stability of
curcumin have been made recently including replacing the β-
diketone moiety of the curcumin structure with a cyclohexanone
or 4-piperidone ring [14-17].
Curcumin is the main active ingredient isolated from
Previously, our group has established the synthesis of
symmetrical curcumin analogues and evaluated their effects on a
Curcuma longa L. also referred to as diarylheptanoid. Previous
———
 Corresponding author. Tel.: +60 3 9289 7031
E-mail: david_lam@ukm.edu.my

[29]. The singlet peak at 7.58 ppm with integration of one found
in the 1H NMR spectra confirmed the presence of olefinic proton
variety of biological activities including anti-inflammatory, anti-
tyrosinase and immunomodulatory [18-21]. The anticancer
properties of these curcumin analogues have also been
intensively studied by others [22-24]. Based on the recent reports,
there were suggestions that the unsymmetrical form of
demethoxycurcumin derivative (Figure 2) might possess greater
biological profile compared to the symmetrical form of
bisdemethoxcurcumin [25, 26]. This was further supported by
our work on the synthesis of unsymmetrical diarylpentadione 1
in the half aldol condensation product of
6 (Fig.S-1,
Supplementary Data). Crude product 6 was coupled with
respective benzaldehydes, 7 in the presence of NaOH at room
temperature to furnish the desired unsymmetrical monocarbonyl
curcumin analogues 8a-e in satisfactory yields. To initiate the
synthesis of compounds 12a-d, Suzuki-Miyaura coupling
reaction was employed utilizing the carbon-carbon single bond
formation between 4-bromobenzaldehyde, 9 and 4-fluroboronic
acid, 10 to afford 4-fluorophenylbenzaldehyde, 11 and reacted
with 6 using the Claisen-Schmidt condensation reaction to afford
12a-d. On the other hand, we have also attempted to replace the
cyclohexanone linker with 4-piperidone ring. Several strategies
have been employed including masking the reactive amine group
of 4-piperidone with tert-butyloxycarbonyl and methyl iodide
and preparing the mono-benzylidenepiperidone using
Mukaiyama aldol reaction as shown in Scheme S-1,
Supplementary Data. However, all attempts failed to yield the
[27],
diarylpentadienone
2
[28]
and
2-benzoyl-6-
benzylidinecyclohexanone 3 [17] which were found to actively
repress the NO production in RAW 264.7 cells (Figure 2). In our
continuing effort to search for new anti-inflammatory agents with
higher efficacy and better safety profiles, we have synthesized a
series of new unsymmetrical monoketone diarylpentanoid
analogues and evaluated their effects on PGE2 production against
the lipopolysaccharide-induced murine RAW 264.7 and human
U937 macrophages cell lines. The active compounds were further
tested on the ability to inhibit human COX-2 activity.
1
target unsymmetrical products. Finally, all H, 13C NMR and
ESI-HRMS spectra were consistent with the assigned structure of
the synthesized compounds [30] (Fig. S-2, Fig. S-3 & Fig. S-4,
Supplementary Data).
Among compounds 8a-e tested for their effects on PGE2
production in LPS/IFN-γ-induced RAW264.7 cells, 8c appeared
to be the frontrunner with excellent inhibition at the screening
concentration. Further testing showed that compound 8c
exhibited an IC50 of 4.86 µM (Table 1). From the MTT assay, it is
clearly shown that the inhibition was not affected by cell viability
as it did not fall in close proximity to the tested concentration.
Therefore, it can be concluded that the reduction of PGE2
secretion level was not due to cell death. It is also noteworthy
that this compound showed approximately 3-fold higher
inhibition than curcumin. Structurally, compound 8c comprises a
five-membered furanyl ring at Ar2 position could be the key
reason that contributes to the activity. This result coincides with
the previous report, in which compound containing furanyl ring
also exhibited a significant PGE2 reduction in vitro [31] and was
further supported that the presence of five-membered
heterocyclic ring system is essential for PGE2 inhibition in vivo
[32]. Besides, compound 8b bearing an adjacent pair of
dimethoxyl groups at Ar2 position also displayed a remarkable
reduction in the PGE2 level at the screening concentration with
IC50 of 12.01 µM. Moreover, compounds containing more than
one methoxyl group also enhanced the PGE2 inhibitory activity in
vitro [33].
Figure 1. Biosynthesis of PGE2 from arachidonic acid by COX-1/2 and
mPGES-1.
Following the positive results obtained, we next addressed the
question of whether the inhibitory action of 8a-e on PGE2
production was restricted to murine cells. Therefore, the study
was repeated using the human U937 macrophage cell line as it
mimics the human in vivo condition sharing many characteristics
of monocytes and is easy to use [34]. Even though murine cells
are the experimental tool of choice for majority pharmacologists
and have yielded tremendous results in the past, undeniably both
murine and human share significant differences in signaling
pathways and protein structures due to genetic and evolution
factors. For example, the mPGES-1 inhibitor, MF63 did not show
any effects on rat possibly due to difference in substitutions of
amino acid residues Thr131, Leu135 and Ala138 (human amino
acid sequence) with bulky aromatic residues. It was suggested
that these residues occlude the entrance to the active site and
therefore prevent some inhibitors from binding [35]. To initiate
the experiment, U937 monocyte was first induced with phorbol
12-myristate 13-acetate (PMA) to start to adhere culture plate and
differentiate into macrophages. The detail of assay procedure is
Figure 2. Naturally isolated unsymmetrical demethoxycurcumin, synthetic
unsymmetrical dicarbonyl, and monocarbonyl curcumin analogues.
The syntheses of monocarbonyl curcumin analogues 8a-e,
12a-d and 15a-e were accomplished by steps outlined in Scheme
1. Initially, mono-benzylidene cyclohexanone, 6 was prepared by
simple aldol condensation reaction using pyrolidine as catalyst in
ethanol solvent as reported by Chimni and co-workers (2005)

different benzaldehydes, 6 in the presence of base. Subsequently,
the THP protecting group was cleaved with p-toluene sulfonic
acid to afford compounds 15a-e in satisfactory yield (Scheme 1).
As expected, treatment of LPS-induced murine and human
macrophages with these compounds strongly suppressed the
PGE2 formation with over 85% inhibition at 25 µM
concentration. In both cell lines, compounds 15a and 15b
displayed even higher potency than curcumin. Figure 3 depicts
the dose-dependent graphs of compounds 15a on the inhibition of
PGE2 and the cytotoxicity against RAW264.7 and U937 cells.
From this finding, it is important to note that the presence of a
single vanillin moiety at Ar2 was sufficient to replicate the
activity of curcumin. This remarkable observation could serve as
important information for the future design of anti-inflammatory
inhibitors.
explained in the experimental section. Overall, the results were
found comparable to the study conducted using RAW264.7
macrophage (Table 2). Compounds 8b and 8c bearing the
dimethoxyl groups and furanyl ring were again able to reduce the
PGE2 secretion level in the cells while compounds 8a, 8d and 8e
only managed to exhibit moderate inhibition. It is assumed that
these compounds could interact in
a relatively similar
mechanistic pathway that is shared between the two species. All
active compounds showed non-cyctotoxic profile against the
U937 human macrophages at the tested concentration indicate
that these compounds may be safe to be implemented in future in
vivo murine model. Figure S-6 depicts the dose-dependent
manner of 8b and 8c towards the secretion of PGE2 with IC50
values of 3.44 µM and 1.65 µM (Fig. S-6, Supplementary Data).
In contrast, the presence of 2-napthyl, 3, 4-dichlorophenyl and 4-
fluorophenylbenzaldehyde in compounds 8a, 8e and 12a-d were
not favorable possibly due to their poor solubility in the buffer
solution (Table 1).
Additionally, the synthesized compounds were tested on the
nitric oxide (NO) inhibition using RAW264.7 cell line at 25 µM
concentration. Unfortunately, we could not evaluate the NO
activity in U937 human macrophages cells due to the reason that
the cells did not produce measurable amount of nitrite following
induction [18]. Most of the compounds exhibited moderate to
strong inhibition except for 12a-d (Table S-1, Supplementary
Data). However, at 25 µM, compounds 8e and 15a-e were
cytotoxic and therefore it is important to reduce the concentration
in the next study.
Previous studies conducted on the symmetrical BHMC (Fig.
S-1, Supplementary Data) showed that it exhibited potent anti-
inflammatory activity in both in vitro and in vivo [20, 36]. To
compare the effects between the symmetrical and unsymmetrical
form of demethoxycurcumin derivatives on PGE2 formation in
both macrophages, compounds 15a-e were designed and
synthesized by coupling the THP vanillin moiety 14 with
Scheme 1. Reagents and conditions: a) pyrolidine, EtOH, r.t.; b) NaOH, EtOH, r.t.; c) p-toluene sulfonic acid, EtOH, r.t.; d) 3,4-dihydro-2H-pyran, pyridinium 4-
toluenesulfonate, DCM, r.t.; e) HCl (g), AcOH, r.t.
Since compounds 8b, 8c, 15a and 15b displayed similar
inhibitory profiles on the PGE2 secretion level in the two cell
lines, we predicted that these compounds might potentially
inhibit the activity of COX-2 enzyme. The reason is simple as
COX-2 in both species share common conserved sequence and
are structurally homology similar [37]. To determine the
selectivity of the compounds on COX isoforms, experiments
were conducted using the ovine COX-1 and human recombinant
COX-2 ELISA kits and the results were compared with the COX
inhibitor, indomethacin which acts as a positive control. At 25
µM concentration, compounds 8b, 8c, 15a and 15b selectively
inhibited COX-2 over COX-1 (Table 2) even though this result
did not justify their strong PGE2 inhibition profile in the cells.
Initial docking simulations revealed that compounds 8b, 8c, 15a
and 15b did not interact with Arg513 (Supplementary Data),
whereas this residue is known to be a crucial requirement for the
time dependent inhibition of COX-2. This implies that the
compounds might work on a different protein target or signaling
pathway.
It is known that the accuracy of docking result is highly
dependent on the initial conformation of the input ligand.
Therefore, two crystals of compounds 15a and 15c were grown
by slowly evaporation from hexane: ethyl acetate (7:3) and were

subjected to single crystal X-ray structural investigation. Both
compounds 15a and 15c crystallized in monoclinic system with
space group a = 15.464(2) Å, b = 8.0410(11) Å, c = 15.537(2) Å,
α= 90°, β= 112.431, γ = 90°, Z=4, V=1785.96(17) Å and
a=10.0643(10) Å, b=10.5045(10) Å, c=17.2687(16) Å, α=90°,
β=94.629(3)°, γ=90°, Z=4, V= 1819.7(3) Å, respectively. It was
difficult to get a quality crystal of 15a despite several attempts of
crystallization. However, the data was solved under monoclinic
system although the unit cell is unusual. No sign of twinning and
pseudo symmetry was detected by PLATON software.
The crystal system and refinement parameters are shown in
Table 3. Figure 4 shows the molecular structure with numbering
scheme for both compounds. Both compounds display the bird-
like molecules consist of a central cyclohexanone ring, (C8-
C13)/O1 with its wings of 3-hydroxy-4-methoxybenzylidine
(C14-C20)/C22//O2/O3 and p-methylbenzylidene, (C1-C7)/C21
groups in 15c whereas in 15a the right wing 4-methylbenzylidene
is replaced by p-methoxybenzylidene, (C1-C7)/O2/C21.
Table 1
In vitro PGE2 production inhibitory activity of synthesized compounds in LPS/IFN-γ-induced RAW264.7 and U937 cells
Percentage (%) of PGE2 inhibition at 25 µM
Percentage (%) of cell viability at 25 µM
IC50 (µM)
Compound
RAW 264.7a
U937b
RAW 264.7a
U937b
RAW 264.7a
U937b
64
83
92
74
73
51
59
53
65
96
96
95
96
95
78
66
68
83
84
66
58
68
76
66
72
86
88
85
87
85
94
89
95
100
83
100
77
90
100
92
100
100
98
94
100
98
n.t
12.01
4.86
n.t
n.t
n.t
n.t
n.t
n.t
0.78
1.9
n.t
n.t
n.t
15.95
<0.078
n.t
3.44
1.65
n.t
n.t
n.t
n.t
n.t
n.t
0.95
0.92
n.t
n.t
n.t
8a
8b
8c
8d
8e
12a
12b
12c
12d
15a
15b
15c
15d
15e
Curcumin
Nimesulide*
n.t = not tested
94
98
94
100
100
100
100
100
100
95
<50
<50
<50
<50
<50
90
1.88
<0.078
100
100
*Concentration of positive control (nimesulide) used in the experiment (5 µM).
a LPS/IFN-γ-induced murine macrophage cell line.
bLPS/IFN-γ-induced human macrophage cell line.
120
100
80
60
40
20
0
2500
RAW 264.7
RAW 264.7
RAW 264.7
100
80
60
40
20
0
#
2000
1500
1000
500
0
**
**
***
***
***
***
5
***
5
8
6
3
5
5
(M)
-7.5
-7.0
-6.5
-6.0
-5.5
-5.0
Control
-
0.078 0.156 0.313 0.625 1.25 2.5
2.5
1.25
0.07
0.15
0.31
0.62
Log [inhibitor], M
Nimesulide
15a
Concentration (M)
IFN-/LPS
15a
120
U937
U937
1500
1000
500
0
#
U937
100
80
60
40
20
0
100
80
60
40
20
0
***
***
***
***
***
***
***
25
***
***
(M)
5
Nimesulide
control
-
0.78 1.56 3.13 6.25 12.5
50
-6.5
-6.0
-5.5
-5.0
-4.5
-4.0
.5
25
50
.78
.56
1
.13
.25
2
1
0
3
6
Log [inhibitor], M
15a
Concentration (M)
IFN-/LPS
15a
Figure 3. Effects of compound 15a on PGE2 production in LPS-stimulated RAW264.and U937 cells. The cells were co-incubated with LPS (2µg/mL) and
different concentrations of compounds ranging from 0.78 to 50µM. The supernatants were then collected for the measurement of PGE2 production. The values
are expressed as the means ± SD of three individual samples. P< 0.001 as compared with the LPS-treated macrophages; significant differences between groups
were determined using one-way ANOVA test followed by a Dunnett’s multiple comparison test. #P< 0.05 solvent control compared with the LPS-treated cells;
significant difference was determined using unpaired student’s-test (n.s. is not significant).

In compound 15c, the central cyclohexyl ring (C8-C13)
adopts a twisted half chair conformation with maximum
deviation of 0.349(3) Ǻ for C12 atom from the least square plane.
The bond lengths and angles are in normal ranges (Table S-3)
with double bond character of C7-C8 and C10-C14 bonds
(1.334(3) and 1.342(3) Ǻ, respectively). Both wings are planar
with maximum deviation of 0.049(4) Ǻ for C21 atom of the least
square plane of the p-methylbenzylidine ring. The dihedral angle
between the two wings is 21.64(10)°. The central cyclohexanone
makes dihedral angles with the 3-hydroxy-4-methoxybenzylidine
(C14-C20)/C22//O2/O3 and p-methylbenzylidene, (C1-C7)/C21
of 48.96(11) and 47.35(13)°, respectively. There is a weak O2-
H2...O3 intramolecular hydrogen bonds. The structure of the
molecule is stabilized by O2-H2...O1 (1/2+x,3/2y,-1/2z, D-A=
2.807(3)Ǻ, D-H-A= 152°) and C7- H7-O2 (-1/2x,3/2-y,1/2+z, D-
A=3.570(4)Ǻ , D-H-A= 168°) intermolecular hydrogen bonds.
The bonding parameters in the analogous 15a are very much
comparable with those in 15c. However the dihedral angles
between the wings 3-hydroxy-4-methoxybenzylidine (C14-
C20)/C22//O3/O4 and p-methoxybenzylidene, (C1-C7)/O1/C21
with the central cyclohexanone, (C8-C13) are slightly smaller
than those in 15c of 23.35(11) and 46.43(13)°, respectively. In
the crystal structure the molecule is stabilized by O3-H13...O1,
C21-H21B...O4 and C22-H22...O2 intermolecular hydrogen
bonds. The crystallographic data for the structural analysis were
deposited with the Cambridge Crystallographic Data Centre No
1446853 (15a) and 1441911 (15c).
binding site, the vanillin moiety could interact by forming a
single hydrogen bond (2.06 Å) with Phe169. Furthermore, the
cyclohexanone carbonyl oxygen of 15a could form a hydrogen
bond (2.01 Å) with Lys53 (Figure 5). On the other hand, a strong
hydrogen bond could be observed between the oxygen atom of
methoxyl moiety of 15a with Cys99 (2.59 Å) in IKK-β binding
site (Figure 5). A weak π-π interaction (5.63 Å) between p-
methoxyphenyl and phenol side chain of Tyr98 could also be
observed. Overall, 15a displayed lower binding affinity in other
target enzymes as compared to their co-crystallized ligands. This
result is in agreement with the previous report, in which the
inhibitory activity of curcumin analogues were more pronounced
on p38α and IKK-β [40, 41]. Thus, it is suggested that 15a could
preferably target p38α or IKK-β, subsequently down-regulated
the COX-2 expression and eventually reduced the PGE2
production.
Table 2
COX-1 and COX-2 inhibition activity
Percentage (%) inhibition at 25 µM
Compound
COX-1
n.a
COX-2
23
8b
n.a
30
8c
n.a
22
15a
n.a
27
15b
n.a
100
24
100
Curcumin
Indomethacin
n.a = not active
Table 3
Crystal and structure refinement parameters of compound 15a
and 15c
Identification code
Empirical formula
Formula weight
15a
C22 H22 O4
350.39
15c
C22 H22 O3
334.39
15a
15c
Temperature
Wavelength
Crystal system
Space group
Unit cell dimensions
301(2) K
0.71073 Å
303(2) K
0.71073 Å
Monoclinic
P21/n
a = 15. 464(2) Å
b = 8.0410 (11) Å
c = 15.5370(2) Å
a= 90°
Monoclinic
P21/n
a = 10.0643(10) Å
b = 10.5045(10) Å
c = 17.2687(16) Å
α= 90°
β= 112.431(3)°
β= 94.629(3)°
γ = 90°
γ = 90°
Figure 4. Molecular structure of 15a and 15c with numbering scheme drawn
3
3
Volume
1785.8(4) Å
1819.7(3) Å
at 50% probability ellipsoids.
Z
4
4
3
3
Density (calculated)
1.303 Mg/m
1.221 Mg/m
Since the compounds did not directly suppress PGE2
production via COX-2, we predicted that it might target the
transcription factor level. LPS mediated the stimulation of TLR4
and subsequently activating NF-κB and MAPK signaling
pathways which have been recognized as one of the crucial
transcription factors known to regulate COX-2 expression.
Interruptions of these signaling pathways may down-regulated
the COX-2 expression, thus reduced the PGE2 production. It has
been reported that the symmetrical monocarbonyl curcumin
analogues could interrupt these signaling pathways in both in
vitro and in vivo [22, 38, 39]. Therefore, the obtained crystal
XRD of 15a was employed in docking simulations using protein
crystal structures of IKK-β (3RZF), ERK (5BVD), JNK2
(3NPC), p38α (1A9U) and p38β (3GP0). The cDOCKER
interaction energies were used as a measurement to compare the
binding affinity of compound 15a with the co-crystallized ligands
(Table 4). Among the target enzymes, compound 15a showed
favorable cDOCKER interaction energy of -38.45 kcal/mol
(p38α) and -42.63 kcal/mol (IKK-β) which closely resembled the
cDOCKER interaction energy of their co-crystallized ligands, -
40.62 kcal/mol and -42.63 kcal/mol, respectively. In the p38α
-1
-1
Absorption coefficient
0.089 mm
0.080 mm
F(000)
Crystal size
744
712
0.500 × 0.180 × 0.090 0.500 × 0.480 × 0.420
3
3
mm
mm
Theta range for data
collection
Index ranges
2.873 to 25.996°
2.983 to 26.498°
-19<=h<=19,
-9<=k<=9,
-12<=h<=12,
-13<=k<=13,
-21<=l<=21
-19<=l<=19
59594
Reflections collected
Independent
reflections
Completeness to theta
= 25.242°
Refinement method
46798
3508 [R(int) = 0.2810]
3765 [R(int) = 0.0971]
99.9 %
99.8 %
Full-matrix
least- Full-matrix least-squares
2
2
squares on F
on F
Data / restraints /
parameters
3508 / 0 / 239
3765 / 0 / 229
2
1.053
1.090
Goodness-of-fit on F
Final R indices
[I>2sigma(I)]
R1 = 0.0707, wR2 = R1
0.1085 0.1166
R1 = 0.1692, wR2 = R1
0.1365 0.1443
=
0.0616, wR2
=
=
R indices (all data)
= 0.1152, wR2

Extinction coefficient
Largest diff. peak and
hole
n/a
n/a
binding (PPB), atom based log P (Alog P98), polar surface area
(PSA), aqueous solubility, blood brain barrier (BBB) penetration,
cytochrome P450 2D6 (CYP2D6) enzyme inhibition and
hepatotoxicity. All of the compounds were predicted to be
efficiently absorbed in the human intestine. Specifically,
compounds 15a, 15b, 15c and 15e bearing the vanillin moiety
showed promising logarithm of the molar solubility value of -
5.403, -4.913, -5.87, -6.183 and -5.854, respectively. On the other
hand, there was no inhibition on cytochrome P450 which means
that these compounds can readily undergo oxidation and
hydroxylation in the first phase of metabolism. All the
compounds were predicted to exhibit ≥90% of plasma-protein
binding. This result coincides with the metabolites studies of
monocarbonyl curcumin analogues reported by Snyder and co-
workers which suggested that the protein binding may serve to
protect the curcumin analogues from full metabolism in vivo
while allowing it to exert a pharmacological effect by means of
slow drug release [42]. Only compounds 15a and 15b were
predicted not to exhibit very high BBB. Notably, all compounds
displayed non-hepatotoxic profile. The data are summarized in
Table 5.
-3
-3
0.171 and -0.166 e.Å
0.211 and -0.188 e.Å
Table 4
Comparison of cDOCKER interaction energy between 15a with
target enzyme inhibitors
cDOCKER interaction energy (kcal/mol)
Enzyme
15a
co-crystallized ligand*
-42.63
-38.45
-45.25
-46.90
-39.46
-45.75
-40.62
-77.52
-74.55
-80.90
IKK-β
p38α
P38β
ERK2
JNK2
*Retrieved from their PDB crystal structure
On the other hand, the ADMET properties of the synthesized
compounds were calculated by the standard descriptors protocol
implemented in Discovery Studio 3.1 to predict the
pharmacokinetic of the compounds when they are administered
and pass through the body. The parameters included in the
analysis were human intestinal absorption (HIA), plasma-protein
Table 5.
ADMET profile prediction of selected compounds
ADMET parameter
Blood Brain
Cytochrome
P450 2D6
Plasma Protein
Binding (PPB)
Compd
Human Intestinal Absorption
Aqueous Solubility
Barrier (BBB)
Penetration
Hepatotoxicity
(CYP2D6)
PSAa
ALogP98b Levelc
Log(Sw)d
-6.502
-6.224
-5.403
-4.913
-5.870
-6.183
-5.854
Levele
LogBBf
1.018
0.970
0.463
0.205
0.760
0.841
0.822
Levelg
Predictionh
Predictioni
Predictionj
35.160
29.855
55.976
67.861
47.046
47.046
50.398
5.590
5.164
4.862
4.637
5.365
5.627
5.738
0
0
0
0
0
0
0
1
1
2
2
2
1
2
0
0
1
1
0
0
0
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8b
8c
15a
15b
15c
15d
15e
a Polar surface area (PSA) (>150: very low absorption).
b Atom-based log P (Alog P98) (≤ 2.0 or P ≥ 0: very low absorption).
c Level of human intestinal absorption prediction; 0 (good), 1 (moderate), 2 (poor), 3 (very poor).
d The based 10logarithm of the molar solubility log (Sw) (25oC, pH = 7.0) (acceptable drug-like compounds:ꢀ6 < log(Sw)≤ 0).
e Level of aqueous solubility prediction; 0 (extremely low), 1 (very low), 2 (low), 3 (good), 4 (optimal), 5 (too soluble), 6 (warning: molecules with one or more
unknown Alog P calculation).
f Very high penetrants (log BBP ≥7).
g Level blood brain barrier penetration prediction; 0 (very high penetrate), 1 (high), 2 (medium), 3 (low), 4 (undefined).
h Prediction Plasma-protein binding (0: <90%; 1≥90%;).
I Prediction cytochrome P450 2D6 enzyme inhibition (0: non-inhibitor; 1: inhibitor).
j Prediction hepatotoxicity (0: non-toxic; 1: toxic).
A
B
Figure 5. Predicted binding poses retrieved from docking simulation of 15a in 1KKβ (A) and p38α (B) putative binding sites. The atom colouring for the
compound is in the following: carbons in blue, oxygen in red, nitrogen in blue, and hydrogen in white. The green line indicates hydrogen-bonding interaction
while the orange line indicates electrostatic interaction, with distance indicated in angstroms, Å.

24. Liu Z., Sun Y., Ren L., Huang Y., Cai Y., Weng Q., Shen X., Li X.,
Liang G., Wang Y., BMC Cancer, 2013, 13, 494.
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Limtrakul P., Badmaev V., Aggarwal B.B., Carcinogenesis, 2007, 28,
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26. Yang J.Y., Zhang L.J., Zhao S.Q., Yuan D., Lian G.N., Wang X.X.,
Zhang H.T., Wang L.H., Wu C.F., Neurosci. Res., 2010, 68, 451.
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Ahmad S, Tham C., Shaari K., Lajis N., Molecules, 2014, 19, 16058.
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K., Lam K.W., Ahmad S., Tham C.L., Shaari K., Lajis N.H.,
MedChemComm, 2015, 6, 1069.
In summary, we have successfully determined the effects of
unsymmetrical monocarbonyl curcumin analogues on PGE2
secretion level in LPS-induced murine and human macrophages.
Structural features revealed that presence of furanyl ring and
vanillin moiety in compounds significantly enhanced the PGE2
inhibition in both macrophages cells. Detailed binding mode
from docking simulations revealed that compounds 8b, 8c, 15a
and 15b could only form only one hydrogen bond in the COX-2
active site. This fully justifies the reason for their low inhibitory
activity in the assay. However, further studies are required to
identify the real molecular target(s) and signaling pathway that
might contribute to the anti-inflammatory properties of the
analogues.
29. Chimni S.S., Mahajan D., Tetrahedron, 2005, 61, 5019.
30. 1.
(a) Representative synthetic procedures and spectral
characterizations. General procedure for the preparation of
unsymmetrical monocarbonyl curcumin analogues. Pyrolidine in EtOH
(30%, 10mL) was added to a stirred solution of respective benzaldehyde
(10 mmol) and cyclohexanone (50 mmol) in absolute EtOH (30mL). The
yellowish solution was then subjected to column chromatography with n-
hexane/EtOAc (9:1) to afford 2-benzylcyclohexanone. Respective
Acknowledgement
This work was financially supported by ScienceFund (02-01-
02-SF00665), Ministry of Science, Technology & Innovation,
Malaysia and FRGS (FRGS/2/2014/ST01/UKM/02/3), Ministry
of High Education, Malaysia. Authors also thank University
Kebangsaan Malaysia for the funds provided under the Research
University Grant UKM-DIP-2014-16.
benzaldehyde (10 mmol) was added to
a stirred solution of 2-
benzylcyclohexanone (10 mmol) in the presence of NaOH (1M) at room
temperature. The yellow precipitate obtained was filtered and rinsed with
distilled water. The precipitate was purified by flash chromatography
with n-hexane/EtOAc (9:1) and recrystallized from absolute ethanol.
(2E,6E)-2-(2,3-dimethoxybenzylidene)-6-(4-methylbenzylidene)
cyclohexanone (8b). Yellow amorphous (yield 70%), mp: 130-131°C; 1H
NMR (500 MHz, CDCl3) δ 7.79 (d, J = 12.1 Hz, 1H), 7.39 (t, J = 13.1
Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 7.14 (dd, J = 8.4, 1.6 Hz, 2H), 7.05 (d,
J = 1.6 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 3.95 (s, 1H), 3.94 (s, 1H), 3.15
– 2.92 (m, 114H), 2.41 (s, 2H), 1.83 (dt, J = 12.4, 6.4 Hz, 55H). 13C
NMR (126 MHz, CDCl3) δ 190.23, 148.72, 138.77, 136.93, 136.84,
135.51, 134.55, 133.26, 130.83, 130.53, 129.13, 123.96, 113.81, 110.97,
55.95, 28.52, 23.04, 21.38. ESI-HRMS: (C23H24O3) calc. [M+H]
349.1725, found 349.1759. (2E,6E)-2-(4-methylbenzylidene)-6-((5-
methylfuran-2-yl) methylene)cyclohexanone (8c). Brownish crystal
(yields 55%), mp: 140-141°C; 1H NMR (500 MHz, CDCl3) δ 7.79 (s,
1H), 7.55 (d, J = 1.9 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 7.9
Hz, 2H), 6.61 (d, J = 3.2 Hz, 1H), 6.16 (d, J = 3.0 Hz, 1H), 3.00 (t, J =
5.4 Hz, 2H), 2.96 – 2.89 (m, 2H), 1.85 (dt, J = 12.5, 6.3 Hz, 2H). 13C
NMR (126 MHz, CDCl3) δ 191.18, 155.55, 151.50, 138.95, 136.33,
135.58, 133.32, 131.53, 130.40, 129.09, 123.94, 117.81, 108.98, 28.29,
28.13, 22.43, 21.38, 14.03. ESI-HRMS: (C20H20O2) calc. [M+H]
293.1463, found 293.1497. (2E,6E)-2-(4-methoxybenzylidene)-6-(3-
methoxy-4-hydroxybenzylidene) cyclohexanone (15a). Yellow crystal
(yields 35%), mp:155-156°C; 1H NMR (600 MHz, CDCl3) δ 7.79 (s, 1H),
7.77 (s, 1H), 7.48 (dd, J = 10.7, 7.9 Hz, 2H), 7.10 (dd, J = 8.3, 1.7 Hz,
1H), 7.02 (d, J = 1.8 Hz, 1H), 6.98 – 6.94 (m, 3H), 5.88 (s, 1H), 3.95 (s,
3H), 3.87 (s, 3H), 3.02 – 2.91 (m, 5H), 1.88 – 1.78 (m, 2H). 13C NMR
(151 MHz, CDCl3) δ 190.19, 159.95, 146.36, 136.90, 136.61, 134.33,
132.25, 128.75, 128.58, 124.43, 114.42, 113.92, 113.25, 55.97, 55.34,
28.54, 23.05. ESI-HRMS: (C28H32O6) calc. [M+H] 351.1518, found
References and notes
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351.1552.
(2E,6E)-2-(4-methylbenzylidene)-6-(3-methoxy-4-
hydroxybenzylidene) cyclohexanone (15c). Yellow crystal (yields
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1
55%), mp: 151-153°C; H NMR (600 MHz, CDCl3) δ 7.80 (s, 1H), 7.77
(s, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.11 (dd, J =
8.3, 1.8 Hz, 1H), 7.02 (d, J = 1.8 Hz, 1H), 7.01 – 6.96 (m, 1H), 5.86 (s,
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min. The cell viability of cultured cells used in the assay was always
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Type Culture Collection (ATCC) (Manassas, VA, United States). U937
mononuclear cell line was grown in the Rosewell Park Memorial
Institute-1640 (RPMI-1640) culture media, enriched with 10% (v/v)
foetal bovine serum (FBS) and 1% (v/v) penicillin G/streptomycin in 96
wells were maintained at 37°C humidified atmosphere with 5% CO2.
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Prior to treatment with the tested compounds, U937 cells (5
cells/well) were incubated with phorbol 12-myristate 13-acetate (PMA)
at concentration 200 nM for 24 hours to allow differentiation from
x
104

monocytes to macrophage-like phenotypes. Subsequently, cells were
incubated with serum free media overnight for recovery phase. Cell
Stimulation and Treatment. RAW264.7 and U937 (5 × 105 cells/well)
were seeded into a tissue culture grade 96-well plate and incubated for 24
h at 37°C, 5% CO2 for cell attachment. The attached cells were stimulated
in 100 U/mL of recombinant IFN-γ and 2µg/mL of LPS with or without
presence of test compound at a final volume of 100µL/well. DMSO was
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placed in calculated positions, allowing them to ride on their parent C
atom with Uiso(H) = xUeq(C) where, x = 1.5 for methyl; 1.2 for non-
methyl groups, except the hydrogen atoms attached to oxygen atoms
were located from Fourier maps and refined isotropically. A summary of
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reduction
of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
using Discovery Studio 3.1 (Accelrys, San Diego, USA) on an Intel® ™
2
bromide (MTT, 5 mg/mL) to formazan salts. After treatment, the
supernatant of each wells were removed followed by addition of 20 µL of
MTT reagents into each well. The mixture of culture media and MTT
were removed after incubated in 37°C for 4 h and the formazan salts were
dissolved by adding 100% DMSO. The absorbance was then measured at
570 nm on a SpectraMax Plus microplate reader (Molecular Devices Inc.,
Sunnyvale, CA, USA) at room temperature. Determination of PGE2. The
cell culture supernatants were collected and analyzed for PGE2 secretion
using PGE2 EIA kits (Cayman Chemical, Ann Arbor, MI, USA). The
protocols provided by the manufacturers were followed to the detail. The
data was obtained using a SpectraMax Plus microplate reader (Molecular
Device, Sunnyvale, CA, USA). The concentration of PGE2 for each
sample was calculated from their respective standard curves.
Cyclooxygenase assay. The COX-1 and COX-2 inhibition of test
compounds were analyzed using COX (ovine/human) inhibitor screening
assay kit (Cayman Chemical, Ann Arbor, MI, USA). The protocols
provided by the manufacturers were followed to the detail. The data was
obtained using a SpectraMax Plus microplate reader (Molecular Device,
Sunnyvale, CA, USA). The concentration of PGEs for each sample was
calculated from their respective standard curves (c) X-ray structure
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performed on Bruker D-QUEST diffractometer using graphite-
monochromated Mo-Kα radiation (λ= 0.71073 Å). Intensity data was
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orientation matrix were determined by the least-squares fit of 25
reflections. Intensity data was collected for Lorentz and polarization
effects. Empirical absorption correction was performed using multiscan.
The structure was solved by direct methods and least-squares refinement
of the structure was carried out by the SHELXL-97 program. All the non-
hydrogen atoms were refined anisotropically. The hydrogen atoms were
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