Synthesis and inhibitory effects of pinosylvin derivatives on prostaglandin E2 production in lipopolysaccharide-induced mouse macrophage cells

Article abstract of DOI:10.1016/j.bmcl.2004.09.022

The inhibitory effects of pinosylvin analogues on the production of COX-2 mediated PGE₂ were evaluated in a cell culture system. A new series of potential inhibitors, including 3-hydroxy-5-benzyloxy-trans-stilbene and 3,5-dimethoxy-trans-stilbene, have been identified. A series of natural stilbenoids, pinosylvin and its derivatives, were synthesized and evaluated for the inhibitory activity of prostaglandin E₂ production in lipopolysaccharide-induced RAW 264.7 cells. Potential inhibitors, including 3,5-dimethoxy-trans-stilbene and 3-hydroxy-5-benzyloxy-trans-stilbene, have been newly identified, and thus providing chemical leads for the further development of anti-inflammatory or cancer chemopreventive agents.

Full text of DOI:10.1016/j.bmcl.2004.09.022

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5895–5898
Synthesis and inhibitory effects of pinosylvin derivatives on
prostaglandin E2 production in lipopolysaccharide-induced mouse
macrophage cells
Eun-Jung Park,a Hye-Young Min,a Yong-Hyun Ahn,b Cheol-Man Bae,b Jae-Ho Pyeeb
and Sang Kook Leea,*
aCollege of Pharmacy, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-ku, Seoul 120-750, Republic of Korea
bCollege of Natural Sciences, Dankook University, 8 Hannam-dong, Yongsan-ku, Seoul 14-714, Republic of Korea
Received 26 July 2004; revised 7 September 2004; accepted 8 September 2004
Available online 28 September 2004
Abstract—A series of natural stilbenoids, pinosylvin and its derivatives, were synthesized and evaluated for the inhibitory activity of
prostaglandin E2 production in lipopolysaccharide-induced RAW 264.7 cells. Potential inhibitors, including 3,5-dimethoxy-trans-
stilbene and 3-hydroxy-5-benzyloxy-trans-stilbene, have been newly identified, and thus providing chemical leads for the further
development of anti-inflammatory or cancer chemopreventive agents.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Naturally occurring stilbenoids have been reported to
possess various biological and pharmacological activi-
ties such as antioxidant, anti-inflammatory, and anticar-
cinogenic effects.1–4 In our continuous efforts to identify
and develop the pharmacologically active stilbenoids
from natural products or synthetic approaches, we have
focused on a natural stilbenoid pinosylvin (3,5-dihyd-
roxy-trans-stilbene) that possesses structural similarity
with biologically active resveratrol. Resveratrol (3,5,40-
trihydroxy-trans-stilbene), one of the representatives of
natural stilbenoid and a major principle of French
Paradox, abundant in grapes, peanuts, and pines, has
demonstrated a variety of biological activities such as
reduction in the incidence of coronary heart disease,
chemoprevention of cancer, growth inhibition of many
cancer cells, and anti-inflammatory effects.5–8 One of
the probable mechanisms for anti-inflammatory and
cancer chemopreventive activity has been suggested with
the inhibition of cyclooxygenase (COX) activity.9,10
constitutively expressed in most of tissues, whereas
COX-2 is negligible in normal conditions, but can be
dramatically induced by pro-inflammatory cytokines,
tumor promoters, growth factors, or oncogens.13–15
Therefore, the overproduction of PGs mediated by
COX-2 involves in the many pathophysiological proc-
esses including inflammation and carcinogenesis. On
this line, reducing the levels of COX-2-mediated PG
productions will be an effective strategy for inhibiting
inflammation and carcinogenesis. Recently, we reported
the styrylheterocyclic compounds might be new lead
candidates for modulating COX-2 activity.16 In this
study, we further extended to envisage the procurement
of natural stilbenoids with pinosylvin derivatives antici-
pating potential anti-inflammatory and cancer chemo-
preventive activity.
Pinosylvin (3,5-dihydroxy-trans-stilbene),
a natural
stilbenoid found in heartwood and leaves of pine, is a
phytoalexin with antioxidant and antifungal activities,
and inhibitory effects of tyrosinase, but it is relatively
not much studied for the biological activity yet.17–19
COX is the rate-limiting enzyme in the biosynthesis of
prostaglandins (PGs) and thromboxane A2 (TXA2)
from arachidonic acid.11,12 Two isozymes, designated
as COX-1 and COX-2, have been identified: COX-1 is
Since the substitution patterns of the trans-stilbene tem-
plate have been shown to be sensitive for various biolog-
ical activities, we primarily investigated the substitutions
of dihydroxy group in pinosylvin with several lipophilic
derivatives. We report here the inhibition of COX-2-
mediated PGE2 production of synthesized pinosylvin
Keywords: Pinosylvin; COX-2; PGE2; Cancer chemoprevention.
*
Corresponding author. Tel.: +82 232773023; fax: +82 232772851;
e-mail: sklee@ewha.ac.kr
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.022

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E.-J. Park et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5895–5898
derivatives and our discovery of 3,5-dimethoxy-trans-
stilbene and 3-hydroxy-5-benzyloxy-trans-stilbene as
new lead compounds.
R1
HO
Sucrose
+
Compounds were prepared by following synthetic path-
way and enzymatic biotransformation (Schemes 1 and
2). The synthesis of pinosylvin and 3-O-a-D-glucopyran-
osyl-(E)-pinosylvin were previously reported.20–22
Briefly, Horner–Wadsworth–Emmons reactions be-
tween phosphonates and aromatic aldehydes were per-
formed to yield 3,5-dimethoxy-trans-stilbene followed
by producing pinosylvin in the catalytic condition
of pyridine hydrochloride at 190ꢁC. After then, we
prepared 3-(2-ethylhexanoyl)-, 3-palmitoyl-, and 3,5-
dipalmitoyl-trans-stilbene from pinosylvin, correspond-
ing acyl chloride, and pyridine in methylene chloride
at room temperature.20 In an attempt to obtain mono-
benzylate compound, the reaction with the correspond-
ing stilbene and benzylbromide gave a 50% yield of the
3-benzyloxy-trans-stilbene. Further reaction for obtain-
ing the second benzyl group gave 98% yield of 3,5-di-
benzyloxy-trans-stilbene. In addition, 3-monomethoxy-
trans-stilbene was synthesized by the reaction between
3,5-dimethoxy-trans-stilbene and boron tribromide
(Scheme 1).21 trans-Piceid and 3-O-a-D-glucopyranos-
yl-(E)-pinosylvin were synthesized by the biological
reaction of Streptococcus mutans (Scheme 2).22,23
Streptococcus mutans
0.1 N phosphate buffer (pH 6.5)
37º C, 48 hr
OH
HO
O
H
R1
HO
HO
OH
O
OH
8
:
R1 = H
trans-Piceid :
R1 = OH
Scheme 2. Synthesis of glucosides of pinosylvin analogues by bio-
transformation. Chemical yields of trans-piceid (18%), 8 (11%).
Primarily, pinosylvin was prepared and evaluated for
the inhibitory activity of PGE2 production in LPS-in-
duced mouse macrophage RAW 264.7 cells by PGE2 en-
zyme immunometric assay (EIA).24 In this assay,
activity is defined as the difference between PGE2 accu-
mulation in the absence and in the presence of LPS for
20h. Treatment of RAW 264.7 cells with LPS (1lg/mL)
increased dramatically the production of PGE2 from
endogeneous arachidonic acid up to 23ng/mL from
the basal level of 0.6ng/mL without LPS. Pinosylvin
was found to inhibit the COX-2-mediated PGE2 produc-
tion, and the potency (IC50 = 10.6lM) was approxi-
mately twofold higher than that of resveratrol
(IC50 = 20.8lM). Based on the potency of pinosylvin,
pinosylvin derivatives were prepared and evaluated. As
shown in Table 1, the effects of substitution were
revealed to be highly dependent on the position and
substituents. For example, compound 1, introduction
of methoxy groups both 3- and 5-position, dramatically
CH Br
2
OEt
H CO
3
P
O
OEt
1
a
b
d
OCH
3
c
HO
R O
1
Pinosylvin
e
OH
OR
2
3: R1= palmitoyl
4: R1= palmitoyl
7: R1= 2-ethylhexanoyl
R2 = H
R2 = palmitoyl
R2 = H
R O
3
Table 1. Inhibitory effects of pinosylvin analogues on the PGE2
production in LPS-stimulated RAW264.7 cells
R
3
OR
4
R O
1
5: R3= benzyl R 4 = H
6: R3= benzyl R 4 = benzyl
OR
2
IC50 (lM)a
HO
Compounds R1
R2
R3
1
Resveratrol
Pinosylvin
trans-Piceid
H
H
H
OH
H
20.8
10.6
13.5
0.1
f
2
H
OCH
3
Glycosyl
Methyl
H
H
OH
H
1
2
3
4
5
6
7
8
Methyl
Methyl
H
Scheme 1. Solution phase synthetic approach employed for the
preparation of pinosylvin and its derivatives described in Table 1.
Reagents and conditions: (a) triethyl phosphite, 130ꢁC; (b) dimethyl
formamide, sodium methoxide, 0ꢁC, followed by 3,5-dimethoxy
benzaldehyde, rt; (c) pyridine hydrochloride, 190ꢁC; (d) RCOCl,
pyridine, methylene chloride, rt; (e) benzylbromide, potassium car-
bonate, acetone; (f) boron tribromide, ꢀ80ꢁC. Chemical yields of
pinosylvin (50%); 1 (50%); 2 (40%); 3 (44%); 4 (28%); 5 (50%); 6 (98%);
7 (91%).
H
10.5
>50
>50
2.0
Palmitoyl
Palmitoyl
Benzyl
Benzyl
H
Palmitoyl
H
H
H
Benzyl
H
H
>50
1.7
12.1
2-Ethylhexanoyl
Glycosyl
H
H
H
a The IC50 values were determined by triplicate tests.

E.-J. Park et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5895–5898
5897
increased the inhibitory activity with 100 times more po-
Acknowledgements
tent than that of pinosylvin and exhibited the most po-
tent activity among test compounds (IC50 = 0.1lM).
However, substitution with only one methoxy group 2
showed the similar activity with pinosylvin. In order to
further elucidate the substitution effects on hydroxy
group the introduction of various aliphatic chains or
benzyl groups were employed. One benzyl-substituent
5 enhanced the activity with fivefold (IC50 = 2.0lM)
compared to pinosylvin, but dibenzyl-substituent 6 did
not show the inhibitory activity (IC50 > 50.0lM), indi-
cating that the introduction of bulky groups might hin-
der the penetration of the compound into cells. In the
introduction of aliphatic chains the short chain of
aliphatic groups exampled by 2-ethylhexyl group 7
increased the activity (IC50 = 1.7lM) with the similar
potency of compound 6, whereas the introduction of
long chain aliphatic group 3 and 4 did not show
the inhibitory activity (IC50 > 50.0lM). In another
variation, the introduction of glucose moiety on the
hydroxy group 8 was found to be similar activity
(IC50 = 12.1lM) with pinosylvin and the inhibitory
This study was supported in part by a grant no 0320220-
1 of the National Cancer Control R&D Program 2003,
Ministry of Health & Welfare, Republic of Korea.
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potential
(IC50 = 13.5lM), which is
resveratrol.
was
also
relevant
to
a glycoside form of
trans-piceid
To clarify the possible underlying molecular mecha-
nisms of pinosylvin derivatives on PGE2 production in
LPS-induced cells, further studies were performed by
examining COX-2 gene expression, using reverse tran-
scription-polymerase chain reaction (RT-PCR) analy-
sis.25 As a result, compound 1 and 7 demonstrated the
suppressive effects of COX-2 mRNA expression (Fig.
1), indicating that the inhibitory effects of PGE2 produc-
tion by pinosylvin derivatives are possibly in part related
to the suppression of COX-2 gene expression.
In conclusion, a series of pinosylvin derivatives has been
prepared and evaluated as to their effects on the activity
of COX with the goal of identifying a potent inhibitor.
We found for the first time that pinosylvin is also one
of active natural stilbenoids exhibiting an appreciable
inhibitory activity against the overproduction of the
inflammatory mediator PGE2, and could serve as a
new lead for further chemical optimization. We have
also gained an insight into the preliminary structure–
activity relationships of pinosylvin derivatives, which is
valuable in the design and development of a new class
of COX inhibitors. Further studies for more potent
inhibitors, based on the above findings, are in progress
in our laboratory.
21. Kubo, I.; Murani, Y. J. Nat. Prod. 1991, 54, 1115.
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24. Production of PGE2 by LPS-induced COX-2 in RAW
264.7 cells: RAW 264.7 mouse macrophage cells applied to
this study were cultured in DMEM supplemented with
10% fetal bovine serum (FBS), penicillin (100U/mL),
and streptomycin (100lg/mL). PGE2 production was
measured by an enzyme-immunometric assay (EIA), as
Figure 1. Effects of compound 1 and 7 on the expression of COX-2
mRNA in LPS-stimulated RAW 264.7 cells using RT-PCR analysis.

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E.-J. Park et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5895–5898
described previously. Briefly, the cells (5 · 105/mL) were
into three phases by chloroform. The aqueous phase was
transferred to a fresh tube, and added the same amount of
isopropanol to precipitate RNA pellet. RNA pellet was
washed with 75% ethanol and dissolved in nuclease free
water. The concentration of RNA was determined by
absorbance at 260nm. RNA (1lg) was performed reverse
transcription (RT) using avian myeloblastosis virus
(AMV) reverse transcriptase. The cDNA products of RT
were amplified by PCR using Taq DNA polymerase.
The sense and antisense primers for COX-2 were
50-GGAGAGACTATCA-AGATAGTGATC-30 and 50-
incubated in 96-well culture plate for 24h. Then, the cells
were washed twice with phosphate buffered saline (PBS),
and changed in the fresh medium with test compounds
and 1lg/mL of LPS. After additional 20h incubation, the
PGE2 in the media were analyzed by the PGE2 enzyme
immunometric assay (Cayman Co., Ann Arbor, MI,
USA). The percentage of inhibition was calculated as
[1 ꢀ (PGE2 level of compound/PGE2 level of vehicle
treated-control)] · 100.
25. Effects of some compounds on COX-2 mRNA expression
in RAW 264.7 cells by reverse transcription-polymerase
chain reaction (RT-PCR) analysis: RAW 264.7 cells
(5 · 105/mL) were grown in 6cm2 dishes for 24h. Then,
compound 1, or 7 was treated with 1lg/mL of LPS for 6h.
After washing with PBS, total RNA was isolated from the
cells. Briefly, cells were lysed by Tri-reagent and separated
ATGGTCAGTAGAC-TTTTACAGCTA-30,
respec-
tively. The sense and antisense primers for b-actin were
50-TGTGATGG-TGGGAATGGGTCAG-30 and 50-
TTTGATGTCA-CGCACGATTTCC-30,
respectively.
The PCR products were run on a 2% agarose gel and
visualized by SYBR Gold staining.