Styrylheterocycles: A novel class of inhibitors on lipopolysaccharide-Induced nitric oxide production

Article abstract of DOI:10.1016/j.bmcl.2003.08.013

A series of styrylheterocycles was prepared and their inhibitory activities against nitric oxide (NO) production were evaluated in a cell culture system using lipopolysaccharide-stimulated RAW264.7 macrophage cells. Our studies have identified a new series of inhibitors on NO production, providing the basis for further development of potent inhibitors. The preliminary structure-activity relationship, to elucidate the essential structural requirements, has been described. Mechanistic studies suggest that the suppression of iNOS mRNA transcription is, at least in part, related to the inhibitory activity of styrylheterocycles.

Full text of DOI:10.1016/j.bmcl.2003.08.013

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3689–3692
Styrylheterocycles: A Novel Class of Inhibitors on
Lipopolysaccharide-Induced Nitric Oxide Production
Sang Kook Lee,a Hye Young Min,a Sun Kyung Huh,a Eun-Young Kim,b Eunjung Lee,b
Soyoung Songb and Sanghee Kimb,*
aCollege of Pharmacy, Ewha Womans University, 11-1 Daehyun, Seodaemun, Seoul 120-750, South Korea
bNatural Products Research Institute, College of Pharmacy, Seoul National University, 28 Yungun, Jongro,
Seoul 110-460, South Korea
Received 12 July 2003; revised 7 August 2003; accepted 7 August 2003
Abstract—A series of styrylheterocycles was prepared and their inhibitory activities against nitric oxide (NO) production were
evaluated in a cell culture system using lipopolysaccharide-stimulated RAW264.7 macrophage cells. Our studies have identified a
new series of inhibitors on NO production, providing the basis for further development of potent inhibitors. The preliminary
structure–activity relationship, to elucidate the essential structural requirements, has been described. Mechanistic studies suggest
that the suppression of iNOS mRNA transcription is, at least in part, related to the inhibitory activity of styrylheterocycles.
# 2003 Elsevier Ltd. All rights reserved.
Endogenous nitric oxide (NO) is an inorganic free radi-
cal that has been implicated in a variety of pathophys-
iological responses, including vasodilation, nonspecific
host defense, ischemia reperfusion injury, chronic
inflammation and carcinogenesis.1ꢀ6 It is enzymatically
synthesized from l-arginine in numerous tissue and cell
types by a nitric oxide synthase (NOS).7ꢀ10 There are
three distinct isoforms of NOS: neural (nNOS), endo-
thelial (eNOS) and inducible (iNOS). Among them, the
iNOS is involved in the pathological responses mediated
by the overproduction of NO, and is overexpressed by
pro-inflammatory and/or carcinogenic stimuli, such as
interleukin-1b, tumor necrosis factor-a and lipopoly-
saccharide (LPS) in macrophages, endothelial cells and
smooth muscle cells.11,12 Thus, inhibitors of iNOS
enzyme activity or its induction have been postulated to
be beneficial in the treatment of numerous disease
mediated by the overproduction of NO.
nitroarginine (l-NA)14 and its methyl ester (l-NAME),
but the low potency and minimal selectivity have limited
their utility. More recently, a diverse class of non-ami-
noacid synthetic NOS inhibitors has appeared in the
literature, including guanidines,15 isothioureas,16 iso-
quinolinamines17 and carboxamidines.18 Even though
some of these exhibited the adequate levels of selectivity
and potency in vitro, their usefulness in vivo has
generally been limited by their poor bioavailability or
toxicity.19
Many natural products that inhibit the production of
NO have also been found, such as flavonoids, coumar-
ins and stilbenoids.20ꢀ22 Of these, stilbenoid has attrac-
ted our attention as a starting point for a new class of
inhibitors, due to its simplicity of structure and ease of
synthesis. It was seen, by comparing the reported activ-
ities of the various natural stilbenoids,22 that the inhib-
itory potency of stilbene compounds, against NO
production, was sensitive to the substitution patterns on
the stilbene template. On the basis of these studies, we
began to investigate the biological evaluation of synthetic
stilbene derivatives to find a potent inhibitor, which
might serve as a new lead for chemical optimization.
Intensive efforts are currently under way to try and dis-
cover potent and selective inhibitors of NOS. Some of
the earliest synthetic inhibitors are analogues of l-argi-
nine, such as N-monomethyl arginine (l-NMMA),13 N-
We designed and prepared a series of compounds, with
the general structure 1 (Fig. 1), where one of the phenyl
*Corresponding author. Tel.: +822-740-8913; fax: +822-762-8322;
e-mail: pennkim@snu.ac.kr
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.013

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S. K. Lee et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3689–3692
Z/E isomers was not possible by silica gel column chro-
matography, the Z/E mixtures 4 were converted to the
E-isomers [4a, 4h, 4o, and 4r (Table 1)] by heating with
catalytic amounts of iodine in refluxing heptane.25
Figure 1. General structure 1.
The compounds were evaluated for their inhibitory
activities on NO production in a cell culture system,
using LPS-activated RAW264.7 macrophage cells,
according to a previously documented procedure.26,27
When the cells had been treated with 1 mg/mL LPS for
20h, the NO production was markedly increased from
the basal level of 2.5ꢁ0.5 to 42.5ꢁ0.7 mM. Under this
experimental condition, the positive control l-NMMA
exhibited an IC50 value of 44.0 mM. The inhibitory
potencies, expressed as the IC50 values, of the synthe-
sized styrylfurans and styrylthiophenes are shown in
Table 1 and are compared with that of prototype nat-
ural stilbene resveratrol22,28 (IC50=15.9 mM). The
results in the present study demonstrated that nine
styrylheterocycles (4a, 4h, 4j and 4o–t) inhibited the
LPS-induced NO production more effectively than
resveratrol, whereas eight compounds (4d–g and 4k–n)
showed little activity (IC50 >50 mM).
rings of the stilbene was replaced by a five-membered
heteroaromatic ring. One of the rationales for this was
the observation of Hamley et al.,23 where the replacement
of the phenyl ring in 3,4-dihydro-1-isoquinolinamines,
with five-membered aromatic heterocycles, resulted in
increased potency. In addition, furanocoumarins have
shown greater inhibitory effects than simple coumar-
ins.20 Thus, we envisaged that the substitution by a five-
membered heteroaromatic ring on the stilbene template
could enhance the activity. Herein, we report the pre-
paration and evaluation of this class of compounds, as
well as the results of the preliminary structure–activity
relationship studies.
The preparation of the styrylheterocycles was carried
out according to Scheme 1. The stilbenoid skeletons of
styrylfuran and styrylthiophene could be constructed by
Wittig reactions, between a five-membered aromatic
heterocyclic aldehyde and an aromatic phosphonium
ylide. Modified Wittig reactions,24 between phospho-
nium bromide 2a–c and commercially available alde-
hydes 3a–d (1.1 equiv) in the presence of freshly
powdered KOH and a catalytic amount of 18-crown-6
in CH2Cl2 at room temperature yielded a mixture of
olefins 4 (>85%) with a Z/E ratio of ca. 1:1 to 5:1 by
TLC intensity. The cis/trans isomers were separated by
silica gel column chromatography, and the structures
The styrylheterocycles having a 3,4,5-trimethoxy group
on the phenyl ring, 4o–t, generally exhibited greater
inhibitory activities than the corresponding compounds
with 3,5-dimethoxy (4h–n) or 4-methoxy groups (4a–g).
As with the 3,4,5-trimethoxyphenyl series, all the fur-
anyl (4o–q) and thiophenyl (4r–t) derivatives showed 2-
to 3-fold greater inhibitory activities than resveratrol.
However, for the 3,5-dimethoxyphenyl (4h–n) or
1
Table 1. Inhibitory effects of styrylfurans and styrylthiophenes on
the NO production in LPS-activated mouse macrophage RAW264.7
cells
were determined from the characteristic H NMR cou-
pling constants of the olefinic protons [16–17 Hz for
(E)-4 and 12 Hz for (Z)-4]. When the separation of the
Compd
R1
R2
Ar
E/Z IC50 (mM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
2-Furanyl
3-Furanyl
3-Furanyl
2-Thiophenyl
2-Thiophenyl
3-Thiophenyl
3-Thiophenyl
2-Furanyl
E
Z
E
Z
E
Z
E
E
Z
E
Z
E
Z
E
E
Z
E
E
Z
E
E
13
34.6
31.6a
>50
>50
>50
>50
4.8a
18.7
13.5
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
3-Furanyl
3-Furanyl
2-Thiophenyl
2-Thiophenyl
3-Thiophenyl
3-Thiophenyl
2-Furanyl
3-Furanyl
3-Furanyl
2-Thiophenyl
3-Thiophenyl
3-Thiophenyl
4-Hydroxyphenyl
>50
>50
>50
>50
5.9
6.6
9.5
5.3
5.6
7.8a
15.9
H
H
4p
4q
4r
4s
4t
Resveratrol
OH
H
aThese compounds exhibited cytotoxicity at 50 mM. At lower con-
centrations the cell viability was maintained over 90%.
Scheme 1. Synthetic approach employed for the preparation of the
styrylheterocycles described in Table 1.

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3691
References and Notes
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Figure 2. Effects of 4r on the expression of iNOS mRNA in LPS-
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4-methoxyphenyl (4a–g) series, only the furanyl deriva-
tives exhibited comparable activities. These results indi-
cate that the potency of these compounds is highly
dependent on the substitution pattern of the phenyl
ring. However, to our surprise, there was no significant
correlation between the double-bond geometry and the
inhibitory activity. Compounds with Z-double bonds
(4b, 4i, 4p and 4s) showed almost equipotent activities
compared with their corresponding E-double bonds
compounds (4c, 4j, 4q and 4t). In the 2-furanyl deriva-
tives (4a, 4h and 4o), the inhibitory activity on NO
production by iNOS was enhanced with respect to their
corresponding 3-furanyl analogues (4c, 4j and 4q).
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McInally, T.; Nicholls, D. J.; Pimm, A. D.; Wallace, A. V. J.
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To elucidate the possible mechanisms of active styryl-
heterocycles, the inhibition of the production of NO by
compound 4r was examined in relation to the suppres-
sion of the iNOS mRNA expression in the LPS-acti-
vated RAW264.7 cells, using RT-PCR analysis.29
Treatment with LPS, for 6 h, dramatically increased the
level of iNOS mRNA expression, and the induction
mRNA was suppressed by treatment with 4r in a dose-
dependent manner, as shown in Figure 2. The RT-PCR
analysis indicated that the inhibition of the NO pro-
duction by active compounds was correlated with the
suppression of the iNOS mRNA gene expression. This
result indicates that active styrylheterocycles, at least in
part, suppress the pathway of stimulation by LPS to
iNOS gene expression, thereby alleviating the produc-
tion of NO.
22. Kageura, T.; Matsuda, H.; Morikawa, T.; Toguchida, I.;
Harima, S.; Oda, M.; Yoshikawa, M. Bioorg. Med. Chem.
2001, 9, 1887.
23. Beaton, H. G.; Boughton-Smith, N.; Hamley, P.; Ghelani,
A.; Nicholls, D. J.; Tinker, A. C.; Wallace, A. V. Bioorg. Med.
Chem. Lett. 2001, 11, 1027.
24. Bellucci, G.; Chiappe, C.; Lo Moro, G. Tetrahedron Lett.
1996, 37, 4225.
25. Kim, S.; Ko, H.; Park, J. E.; Jung, S.; Lee, S. K.; Chun,
Y.-J. J. Med. Chem. 2002, 45, 160.
In conclusion, we have designed and evaluated a series
of styrylheterocycles as inhibitors on NO production by
iNOS. Several compounds have shown potent inhibi-
tory activity towards the LPS-induced NO production.
The present study suggests that the suppression of iNOS
mRNA transcription is, at least in part, related to the
inhibitory activity of styrylheterocycles. This new series
of inhibitors are suggested as lead compounds for the
development of potent and selective inhibitors for
potential therapeutic use. Further studies toward more
potent inhibitors, based on these findings, are currently
in progress in our laboratory.
26. Hong, C. H.; Noh, M. S.; Lee, W. Y.; Lee, S. K. Planta
Med. 2002, 68, 545.
27. Measurements of Nitric oxide formation by iNOS in cul-
tured LPS-induced RAW264.7 cells: RAW264.7 macrophage
cells, in 10% FBS-DMEM without phenol red media, were
plated in 24-well plates (8ꢂ105 cells/mL), and then incubated
for 24 h. The cells were replaced with new media, and then
incubated in the medium, with 1 mg/mL of LPS, in the pres-
ence or absence of test samples. After an additional 20h of
incubation, the media were removed and analyzed for nitrite
accumulation, as an indicator of NO production, using the
Griess reaction.26 Briefly, 150 mL of Griess reagent (0.1%
naphthylethylenediamine and 1% sulfanilamide in 5% H3PO4
solution) was added to 100 mL of each LPS supernatant, or
sample-treated cells, in triplicate. The absorbance was read at
540nm, and compared against a standard curve of sodium
Acknowledgements
This study was supported by a grant of the Korea
Health 21 R&D Project, Ministry of Health & Welfare,
Republic of Korea (HMP-00-CH-15-0014).

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S. K. Lee et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3689–3692
nitrite. The percentage inhibition was expressed as [1ꢀ(NO
level of sample/NO level of vehicle-treated control)]ꢂ100.
Dose-response curves were prepared, and the results typically
expressed as the IC50 values.
28. For a good review with citations, see: Gusman, J.; Mal-
onne, H.; Atassi, G. Carcinogenesis 2001, 22, 1111.
29. Reverse transcription-polymerase chain reaction (RT-PCR)
analysis of iNOS mRNA expression: RAW264.7 cells (5ꢂ106
cells—10cm dish) were incubated for 6 h with, and without,
various concentrations of test samples and LPS (1 mg/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 the absorbance at 260nm. One
microgram (1 mg) of RNA was reverse transcribed into cDNA
using an avaian myeloblastosis virus (AMV) reverse tran-
scriptase and oligo(dT)15 primers (Promega Co., Madison,
WI, USA). The PCR samples, contained in 50 mL of the reac-
tion mixture, comprised of 50mM KCl, 5 mM MgCl , 0.16
2
mM dNTP, 5.0units of Taq DNA polymerase (Qiagen,
Valencia, CA, USA) and 20pmol of sense and antisense pri-
mers in 10mM Tris–HCl (pH 8.3). The sense and antisense
primers for iNOS were 50-ATGTCCGAAGCAAACATCAC-
30 and 50-TAATGTCCAGGAAGTAGGTG-30, respectively.
The sense and antisense primers for b-actin were 50-
TGTGATGGTGGGAATGGGTCAG-30
and
50-
TTTGATGTCACGCACGATTTCC-30, respectively. The
PCR amplification was performed under the following condi-
tions: 25 cycles of denaturation at 94 ꢃC for 30s, annealing at
55 ꢃC for 30s and extension at 72 ꢃC for 30s, using a thermal
cycler (GeneAmp PCR Systems 2400; PE Applied Biosystems,
USA). The amplified PCR products were separated on a 2%
agarose gel and visualized by SYBR Gold staining. The bands
in the gel were photographed, and the fluorescence intensities
analyzed using an Alpha ImagerTM (Alpha Innotech Corp.,
USA).