Styrylheterocycles as a novel class inhibitor of cyclooxygenase-2-mediated prostaglandin E2 production

Article abstract of DOI:10.1016/j.bmcl.2004.02.038

The inhibitory effects of a series of styrylheterocycles on the production of cyclooxygenase-2-mediated prostaglandin E₂ (PGE₂) were evaluated in lipopolysaccharide-stimulated RAW264.7 murine macrophages. A new series of potential inhibitors, including 3-[2-(4-methoxy-phenyl)-vinyl]- thiophene, have been identified, thus providing novel chemical leads for the further development of potential inhibitors in this capacity. The suppression of COX-2 mRNA expression by the active styrylheterocycles, in part, was involved in the inhibitory activity against the overproduction of PGE₂.

Full text of DOI:10.1016/j.bmcl.2004.02.038

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2105–2108
Styrylheterocycles as a novel class inhibitor of cyclooxygenase-2-
mediated prostaglandin E production
2
a
a
b
a
Sang Kook Lee, Eun-Jung Park, Eunjung Lee, Hye-Young Min, Eun-Young Kim,
b
b
Taeho Lee and Sanghee Kim
b,
*
a
College of Pharmacy, Ewha Womans University, 11-1 Daehyun, Seodaemun, Seoul 120-750, South Korea
b
Natural Products Research Institute, College of Pharmacy, Seoul National University,
8 Yungun, Jongro, Seoul 110-460, South Korea
2
Received 27 December 2003; revised 7 February 2004; accepted 10 February 2004
Abstract—The inhibitory effects of a series of styrylheterocycles on the production of cyclooxygenase-2-mediated prostaglandin E
PGE ) were evaluated in lipopolysaccharide-stimulated RAW264.7 murine macrophages. A new series of potential inhibitors,
2
(
2
including 3-[2-(4-methoxy-phenyl)-vinyl]-thiophene, have been identified, thus providing novel chemical leads for the further
development of potential inhibitors in this capacity. The suppression of COX-2mRNA expression by the active styrylheterocycles,
in part, was involved in the inhibitory activity against the overproduction of PGE
2004 Elsevier Ltd. All rights reserved.
2
.
ꢀ
Several lines of evidence suggest that the overproduction
of prostaglandins (PGs) mediates a variety of patho-
physiological processes, including inflammation, Alz-
heimerÕs disease, hypertension, heart failure and
carcinogenesis, and inducible isoform of cyclooxygenase
inhibitors, or suppressors, for anti-inflammatory or
cancer chemopreventive agents from natural products,
or synthetic chemicals, our study was extended to styr-
ylheterocycles, based on the effects of stilbenoids in these
capacities.
(
large amounts of the mediator.
COX)-2is mainly responsible for the production of
Two isoforms of
1–3
A number of naturally occurring trans-stilbenoids have
been reported as inhibitors of COX, although these
compounds were somewhat less active than the repre-
sentative COX inhibitors, such as celecoxib and indo-
methacin. For examples, resveratrol (1, Fig. 1) exhibited
in vitro inhibitory activity against both COX-1 and
COX, designated COX-1 and COX-2, at least, have
been identified, and catalyze the biosynthesis of PGs
from arachidonic acid. Especially, COX-2is an induc-
ible enzyme, and is expressed in response to growth
factors, tumor promoters, or various physiological and
4;5
6
inflammatory stimuli. With exposure to lipopolysac-
charide (LPS), macrophages express the COX-2gene
and protein, and COX-2-mediated metabolites,
COX-2. Aiphanol (4), a natural stilbenolignan, showed
significant inhibitory activity against COX-1 and COX-
7
2. Isorhapontigenin (2), a natural hydroxystilbene,
showed a COX inhibition, whereas piceatannol (3), the
including PGE
inflammation, pain, and carcinogenesis. Since COX-
-mediated PGE modulates inflammation and tumor
2
, are also involved in the induction of
1–3
7
demethyl derivative of isorhapontigenin, was inactive.
Recently, some prenylated stilbenoid natural products
2
2
growth, the inhibitors of COX-2activity, or suppressors
of COX-2expression, have recently received much
attention as effective anti-inflammatory or cancer
chemopreventive agents, with anticipated lower side
effects. In our continuous efforts to develop COX-2
(5 and 6) have been found to exhibit moderate COX-1-
8
selective COX inhibiting activity.
It was seen by comparing the reported activities of the
7
–9
various natural trans-stilbenoids
that the selectivity
and inhibitory potency of stilbene compounds against
COX were sensitive to the substitution patterns on the
trans-stilbene template. On the basis of these studies, we
investigated the biological evaluation of various syn-
thetic trans-stilbene derivatives in order to establish the
preliminary structure–activity relationships of stilbenes,
Keywords: Styrylheterocycles; Cyclooxygenase-2; Prostaglandin E2;
Stilbenoids.
*
Corresponding author. Tel.: +82-2-7408913; fax: +82-2-7628322;
e-mail: pennkim@snu.ac.kr
0
960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.038

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S. K. Lee et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2105–2108
Table 1. Inhibitory effects of the 3,5-dimethoxystilbene analogues on
a
OH
R
resveratrol (1) R = H
isorhapontigenin (2) R = OMe
piceatannol (3) R = OH
2
the PGE production in LPS-stimulated RAW264.7 cells
HO
MeO
Ar
OH
O
O
10a - 10j
OMe
OH
HO
OMe
b
c
Compounds
Ar
IC50 (lM)
1
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
3,5-Dimethoxyphenyl
2,4,5-Trimethoxyphenyl
2,5-Dimethoxyphenyl
2-Hydroxy-4-methoxyphenyl
2,3-Dimethoxyphenyl
4-Bromophenyl
4-Pyridyl
>33.3
7.4
5.8
4.1
1.5
3.1
1.2
1.0
1.3
1.5
4.0
OH
aiphanol (4)
OH
OMe
OH
HO
0g
R
5
6
R = H
R = OH
10h
2-Furyl
3-Furyl
1
1
1
0i
0j
OH
3-Thienyl
Resveratrol
Figure 1.
a
b
c
12;13
The activities were measured as described previously.
Compounds 10a–e and 10h were previously known.
The IC50 values were determined from triplicate tests.
and find a potent COX inhibitor that could serve as a
new lead for chemical optimization.
We designed and prepared several trans-stilbenoids in
which one phenyl ring contains dimethoxy groups on
the 3 and 5 positions instead of the corresponding
dihydroxyl groups of the natural products 1–6. We
envisaged that the substitution by dimethoxy groups
could enhance the activity since previously reported
potent COX inhibitors or suppressors generally have at
(1.1 equiv) in the presence of KOH and a catalytic
amount of 18-crown-6 in CH Cl yielded a mixture of Z
2
2
and E olefins 9. After standard aqueous workup, the
excess aldehydes 8 was removed from the mixture by
treatment with GirardÕs reagent T and acetic acid. The
Z/E mixture 9 was converted to the E-isomers 10 by
heating with catalytic amounts of iodine in refluxing
heptane. The reaction mixtures were diluted with diethyl
ether and washed with saturated aqueous sodium
bisulfite and water to remove iodine from the trans-
stilbene derivatives. The NMR spectra of the synthe-
sized trans-stilbenes showed that these were very clean
and contained no notable impurities.
10
least one lipophilic aromatic ring. We report here the
inhibition of COX-2-mediated PGE production of
2
synthesized trans-stilbene analogues and our discovery
of 3-[2-(4-methoxy-phenyl)-vinyl]-thiophene as a new
lead compound.
Compounds were prepared through our previously
11
reported solution-phase synthetic pathway (Scheme 1).
Briefly, Horner–Wadsworth–Emmons reactions
between phosphonates 7 and aromatic aldehydes 8
Initially, the compounds, in which one phenyl ring
contains 3,5-dimethoxy groups, were prepared and
evaluated for their inhibitory activities on the PGE
2
12;13
production in LPS-stimulated RAW264.7 cells.
As
shown in Table 1, the effects of substituents introduced
into the other phenyl ring were revealed to be highly
dependent, as expected, on the position of substitution
(compounds 10a–f). For example, compound 10a, which
contains dimethoxy groups on the 3 and 5 positions, did
not show any inhibitory activities up to 33 lM. How-
ever, the inhibitory activities of 10e (IC50 ¼ 1.5 lM) and
R1
R2
(OMe)2
R1
R2
Ar
Ar
P
O
ArCHO 8
a, b
R1
9
7
R1
c, d
1
0f (IC ¼ 3.1 lM) were increased compared to resve-
50
7
7
a R1 = OMe, R2 = H
b R1 = H, R2 = OMe
ratrol (1) (IC50 ¼ 4.0 lM). In another variation, we
replaced the phenyl ring with 4-pyridyl, 2-furyl, 3-furyl,
and 3-thienyl rings (compounds 10g, 10h, 10i, and 10j,
respectively). Comparison of the styrylheterocycles 10g–
j with resveratrol (1) showed that the inhibitory activi-
ties were improved more than twofold by the introduc-
tion of the heteroaromatic rings.
R1
R2
R1
10
Scheme 1. Solution-phase synthetic approach employed for the prep-
aration of the trans-stilbenoids described in Tables 1 and 2. Reagents
and conditions: (a) 8, 18-crown-6, KOH, CH
b) GirardÕs reagent T, AcOH, CH Cl , rt; aqueous workup; (c) I
heptane, reflux; (d) NaHSO , aqueous workup. Chemical yields of 10a
94%); 10b (93%); 10c (94%); 10d (90%); 10e (92%); 10f (90%); 10g
2 2
Cl , rt; aqueous workup;
Taking compounds 10h–j as leads, we explored the
modification of 3,5-dimethoxyphenyl moiety to 4-meth-
oxyphenyl, in view of the fact that many potent
COX inhibitors have characteristic para-substituted aryl
(
2
2
2
,
3
(
(
(
90%); 10h (92%); 10i (91%); 10j (88%); 10k (89%); 10l (87%); 10m
89%).
11
groups. As seen in Table 2, this replacement led to

S. K. Lee et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2105–2108
2107
Table 2. Inhibitory effects of the 4-methoxystilbene analogues on the
a
the activity of COX with the goal of identifying a potent
inhibitor. We found that 3-[2-(4-methoxy-phenyl)-
vinyl]-thiophene 10m had an appreciable inhibitory
activity against the overproduction of the inflammatory
PGE
2
production in LPS-stimulated RAW264.7 cells
Ar
2
mediator PGE , and could serve as a new lead for fur-
MeO
ther chemical optimization. We have also gained an
insight into the preliminary structure–activity relation-
ships of stilbenoids, 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.
1
0k - 10m
b
Compounds
Ar
IC50 (lM)
1
1
1
0k
2-Furyl
3-Furyl
3-Thienyl
0.4
0.5
0.1
0l
0m
a
12;13
The activities were measured as described previously.
The IC50 values were determined from triplicate tests.
b
Acknowledgements
significant improvements in the inhibitory activity of the
COX-2-mediated PGE production. Compounds 10k–m
were found to be almost three to fifteen times more
potent than their corresponding parent compounds
2
This study was supported by a grant from the Korea
Health 21 R&D Project, Ministry of Health & Welfare,
Republic of Korea (01-PJ2-PG6-01NA01-0002).
(
10h–j). Among these styrylheterocycles, 3-[2-(4-meth-
oxy-phenyl)-vinyl]-thiophene 10m exhibited a most
appreciable inhibitory activity in the sub-micromolar
range (IC50 ¼ 0.1 lM). This result led us to consider it as
a lead compound, with the view of developing a new
series of potent inhibitors of the COX-2-mediated PGE2
production.
References and notes
1
2
. Dubois, R. N.; Abramson, S. B.; Crofford, L.; Gupta, R.
A.; Simon, L. S.; Van de Putte, L. B. A.; Lipsky, P. E.
FASEB J. 1998, 12, 1063–1073.
. Vane, J. R.; Bakhle, Y. S.; Botting, R. M. Annu. Rev.
Pharmacol. Toxicol. 1998, 38, 97–120.
3. Prescott, S. M.; Fitzpatrick, F. A. Biochim. Biophys. Acta
2000, 1470, M69–M78.
4. Smith, W. L.; Garavito, R. M.; DeWitt, D. L. J. Biol.
Chem. 1996, 271, 33157–33160.
. Morita, I. Prostag. Other Lipid Mediat. 2002, 68-69, 165–
75.
. For a good review with citations, see: Gusman, J.;
Malonne, H.; Atassi, G. Carcinogenesis 2001, 22, 1111–
1
. Lee, D.; Cuendet, M.; Vigo, J. S.; Graham, J. G.;
Cabieses, F.; Fong, H. H. S.; Pezzuto, J. M.; Kinghorn,
A. D. Org. Lett. 2001, 3, 2169–2171.
8. Su, B.-N.; Cuendet, M.; Hawthorne, M. E.; Kardono, L.
B. S.; Riswan, S.; Fong, H. H. S.; Mehta, R. G.; Pezzuto,
J. M.; Kinghorn, A. D. J. Nat. Prod. 2002, 65, 163–169.
To obtain a clearer knowledge of the inhibitory mech-
anism of PGE
representative active styrylheterocycle 10h on the COX-
mRNA expression was investigated using RT-PCR
analysis as described previously with the minor modifi-
2
production, the suppressive effect of one
2
12;14
cations.
Treatment of LPS for 4 h dramatically in-
5
creased the expression of the COX-2mRNA levels, and
the induction was suppressed by the treatment of 10h in
a concentration-dependent manner, as depicted in Fig-
ure 2. This result suggests that the inhibitory effects of
active styrylheterocycles, against LPS-stimulated PGE2
production, might possibly be, at least, in part corre-
lated with the suppression of COX-2mRNA expression.
However, since the suppressive effect of COX-2mRNA
expression was not reached completely at the IC50 value
1
6
117.
7
2
of PGE production by the representative compound,
other mechanisms such as direct COX-2inhibitory
activity could not be excluded. Therefore, the inhibition
9
. Likhitwitayawuid, K.; Sawasdee, K.; Kirtikara, K. Planta
Med. 2002, 68, 841–843.
1
0. (a) Desiraju, G. R.; Gopalakrishnan, B.; Jetti, R. K.;
Nagaraju, A.; Raveendra, D.; Sarma, J. A.; Sobhia, M. E.;
Thilagavathi, R. J. Med. Chem. 2002, 45, 4847–4857; (b)
Chavatte, P.; Yous, S.; Marot, C.; Baurin, N.; Lesieur, D.
J. Med. Chem. 2001, 44, 3223–3230.
2
of PGE production in LPS-activated RAW264.7 cells
by styrylheterocyles might be due to either suppression
of COX-2expression or direct inhibition of COX-2
enzyme activity.
1
1. Kim, S.; Ko, H.; Park, J. E.; Jung, S.; Lee, S. K.; Chun,
Y.-J. J. Med. Chem. 2002, 45, 160–164.
12. Kim, S. S.; Oh, O.-J.; Min, H.-Y.; Park, E.-J.; Kim, Y.;
In conclusion, a series of lipophilic trans-stilbenoids
have been prepared and evaluated as to their effects on
Park, H. J.; Han, Y. N.; Lee, S. K. Life Sci. 2003, 73, 337–
3
48.
3. Measurements of PGE
cultured LPS-induced RAW264.7 cells: The level of
prostaglandin E (PGE ) production was measured by
an enzyme-immunometric assay, as described previously.
1
2
accumulation by COX-2in
2
2
1
2
RAW264.7 macrophage cells were maintained in DMEM,
supplemented with penicillin–streptomycin and 10% FBS,
at 37 ꢁC in 5% CO
2
humidified air. To evaluate the
inhibitory activity of the test materials on COX-2, the cells
were allowed to adhere, for 2h in the presence of aspirin
(250 lM) in a 96-well culture plate, changed with fresh
Figure 2. Effects of 10h on the expression of COX-2mRNA in LPS-
stimulated RAW264.7 cells, using RT-PCR analysis.

2108
S. K. Lee et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2105–2108
media, and further incubated for 22 h. The attached cells
(1 lg) of RNA was reverse transcribed into cDNA using
an avaian myeloblastosis virus (AMV) reverse transcrip-
tase and oligo(dT)15 primers (Promega Co., Madison, WI,
USA). The PCR samples, contained in 50 lL of the
were washed twice with PBS, and then incubated in the
fresh medium, with 1 lg/mL of LPS. The test materials
were simultaneously added to each well. After an addi-
tional 20 h incubation, the media were removed and
2
reaction mixture, comprised of 50 mM KCl, 5 mM MgCl ,
analyzed by the PGE
In these assays, 100% activity is defined as the difference
between the PGE accumulation in the absence and
presence of LPS for 20 h from triplicate determinations.
The percentage inhibition was expressed as [1)(PGE level
of sample/PGE
level of vehicle treated-control)] · 100.
4. Reverse transcription-polymerase chain reaction (RT-
2
enzyme immunometric assay (EIA).
0.16 mM dNTP, 5.0 units of Taq DNA polymerase
(Qiagen, Valencia, CA, USA), and 20 pmol of sense and
antisense primers in 10 mM Tris–HCl (pH 8.3). The sense
2
0
and antisense primers for COX-2were 5 -GGAGAGA-
0
0
2
CTATCAAGATAGTGATC-3 and 5 -ATGGTCAGTA-
0
2
GACTTTTACAGCTA-3 , respectively. The sense and
0
1
antisense primers for b-actin were 5 -TGTGATGGTG-
GGAATGGGTCAG-3 and 5 -TTTGATGTCACGCAC-
0
0
PCR) analysis of COX-2mRNA in RAW 62 4.7 cells:
6
0
RAW264.7 cells (5 · 10 cells––10 cm dish) were incubated
GATTTCC-3 , respectively. The PCR amplification was
for 4 h with or without various concentrations of test
samples and LPS (1 lg/mL). After washing with twice
PBS, total RNA was isolated from the cell pellet, using an
RNA isolation kit (Tri-reagent, Sigma Chemical Co., St.
Louis, MO, USA). The total amount of RNA was
determined by absorbance at 260 nm. One microgram
performed under the following conditions: 38 cycles of
denaturation at 94 ꢁC for 15 s, annealing at 55 ꢁC for 1 min
and extension at 72 ꢁC for 1 min, using a thermal cycler
(GeneAmp PCR Systems 2400; PE Applied Biosystems,
USA). The amplified PCR products were run on a 2%
agarose gel and visualized by SYBR Gold staining.