Inhibition of prostaglandin E2 production by synthetic minor prenylated chalcones and flavonoids: Synthesis, biological activity, crystal structure, and in silico evaluation

Article abstract of DOI:10.1016/j.bmcl.2014.06.061

The discovery of potent inhibitors of prostaglandin E₂ (PGE ₂) synthesis in recent years has been proven to be an important game changer in pharmaceutical industry. It is known that excessive production of PGE₂ triggers a

Full text of DOI:10.1016/j.bmcl.2014.06.061

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3826–3834
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of prostaglandin E2 production by synthetic minor
prenylated chalcones and flavonoids: Synthesis, biological activity,
crystal structure, and in silico evaluation
Kamal Rullah a,f, Mohd Fadhlizil Fasihi Mohd Aluwi a, Bohari M. Yamin b, Mohd Nazri Abdul Bahari c,
Leong Sze Wei d, Syahida Ahmad c, Faridah Abas d, Nor Hadiani Ismail e, Ibrahim Jantan a, Lam Kok Wai a,
⇑
a Drugs and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia
b School of Chemical Sciences and Food Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
c Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
d Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
e Faculty of Science, Universiti Teknologi MARA, 50400 Shah Alam, Selangor, Malaysia
f Sekolah Tinggi Ilmu Farmasi Riau, Universitas Riau, Kampus Bina Widya Km 12.5, Simpang baru-Pekanbaru, Indonesia
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery of potent inhibitors of prostaglandin E2 (PGE2) synthesis in recent years has been proven to
be an important game changer in pharmaceutical industry. It is known that excessive production of PGE2
triggers a vast array of biological signals and physiological events that contributes to inflammatory dis-
eases such as rheumatoid arthritis, atherosclerosis, cancer, and pain. In this Letter, we report the synthe-
sis of a series of minor prenylated chalcones and flavonoids which was found to be significantly active in
suppressing the PGE2 production secreted by lipopolysaccharide-induced mouse macrophage cells (RAW
264.7). Among the compounds tested, 14b showed a dose-response inhibition of PGE2 production with an
Received 25 April 2014
Revised 11 June 2014
Accepted 20 June 2014
Available online 27 June 2014
Keywords:
Prenylated chalcone
Minor flavonoids
Single-crystal XRD
Prostaglandin E2
COX-2 inhibitor
ADMET prediction
IC50 value of 2.1 lM. The suppression upon PGE2 secretion was not due to cell death since 14b did not
reduce the cell viability in close proximity to the PGE2 inhibition concentration. The obtained atomic
coordinates for the single-crystal XRD of 14b was then applied in the docking simulation to determine
the potential important binding interactions with murine COX-2 and mPGES-1 putative binding sites.
Ó 2014 Elsevier Ltd. All rights reserved.
Prostaglandin E2 (PGE2), a product of the cyclooxygenase (COX)
pathway is well identified as the lipid mediator that contributes to
inflammatory diseases such as cancer, rheumatoid arthritis, ath-
erosclerosis, and pain.1 The conversion of arachidonic acid into
prostaglandin H2 (PGH2) is tightly regulated by cyclooxygenases
and subsequently transformed to PGE2 by either of the PGE
dom and possess many interesting biological activities. Addition-
ally, the presence of prenyl group in the flavonoid ring system
increases lipophilicity and bestows to the molecule a strong affin-
ity for biological membranes. It also improves the pharmacokinetic
profile of a compound leading to a broader range of interesting
pharmacological activities.7 As an example, xanthohumol (1), a
prenylated chalcone, was found to be active in a wide range of
anti-inflammatory activities.8,9 Furthermore, a prenylated flavo-
noid namely sophoraflavanone G (2) inhibited prostaglandin E2
(PGE2) production from lipopolysaccharide (LPS)-treated RAW cells
synthases including microsomal prostaglandin
E synthase-1
(mPGES-1), microsomal prostaglandin E synthase-2 (mPGES-2),
and cytosolic prostaglandin E synthase (cPGES). Both cPGES and
mPGES-2 are constitutively expressed in various organs/tissues,
whilst mPGES-1, like COX-2, is up-regulated in response to various
inflammatory stimuli (Fig. 1).2–5
by down-regulating the COX-2 expression at 1–50
lM (IC50 =
2.7
l
M).10 Some of the aurones such as sulfuretin (3) derivatives
Dihydrochalcones, prenylated chalcones, prenylated flavones,
aurones, and prenylated aurones are categorised into the group
of minor flavonoids.6 These compounds have a unique and defined
chemical structure, which are commonly found in the plant king-
have been synthesized and displayed remarkable inhibition results
on PGE2 production in LPS-induced RAW 264.7 cells to reveal the
SAR of the compounds.11 On the other hand, artocarpaurone (4),
a newly isolated prenylated aurone from the methanol extract of
Artocarpus altilis was found to exhibit NO radical scavenging activ-
ity (Fig. 2).12
⇑
Corresponding author. Tel.: +60 3 9289 7031.
E-mail address: david_lam_98@yahoo.com (L.K. Wai).
http://dx.doi.org/10.1016/j.bmcl.2014.06.061
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

K. Rullah et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3826–3834
3827
chromatography and recrystallised from EtOH to afford the corre-
sponding aurones (8a–c) in low yields (22–27%). To produce the
corresponding flavonols (9a–c), the 20-hydroxychalcone deriva-
tives were cyclised via a modified Algar–Flynn–Oyamada (AFO)
reaction, using an aqueous hydrogen peroxide (30%) and NaOH
(40%) in EtOH.15,16 The conversion of the 20-hydroxychalcone
derivatives to the flavonols were successful using the conventional
AFO reaction although the yields varied depending on the type of
substituents in the aryl aldehydes used after flash chromatography
and recrystallisation from EtOH. AFO reaction is a convenient
method used in the synthesis of dihydroflavonols, aurones, and
other flavonoid compounds.17 In spite of this, we did not manage
to isolate 9c derived from chalcone bearing electron withdrawing
dichloro groups; the product yield was not determined.
N
O
P
Phospholipid
O
Hydrophilic group
O
O
O
O
O
O
Hydrophobic group
Cell membrane
Phospolipases A2
O
Several strategies were employed to introduce the prenyl group
into the corresponding chalcones, flavonols, and aurones in this
Letter. Addition of the prenyl group to the 3-hydroxyflavones
was achieved by treating 9a–b with 1-chloro-3-methyl-2-butene
in the presence of K2CO3 in acetone under reflux condition. The
desired products, 10a–b were obtained in moderate to high yields
(62–80%). Insertion of the geranyl group to the 3-position of the 3-
hydroxyflavones successfully produced compounds 10c–d in mod-
erate yields (42–62%). Attempt to prenylate the ring system of 5 by
direct alkylation using KOH,18 however failed to yield any product.
To facilitate the migration, a [3,3]-sigmatropic rearrangement
approach was then adopted by refluxing compound 11 in N,N-
dimethylaniline at 200 °C under nitrogen atmosphere to give the
desired product in moderate yield. Based on the NMR result19a
(Supplementary data), the prenyl group effectively migrated to
the para position to give the C-prenylated product, 12 with 36%
of yield after flash chromatography (Scheme 2). Replacing 3,3-
dimethylallyl bromide with 1-chloro-3-methyl-2-butene, how-
ever, resulted in a low yield of 12. It is important to mention that
11 was used directly in the subsequent reaction after the removal
of K2CO3 by filtration which could resulted in the presence of 3,3-
dimethylallyl bromide or 1-chloro-3-methyl-2-butene residue in
the reaction mixture. It is possible that 3,3-dimethylallyl bromide
residue might participate in the [3,3]-sigmatropic rearrangement
reaction to form 12 based on our observation. On the other hand,
we failed to introduce the prenyl group to the ortho position of
compound 5 ring system by [1,3]-sigmatropic rearrangement by
using montmorillonite K10 as a catalyst in CH2Cl2 at 0 °C, even-
though a similar method was reported previously by Sugamoto
et al.20
HO
Arachidonic acid
COX-2
COX-1
TXA2
PGI2
PGH2
cPGES
PGD2
PGF2a
mPGES-1
mPGES-2
PGE2
Figure 1. Biochemical conversion of PGE2 from arachidonic acid.
OH
HO
OH
HO
OH
HO
O
OCH3O
1
OH
O
O
2
HO
OH
HO
O
HO
O
OH
O
O
3
4
The Claisen–Schmidt condensation between 12 and aryl alde-
hyde derivatives (6) was performed in the presence of KOH base
in EtOH under ultrasound irradiation at 40 °C to give the corre-
sponding C-prenylated chalcones, (14a–e) in the range of low to
high yields (12–80%). It is interesting to note that the cyclisation
of 14a–b in the presence of Hg(OAc)2 in DMSO gave the desired
C-prenylated aurones in moderate yields (33–44%) without affect-
ing the stability of the prenyl group (Scheme 3). Only a few litera-
tures have reported the synthesis of C-prenylated aurones up to
date.21,22 An alternative method is proposed here for the synthesis
of C-prenylated aurones. On the other hand, two new O-prenylated
aurone derivatives, 17a–b were obtained in reasonable yields as
shown in Scheme 4. Besides, we did not manage to isolate 22a–
b, the C-prenylated flavonols derivatives via the AFO reaction
(Scheme S2, Supplementary data).
Preliminary screening results on PGE2 production in
LPS-induced RAW 264.7 cells showed that compounds 14a–e
demonstrated good PGE2 inhibitory activity have prompted us to
introduce bulky phenyl group to both R1 and R2 as shown in
Scheme 5. A Suzuki coupling reaction was adopted utilising a sim-
ple formation of carbon-carbon single bond to give the new preny-
lated chalcone derivatives, 21a–b.23 The reactants (20a–b) were
Figure 2. Selected minor flavonoids as potential anti-inflammatory agents.
In the present Letter, we have successfully synthesized 20-
hydroxychalcones, prenylated chalcones, flavonols, prenylated
flavonols, aurones, and prenylated aurones. The preparation of
the 20-hydroxychalcone derivatives were carried out via the Clais-
en–Schmidt condensation (Scheme 1). Initially, 20-hydroxy-40-
methoxy-acetophenone (5) was reacted with the commercially
available appropriate aryl aldehyde derivatives (6) in the presence
of KOH in EtOH to afford the corresponding crude 20-hydroxychal-
cone derivatives. Subsequent neutralization with HCl yielded the
desired products with average yields of 55–98%. The yield of the
products could be further improved when the reaction was carried
out under ultrasound irradiation at 40 °C.13 On the other hand, the
aryl aldehydes with hydroxyl substituent were first converted to
the corresponding tetrahydropyranyl ethers to afford the final
products with an improved overall percentage of yields
(Scheme S1, Supplementary data). Next, the aurones were pre-
pared via oxidative cyclisation method by reacting the 20-hydroxy-
chalcone derivatives (7a–g) with mercury (II) acetate in DMSO
under reflux condition.14 All products were purified using flash

3828
K. Rullah et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3826–3834
O
b
O
H3CO
R1
8a
8b
8c
R1 = H; R2 = Methyl (27%)
R1 = Methoxyl; R2 = Methoxyl (22%)
R1 = Cl; R2 = Cl (25%)
R2
O
OH
O
OH
O
R1
R2
R1
R2
H
a
CH3
+
H3CO
H3CO
5
6
7a R1 = H; R2 = Methyl (97%)
7b
R
1 = Methoxyl; R2 = Methoxyl (95%)
O
O
O
O
7c R1 = Cl; R2 = Cl (96%)
7d R1 = H; R2 = OTHP (63%)
7e R1 = H; R2 = OH (98%)
OH
c
d
f
R1
R2
R1
R2
H3CO
H3CO
O
7f R1 = OTHP; R2 = OTHP (55%)
7g
f
R1 = OH; R2 = OH (95%)
10a
10b
R1 = H; R2 = Methyl (80%)
R1 = Methoxyl; R2 = Methoxyl (62%)
9a R1 = H; R2 = Methyl (43%)
9b R1 = Methoxyl; R2 = Methoxyl (31%)
9c R1 = Cl; R2 = Cl (n.d)@
O
O
e
R1
H3CO
O
R2
10c R1 = H; R2 = Methyl (62%)
10d R1 = Methoxyl; R2 = Methoxyl (42%)
Scheme 1. Reagents and conditions of synthesis: (a) KOH 40%, EtOH, (i) sonicate 1 h at 40 °C (ii) stir at rt overnight; (b) Hg(OAc)2, DMSO, reflux 160 °C, 6 h; (c) KOH 40%, H2O2,
EtOH, rt 3–4 h; (d) 1-Chloro-3-methyl-2-butene, K2CO3, Acetone, reflux 75 °C, 20–24 h; (e) Geranyl bromide, K2CO3, Acetone, reflux 75 °C, 20–24 h (f) TsOH, MeOH, stir at rt
2 h. @Not determined due to purification problem.
R2
R2
OH
O
O
O
OH
O
a
R1
CH3
O
CH3
CH3
a
b
R1
O
H3CO
H3CO
H3CO
O
5
11 (92%)
12 (36%)
O
16a
R
1 = OH; R2 = Methyl
17a
R1 = OPrenyl; R2 = Methyl (33%)
16b R1 = H; R2 = OH
17b R1 = H; R2 = OPrenyl (48%)
Scheme 2. Reagents and conditions of synthesis: (a) 3,3-dimethylallyl bromide,
K2CO3, acetone, reflux 75 °C, 20 h; (b) DMA, reflux 200 °C under N2 atmosphere.
Scheme 4. Reagents and conditions of synthesis: (a) 1-Chloro-3-methyl-2-butene,
K2CO3, acetone, reflux 75 °C, 20–24 h.
obtained in reasonable yields (70% and 79%, respectively) accord-
ing to the modified method using the Suzuki coupling reaction cat-
alysed by in situ generated palladium nanoparticles in PEG-400
solvent under aerobic condition (Scheme 5). This oxygen-pro-
moted ligand-free Suzuki coupling reaction was previously
reported by Han et al.. The reaction is highly efficient for coupling
aryl chlorides with phenylboronic acid in a short time under mild
condition.24 The Claisen–Schmidt condensation between 20a–b
and 12 yielded the desired products 21a–b in moderate yields
(34% and 48%, respectively) (Scheme 6).
OH
B
O
R1
a
OH
H
R2
Br
R2
19
20a R1 = 3-Formyl; R2 = 4'-Fluoro (70%)
20b
18
R1 = 4-Formyl; R2 = 4'-Fluoro (79%)
Scheme 5. Reagents and conditions of synthesis: (a) 4-fluorophenylboronic acid,
Pd(OAc)2, PEG 400, K2CO3, 60 °C 20 h.
The PGE2 inhibitory activity of the synthesized compounds was
evaluated using the mouse macrophage (RAW 264.7) cell line,
stimulated by lipopolysaccharide (LPS) at the concentration of
of the synthesized compounds exhibited moderate to strong
PGE2 production inhibition (41–90%) except for compounds 7a,
15a, and 21b, which displayed poor activity. Compound 7a was
50 l
M.19b The results summarised in Table 1, showed that most
O
O
OH
O
OH
O
R1
R2
R1
R2
14a R1 = H; R2 = Methyl (65%)
14b R1 = Methoxyl; R2 = Methoxyl (60%)
14c R1 = H; R2 = Br (55%)
14d
14e
H
b
CH3
a
O
H3CO
H3CO
H3CO
R1
12
6
R2
15a R1 = H; R2 = Methyl (33%)
15b
R1 = Methoxyl; R2 = Methoxyl (44%)
R1 = OTHP; R2 = OTHP (12%)
1 = OH; R2 = OH (84%)
c
R
Scheme 3. Reagents and conditions of synthesis: (a) KOH 40%, EtOH, (i) sonicate 1 h at 40 °C (ii) stir at rt overnight; (b) Hg(OAc)2, DMSO, reflux 160 °C, 6 h; (c) TsOH, MeOH,
stir at rt 2 h.

K. Rullah et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3826–3834
OH
3829
O
OH
O
R1
CH3
H3CO
R1
a
R2
H3CO
R2
12
20a-b
21a R1 = Fluorophenyl; R2 = H (34%)
21b R1 = H; R2 = Fluorophenyl (48%)
Scheme 6. Reagents and conditions of synthesis: (a) KOH 40%, EtOH, (i) sonicate 1 h at 40 °C (ii) stir at rt overnight.
Table 2
Table 1
Crystal and structure refinement parameters of compound 14b
In vitro PGE2 production inhibitory activities of synthesized compounds in LPS-
induced RAW 267.4 cells
Identification code
Compound Percentage (%) inhibition of PGE2 at
IC50
M)
Viability
(%)
Empirical formula
Formula weight
C23H26O5
382.44
50
lM
(
l
7a*
7b
8a
8b
9a
9b
10a
10b
14a
14b#
14c
14e
15a⁄
15b
17a
17b
21a
21b*
NS-3981
na
75
78
76
70
80
41
58
64
90
70
61
na
83
43
77
63
na
97
97
Temperature
Wavelength
Crystal system, space group
Unit cell dimensions
296(2) K
0.71073 Å
Triclinic, Pı
¯
a = 8.4547(13) Å
b = 13.816(2) Å
c = 18.621(3) Å
>100
>100
>100
78
82
62
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
a
= 97.590(5)°
b = 92.128(5)°
c
= 105.061(5)°
Volume
2076.1(5) Å3
2.1
Z, calculated density
Absorption coefficient
F(0 0 0)
Crystal size
Theta range for data collection
Limiting indices
Reflections collected/unique
Completeness to theta = 25.00
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2sigma(I)]
R indices (all data)
4, 1.224 Mg/m3
0.085 mmꢀ1
816
0.330 ꢁ 0.260 ꢁ 0.150 mm3
3.087–24.998°
ꢀ10 6 h 6 10, ꢀ16 6 k 6 16, ꢀ22 6 l 6 21
7281 [R(int) = 0.1322]
96.9%
Full-matrix least-squares on F2
7281/3/514
0.1
*
#
na is not active.
Most active compound.
NS-398 (N-(2-cyclohexyloxy)-4-nitromethanesulfonamide) was used as
reference compound.
1.119
R1 = 0.1308, wR2 = 0.3994
R1 = 0.1763, wR2 = 0.4348
0.018(4)
1
a
Extinction coefficient
Largest diff. peak and hole
0.508 and ꢀ0.363 e Åꢀ3
IC50 of the most active compound and the positive control, NS-398.
inactive against PGE2 production though 8a (aurone) and 9a (flavo-
nol) which are the cyclised products of the ,b-unsaturated chal-
a
cone were found to be active. Insertion of the prenyl group to
the 3-hydroxyflavone series was equally bad for the activity. On
the other hand, compound 14b (E)-3-(3,4-dimethoxyphenyl)-1-
(4-methoxy-3-(3-methylbut-2-enyl)phenyl)-prop-2-en-1-one),
the prenylated chalcone, significantly suppressed PGE2 production
with 90% of inhibition was found to be comparable to the result of
the positive inhibitor, NS-398. Further test showed that 14b sup-
pressed the production of PGE2 with an IC50 value of 2.1 lM. Based
on the preliminary results, the prenylated chalcones (14a–e) were
found to be highly potent against PGE2 synthesis. Currently, we are
synthesizing and optimising this series of compounds and a full
SAR study will be reported in the near future. Nevertheless, it is
critical to note that the position of fluorophenyl group of com-
pounds 21a and 21b was also decisive for the inhibitory activity.
Apparently, the presence of the fluorophenyl group at para position
resulted in the moderate inhibition as displayed by 21a while at
meta position, compound 21b did not demonstrate any inhibition.
This remarkable observation will definitely serve as an important
point in the future design of PGE2 synthesis inhibitors using the
template structure of 21a. The cytotoxic effect of the synthetic
compounds was also evaluated using the MTT assay to test
whether the inhibitory effect on the production of PGE2 was due
to cell cytotoxicity. Most of the synthesized compounds were
Figure 3. ORTEP diagram of the compound 14b with thermal ellipsoids drawn at
50% probability.
found to exhibit an IC50 value of 13.8 lM. Figure 5 shows the
dose–response of 14b towards the secretion of PGE2 and NO.
All 1H NMR, 13C NMR and ESI-HRMS spectra were consistent with
the assigned structures of the synthesized compounds (Supplemen-
tary data).19a The characterisation of the most active compound 14b
was conducted by a single crystal X-ray structural analysis.19c It was
difficult to obtain a good quality crystal but after several attempts of
recrystallisation, sufficient reflections for structural solution with
R1 value of 13% was finally achieved. The compound crystallized
non-cytotoxic to RAW 264.7 cells at 50
lM concentration. Addi-
tionally, compound 14b was tested for the NO inhibition and was

3830
K. Rullah et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3826–3834
Table 3
Selected geometric parameter of compound 14b (Å, °)
C1–C2
C2–C3
C3–C4
C4–C5
C5–C6
C1–C6
C18–C19
C41–C42
O3–C7
1.371(10)
1.411(10)
1.368(11)
1.392(11)
1.431(10)
1.406(10)
1.321(11)
1.332(11)
1.268(9)
1.341(9)
1.351(9)
1.353(9)
1.350(9)
1.383(10)
1.388(11)
1.367(11)
1.401(10)
1.385(10)
1.392(10)
117.5(7)
118.0(6)
118.3(7)
120.5(6)
122.4(7)
O1–C3
O2–C5
O3–C7
O4–C13
O5–C14
C6–C7
C7–C8
C8–C9
C18–C19
O6–C26
O7–C28
O8–C36
O9–C37
O10–C30
C29–C30
C31–C32
C32–C33
1.351(6)
1.341(10)
1.269(10)
1.361(10)
1.353(10)
1.446(10)
1.473(11)
1.327(11)
1.319(12)
1.356(10)
1.329(10)
1.375(9)
1.354(9)
1.258(9)
1.457(10)
1.318(10)
1.466(10)
O2–C5
O1–C3
O5–C14
O4–C13
C10–C11
C11–C12
C12–C13
C13–C14
C14–C15
C10–C15
C13–O4–C22
C14–O5–C23
C3–O1–C21
C6–C7–C8
C7–C8–C9
C41–C42
C8–C9–C10
C29–C30–C31
C30–C31–C32
1.333(11)
126.8(7)
120.2(6)
122.9(7)
C31–C32–C33
126.8(7)
in a triclinic system with space group Pı, a = 8.4547(13) Å,
¯
b = 13.816(2) Å, c = 18.621(3) Å,
c
a = 97.590(5)°, b = 92.128(5)°,
= 105.061(5)°, Z = 4 and V = 2076.1(5) Å3. The crystal system and
refinement data are shown in Table 2. The asymmetric unit consists
of two independent molecules (Fig. 3). The molecules are apparently
planar. However, the dihedral angles between 1-(2-hydroxy-4-
methoxy-5-(3-methylbut-2-enyl)phenyl)propanyl-1-one, O1/O2/
O3/(C1-C9)/C20/C21 and 1,2-dimethoxy-4-methylbenzene, O4/
O5/(C9-C15)/C22/C23 fragments in the first molecule, and O6/O7/
O10/(C24-C32)/C43/C44 and O8/O9/(C32-C38)/C45/ C46 in the sec-
ond molecule are 7.32(19)° and 6.61(18)°, respectively. All the frag-
ments are planar with maximum deviation of 0.072(6) Å for O10
atom from the least square plane of the O6/O7/O10/(C24-C32)/
C43/C44 fragment. Both molecules adopt trans configuration with
Figure 5. Effects of 14b on (a) PGE2 and (b) NO production in LPS-stimulated RAW
264.7 cells. The cells were co-incubated with LPS (5 g/mL) and different
concentrations of 14b ranging from 0.78 to 50 M. The supernatants were then
l
l
collected for the measurement of PGE2 and NO production using EIA kit and a Griess
reagent, respectively. 14b significantly inhibited PGE2 and NO levels in LPS
stimulated macrophages. 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 t-test (ns is not significant).
R
NH
VAL349
b
d
Hydrophilic
pocket 1
Hydrophobic
pocket 2
a
O
LEU352
NH2
R
+
N
6.5Å
H
H
2
ARG120
O
O
N
PHE518
2.3Å
O
6.0Å
R
NH
R
VAL116
1.8Å
+
NH2
NH2
O
O
Hydrophobic
pocket 1
H
LEU93
N
H
O
1.9Å
O-
ARG513
O
VAL89
Hydrophilic
pocket
GLU524
2
O
NH
R
R
R
O
ARG192
NH
R
c
NH2
HN
+HN
ARG127
4.0Å
ARG207
O
O
O
LEU193
O
HO
ARG74
Figure 4. Predicted binding poses retrieved from flexible docking of 14b in (a) the murine COX-2 and (b) murine mPGES-1 (homology model) putative binding sites. The atom
colouring for the compounds is the following: carbons in grey, oxygen in red, nitrogen in blue, and hydrogen in white. (b) and (d) represent the 2D diagram of binding
interactions between 14b with the active site of murine COX-2 and murine mPGES-1(homology model), respectively. Hydrogen atoms have been omitted in the 2D diagram
for clarity. The orange line indicates the p-interaction, while the green line indicate the hydrogen-bonding interactions, with distance indicated in angstroms, Å.

K. Rullah et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3826–3834
3831
respect to the position of the 1-(2-hydroxy-4-methoxy-5-(3-meth-
ylbut-2-enyl)phenyl)-propanyl-1-one and 1,2-dimethoxy-4-ben-
zene groups across their C8-C9 and C31-C32 double bonds of
1.321(10) and 1.318(10) Å, respectively. Other bond lengths and
angles are in normal ranges as shown in Table 3. There are O2-
H2A. . .O3 and O7-H7A. . .O10 intramolecular hydrogen bonds in
the molecules. The structure is stabilised by C23-H23A. . .O4
(1 ꢀ x, 2 ꢀ y, ꢀz; D. . .A = 3.469(12) Å and D. . .H. . .A = 165°) and
C46-H46A. . .O8 (1 ꢀ x, 1 ꢀ y, 1 ꢀ z; D-H= 3.427(11) Å and D-
H. . .A = 162°) intermolecular hydrogen bonds. The crystallographic
data for the structural analysis were deposited with the Cambridge
Crystallographic Data Centre No 997318.
To elucidate the possible binding interactions of compound 14b,
the protein crystal structures of murine COX-2 (6COX: 2.8 Å) and
human mPGES-1 (4AL0: 1.2 Å) were retrieved from BrookhavenPro-
tein Data Bank. The obtained single crystal-XRD atomic coordinates
for 14b was then employed in the docking study (see Fig. 3). Since
the complete crystallised three-dimensional structure of murine
mPGES-1 is currently not available, we attempted to build a homol-
ogy model from the human protein crystal structure mPGES-1
(4AL0: 1.2 Å).25 We selected the non-conserved region in human
mPGES-1 as the putative binding site for compound 14b based on
the fact that MK-886 inhibitor selectively inhibit human mPGES-1
activity while weakly against murine mPGES-1. The murine
mPGES-1 homology model built was used as a molecular target in
the docking experiment. Furtheranalysis on the sequence alignment
revealed that human and murine mPGES-1 share 77.1% of their
sequence identity and have 84.3% of sequence similarity, which
supports the use of 4AL0 as a suitable template for building the mur-
ine homology model, and these proteins potentially share a con-
served common folding (Fig. S1, Supplementary Data). The quality
of the refined homology model was then assessed by Ramachandran
plots and PROCHECK analyses, which revealed that most of the res-
idues were in favored regions: 93.7% most favored, 5.3% additional
allowed, 0.3% generously allowed, and 0.8% disallowed. All the crit-
ical points were located away from the putative binding site (Figs. S2
& S3 and Table S1, Supplementary Data).
In order to check the CHARMm flexible docking efficiency in
reproducing the X-ray structure, the co-crystallised ligand of the
pdb file (6COX) was extracted and redocked using the default set-
tings (Fig. S4, Supplementary Data). The RMSD between the top
ranked pose and the crystal structure was found consistent (RMSD
value 0.65 Å). According to the docking results, compound 14b par-
ticipates in a number of important binding interactions in the
active site of murine COX-2 than murine mPGES-1 (homology
model) (Fig. 4). Near the surface active site of murine COX-2, com-
pound 14b interacts with the side chains of Arg120, Arg513, and
Glu524. The docking results showed that both the aromatic rings
of 14b form weak cation-p interaction with the protonated
amino-side chain of residue Arg120, which is located in the hydro-
philic pocket 1 by about 6.5 Å and 6.0 Å (aromatic ring—+H2N–),
respectively. In the hydrophilic pocket 2, the hydroxyl group of
14b engaged with Arg513 and Glu524 residues through H-bonding
molecular interaction within distances of 1.8 Å and 1.9 Å (HO—+H2-
N– and HO—ꢀOOC–), respectively. Such interactions are almost
essential for the COX-2 inhibitory activity based on the binding
interactions displayed by the ligand co-crystal of SC-558, an ana-
logue of celecoxib, in the COX-2 active site.26,27 Nonetheless, resi-
due Arg513 has been identified as the key contributor to COX-2
specificity while in COX-1, this residue is replaced by a histidine
residue. The presence of Arg513 residue results in a stable positive
charge group being placed at the center of the pocket.28 It is also
known that, this residue is a crucial requirement for the time-
dependent inhibition of COX-2. This arginine residue appears to
Table 4
ADMET profile prediction of the synthesized compounds
Compd
ADMET parameter
Human Intestinal
Absorption
Aqueous
Solubility
Blood Brain Barrier (BBB)
Penetration
Plasma Protein
Binding (PPB)
Cytochrome P450
2D6 (CYP2D6)
Hepatotoxicity
Predictionj
PSAa
AlogP98b
Levelc
Log (Sw)d
Levele
LogBBf
Levelg
Predictionh
Predictioni
7a
7b
8a
8b
9a
9b
10a
10b
14a
14b#
14c
14e
15a
15b
17a
17b
21a
21b
47.046
64.906
35.160
53.021
55.976
73.836
44.091
61.951
47.046
64.906
47.046
88.677
35.160
53.021
35.160
35.160
47.046
47.046
3.929
3.410
3.606
3.087
3.068
2.549
4.759
4.240
5.786
5.267
6.048
4.816
5.462
4.943
5.071
4.585
7.024
7.024
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
3
3
ꢀ4.195
ꢀ3.821
ꢀ4.829
ꢀ4.301
ꢀ4.031
ꢀ3.664
ꢀ5.789
ꢀ5.188
ꢀ5.781
ꢀ5.313
ꢀ6.095
ꢀ4.503
ꢀ6.433
ꢀ5.811
ꢀ5.989
ꢀ5.490
ꢀ6.855
ꢀ6.915
2
3
2
2
2
3
2
2
2
2
1
2
1
2
2
2
1
1
0.316
ꢀ0.127
0.404
ꢀ0.039
ꢀ0.091
ꢀ0.534
0.619
0.176
0.890
0.447
0.971
—
0.978
0.535
0.857
0.707
—
1
2
1
2
2
3
1
1
0
1
0
4
0
1
0
0
4
4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
0
1
1
1
1
1
1
0
0
1
1
—
#
a
b
c
Most active compound.
Polar surface area (PSA) (>150: very low absorption).
Atom-based logP (AlogP98) (6ꢀ2.0 or P7.0: very low absorption).
Level of human intestinal absorption prediction; 0 (good), 1 (moderate), 2 (poor), 3 (very poor).
d
e
The based 10logarithm of the molar solubility log(Sw) (25 °C, pH = 7.0) (acceptable drug-like compounds: ꢀ6 < log(Sw) 6 0).
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
AlogP calculation).
f
Very high penetrants (logBB P 0.7).
Level blood brain barrier penetration prediction; 0 (very high penetrate), 1 (high), 2 (medium), 3 (low), 4 (undefined).
Prediction Plasma-protein binding (0: <90%; 1: P90%;).
Prediction cytochrome P450 2D6 enzyme inhibition (0: non-inhibitor; 1: inhibitor).
g
h
i
j
Prediction hepatotoxicity (0: non-toxic; 1: toxic).

3832
K. Rullah et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3826–3834
6. Ninomiya, M.; Koketsu, M. In Natural Products; Ramawat, K. G., Mérillon, J.-M.,
Eds.; Springer: Berlin, Heidelberg, 2013; p 1867.
7. Botta, B.; Vitali, A.; Menendez, P.; Misiti, D.; Monache, G. D. Curr. Med. Chem.
2005, 12, 713.
8. Lee, I.-S.; Lim, J.; Gal, J.; Kang, J. C.; Kim, H. J.; Kang, B. Y.; Choi, H. J. Neurochem.
Int. 2011, 58, 153.
9. Cho, Y.-C.; Kim, H. J.; Kim, Y.-J.; Lee, K. Y.; Choi, H. J.; Lee, I.-S.; Kang, B. Y. Int.
Immunopharmacol. 2008, 8, 567.
10. Kim, D.; Chi, Y.; Son, K.; Chang, H.; Kim, J.; Kang, S.; Kim, H. Arch. Pharmacal Res.
2002, 25, 329.
11. Shin, S. Y.; Shin, M. C.; Shin, J.-S.; Lee, K.-T.; Lee, Y. S. Bioorg. Med. Chem. Lett.
2011, 21, 4520.
12. Huong, T. T.; Cuong, N. X.; Tram, L. H.; Quang, T. T.; Duong, L. V.; Nam, N. H.;
Dat, N. T.; Huong, P. T. T.; Diep, C. N.; Kiem, P. V.; Minh, C. V. J. Asian Nat. Prod.
Res. 2012, 14, 923.
13. Li, J.-T.; Yang, W.-Z.; Wang, S.-X.; Li, S.-H.; Li, T.-S. Ultrason. Sonochem. 2002, 9,
237.
14. Narsinghani, T.; Sharma, M.; Bhargav, S. Med. Chem. Res. 2013, 22, 4059.
15. Li, Z.; Ngojeh, G.; DeWitt, P.; Zheng, Z.; Chen, M.; Lainhart, B.; Li, V.; Felpo, P.
Tetrahedron Lett. 2008, 49, 7243.
16. Pandurangan, N.; Bose, C.; Banerji, A. Bioorg. Med. Chem. Lett. 2011, 21, 5328.
17. Bennett, M.; Burke, A. J.; Ivo O’Sullivan, W. Tetrahedron 1996, 52, 7163.
18. Diller, R. A.; Riepl, H. M.; Rose, O.; Frias, C.; Henze, G.; Prokop, A. Chem.
Biodivers. 2005, 2, 1331.
interact with the hydroxyl substituent of 14b. On the other hand,
the prenyl substituent of 14b is well-positioned in the hydrophobic
pocket 1, constituting of hydrophobic residues Val116, Leu93, and
Val89 in the COX-2 active site. The presence of this group could
possibly increase the ligand binding affinity and thus resulted in
higher inhibitory activity of 14b. However, 14b were predicted to
bind poorly to the murine mPGES-1 active site. The docking result
in Figure 4, showed that the aromatic ring of 14b could only form a
weak cation-p interaction with the protonated amino-side chain of
residue Arg192 of about 4.0 Å (aromatic-ring—+H2N–).
Pharmacokinetic is the study of what happens to drugs as they
are administered and pass through the body. Poor clinical safety
and toxicity contributes significantly to clinical failures in drug dis-
covery. The ADMET profile of the synthesized compounds was
assessed by the standard descriptors protocol implemented in Dis-
covery StudioÒ 3.1. The parameters included in the analysis were
human intestinal absorption (HIA), aqueous solubility, blood brain
barrier (BBB) penetration, cytochrome P450 2D6 (CYP2D6) enzyme
inhibition, hepatotoxicity, plasma-protein binding (PPB), atom-
based logP (AlogP98), and polar surface area (PSA). According to
the values presented in Table 4, all of the compounds except 21a
and 21b, can be efficiently absorbed in the human intestine. The
most potent compound, 14b also showed promising logarithm of
the molar solubility value of ꢀ5.313 for the aqueous solubility.
On a positive note, 14b does not exhibit very high BBB penetration
and it does not inhibit the cytochrome P450, which means that the
compound can readily undergo oxidation and hydroxylation in the
first phase of metabolism. Although the in silico prediction reveals
that 14b is hepatotoxic, further structural modifications can be
done to improve the toxicity profile of the compound.
19. (a) Representative synthetic procedures and spectral characterizations. Procedure for
preparation of 1-(4-methoxy-2-(3-methylbut-2-enyloxy)-phenyl)ethanone
(11). A mixture of 5 (3 g, 18.05 mmol), anhydrous K2CO3 (9.98 g, 72.20 mmol),
and 3,3-dimethylallyl bromide (5.38 g, 36.10 mmol) was stirred in acetone
(30 mL) and heated at 75 °C for 20 h. The reaction mixture was monitored by TLC.
After completion of the reaction, the reaction mixture was filtrated and
evaporated under vacuum. The residue was purified by silica gel flash column
chromatography with n-hexane/EtOAc (gradient 0–30% EtOAc) to afford
compound 11 (3.89 g, 92%) as colorless oil; Rf (10% EtOAc/n-hexane) 0.325; 1H
NMR (500 MHz, CDCl3) d 7.85 (d, J = 8.7 Hz, 1H), 6.53 (dd, J = 8.7, 2.3 Hz, 1H), 6.47
(d, J = 2.2 Hz, 1H), 5.53 (t, 1H), 4.61 (d, J = 6.6 Hz, 2H), 3.87 (s, 3H), 2.60 (s, 3H), 1.82
(s, 3H), 1.78 (s, 3H). 13C NMR (126 MHz, CDCl3) d 198.04, 164.38, 160.44, 138.34,
132.62, 119.06, 105.08, 100.86, 99.33, 65.42, 55.49, 32.01, 25.72, 18.26.
Procedure for preparation of 1-(2-hydroxy-4-methoxy-5-(3-methylbut-2-
enyl)phenyl)ethanone (12). Intermediate 11 (3 g, 12.8 mmol) was dissolved in
10 mL of dimethylaniline and heated at 200 °C for 4 h. Ethyl acetate (50 mL) was
added and the mixture was washed with 10% aq HCl (3 ꢁ 50 mL). The organic
layer was dried over magnesium sulfate anhydrous, filtered, and evaporated. The
residue was purified by silica gel flash column chromatography with n-hexane/
EtOAc (gradient 0–50% EtOAc) to afford compound 12 (3.89 g, 36%) as colorless
yellow oil; Rf (10% EtOAc/n-hexane) 0.45; 1H NMR (500 MHz, CDCl3) d 12.72 (s,
1H), 7.42 (s, 1H), 6.41 (s, 1H), 5.28 (t, 1H), 3.88 (s, 3H), 3.24 (d, J = 7.2 Hz, 2H), 2.56
(s, 3H), 1.78 (s, 3H), 1.73 (s, 3H). 13C NMR (126 MHz, CDCl3) d 202.52, 164.07,
163.89, 133.05, 130.73, 122.03, 121.68, 113.18, 99.05, 55.68, 27.77, 26.17, 25.76,
17.76. ESI-HRMS: (C14H18O3) calc. [M+H] 235.1335, found 235.1320.
General procedure for preparation of 20-hydroxychalcone derivatives. aq KOH
(40%, 15 mL) wasaddedtoa stirred solution ofacetophenonederivates(10 mmol)
and (10 mmol) in absolute EtOH (30 mL). The mixture was sonicated for 1 h and
was then stirred at room temperature overnight. The dark red solution was
acidified with 2 M aq HCl to form a yellow precipitate, which was filtered and
rinsed with distilled water. The precipitate was purified by flash chromatography
with n-hexane/EtOAc (gradient 10–60% EtOAc) and recrystallisation using
absolute EtOH. (E)-1-(2-hydroxy-4-methoxyphenyl)-3-p-tolylprop-2-en-1-
one (7a). Yellow solid, yield 97%, mp 120–121 °C; 1H NMR (500 MHz, CDCl3) d
13.50 (s, 1H), 7.90 (d, J = 15.4 Hz, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.57 (dd, J = 15.5,
8.0 Hz, 3H), 7.26 (d, J = 7.9 Hz, 2H), 6.53–6.49 (m, 2H), 3.88 (s, 3H), 2.43 (s, 3H).
In conclusion, a series of novel minor chalcone and flavonoid
compounds have been synthesized and their effects on PGE2 pro-
duction in LPS-induced RAW 264.7 cells were evaluated. Prenylat-
ed chalcone (14b) exhibited the highest inhibition on PGE2
production with an IC50 value of 2.1 lM and was non-cytotoxic
at the tested concentration. Docking study revealed that 14b could
bind favorably to COX-2 and will serve as an important template
for future chemical modification. Future studies will address on
the SAR study of 14b as a potential PGE2 synthesis inhibitor.
Acknowledgments
This work was financially supported by Science Fund (02-01-
02-SF00665), Ministry of Science, Technology & Innovation, Malay-
sia. Authors also thank University Kebangsaan Malaysia for the
funds provided under the Research University Grant (GUP)
(UKM-GUP02011-014) and UKM-DIP-2012-11. We thank Prof. Dr.
Simon Gibbons and Mr. Mohd Syukri Baharuddin for valuable com-
ments on different parts of the manuscript.
13C NMR (126 MHz, CDCl3)
d 191.97, 166.69, 166.17, 144.51, 141.25,
132.11, 131.21, 129.75, 128.58, 119.31, 114.18, 107.67, 101.11, 55.58, 21.53.
ESI-HRMS: (C17H16O3) calc. [M+Na+] 291.0997, found 291.0984. (E)-3-(3,4-
dimethoxyphenyl)-1-(2-hydroxy-4-methoxy-phenyl)prop-2-en-1-one (7b).
Yellow solid, yield 95%, mp 157–159 °C; 1H NMR (500 MHz, CDCl3) d 13.56 (s,
1H), 7.88 (d, J = 7.4 Hz, 1H), 7.85 (s, 1H), 7.46 (d, J = 15.4 Hz, 1H), 7.27 (dd, J = 8.3,
1.8 Hz, 1H), 7.18(d,J = 1.9 Hz, 1H), 6.93(d, J = 8.3 Hz, 1H), 6.51(dd, J = 11.5, 2.5 Hz,
2H), 3.99 (s, 3H), 3.96 (s, 3H), 3.88 (s, 3H). 13C NMR (126 MHz, CDCl3) d 191.95,
166.81, 166.24, 151.79, 149.50, 144.72, 131.26, 127.99, 123.44, 118.23, 114.31,
111.38, 110.51, 107.78, 101.23, 56.17, 55.71. ESI-HRMS: (C18H18O5) calc. [M+Na+]
337.1052, found 337.1078. (E)-1-(2-hydroxy-4-methoxy-5-(3-methylbut-2-
enyl)phenyl)-3-p-tolyl prop-2-en-1-one (14a). Needle-yellow crystals, yield
65%, mp 119–120 °C; 1H NMR (500 MHz, CDCl3) d 13.46 (s, 1H), 7.88 (d,
J = 15.5 Hz, 1H), 7.62 (s, 1H), 7.56 (t, 3H), 7.27 (d, J = 8.0 Hz, 2H), 6.47 (s, 1H),
5.31 (t, 1H), 3.90 (s, 3H), 3.29 (d, J = 7.1 Hz, 2H), 2.43 (s, 3H), 1.80 (s, 3H), 1.76 (s,
3H). 13C NMR (126 MHz, CDCl3) d 192.05, 165.54, 164.36, 144.24, 141.27, 133.14,
132.41, 129.90, 128.66, 122.43, 121.79, 119.76, 113.58, 99.53, 55.86, 28.15, 25.91,
21.69, 18.00. ESI-HRMS: (C22H24O3) calc. [M+H] 337.1804, found 337.1811. (E)-3-
(3,4-dimethoxyphenyl)-1-(2-hydroxy-4-methoxy-5-(3-methylbut-2-enyl)
phenyl)prop-2-en-1-one (14b). Needle-yellow crystals, yield 60%, mp 132–
133 °C; 1H NMR (500 MHz, CDCl3) d 13.51 (s, 1H), 7.85 (d, J = 15.4 Hz, 1H), 7.62 (s,
1H), 7.45 (d, J = 15.4 Hz, 1H), 7.27 (d, J = 8.4 Hz, 1H), 7.19 (s, 1H), 6.94 (d, J = 8.3 Hz,
1H), 6.47 (s, 1H), 5.32 (t, 1H), 3.99 (s, 3H), 3.97 (s, 3H), 3.90 (s, 3H), 3.29 (d,
J = 7.1 Hz, 2H), 1.80 (s, 3H), 1.76 (s, 3H). 13C NMR (126 MHz, CDCl3) d 191.83,
165.51, 164.26, 151.68, 149.47, 144.23, 133.18, 129.71, 128.12, 123.35, 122.41,
121.62, 118.52, 113.54, 111.39, 110.38, 99.51, 56.17, 56.08, 55.85, 27.99, 25.90,
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
06.061. These data include MOL files and InChiKeys of the most
important compounds described in this article.
References and notes
1. Kawabata, A. Biol. Pharm. Bull. 2011, 34, 1170.
2. Legler, D. F.; Bruckner, M.; Uetz-von Allmen, E.; Krause, P. Int. J. Biochem. Cell
Biol. 2010, 42, 198.
3. Funk, C. D. Science 1871, 2001, 294.
4. Fahmi, H. Curr. Opin. Rheumatol. 2004, 16, 623.
5. Jegerschöld, C.; Pawelzik, S.-C.; Purhonen, P.; Bhakat, P.; Gheorghe, K. R.;
Gyobu, N.; Mitsuoka, K.; Morgenstern, R.; Jakobsson, P.-J.; Hebert, H. Proc. Natl.
Acad. Sci. 2008, 105, 11110.

K. Rullah et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3826–3834
17.98. ESI-HRMS: (C23H26O5) calc. [M+H] 383.1859, found 383.1856. (E)-3-(4-
3833
dimethoxyphenyl)-3-hydroxy-7-methoxy-4H-chromen-4-one (9b). Pale
yellow solid, yield 31%, mp 188–189 °C; 1H NMR (500 MHz, CDCl3) d 8.16 (d,
J = 8.9 Hz, 1H), 7.88 (dd, J = 8.5, 1.9 Hz, 1H), 7.86 (s, J = 1.8 Hz, 1H), 7.03 (dd,
J = 12.5, 5.5 Hz, 2H), 6.98(d,J = 2.2 Hz, 1H), 4.00 (t, 9H). 13C NMR (126 MHz, CDCl3)
d 164.33, 157.36, 150.76, 149.08, 144.65, 132.31, 126.91, 124.09, 121.29, 114.82,
111.17, 110.90, 100.16, 100.11, 68.00, 56.25, 56.14, 56.02. ESI-HRMS: (C18H16O6)
calc. [M+H] 329.1026, found 329.1036. General procedure for the preparation of
prenylated and geranylated flavonols and aurone derivatives. A mixture of
flavonol or aurone derivates (1.5 mmol), 1-chloro-3-methyl-2-butene or geranyl
bromide (2 equiv) and anhydrous K2CO3 (3 equiv), was stirred in acetone (30 mL)
and heated at 75 °C for 20 h. The reaction mixture was monitored by TLC. After
completion of the reaction, the reaction mixture was filtrated and evaporated
under vacuum. The residue was purified by silica gel flash column
chromatography with n-hexane/EtOAc (gradient 20-60% EtOAc). 7-methoxy-3-
(3-methylbut-2-enyloxy)-2-p-tolyl-4H-chromen-4-one (10a). Pale yellow
solid, yield 80%, mp 97–98 °C; 1H NMR (500 MHz, CDCl3) d 8.17 (d, J = 8.9 Hz,
1H), 8.03 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 6.97 (dd, J = 8.9, 2.3 Hz, 1H),
6.92 (d, J = 2.3 Hz, 1H), 5.43 (t, 1H), 4.61 (d, J = 7.3 Hz, 2H), 3.93 (s, 3H), 2.45 (s, 3H),
1.69 (s, 3H), 1.64 (s, 3H). 13C NMR (126 MHz, CDCl3) d 175.24, 164.38, 157.48,
156.29, 141.16, 140.22, 139.52, 129.51, 129.02, 128.93, 127.57, 120.58, 118.58,
114.70, 100.41, 69.18, 56.26, 26.20, 21.97, 18.44. ESI-HRMS: (C22H22O4) calc.
[M+H] 351.1597, found 351.1620. (E)-3-(3,7-dimethylocta-2,6-dienyloxy)-7-
methoxy-2-p-tolyl-4H-chromen-4-one (10c). Colorless oil, yield 62%; 1H NMR
(500 MHz, CDCl3) d 8.18 (d, J = 8.9 Hz, 1H), 8.04 (d, J = 8.3 Hz, 2H), 7.31 (d,
J = 8.1 Hz, 2H), 6.98 (dd, J = 8.9, 2.3 Hz, 1H), 6.93 (d, J = 2.2 Hz, 1H), 5.42 (t, 1H),
5.06 (t, 1H), 4.66 (d, J = 7.2 Hz, 2H), 3.93 (s, 3H), 2.45 (s, 3H), 2.04–1.93 (m, 4H),
1.68–1.54 (m, 9H). 13C NMR (126 MHz, CDCl3) d 174.80, 163.91, 157.00, 155.80,
142.34, 140.70, 139.73, 131.57, 129.04, 128.56, 128.50, 127.13, 123.96, 119.71,
118.10, 114.24, 99.94, 68.71, 55.80, 39.52, 26.31, 25.63, 21.51, 17.63, 16.43. ESI-
HRMS: (C27H30O4) calc. [M+H] 419.2223, found 419.2243. (E)-2-(3,4-
dimethoxyphenyl)-3-(3,7-dimethylocta-2,6-dienyloxy)-7-methoxy-4H-
chromen-4-one (10d). Colorless yellow oil, yield 42%; 1H NMR (500 MHz, CDCl3)
d 8.18 (d, J = 8.9 Hz, 1H), 7.85 (d, J = 2.0 Hz, 1H), 7.76 (dd, J = 8.5, 2.1 Hz, 1H), 7.01–
6.97 (m, 2H), 6.93 (d, J = 2.3 Hz, 1H), 5.46 (t, 1H), 5.06 (t, 1H), 4.66 (d, J = 7.2 Hz,
2H), 3.99 (s, 3H), 3.97 (s, 3H), 3.95 (s, 3H), 2.06–1.94 (m, 4H), 1.67 (s, 3H), 1.63 (s,
3H), 1.58 (s, 3H). 13C NMR (126 MHz, CDCl3) d 174.82, 164.06, 157.06, 155.52,
151.05, 148.71, 142.44, 139.60, 131.81, 127.32, 124.13, 124.02, 122.04, 119.92,
118.25, 114.28, 112.11, 110.85, 100.15, 68.95, 56.17, 56.11, 55.97, 39.71, 26.49,
25.76, 17.77, 16.61. ESI-HRMS: (C28H32O6) calc. [M+H] 464.2278, found 464.2290.
(b) Representative biological activity procedures. Cell Culture. The murine
macrophages-like cell line (RAW 264.7) from the European Collection of Cell
Cultures (Porton Down, UK) were maintained in DMEM supplemented with 10%
bromophenyl)-1-(2-hydroxy-4-methoxy-5-(3-methylbut-2-enyl)
phenyl)-
prop-2-en-1-one (14c). Needle-yellow crystals, yield 55%, mp 138–140 °C; 1H
NMR (500 MHz, CDCl3) d 13.35 (s, 1H), 7.81 (d, J = 15.5 Hz, 1H), 7.60 (s, 1H), 7.59
(d, J = 2.7 Hz, 2H), 7.57 (d, J = 15.7 Hz, 1H), 7.53 (d, J = 8.5 Hz, 2H), 6.47 (s, 1H), 5.30
(t, 1H), 3.91 (s, 3H), 3.28 (d, J = 7.1 Hz, 2H), 1.80 (s, 3H), 1.76 (s, 3H). 13C
NMR (126 MHz, CDCl3)
d 191.59, 165.68, 164.60, 142.68, 134.06, 133.20,
132.41, 131.82, 129.94, 129.84, 124.95, 122.35, 122.01, 121.41, 113.47, 100.15,
99.57, 55.90, 28.17, 25.92, 18.01. ESI-HRMS: (C21H21BrO3) calc. [M+H] 401.0753,
found 401.0735. (E)-3-(3,4-dihydroxyphenyl)-1-(2-hydroxy-4-methoxy-5-(3-
methylbut-2-enyl)phenyl)prop-2-en-1-one (14e). Brown solid, 10%, mp 166–
168 °C; 1H NMR (500 MHz, MeOD) d 7.71 (t, 2H), 7.48 (d, J = 15.3 Hz, 1H), 7.17 (d,
J = 2.0 Hz, 1H), 7.10 (dd, J = 8.2, 1.9 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 6.46 (s, 1H),
5.29 (t, J = 7.3, 5.9, 1.4 Hz, 1H), 3.87(s, 3H), 3.27(d, J = 7.2 Hz, 2H), 1.76(s, 3H), 1.74
(s, 3H). 13C NMR (126 MHz, MeOD) d 192.22, 164.85, 164.16, 148.60, 145.50,
144.77, 132.06, 129.73, 127.01, 122.33, 122.13, 121.59, 116.93, 115.24, 114.44,
113.13, 98.70, 54.85, 27.60, 24.50, 16.47. ESI-HRMS: (C21H22O5) calc. [M+H]
355.1546, found 355.1534. (E)-3-(4’-Fluoro-4-biphenylyl)-1-(2-hydroxy-4-
methoxy-phenyl)-2-propen-1-one (21a). Yellow solid, yield 34%, mp 150–
151 °C; 1H NMR (500 MHz, CDCl3) d 13.44 (s, 1H), 7.93 (d, J = 15.5 Hz, 1H), 7.74 (d,
J = 8.2 Hz, 2H), 7.66–7.61 (m, 6H), 7.20–7.16 (m, 2H), 6.48 (s, 1H), 5.32 (t, 1H), 3.91
(s, 3H), 3.30 (d, J = 7.2 Hz, 2H), 1.81 (s, 3H), 1.77 (s, 3H). 13C NMR (126 MHz, CDCl3)
d 191.82, 165.65, 164.50, 143.53, 142.36, 136.43, 134.11, 133.18, 130.89, 129.89,
129.19, 128.87, 128.81, 127.64, 122.41, 121.92, 120.75, 116.11, 115.94, 113.58,
99.57, 68.00, 55.89, 28.17, 25.93, 18.02. ESI-HRMS: (C27H25FO3) calc. [M+H]
417.1867, found 417.1856. (E)-3-(4’-Fluoro-3-biphenylyl)-1-(2-hydroxy-4-
methoxy-phenyl)-2-propen-1-one (21b). Yellow crystals, yield 48%, mp 125–
126 °C; 1H NMR (500 MHz, CDCl3) d 13.41 (s, 1H), 7.95 (d, J = 15.5 Hz, 1H), 7.82 (s,
1H), 7.68–7.59 (m, 6H), 7.53 (t, J = 7.7 Hz, 1H), 7.19 (t, J = 8.6 Hz, 2H), 6.48 (s, 1H),
5.32 (t, 1H), 3.91 (s, 3H), 3.29 (d, J = 7.2 Hz, 2H), 1.81 (s, 3H), 1.76 (s, 3H). 13C NMR
(126 MHz, CDCl3) d 191.79, 165.67, 164.55, 161.93, 143.83, 141.30, 135.72,
133.29, 129.87, 129.66, 129.31, 128.96, 128.90, 127.44, 127.17, 122.35, 121.90,
121.28, 116.07, 115.90, 113.54, 99.56, 55.90, 28.06, 25.90, 18.01. ESI-HRMS:
(C27H25FO3) calc. [M+H] 417.1867, found 417.1846. General procedure for
preparation of aurones derivatives. To a solution of mercuric acetate (Hg(OAc)2)
(3 mmol) in DMSO (10 mL) was added 2’-hydroxychalcone (2 mmol) at room
temperature and the mixture was stirred at 160 °C for 6 h. The cooled reaction
mixture was poured into ice cold water and acidified with 10% aq HCl. The
precipitated solid was extracted with dichloromethane or ethyl acetate, the
extracts were dried over magnesium sulfate anhydrous and the solvent was
evaporated to give
column chromatography with n-hexane/EtOAc (gradient 20–70% EtOAc)
and recrystallisation using absolute EtOH. (2Z)-6-methoxy-2-(p-
a solid which was further purified either by flash
FBS, 4.5 g/L glucose, sodium pyruvate (1 mM),
L-glutamine (2 mM), streptomycin
tolylmethylene)benzofuran-3-one (8a). Needle-pale yellow crystals, yield
27%, mp 156–157 °C; 1H NMR (500 MHz, CDCl3) d 7.82 (d, J = 8.1 Hz, 2H), 7.73
(d, J = 8.5 Hz, 1H), 7.28 (d, J = 7.0 Hz, 2H), 6.84 (s, 1H), 6.80 (d, J = 2.0 Hz, 1H), 6.78
(dd, J = 8.5, 2.1 Hz, 1H), 3.96 (s, 3H), 2.43 (s, 3H). 13C NMR (126 MHz, CDCl3) d
183.02, 168.47, 167.36, 147.49, 140.11, 131.35, 129.68, 129.64, 125.78, 115.04,
112.15, 112.08, 96.66, 56.01, 21.58. ESI-HRMS: (C17H14O3) calc. [M+H] 267.1022,
(50 g/mL) and penicillin (50 U/mL) at 37 °C and 5% CO2. When RAW 264.7 cells
l
reachedconfluencesof80–90%, the cellswere scraped out andcentrifuged at110g
in 4 °C for 10 min. The cell viability of cultured cells used in the assay was always
>95% as determined by trypan blue dye exclusion. Cell Stimulation and
Treatment. RAW 264.7 (4 ꢁ 105 cells/well) were seeded into a tissue culture
grade 96-well plate except for blank and incubated for 2 h at 37 °C, 5% CO2 for cell
attachment. Theattachedcells were activated with 100U/mL of recombinant IFN-
found 267.1018.
(2Z)-2-[(3,4-dimethoxyphenyl)methylene]-6-methoxy-
benzofuran-3-one (8b). Yellow solid, yield 22%, mp. 186-188 °C; 1H NMR
(500 MHz, CDCl3) d 7.73 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 1.9 Hz, 1H), 7.49 (dd, J = 8.3,
1.9 Hz, 1H), 6.96 (d, J = 8.3 Hz, 1H), 6.81 (s, 1H), 6.78 (dd, J = 10.5, 2.1 Hz, 2H), 4.00
(s, 3H), 3.96 (s, 3H), 3.95 (s, 3H). 13C NMR (126 MHz, CDCl3) d 182.94, 168.34,
167.38, 150.84, 149.26, 147.02, 125.91, 125.79, 125.59, 115.31, 113.96, 112.55,
112.15, 111.43, 96.79, 56.17, 56.15, 56.11. ESI-HRMS: (C18H16O5) calc. [M+H]
313.1077, found 313.1098. (2Z)-6-methoxy-5-(3-methylbut-2-enyl)-2-(p-
tolylmethylene)benzofuran-3-one (15a). Pale yellow solid, yield 33%, mp.
149-150 °C; 1H NMR (500 MHz, CDCl3) d 7.82 (d, J = 8.1 Hz, 2H), 7.57 (s, 1H), 7.27
(d, J = 8.1 Hz, 2H), 6.82 (s, 1H), 6.77 (s, 1H), 5.32 (t, 1H), 3.99 (s, 3H), 3.31 (d,
J = 7.4 Hz, 2H), 2.43 (s, 3H), 1.79 (s, 3H), 1.71 (s, 3H). 13C NMR (126 MHz, CDCl3) d
167.76, 165.45, 147.79, 140.10, 133.94, 131.45, 129.99, 129.76, 127.16, 124.31,
121.38, 114.23, 111.96, 100.16, 94.40, 56.23, 28.17, 26.00, 21.73, 17.87. ESI-
HRMS: (C22H22O3) calc. [M+H] 335.1648, found 335.1654. (2Z)-2-[(3,4-
dimethoxyphenyl)methylene]-6-methoxy-5-(3-methylbut-2-
c
and 5
l
g/mL of LPS with or without presence of synthetic compound at a final
volumeof100
lL/well.DMSOwas usedasvehicletoaddsyntheticcompoundinto
the culture medium and the final concentration of DMSO was 0.1% in all cultures.
Cells were then incubated at 37 °C, 5% CO2 for 17–20 h. The level of PGE2 was
determined by using PGE2 EIA kit (Item No. 500141). Cell Viability. The
cytotoxicity of synthetic compound on cultured cells was determined by
assaying the reduction of MTT reagents to formazan salts. After treatment, the
supernatant of 96-wells plate containing cells were removed and MTT reagents
(0.05 mg/mL) were added into each well. The cells were incubated in 37 °C for 4 h
andtheformazansaltsweredissolvedbyadding100%DMSO. Theabsorbance was
then measured at 570 nm on a SpectraMax Plus microplate reader (Molecular
Devices Inc., Sunnyvale, CA, USA). Determination of PGE2. The cell culture
supernatants were collected and analyzed for PGE2 secretion PGE2 EIA kits
(Cayman Chemical, Ann Arbor, MI, USA). The protocols provided by the
manufacturers were followed to the detail. The data was acquired 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.
enyl)benzofuran-3-one (15b). Pale yellow solid, yield 44%, mp 151–152 °C; 1
H
NMR (500 MHz, CDCl3) d 7.57 (s, 1H), 7.52 (s, 1H), 7.50 (d, J = 8.4 Hz, 1H), 6.96 (d,
J = 8.4 Hz, 1H), 6.80 (s, 1H), 6.73 (s, 1H), 5.32 (t, 1H), 4.00 (s, 3H), 4.00 (s, 3H), 3.97
(s, 3H), 3.31 (d, J = 7.4 Hz, 2H), 1.79 (s, 3H), 1.71 (s, 3H). 13C NMR (126 MHz, CDCl3)
d 183.24, 167.50, 165.33, 150.74, 149.24, 147.16, 133.96, 127.16, 125.75, 125.70,
124.28, 121.36, 114.32, 113.96, 112.19, 111.41, 94.30, 56.24, 56.18, 56.11, 28.16,
26.00, 17.86. ESI-HRMS: (C23H24O5) calc. [M+H] 381.1703, found 381.1703.
General procedure for preparation of flavonols derivatives. To a well-stirred
solution of 2’-hydroxychalcone derivates (10 mmol) in EtOH (30 mL) and aq KOH
(40%, 15 mL), H2O2 (30%, 10 mL) was added drop wise for 30 min at room
temperature. The reaction mixture was further stirred for 3–4 h. The resulting
light-yellow reaction mixture was poured on crushed ice and neutralized with
2 M aq HCl. The light-yellow solid thus obtained was filtered, washed with water
and dried. The crude product was purified by flash chromatography with n-
hexane/EtOAc (gradient 20-70% EtOAc) andrecrystallisation usingabsolute EtOH.
3-hydroxy-7-methoxy-2-p-tolyl-4H-chromen-4-one (9a). Pale brown solid.
Yield 43%, mp 220–221 °C; 1H NMR (500 MHz, CDCl3) d 8.16 (dd, J = 8.6, 3.1 Hz,
3H), 7.36 (d, J = 8.1 Hz, 2H), 7.02 (dd, J = 8.8, 2.2 Hz, 1H), 6.98 (d, J = 2.2 Hz, 1H),
3.96 (s, 3H), 2.46 (s, 3H). 13C NMR (126 MHz, CDCl3) d 164.21, 157.34, 144.61,
140.28, 132.15, 129.32, 128.42, 127.45, 126.74, 114.77, 99.91, 67.84, 55.85, 21.51.
ESI-HRMS: (C17H14O4) calc. [M+H] 283.0971, found 283.0970. 2-(3,4-
(c) X-ray structure determination. Single crystal X-ray experiment of 14b was
performed on Bruker D-QUEST diffractometer using graphite-monochromated
Mo-K
a radiation (k = 0.71073 Å). Intensity data was measured at 301(2)°K by the
-scan. Accurate cell parameters and orientation matrix were determined by the
x
least-squares fit of 25 reflections. Intensity data was collected for Lorentz and
polarization effects. Empirical absorption correction was carried out using multi-
scan. The structure was solved by direct methods and least-squares refinement of
the structure was performed by the SHELXL-97 program. All the non-hydrogen
atoms were refined anisotropically. The hydrogen atoms were 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 the data collections and details of the
structure refinement is given in Tables 2 and 3. Crystallographic data for the
structural determination has been deposited with the Cambridge
Crystallographic Data Centre, CCDC No 997318. This information may be
obtained free of charge from the Director, CCDC, 12 Union Road, Cambridge
CB2, 1EZ, UK; http://www.ccdccam.ac.uk/const/retrieving.html).

3834
K. Rullah et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3826–3834
(d) Molecular Modeling. All molecular modeling methods were performed using
24. Han, W.; Liu, C.; Jin, Z.-L. Org. Lett. 2007, 9, 4005.
25. Sjögren, T.; Nord, J.; Ek, M.; Johansson, P.; Liu, G.; Geschwindner, S. Proc. Natl.
Acad. Sci. 2013, 110, 3806.
26. Kurumbail, R. G.; Stevens, A. M.; Gierse, J. K.; McDonald, J. J.; Stegeman, R. A.;
Pak, J. Y.; Gildehaus, D.; Iyashiro, J. M.; Penning, T. D.; Seibert, K.; Isakson, P. C.;
Stallings, W. C. Nature 1996, 384, 644.
27. Wang, J. L.; Limburg, D.; Graneto, M. J.; Springer, J.; Hamper, J. R. B.; Liao, S.;
Pawlitz, J. L.; Kurumbail, R. G.; Maziasz, T.; Talley, J. J.; Kiefer, J. R.; Carter, J.
Bioorg. Med. Chem. Lett. 2010, 20, 7159.
Discovery Studio 3.1 (Accelrys, San Diego, USA) on an IntelÒ (TM)2 Quad CPU
Q8200 @2.33 GHz running under a Windows XP Professional environment.
20. Sugamoto, K.; Matsusita, Y.-I.; Matsui, K.; Kurogi, C.; Matsui, T. Tetrahedron
2011, 67, 5346.
21. Dong, X.; Qi, L.; Jiang, C.; Chen, J.; Wei, E.; Hu, Y. Bioorg. Med. Chem. Lett. 2009,
19, 3196.
22. Chu, W. L. A.; Jensen, F. R.; Jensen, T. B.; McAlpine, J. B.; Søkilde, B.; SantAna-
Sørensen, A. M.; Ratnayake, S.; Jiang, J. B.; Noble, C.; Stafford, A. M.; Google
Patents: 2001.
28. Garavito, R. M.; DeWitt, D. L. Biochimica et Biophysica Acta (BBA)-Mol. Cell Biol.
Lipids 1999, 1441, 278.
23. Suzuki, A. Angew. Chem., Int. Ed. 2011, 50, 6722.