Vol.2, No.3, 37-45 (2013) Modern Research in Inflammation
http://dx.doi.org/10.4236/mri.2013.23005
Synthesis and bioevaluation of novel heterostilbenes
as potential anti-inflammatory and anti-angioge nic
agents
Chien-Ming Huang1,2, An-Rong Lee3, Jiajiu Shaw4, Wen-Hsin Huang3*
1Division of Pharmacy, Cheng-Hsin General Hospital, Taipei, China
2Department of Nursing, Cardinal Tien College of Healthcare & Management, Taipei, China
3School of Pharmacy, National Defense Medical Center, Taipei, China;
*Corresponding Author:
4JAK3 Pharma, Inc., Ann Arbor, USA
Received 10 April 2013; revised 10 May 2013; accepted 10 June 2013
Copyright © 2013 Chien-Ming Huang et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Novel heterocyclic analogs of resveratrol, (E)-
stilbene analogs, were readily prepared by con-
jugation of a heterocyclic benzothiazolium moi-
ety with the core styrene structure of resveratrol
and evaluated for their biological properties. The
result s sho wed that these analogs were superior
to resveratrol in 1) anti-angiogenesis in vitro, 2)
nitric oxide inhibition in vitro, and 3) inhibition of
carrageenan-induce d edema in vivo. In summary,
introduction of a heterocyclic benzothiazolium
moiety to replace one of two aromatic rings from
the core stilbene structure of resveratrol pro-
vided beneficial biological properties and is wor-
thy of further investigation.
Keywords: Resveratrol Analogs; Benzothiazolium;
Anti-Angiogenesis; Carrageenan-Induced Edema
1. INTRODUCTION
Inflammation is a symptom standing for cell injury
that plays a housekeeping role in diseases triggered by
inflammatory cells such as macrophages, microglial, and
mast cells that acutely or chronically mediate and/or ag-
gravatedly activate pathology inducing the primary re-
sponse of the innate immune system to damage and to
promote disease progression.
As an effective defense in biology, the inflammatory
response usually lacks specificity and overreacts to cause
significant bystander damage which activated in resp-
onse to pathogens or tissue damage and amplified the
inflammatory response to induce production of inflam-
matory mediators including nitric oxide, reactive oxygen
species, proinflammatory cytokine s (such as IL-1, NFκB,
AP-1, IL-6, IL-17, IL-18, TGFβ, and TNFα), chemokines
(MMPs, MCP-1) and prostaglandins, associated with
vicious circle of cell death, and so on [1,2].
Nitric oxide (NO), one of reactive nitrogen species, is
an endothelial survival factor, it inhibits apoptosis, en-
hances endothelial cell proliferation and/or migration,
and might increase the VEGF or fibroblast growth factor
expressions in part, but inducible nitric oxide (NO) pro-
duction by activated inflammatory cells which further
contribute to tissue damage or degradation, and/or ulti-
mate cell death. NO is also one of angiogenesis media-
tors and vascular endothelial growth factor (VEGF) mu-
tually triggers the release of NO from cultured human
umbilical venous endothelial cells and even up-regulates
the expression of nitric oxide synthase (NOS) [3,4].
The bio-complexities of NO-angiogenesis pathway be-
come pathological evidences related to neovasculariza-
tion in the convoluted biological context of arthritis,
atherosclerosis, and tumor ever more, whereas angio-
genic factors accelerate it [5,6].
Most biological processes such as angiostatin gene-
rated from the degradation of plasminogen by matrix
metalloproteinases (MMPs), a known endogenous in-
hibitor of angiogenesis, modulate response to keep ho-
meostasis, however, an issued that NO may trigger
countervailing effects to suppress the production of an-
giostatin [7]. Investigations issued that angiogenesis is
attenuated when NO bioactivity is reduced. Furthermore,
anti-inflammatory mechanisms include repression of pro-
inflammatory mediators including NO, cytokines, chemo-
kines, matrix metalloproteases, and prostaglandins in in-
flammatory cells [5,6].

C.-M. Huang et al. / Modern Research in Inflammation 2 (2 013) 37-45
38
Resveratrol, 3,5,4’-trihydroxy-trans-stilbene (1), is a
polyphenol found in grapes, blue berries, peanuts, and a
number of other plants [8-10]. Resveratrol exhibits a
variety of useful biological properties including antileu-
kemic, antibacterial, antifungal, antiplatelet aggregation,
coronary vasodilator, antioxidative, and antiinflamma-
tory activities and so on [11-13]. In the specific field of
arthritis, it was recently reported that resveratrol inhibits
the proliferation of synoviocytes in rheumatoid arthritis
(RA) in vitro [14]. Elmali et al. suggested that intra-arti-
cular injection of resveratrol may protect cartilage against
the development of experimentally induced inflamma-
tory arthritis [15]. Resveratrol has been reported to in-
hibit nitric oxide production by lipopolysaccharide-acti-
vated brain microglia [16]. Resveratrol revealed effec-
tively anti-angiogenic action as well [17].
Due to the beneficial effects of resveratrol, many at-
tempts to modify its structure have been made by scien-
tists to further improve its water-solubility and biological
activities. A number of hetercyclic resveratrol analogs
have been reported and some of them have shown inter-
esting biological activities [18-22]. However, for most of
the reported analogs, the (E)-stilbene core structure is
either maintained intact or only slightly modified.
We hypothesized that it is not necessary to maintain
the core structure of resveratrol in order to improve its
biological activities. Based on this hypothesis, we intro-
duced a novel heterocyclic benzothiazolium moiety into
the (E)-stilbenes, made four analogs first, and investi-
gated their biological properties.
2. METHODS
2.1. Chemistry
Research chemicals were purchased from Sigma-Al-
drich or Alfa Aesar and used at least 95% purity without
further purification. The reference compound, resveratrol
(99% GC purity), was purchased from Sigma-Aldrich.
Reactions were monitored by thin-layer chromatography
(TLC) on silica gel plates (60 F254; Merck) visualizing
with ultraviolet light or iodine. Melting points were taken
in open capillary tubes on a Buchi-530 melting point
apparatus and are uncorrected. 1H-NMR spectra were
determined on a Varian Gemini-300 NMR instrument.
Chemical shifts (  ) are reported in parts per million (ppm)
relative to tetramethylsilane (TMS) as an internal stan-
dard; coupling constants (J) are shown in hertz (Hz) and
signals are described as s (singlet), d (doublet), t (triplet),
and m (multiplet). IR spectra were recorded on a Per-
kin-Elmer FTIR 1610 series infrared spectrophotometer
in KBr discs. Fast atom bombardment (FAB) mass spec-
tra were recorded using a JEOL-SX102A (GC/LC/MS)
spectrometer. Only peaks of significant relative intensity
are presented in the form of m/z (intensity relative to
base peak). Elemental analyses for carbon, hydrogen,
nitrogen and sulfur were performed in the Instrument
Center of the National Science Counsel at the National
Taiwan University using HERAEUS VarioEL analyzer.
3-Ethyl-2-methylbenzothiazolium bromide (2).
2-Methylbenzothiazole (1.5 g, 10 mmol) was reacted
with ethyl bromide (1.5 ml, 20 mmol) over 5 h in a spe-
cial closed glass tube (explosive-proof) using CEM mi-
crowave reactor under 250 watts, 120˚C, to obtain the
product as precipitate. Filter and wash the precipitate
with ethyl ether or dichloromethane to obtain the pure
product (1.47 g, 57% yield). The product is soluble in
acetone and in water. Alternative method without the
microwave reactor: 2-methylbenzothiazole (1.5 g, 10
mmol) was reacted with ethyl bromide (15 ml, 200 mmol)
in 100 ml of acetonitrile (as the solvent) under reflux
over 72 h to obtain the product as precipitate. Filter and
wash the precipitate with ethyl ether or dichloromethane
to obtain the title compound (0.89 g, 32% yield). ); mp
240˚C - 241˚C; 1H-NMR (300 MHz, DMSO-d6)
 : 1.44
(3H, t, J = 7.2 Hz), 3.19 (3H, s), 4.75 (1H, q, J = 7.2 Hz),
7.78 (1H, t, J = 8.4 Hz), 7.88 (1H, t, J = 8.4 Hz), 8.32
(1H, d, J = 8.4 Hz), 8.43 (1H, d, J = 8.4 Hz), FAB-MS
m/z (%): 178 (M+-Br, 19%).
N-Ethyl-2-[(E)-2-(2’-hydroxy-4’-methoxyphenyl)viny
l] benzothiazolium bromide (3).
To 15 ml of n-propanol was added 258 mg (1 mmol)
of 2, 228 mg (1.5 mmol) of 2-hydroxy-4-methoxyben-
zaldehyde, and catalytic amount of zinc chloride. The
mixture was refluxed for 3 h. At the completion of the
reaction, 30 ml of ether/hexane (1/1, v/v) was added to
the solution to precipitate the crude product. H2O/Me-
OH/acetone (1/1/2, v/v/v) was used to recrystallize
highly purified product. Bricky red powder; yield 0.36 g
(92%); mp 168˚C - 170˚C; 1H-NMR (300 MHz, DM-
SO-d6)  : 1.44 (3H, t, J = 7.2 Hz), 3.81 (3H, s), 4.83 (1H,
q, J = 7.2 Hz), 6.52 (1H, d, J = 2.1 Hz), 6.60 (1H, dd, J =
8.7, 2.1 Hz), 7.23 (1H, t, J = 7.2 Hz), 7.81 (1H, d, J =
15.6 Hz), 7.82 (1H, t, J = 7.2 Hz), 8.00 (1H, d, J = 8.7
Hz), 8.23 (1H, d, J = 15.6 Hz), 8.24 (1H, d, J = 8.7 Hz),
8.33 (1H, d, J = 8.1 Hz), 10.59 (1H, s); IR (KBr) cm−1:
3150 (br, OH), 1597, 1573, 1513, 1440; FAB-MS m/z
(%): 313 (M+-Br, 27%). Anal. (C18H18BrNO2S·3.5H2O)
C, H, N, S.
N-Ethyl-2-[(E)-2-(3’,4’,5’- trimethoxyph enyl)vin yl]be
nzothiazolium bromide (4).
The same method for 3 was used except 2-hydroxy-
4-methoxybenzaldehyde was replaced by 247.5 mg (1.5
mmol) of 3,4,5-trimethoxybenzaldehyde. Earth yellow
powder; yield 0.41 g (94%); mp 240˚C - 242˚C;
1H-NMR (300 MHz, DMSO-d6)
 : 1.48 (3H, t, J = 7.2
Hz), 3.77 (3H, s), 3.90 (6H, s), 5.00 (1H, q, J = 7.2 Hz),

C.-M. Huang et al. / Modern Research in Inflammation 2 (2 013) 37-45 39
7.42 (2H, s), 7.79 (1H, t, J = 8.1 Hz), 7.88 (1H, t, J = 8.1
Hz), 7.93 (1H, d, J = 15.9 Hz), 8.19 (1H, d, J = 15.9 Hz),
8.30 (1H, d, J = 8.7 Hz), 8.45 (1H, d, J = 8.7 Hz). IR
(KBr) cm−1: 3421 (br, OH), 1610, 1576, 1503, 1452.
FAB-MS m/z (%):357 (M+-Br, 57%). Anal. (C20H22Br-
NO3S·3H2O) C, H, N, S.
N-Ethyl-2-[(E)-2-(3’,5’-d imethoxy-4’-h ydroxyphen yl)
vinyl]benzothiazolium bromide (5).
The same method for 3 was used except 2-hydro-
xy-4-methoxybenzaldehyde was replaced by 273 mg (1.5
mmol) of 3, 5-dimethoxy-4-hydroxybenzalde hyde (mm-
ol). Scarlet powder; yield 0.32 g (89%); mp 238˚C -
240˚C. 1H-NMR (300 MHz, DMSO-d6)
 : 1.46 (3H, t, J
= 6.9 Hz), 3.88 (6H, s), 4.95 (1H, q, J = 6.9 Hz), 7.41
(2H, s), 7.75 (1H, t, J = 7.2 Hz), 7.80 (1H, d, J = 15.9
Hz), 7.84 (1H, t, J = 7.2 Hz), 8.15 (1H, d, J = 15.9 Hz),
8.24 (1H, d, J = 8.1 Hz), 8.40 (1H, d, J = 8.1 Hz), 9.75
(1H, s). IR (KBr) cm−1: 3427 (br, OH), 1585, 1508, 1443.
FAB-MS m/z (%): 343 (M+-Br, 48%). Anal. (C19H20Br-
NO3S·3H2O) C, H, N, S.
N-Ethyl-2-[(E)-2-(4’-hydroxy-3’-methoxyphenyl)viny
l]benzothiazolium bromide (6).
The same method for 2 was used except 2-hydro-
xy-4-methoxybenzaldehyde was replaced by 228 mg (1.5
mmol) of 3-methoxy-4-hydroxybenzaldehyde. Brick red
powder; yield 0.35 g (89%); mp 148˚C - 150˚C.
1H-NMR (300 MHz, DMSO-d6)
 : 1.46 (3H, t, J = 7.2
Hz), 3.83 (3H, s), 4.94 (1H, q, J = 7.2 Hz), 6.94 (1H, d, J
= 8.1 Hz), 7.57 (1H, dd, J = 8.1, 2.1 Hz), 7.66 (1H, d, J =
2.1 Hz), 7.75 (1H, t, J = 7.5 Hz), 7.80 (1H, d, J = 15.3
Hz), 7.84 (1H, t, J = 7.5 Hz), 8.15 (1H, d, J = 15.3 Hz),
8.24 (1H, d, J = 8.4 Hz), 8.39 (1H, d, J = 7.8 Hz), 10.29
(1H, s). IR (KBr) cm−1: 3434 (br, OH), 1583, 1511, 1440.
FAB-MS m/z (%): 313 (M+-Br, 43%). Anal. (C18H18Br-
NO2S·4H2O) C, H, N, S.
2.2. In-Vitro Biological Studies
2.2.1. Nitric Oxide Inhibition
The mouse BALB/c macrophage cell line, RAW 264.7
BCRC No. 60001 (identical to ATCC number TIB-71),
was obtained from Bioresource Collection and Research
Center, Taiwan. The cells were maintained according to
the protocols being cultured in 50 cm2 plastic flasks
(Nunc, Roskilde, Denmark) with the medium renewed
every 3 days. LPS (from Escherichia coli, serotype
0127:B8), trypan blue and all the other chemicals, unless
otherwise specified, were purchased from Aldrich-Sigma
Chemical Company (St. Louis, MO, USA).
Determination of cell viability by an MTT assay. To
evaluate the cell viability, a methylthiazoletetrazolium
bromide (MTT) assay was conducted by the standard
method in our laboratory. Incubation was performed after
pretreating with a combination of the test compounds, in
stock concentrations of 25, 50, 100, and up to 200 μM in
dimethyl sulfoxide, and LPS (100 ng/ml) in normal sa-
line for 24 h. Untreated cells were used as the control.
Nitrite quantification. The cells were cultured with or
without a pretreatment by the test compounds and res-
veratrol of 25, 50, 100, and up to 200 μM as already de-
scribed. Each value is the mean ± SEM of three determi-
nations. *p < 0.05, each value was compared with or
without the LPS-stimulated group.
The production of NO was determined by measuring
the accumulated level of nitrite in the culture supernatant
with the Griess reagent in LPS-stimulated macrophage
cells. A quantity of 100 μl of a sample aliquot were
mixed with 100 μl of the Griess reagent (0.1% N-(1-na-
phthyl)ethylenediamine, 1% sulfanilamide, and 2.5%
phosphoric acid) in a 96-well plate and then incubated at
25˚C for 10 min. The absorbance at 550 nm was mea-
sured with an ELISA reader (MR 700, Dynatech Labo-
ratories, Alexandria, VA, USA). NaNO2 was used as the
standard to calculate the nitrite concentration.
2.2.2. Anti-Angiogenesis Study
Capillary-tube formation was assayed using the angi-
ogenesis kits (Kurabo, Okayama, Japan) according to the
manufacturer’s instruction. Briefly, human umbilical vein
endothelial cells (HUVECs) cocultured with normal hu-
man dermal fibroblasts (NHDF), which a cultural kit
originally developed by Kurabo Co. Ltd., Okayama, Ja-
pan, onto 24-well plate were incubated either with or
without test compounds and resveratrol respectively at
appropriate concentrations (1 - 100 µM) for 11 d (media
replacement was performed at d 1, 4, 7, and 9). The cells
were thereafter fixed with 70% cold ethanol, incubated
with mouse antihuman CD31 antibody followed by in-
cubation with an alkaline phosphatase conjugate goat
antimouse antibody, and then stained with 5-bromo-4-ch-
loro-3-indolyl phosphate/nitro blue tetrazolium to visu-
alize tube network at d-11, respectively. In each assay,
randomly selected five fields of view in each well were
captured by digital camera under the light microscopy on
x40 magnification. The analysis of tube formation was
then carried out by measuring total area and length of
tubes per well using analytical software (by Kurabo).
2.2.3. In-Vivo Carrageenan-Induced Paw Edema
Study
In this study, λ-carrageenan was used to stimulate the
inflammation on the rear footpad of the rats. Male albino
Wistar rats (183 - 218 g) 4 to 6 weeks old were housed
and cared under the guidelines of the Institutional Ani
mal Care and Use Committee at the National Defense
Medical Center, Taiwan. The rats were assigned to
groups. For each group, 3 rats were used and marked on

C.-M. Huang et al. / Modern Research in Inflammation 2 (2 013) 37-45
40
the tails. one of them being the control. In order to in-
duce inflammation, 50 μl of a 1% λ-carrageenan solution
in normal saline was injected into the right hind paw
subplantar tissue. The development of paw edema was
measured plethysmographically (Basile 7140 plethys-
mometer, Ugo, Varese, Italy) and recorded prior to this
administration. One hour before the λ-carrageenan chal-
lenge, a sample preparation (20 mg/kg in CMC) includes
resveratrol (1) and its analogs 3, 4, 5 and 6. Before carra-
geenan treatment, the volume of each left footpad was
measured (0
V). Each rat was injected i.p. with the fol-
lowings: 1) Control Group: medium only; 2) Testing
Groups (one group for each compound), compound sus-
pension in carboxymethyl cellulose (CMC) (20 mg/kg);
and 3) Comparison group: resveratrol suspension in CMC
(20 mg/kg) was injected i.p. into the rat in the test group.
After the λ-carrageenan challenge, each paw volume (ml)
was measured hourly up to 5 h. The percentage of paw
edema and the inhibition of inflammation were calcu-
lated. Edema rate (E%) was calculated as follows:

00
%

100EVVV 
t: volume of hind paw before 1%
λ-carrageenan administration; V: volume of hind paw
after 1% carrageenan administration at t h. Percentage of
inhibition (I%) was determined as follows:
%1E00
ctc ; c: edema rate of control
group; Et: edema rate of the respective test compound at
t h.
IEEE 
2.3. Statistical Analysis
Each experimental data value is expressed as the mean
± SEM. The statistical significance of differences was
assessed with an analysis of variance (ANOVA), fol-
lowed by Tukey’s test or Student’s test between two
groups. Differences with p values of less than 0.05 are
considered statistically significant. The statistical sig-
nificance of differences was assessed with an analysis of
variance (ANOVA), followed by Tukey’s test or Stu-
dent’s test between two groups. Each value represents the
mean ± SEM, n = 5, *P < 0.05 compared with the control
(0.1 ml normal saline), **P < 0.05 compared with posi-
tive control (ibuprofen), respectively.
3. RESULTS
3.1. Synthesis
Two methods were utilized for the synthesis of
3-ethyl-2-methylbenzothiazolium bromide 2): a) in a
CEM Discover microwave reactor, and b) by a tradi-
tional reflux, in 57% and 32% yields, respectively.
Compound 2 was then used as the key starting material
to react with the respective benzaldehydes to synthesize
four heterocyclic resveratrol analogs 3 - 6 (Scheme 1 and
Figure 1).
N
SCH3
(a)
N
SCH3
+
Br
(b)
N
S
+
Br
R
23-6
Scheme 1. Synthesis of resveratrol analogsa. aReagents and
conditions: (a) C2H5Br in CEM microwave under 250 watts,
120˚C. for 5 hr. (b) reacted with respective benzaldehyde and
trace amount of ZnCl2 in n-propanol and reflux for 3 hr.
Figure 1. Structures of resveratrol, (E)-stilbene, and
compounds 3 - 6.
3.2. Biological Evaluation
3.2.1. In-Vitro Nitric Oxide Inhibition (Based on
Nitrite)
These analogs 3 - 6 compared to resveratrol were
evaluated for the inhibition of nitric oxide (NO) based
on the accumulation of nitrite by activated macro-
phages in vitro. The results of NO inhibition in vitro as
indicated by the reduction of nitrite are shown in Table
1. The results indicated that, under current experi-
mental condition, all the four compounds showed bet-
ter inhibition on NO production below at a cut-off
concentration (25 μM) in vitro as compared to res-
veratrol (1) (40 μM), a natural polyphenol.
3.2.2. In-Vitro Anti-Angiogenesis
As an initial screen for biological activity, compounds
3 - 6 were evaluated for anti-angiogenesis in vitro against
resveratrol. The IC50 values of anti-angiogenesis in vitro
(by Kurabo software) were shown in Table 1. Represen-
tative images of the anti-angiogenesis were shown in
Figure 2.
The results indicate that, under current experimental
condition, all four resveratrol analogs are more effective
anti-angiogenic agents in vitro as compared to resveratrol
(1) (IC50 at 47.7 ± 1.1 μM fr HUVEC tube formation o

C.-M. Huang et al. / Modern Research in Inflammation 2 (2 013) 37-45 41
Table 1. IC50 values of in vitro anti-angiogenesis activity and NO.
Compound Anti-angiogenesis IC50 (µM) NO inhibition IC50 (µM)
Control (Medium only) N/A N/A
Resveratrol (1) 47.7 ± 1.1 40
3 31.2 ± 2.0 <25
4 18.8 ± 1.3 <25
5 21.2 ± 1.6 <25
6 19.9 ± 2.3 <25
inhibition); analogs 4, 5 and 6 (comparable IC50s = 18.8
± 1.3, 21.2 ± 1.6, and 19.9 ± 2.3 μM, respectively) ap-
peared to be better than analog 3 (31.2 ± 2.0 μM).
3.2.3. In-Vivo Carrageenan-Induced Edema
Study
A state of local acute inflammation was evoked by in-
jecting 1% (w/v) λ-carrageenan (0.1 ml/paw) s.c. into the
plantar surface of the right hind paw of the rat, with the
left paw (CMC treated) serving as a control. These ana-
logs of resveratrol were evaluated for their in vivo
anti-inflammatory activity at a single dose of 20 mg/kg
using the carageenan-induced paw edema method in rats.
In the vehicle-treated control group, the mean volume of
the right hind paws increased by 0.71 ± 0.07 ml equiva-
lent to 82.8% edema at 5 h after a carrageenan challenge
(Figure 3). Our results obviously showed that the syn-
thetic resveratrol analogs 3 - 6 exerted a striking anti-in-
flammatory effect with a significant reduction in swell-
ing compared to resveratrol at rats. Among them, com-
pound 4 manifested the greatest inhibition of 69.5% (paw
edema, 0.21 ± 0.06 ml, P < 0.05) after 5 h.
4. DISCUSSION
4.1. Synthesis
At first, we prepared a key heterocyclic starting mate-
rial, 3-ethyl-2-methylbenzothiazolium bromide (2), by a
traditional reflux over 72 h in a low yield (32%) and us-
ing a large amount of solvent. Alternatively, it appeared
that microwave-assistant synthesis for compound 2 in
higher yield (57%) was more efficient and a saving to
take a short time from 72 h to 5 h and to use a little
amount solvent in a closed reaction system. Compound 2
was then used as the key starting material to react with
the respective benzaldehydes to synthesize four hetero-
cyclic resveratrol analogs 3 - 6 (Scheme 1 and Figure 1)
in high yields about or over 90%.
4.2. Biological Evaluation
4.2.1. In-Vitro Nitric Oxide Inhibition (Based On
Nitrite)
The effects of the resveratrol analogs on cell viability
were evaluated by RAW 264.7 macrophages at first. In
the MTT assay, none of the analogs including resveratrol
itself were toxic at various concentrations of 25, 50, 100,
and up to 200 μM on LPS-activated murine RAW 264.7
over 24 h. The cell viability was in the 85% - 95% range.
On the basis of their non-toxicity to macrophages, the
effects of the test compounds on NO production were
further evaluated.
In order to evaluate the cytoprotective ability at the
NO production level at the 24-h time point, one of the
oxidative stress mediators, LPS-elicited RAW 264.7
macrophages were used in our experiments, The IC50
values are shown in Tabl e 1, compounds 3 - 6 showed
better inhibition on NO production below a cut-off con-
centration (25 μM) in vitro currently as compared to res-
veratrol (1) (40 μM), yet no more further screenings at
NO inhibition were conducted but furthermore focused
on anti-angiogenic testings.
Interestingly resveratrol itself has a paradoxical effect
on the NO production in biological system that might be
no effect at low concentration or short-term incubation
with resveratrol but differentially boosts the NO produc-
tion at high concentration over 50 μM and after long-te-
rm use of resveratrol in a time- and concentration-depen-
dent manner of endothelial cells. [23]
However, in macrophage cells (RAW 264.7), resvera-
trol for the inhibition of LPS-induced nitric oxide pro-
duction by considering the involvement of an estrogen
receptor was inhibited only when cells were treated with
resveratrol prior to LPS-stimulation at a higher concen-
tration of resveratrol [24].
4.2.2. In-Vitro Anti-Angiogenesis
It is now accepted that angiogenesis is central to
maintaining and promoting rheumatoid arthritis (RA)
and several studies in human have suggested that block-
ing angiogenesis during the course of RA may be of
therapeutic benefits [25-27]. As an initial screen for
anti-angiogenesis in vitro, compounds 3 - 6 compared to
resveratrol were conducted. Among four resveratrol ana-
logs, analogs 4, 5 and 6 are comparable and more effec-
tive anti-angiogenic agents in vitro than resveratrol (1),

C.-M. Huang et al. / Modern Research in Inflammation 2 (2 013) 37-45
42
(a)
(b)
Figure 2. Representative images of the anti-angiogenesis (40×) by the test compounds.
in consistence with an antiangiogenic action at 20 - 80
μM for HUVEC tube formation on Matrigel [28] and ap-
peared to be a little better than analog 3.
4.2.3. In-Vivo Carrageenan-Induced Edema
Study
Carrageenan-induced rat paw edema, an in-vivo model

C.-M. Huang et al. / Modern Research in Inflammation 2 (2 013) 37-45 43
of acute inflammation for a variety of inflammatory
and/or oxidative stress mediators with biphasic inflam-
matory nature, is the standard and most commonly used
technique to screen inflammatory and/or anti-inflamma-
tory activity. A state of local acute inflammation was
evoked by injecting 1% (w/v) λ-carrageenan (0.1 ml/paw)
s.c. into the plantar surface of the right hind paw of the
rat, with the left paw (vehicle treated) serving as a con-
trol. In the vehicle-treated control group, the mean vol-
ume of the right hind paws increased by 0.71 ± 0.07 ml
at 5 h after a carrageenan challenge. Our results (Figure
3) obviously showed that the synthetic resveratrol ana-
logs 3 - 6 exerted a strikingly strong anti-inflammatory
effect with a significant reduction in swelling compared
to resveratrol at rats. Among them, compound 4 mani-
fested the greatest inhibition of 69.5% (in turn of 25.3%
paw edema, 0.21 ± 0.06 ml, compared to vehicle control,
82.8% paw edema, P < 0.05) after 5 h.
On contrary, resveratrol has a slight anti-inflammatory
activity at first 2-h but potentiate inflammatory action to
boost rat paw edema after 3-h carrageenan challenge.
Overall resveratrol has no anti-inflammatory effect that
this result is consistent with the report by Gentilli et al.
[29].
However, based on the results the antiinflammatory
activities of compounds 3 - 6 were better as compared to
resveratrol (1); compound 4 is the best among these four
analogs. In the in vivo carrageenan-induced paw edema
test, the anti-inflammatory activities of all the four ana-
logs were better than that of resveratrol. Analog 4 re-
duced the edema to only about one of third that was the
best among these four compounds. Therefore, these
compounds may be worthy of further investigation as
potential therapeutics.
5. CONCLUSIONS
In this study, we have made four novel analogs of res-
veratrol (1) basically by insertion of heterocyclic moie-
ties into (E)-stilbene. These four new compounds were
then studied for their biological properties.
In the in vitro studies, all the four analogs showed sig-
nificantly better in vitro anti-angiogenesis effect as com-
pared to resveratrol (1). Inhibition of LPS-induced NO
production in vitro for each of these analogs was also
significantly better than that of resveratrol (1).
In summary, this study showed that insertion of hete-
rocyclic moieties into the (E)-stilbene core structure may
be a new direction in term of modifying resveratrol and
improving its biological properties. On the other hand,
these preliminary biological properties do not guarantee
that this type of analogs of resveratrol will result in ac-
tual therapeutics. Many more studies will have to be
conducted in order to verify their potential to become
Figure 3. Anti-inflammatory activities of compounds 3 - 6 at 20 mg/kg i.p. single dose in carrageenan-induced
paw edema model in rats (n = 3 animals/group). Each value represents the average ± SEM of 3 animals.

C.-M. Huang et al. / Modern Research in Inflammation 2 (2 013) 37-45
44
suitable therapeutics.
6. ACKNOWLEDGEMENTS
We gratefully acknowledge the research grant supported from the
Cheng-Hsin Rehabilitation Medical Center (CHGH 97-63), the Repub-
lic of China and JAK3 Pharma, Inc., Ann Arbor, Michigan, USA. We
also gratefully thank for Dr. Ben Chen’s helpful comments.
REFERENCES
[1] Bingham III, C.O. (2002) The pathogenesis of rheuma-
toid arthritis: Pivotal cytokines involved in bone degrada-
tion and inflammation. The Journal of Rheumatology, 29,
3-9.
[2] Nanjundaiah, S.M., Astry, B. and Moudgil, K.D. (2013)
Mediators of inflammation-induced bone damage in ar-
thritis and their control by herbal products. Evidence-
Based Complementary and Alternative Medicine, 2013,
1-20.
[3] Ziche, M. and Morbidelli, L. (2000) Nitric oxide and an-
giogenesis. Journal of Neuro-Oncology, 50, 139-148.
doi:10.1155/2013/518094
[4] Cooke, J.P. and Losordo, D.W. (2002) Nitric oxide and
angiogenesis. Circulation, 105, 2133-2135.
doi:10.1161/01.CIR.0000014928.45119.73
[5] Tamura, T., Nakanishi, T., Kimura, Y., Hattori, T., Sasaki,
K., Norimatsu, H., Takahashi, K. and Takigawa, M. (1996)
Nitric oxide mediates interleukin-1-induced matrix degra-
dation and basic fibroblast growth factor release in cul-
tured rabbit articular chondrocytes: a possible mechanism
of pathological neovascularization in arthritis. Endocri-
nology, 137, 3729-3737. doi:10.1210/en.137.9.3729
[6] Moulton, K.S., Heller, E., Konerding, M.A., Flynn, E.,
Palinski, W. and Folkman, J. (1999) Angiogenesis inhibi-
tors endostatin or TNP-470 reduce intimal neovasculari-
zation and plaque growth in apolipoprotein E-deficient
mice. Circulation, 99, 1726-1732.
doi:10.1161/01.CIR.99.13.1726
[7] Matsunaga, T., Weihrauch, D.W., Moniz, M.C., Tessmer,
J., Warltier, D.C. and Chilian, W.M. (2002) Angiostatin
inhibits coronary angiogenesis during impaired produc-
tion of nitric oxide. Circulation, 105, 2185-2191.
doi:10.1161/01.CIR.0000015856.84385.E9
[8] Chun, Y.J., Kim, M.Y. and Guengerich, F.P. (1999) Res-
veratrol is a selective human cytochrome P450 1A1 inhi-
bitor. Biochemical and Biophysical Research Communi-
cations, 262, 20-24. doi:10.1006/bbrc.1999.1152
[9] Orsini, F., Pelizzoni, F., Verotta, L., Aburjai, T. and
Rogers, C.B. (1997) Isolation, synthesis, and antiplatelet
aggregation activity of resveratrol 3-O-beta-D-glucopy-
ranoside and related compounds. Journal of Natural Pro-
ducts, 60, 1082-1087. doi:10.1021/np970069t
[10] Arichi, H., Kimura, Y., Okuda, H., Baba, K., Kozawa, M.
and Arichi, S. (1982) Effects of stilbene components of
the roots of Polygonum cuspidatum Sieb. et Zucc. on lipid
metabolism. Chemical Pharmaceutical Bulletin, 30, 1766-
1770. doi:10.1248/cpb.30.1766
[11] Goldberg, D.M. (1996) More on antioxidant activity of
resveratrol in red wine. Clinical Chemistry, 42, 113-114.
[12] Pace-Asciak, C.R., Hahn, S., Diamandis, E.P., Soleas, G.
and Goldberg, D.M. (1995) The red wine phenolics trans-
resveratrol and quercetin block human platelet aggrega-
tion and eicosanoid synthesis: Implications for protection
against coronary heart disease. Clinica Chimica Acta, 235,
207-219. doi:10.1016/0009-8981(95)06045-1
[13] Inamori, Y., Inamori, Y., Kubo, M., Tsujibo, H., Ogawa,
M., Saito, Y., Miki, Y. and Takemura, S. (1987) The ich-
thyotoxicity and coronary vasodilator action of 3,3’-dihy-
droxy-alpha,beta-diethylstilbene. Chemical and Pharma-
ceutical Bulletin, 35, 887-890. doi:10.1248/cpb.35.887
[14] Tang, L.L., Gao, J.S., Chen, X.R. and Xie, X. (2006)
Inhibitory effect of resveratrol on the proliferation of syn-
oviocytes in rheumatoid arthritis and its mechanism in vi-
tro. Zhong Nan Da Xue Xue Bao Yi Xue Ban, 31, 528-533.
[15] Elmali, N., Baysal, O., Harma, A., Esenkaya, I. and Miz-
rak, B. (2007) Effects of resveratrol in inflammatory ar-
thritis. Inflammation, 30, 1-6.
doi:10.1007/s10753-006-9012-0
[16] Bi, X.L., Yang, J.Y., Dong, Y.X., Wang, J.M., Cui, Y.H.,
Ikeshima, T., Zhao, Y.Q. and Wu, C.F. (2005) Resveratrol
inhibits nitric oxide and TNF-alpha production by lipo-
polysaccharide-activated microglia. International Immu-
nopharmacology, 5, 185-193.
doi:10.1016/j.intimp.2004.08.008
[17] Tseng, S.H., Lin, S.M., Chen, J.C., Su, Y.H., Huang, H.Y.,
Chen, C.K., Lin, P.Y. and Chen, Y. (2004) Resveratrol
suppresses the angiogenesis and tumor growth of gliomas
in rats. Experimental Therapeutic Preclinical Pharma-
cology, 10, 2190-2202.
[18] Ghai, G., Chen, K.Y., Rosen, R.T., Wang, M., Telang, N.,
Lipkin, M. and Ho, C.-T. (2004) Resveratrol analogs for
prevention of disease, US Patent 6790869. The State
University of New Jersey, Rutgers.
[19] Pettit, G.R., Grealish, M.P., Jung, M.K., Hamel, E., Pettit,
R K., Chapuis, J.C. and Schmidt, J.M. (2002) Antineo-
plastic agents. 465. Structural modification of resveratrol:
Sodium resverastatin phosphate. Journal of Medicinal
Chemistry, 45, 2534-2542. doi:10.1021/jm010119y
[20] Ohyama, M., Ohyama, M., Tanaka, T., Ito, T., Iinuma, M.,
Bastow, K.F. and Lee, K.H. (1999) Antitumor agents 200.
Cytotoxicity of naturally occurring resveratrol oligomers
and their acetate derivatives. Bioorganic Medicinal Che-
mistry Letters, 9, 3057-3060.
doi:10.1016/S0960-894X(99)00520-X
[21] Gosslau A., Chen, M., Ho, C.T. and Chen, K.Y. (2005) A
methoxy derivative of resveratrol analogue selectively
induced activation of the mitochondrial apoptotic path-
way in transformed fibroblasts. British Journal of Cancer,
92, 513-521.
[22] Gosslau, A., Pabbaraja, S., Knapp, S. and Chen, K.Y.
(2008) Trans- and cis-stilbene polyphenols induced rapid
perinuclear mitochondrial clustering and p53-independent
apoptosis in cancer cells but not normal cells. European
Journal of Pharmacology, 587, 25-34.
doi:10.1016/j.ejphar.2008.03.027
[23] Takahashi, S., Uchiyama, T. and Toda, K. (2009) Differ-

C.-M. Huang et al. / Modern Research in Inflammation 2 (2 013) 37-45 45
ential effect of resveratrol on nitric oxide production in
endothelial f-2 cells. Biological and Pharmaceutical Bul-
letin, 32, 1840-1843. doi:10.1248/bpb.32.1840
[24] Cho, D.I., Koo, N.Y., Chung, W.J., Kim, T.S., Ryu, S.Y.,
Im, S.Y. and Kim, K.M. (2002) Effects of resveratrol-re-
lated hydroxystilbenes on the nitric oxide production in
macrophage cells: Structural requirements and mechanism
of action. Life Sciences, 71, 2071-2082.
doi:10.1016/S0024-3205(02)01971-9
[25] FitzGerald, O., Soden, M., Yanni, G., Robinson, R. and
Bresnihan, B. (1991) Morphometric analysis of blood
vessels in synovial membranes obtained from clinically
affected and unaffected knee joints of patients with
rheumatoid arthritis. Annals of the Rheumatic Diseases,
50, 792-796. doi:10.1136/ard.50.11.792
[26] Ceponis, A., Konttinen, Y.T., Imai, S. Tamulaitiene, M.,
Li, T.F., Xu, J.W., Hietanen, J., Santavirta, S. and Fass-
bender, H.G. (1998) Synovial lining, endothelial and
inflammatory mononuclear cell proliferation in synovial
membranes in psoriatic and reactive arthritis: A compa-
rative quantitative morphometric study. British Journal of
Rheumatology, 37, 170-178.
doi:10.1093/rheumatology/37.2.170
[27] Walsh, D.A., Wade, M., Mapp, P.I. and Blake, D.R. (1998)
Focally regulated endothelial proliferation and cell death
in human synovium. American Journal of Pathology, 152,
691-702.
[28] Cao, Y., FU, Z.-D., Wang, F., Liu, H.-Y. and Han, R.
(2005) Anti-angiogenic activity of resveratrol, a natural
compound from medicinal plants. Journal of Asian Natu-
ral Products Research, 7, 205-213.
doi:10.1080/10286020410001690190
[29] Gentilli, M., Mazoit, J.X., Bouaziz, H., Fletcher, D.,
Casper, R.F., Benhamou, D. and Savouret, J.-F. (2001)
Resveratrol decreases hyperalgesia induced by carragee-
nan in the rat hind paw. Life Science, 68, 1317-1321.
doi:10.1016/S0024-3205(00)01018-3