Synthesis of tricyclic fused coumarin sulfonates and their inhibitory effects on LPS-induced nitric oxide and PGE2 productions in RAW 264.7 macrophages

Article abstract of DOI:10.1016/j.bmcl.2013.12.018

The regulations of NO and PGE₂ productions are research topics of interest in the field of antiinflammatory drug development. In the present study, a series of tricyclic fused coumarin sulfonate derivatives was synthesized and evaluated for their abilities to inhibit NO and PGE₂ productions in LPS-induced RAW 264.7 macrophages. Among all the target compounds, compound 1g possessing p-(trifluoromethyl)phenyl and fused cycloheptane moieties showed the highest inhibitory effects on NO and PGE ₂ productions. Compound 1g not only inhibited COX-2 activity but also reduced expressions of COX-2 and iNOS. Furthermore, ADME profiling showed that compounds 1g, 1j, 1m, and 1n are estimated to be orally bioavailable.

Full text of DOI:10.1016/j.bmcl.2013.12.018

Bioorganic & Medicinal Chemistry Letters 24 (2014) 571–575
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of tricyclic fused coumarin sulfonates and their inhibitory
effects on LPS-induced nitric oxide and PGE2 productions in RAW
264.7 macrophages
Hyeon-Lok Jang a, Mohammed I. El-Gamal b,c,d, Hye-Eun Choi e, Ho-Yeong Choi a, Kyung-Tae Lee e,
,
⇑
Chang-Hyun Oh b,c,
⇑
a Korean Medical School, Kyung Hee University, Hoegi-dong, Dongdaemoon-ku, Seoul 130-701, Republic of Korea
b Center for Biomaterials, Korea Institute of Science and Technology (KIST), PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
c Department of Biomolecular Science, University of Science and Technology (UST), 113 Gwahangno, Yuseong-gu, Daejeon 305-333, Republic of Korea
d Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
e Department of Pharmaceutical Biochemistry, Department of Life and Nanopharmaceutical Science, College of Pharmacy, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu,
Seoul 130-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
The regulations of NO and PGE2 productions are research topics of interest in the field of antiinflamma-
tory drug development. In the present study, a series of tricyclic fused coumarin sulfonate derivatives
was synthesized and evaluated for their abilities to inhibit NO and PGE2 productions in LPS-induced
RAW 264.7 macrophages. Among all the target compounds, compound 1g possessing p-(trifluoro-
methyl)phenyl and fused cycloheptane moieties showed the highest inhibitory effects on NO and PGE2
productions. Compound 1g not only inhibited COX-2 activity but also reduced expressions of COX-2
and iNOS. Furthermore, ADME profiling showed that compounds 1g, 1j, 1m, and 1n are estimated to
be orally bioavailable.
Received 29 August 2013
Revised 25 November 2013
Accepted 4 December 2013
Available online 10 December 2013
Keywords:
Antiinflammatory
Coumarin
Nitric oxide
PGE2
Ó 2013 Elsevier Ltd. All rights reserved.
Sulfonate
Tricyclic fused coumarin
Inflammation is a complex physiological and pathological pro-
cess accompanied by the activation of the immune system, local
vascular system, and various cells within the damaged tissue.1 It
is a normal protective response to tissue injury caused by physical
trauma (cut, burn or bruise), noxious chemicals, microbiologic
agents, or even autoimmune disease. Acute inflammation is a part
of the defense response by organisms to remove injurious stimuli,
such as pathogens, irritants, or physical injury, from tissues and to
initiate the healing process. However, persistent and excessive im-
mune response can promote tissue damage, resulting in chronic
inflammation. This chronic inflammation is a part of many human
diseases including arteriosclerosis,2 inflammatory bowel disease,3
arthritis,4 cancer,5 and Alzheimer’s disease.6
various pathological conditions.7,8 Accordingly, control of the pro-
duction of NO and PGE2 in macrophages are current research topics
for the development of new antiinflammatory agents.
Coumarins and their derivatives have acquired much attention
from the pharmacological and pharmaceutical arena due to their
broad range of therapeutic qualities. Several coumarin analogs have
been reported as potential antiinflammatory agents.9–11 In the
present study, various fused tricyclic coumarin sulfonate analogs
1a–n (Fig. 1) were synthesized with the aim to study structure–
activity relationships, and thereby provide new lead compounds
possessing enhanced antiinflammatory activity and reduced cyto-
toxicity. The antiinflammatory activities of the target compounds
In the inflammatory state, activated immune cells, such as mac-
rophages secrete large amounts of proinflammatory cytokines, ni-
tric oxide (NO), and prostaglandin E2 (PGE2). However, high levels
of NO and PGE2 in a chronic inflammation state can result in
n
O
O
S
R
O
O
O
1a-n
n = 1, 2
⇑
R = Me, Et, n-Pr, c-Pr, Ph, p-tolyl, p-(CF3)C6H4
Figure 1. Structures of the target compounds.
Corresponding authors. Tel.: +82 2 961 0860; fax: +82 2 966 3885 (K.-T.L.); tel.:
+82 2 958 5160; fax: +82 2 958 5189 (C.-H.O.).
E-mail addresses: ktlee@khu.ac.kr (K.-T. Lee), choh@kist.re.kr (C.-H. Oh).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.018

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H.-L. Jang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 571–575
n
n
ii
i
iii
O
O
O
O
S
n
n
n
HO
O
O
R
O
O
O
O
O
O
HO
O
4a,b
1a-n
2a,b
3a,b
n = 1, 2
R = Me, Et, n-Pr, c-Pr, Ph, p-tosyl, p-(CF3)C6H4
Scheme 1. Reagents and conditions: (i) diethyl carbonate, NaH, benzene, reflux, 90% (3a, n = 1), 85% (3b, n = 2); (ii) resorcinol, CF3COOH, concd H2SO4, 0 °C; rt, 3 h; (iii)
appropriate sulfonyl chloride derivative, triethylamine, CH2Cl2, 0 °C; rt, 1 h.
were evaluated by measuring the inhibitions of the productions of
lipopolysaccharide (LPS)-induced NO and PGE2 productions in RAW
264.7 macrophages.
respectively. The cytotoxic effects of the coumarins on RAW
264.7 macrophages were also evaluated using the MTT assay17 to
test whether the inhibitory effects on the productions of NO and
PGE2 were caused by nonspecific cytotoxicity (Table 2). All the tar-
The target compounds were synthesized by the pathway illus-
trated in Scheme 1. Cycloheptanone (2a) or cyclooctanone (2b)
was reacted with diethyl carbonate in refluxing benzene in the pres-
ence of sodium hydride to produce the corresponding ethoxycar-
bonate derivatives 3a,b which exist in keto–enol tautomers.12
Cyclizationto the phenolictricyclicintermediates 4a,b was achieved
by reaction of compounds 3a,b with resorcinol in the presence of
concentrated sulfuric acid and trifluoroacetic acid.13 Treatment of
the phenolic compounds 4a,b with the appropriate sulfonyl chloride
derivatives in the presence of triethylamine yielded the target sulfo-
nates 1a–n. Table 1 illustrates structures of the final compounds,
their yield percentages, and their melting points.
Inflammation is a protective immune response against tissue
damage induced by external stimulants in the body.2 However,
the inflammatory response can also result in considerable damage
to the host, because microbial components such as LPS stimulate
macrophages to produce pro-inflammatory cytokines, iNOS, and
COX-2. In particular, the large amounts of NO and PGE2 secreted
by activated immune cells in the inflammatory state can induce
various pathological conditions. Thus, inhibition of the production
of inflammatory mediators is a potential strategy for the treatment
of many acute and chronic inflammatory disorders. The synthe-
sized coumarin sulfonates 1a–n were assessed in terms of their
ability to inhibit the production of inflammatory mediators, NO
and PGE2, in LPS-induced RAW 264.7 macrophages.14–16
get compounds showed IC50 values P9.2
and 1l–n with aromatic sulfonate moieties did not inhibit 50% of
RAW 264.7 macrophages growth up to 100 M. After that, the tar-
get compounds were tested for NO and PGE2 production inhibi-
tions at 5 M.
lM. Compounds 1e–g
l
l
The target coumarin compounds 1a–n expressed varying inhib-
itory activities on the LPS-induced NO and PGE2 productions (Figs. 2
and 3). Compounds 1f and 1g possessing p-toluenesulfonate
and p-(trifluoromethyl)benzenesulfonate moieties, respectively,
showed higher inhibitory effect on NO production than compounds
1a–e. Among cyclooctane-fused derivatives, compound 1j with n-
propylsulfonate terminal moiety demonstrated the strongest
inhibitory effect on NO production. Compounds 1b, 1d, and 1g
with fused cycloheptane ring were more active as NO production
Table 2
Cytotoxicty (IC50, MTT assay) of the target compounds 1a–n over RAW 264.7 cells
a
Compound no.
IC50
(
lM)
1a
1b
1c
1d
1e
1f
90.80 3.25
23.40 2.36
23.90 1.25
20.30 2.56
>100
>100
1g
1h
1i
1j
1k
1l
>100
N6-(1-Iminoethyl)-L-lysine (L-NIL) and N-(2-cyclohexyloxy-4-
nitrophenyl)methanesulfonamide (NS398) were used as reference
compounds for screening of NO and PGE2 production inhibitions,
24.50 2.89
21.50 3.69
9.20 1.20
10.50 3.22
>100
1m
1n
>100
>100
Table 1
Structures of the target compounds, and their yield percentages and melting points
a
Data are presented as the means SD of three independent experiments.
n
O
O
S
100
80
60
40
20
0
R
O
O
O
Compound no.
R
n
Yield%
Melting point (°C)
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
Me
Et
n-Pr
Cyclo-Pr
Ph
p-Tolyl
p-(CF3)C6H4
Me
Et
n-Pr
Cyclo-Pr
Ph
1
1
1
1
1
1
1
2
2
2
2
2
2
2
95
92
93
85
93
95
90
94
90
88
87
92
95
90
172–3
169–72
133–5
128–30
125–8
149–52
137–40
146–8
157–60
114–7
133–6
108–11
107–10
154–7
1c 1d
1a
1b
1e 1f
1i 1j
L-NIL (20 µM)
1n
1g
1m
1h
1k 1l
Compound (5 µM)
Figure 2. Inhibitory effect of the target compounds 1a–n on LPS-induced NO
production in RAW 264.7 cells. Compounds 1a–n were tested at 5 M concentra-
tion, while the reference compound L-NIL was tested at 20 M concentration. Data
are presented as the means SD of three independent experiments.
l
p-Tolyl
p-(CF3)C6H4
l

H.-L. Jang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 571–575
573
100
80
inhibitors than the corresponding cyclooctane-containing com-
pounds 1i, 1k, and 1n. On the other hand, cyclooctane-possessing
compounds 1h, 1j, and 1l showed higher inhibitory effects on NO
production compared to the corresponding compounds 1a, 1c,
and 1e with cycloheptane ring. Compounds 1f and 1m possessing
p-toluenesulfonate moiety were more active than compounds 1e
and 1l with benzenesulfonate group.
60
40
Regarding PGE2 production inhibition results, compounds 1h,
1i–k, and 1m with cyclooctane ring showed higher inhibitory effect
than the corresponding compounds 1a, 1b–d, and 1f possessing
fused cycloheptane ring. This may be rationalized that the cyclooc-
tane ring may perform stronger hydrophobic interaction at the
receptor site. Or the steric factor may influence the affinity with
the receptor and therefore the inhibitory effect of the compounds.
The results of compounds 1e (cycloheptane, benzenesulfonate),
20
0
1c 1d
1a
1b
1e 1f
1i 1j
NS-398 (3 µM)
1n
1g
1m
1h
1k 1l
Compound (5 µM)
Figure 3. Inhibitory effect of the target compounds 1a–n on LPS-induced PGE2
production in RAW 264.7 cells. Compounds 1a–n were tested at 5 M concentra-
tion, while the reference compound NS398 was tested at 3 M concentration. Data
l
l
are presented as the means SD of three independent experiments.
Table 3
NO and PGE2 production inhibitory potency (IC50) and calculated Lipinski’s rule of five for the most active target compounds
Compd. no.
NO production inhibitiona (IC50
,
lM)
PGE2 production inhibitiona (IC50, nM)
LogPb
TPSAc
MWd
nONe
nOHNHf
nViolations
1g
1j
1m
1n
NS398
L-NIL
3.84 0.26
10.00 1.84
40.69 5.30
75.32 5.01
—
6.45 1.02
4.02 1.09
6.75 1.10
6.04 0.92
7.01 0.66
—
5.11
3.42
5.09
5.53
2.79
—
73.59
73.59
73.59
73.59
101.23
—
438.42
350.43
398.47
452.44
314.36
—
5
5
5
5
7
—
0
0
0
0
1
—
1
0
1
1
0
—
20.14 2.32
a
b
c
d
e
f
Data are presented as the means SD of three independent experiments.
Calculated lipophilicity.
0
Total polar surface area (AÅ2).
Molecular weight.
Number of hydrogen bond acceptors.
Number of hydrogen bond donors.
(A)
120
100
80
60
40
20
0
***
***
***
***
***
-
-
5
-
-
0.01
-
0.1
-
1
-
1g (µM)
+
Dup-697 (10µM)
120
100
80
60
40
20
0
(B)
(C)
ꢁ
ꢀꢀꢀ
LPS (10ng/ml)
-
-
-
+
+
-
+
1
-
+
5
-
+
10
-
1g (µM)
-
-
L-NIL (20µM)
+
Figure 4. Inhibitory effects of compound 1g on LPS-induced COX-2 and iNOS enzyme activity and protein expressions in RAW 264.7 macrophages. (A) Recombinant COX-2
enzyme was in vitro treated with the indicated concentrations of 1g for 10 min. As a negative control, these enzymes were inactivated by boiling for 3 min. Dup-697 (10 M)
were used as a positive COX-2 inhibitor controls. (B) Cellular lysates were prepared from the pretreated with/without 1g (0.010.1, 0.5, 1, 5 or 10 M) for 1 h and then with LPS
(10 ng/mL) for 24 h. Total cellular proteins were resolved by SDS–PAGE, transferred to PVDF membranes, and detected with specific iNOS and COX-2 antibody. b-actin was
used as an internal control. (C) Following pretreatment with LPS (10 ng/ml) for 12 h and wash PBS, cells were treated with 1g (1, 5, or 10 M) for 12 h. L-NIL (20 M) was used
l
l
l
l
as positive control in the assay. Levels of NO in culture media were quantified using Griess reaction assay. Data are presented as the means SDs of three independent
experiments. #p <0.05 versus the control cells; ⁄⁄⁄p <0.001 versus LPS-stimulated cells; statistical significances were compared using ANOVA and Dunnett’s post hoc test.

574
H.-L. Jang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 571–575
1g (cycloheptane, p-(trifluoromethyl)benzenesulfonate), 1h (cyclo-
octane, methanesulfonate), 1j (cyclooctane, n-propanesulfonate),
and 1m (cyclooctane, p-toluenesulfonate) were near the inhibitory
effect expressed by the reference compound, NS398.
Among all the target compounds, compound 1g possessing
fused cycloheptane ring and p-(trifluoromethyl)benzenesulfonate
moieties showed the highest inhibitory effects on NO and PGE2
productions. It showed higher efficacy and potency than the refer-
ence NO production inhibitor, L-NIL. It inhibited NO production by
#
40
35
30
25
20
15
10
5
IC 50:85.76 3.58 µM
**
***
65.76% at 5
lM concentration, while L-NIL inhibited it by 50% at
20 M. It also inhibited the PGE2 production in RAW 264.7 macro-
phages by 91.48%, which is very near to the inhibition effect pro-
duced by NS398.
l
0
LPS (10ng/ml)
-
-
+
+
1
+
5
+
+
+
-
STX-64 (µM)
-
10
50
100
100
Based on NO and PGE2 production inhibition% data, compounds
1g, 1j, 1m, and 1n with the most promising results were selected
for further screening of their potencies. Their IC50 values were cal-
culated and are summarized in Table 3. Compounds 1g and 1j were
more potent than L-NIL as NO production inhibitor. Compound
1g possessing fused cycloheptane ring showed higher potency
than the corresponding analog with fused cyclooctanone ring,
compound 1n. Among the four tested compounds, compound 1g
demonstrated the highest potency. It is 5.24 times more potent
than L-NIL. Regarding PGE2 inhibitory potency, the four com-
pounds 1g, 1j, 1m, and 1n showed higher potency than NS398.
Compound 1j possessing fused cyclooctanone and n-propanesulfo-
nate moieties was the most potent, and it was 1.74-fold more po-
tent than NS398. We further investigated whether the inhibitory
20
15
10
5
#
IC50:81.04 0.92 nM
***
***
***
***
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
+
***
+
0
LPS (10 ng/ml)
-
+
-
+
+
1
+
-
effects of compound 1g (0.1, 1 or 5 lM) on PGE2 production are re-
STX-64 (nM)
-
0.1
10
100
1000 1000
lated to COX-2 enzyme activity and protein expression. We ob-
served that compound 1g significantly inhibited COX-2 enzyme
activity (Fig. 4A). Because iNOS expression induced by LPS produce
NO, the protein expression of iNOS was determined. As shown in
Figure 4B, COX-2 protein levels were found to be markedly up-reg-
ulated in response to LPS, and compound 1g dose-dependently
inhibited LPS-induced COX-2 protein expression. Next, we investi-
gated whether inhibitory effects of compound 1g on LPS-induced
NO production are related to iNOS enzyme activity or iNOS expres-
sion. We found that 1g exhibited no effect on the iNOS enzyme
activity (Fig. 4C). However, 1g suppressed the LPS-induced iNOS
expression (Fig. 4B). Thus, we suggest that the inhibition of PGE2
and NO production by 1g are due to its inhibition of the COX-2 en-
zyme activity and protein expressions of COX-2 and iNOS. Thus,
the marked inhibitory effect of 1g on PGE2 production can be
caused by simultaneously suppressed COX-2 enzyme activity and
protein synthesis. However, 1g did not affect iNOS enzyme activity.
Although the present study does not clarify the mechanism
responsible for the inhibition of iNOS and COX-2 expression by
1g, we suggest that 1g could interferes the LPS-induced activation
Figure 5. Inhibitory effects of STX-64 on LPS-induced NO and PGE2 production in
RAW 264.7 macrophages. Following pretreatment with STX-64 (1, 5, 10, 50 or
100 lM) for 1 h, cells were treated with LPS (10 ng/mL) for 24 h. Levels of NO in
culture media were quantified using Griess reaction assay. Following pretreatment
with STX-64 (0.1, 1, 10, 100 or 1000 nM) for 1 h, cells were treated with LPS (10 ng/
mL) for 24 h. Levels of PGE2 in culture media were quantified using EIAs. Data are
presented as the means SDs of three independent experiments. #p <0.05 versus
the control cells; ⁄⁄p <0.01, ⁄⁄⁄p <0.001 versus LPS-stimulated cells; statistical
significances were compared using ANOVA and Dunnett’s post hoc test.
Lipinski’s rule of five.19 This approach has been widely used as a fil-
ter for substances that would likely be further developed in drug
design programs. In addition, we calculated the total polar surface
area (TPSA) since it is another key property that has been linked to
drug bioavailability. Thus, passively absorbed molecules with a
TPSA >140 are thought to suffer from low oral bioavailability.20
Molecules violating more than one of these rules may have prob-
lems with bioavailability. Predictions of ADME properties for the
studied compounds are summarized in Table 3. The results showed
that all the four tested compounds and the reference compound,
NS398, comply with these rules. Theoretically, compounds 1g, 1j,
1m, and 1n should present good passive oral absorption and differ-
ences in their bioactivity cannot be attributed to this property.
In conclusion, we synthesized a series of tricyclic coumarin sul-
fonates in order to explore the relationship between their struc-
tures and their antiinflammatory activities. The compounds
showed varying cytotoxicities and inhibitory activities on the
LPS-induced productions of NO and PGE2. The biological screening
of this series of coumarins led to discovery of a new potential
lead compound 1g. This compound with fused cycloheptane and
p-(trifluoromethyl)benzenesulfonate moieties exhibited the high-
est inhibitory activities on both NO and PGE2 productions in LPS-
induced RAW 264.7 macrophages with low cytotoxicity. It showed
superior potencies to the reference compounds, L-NIL and NS398,
respectively. In silico ADME prediction showed that compound
1g complies with Lipinski’s rule of five and it can be passively ab-
sorbed orally. Compound 1g can be utilized as a promising lead
of TAK1/IKK-a/b/NF-jB through TLR4.
Since STX-64 (Irosustat, BN83495), the first steroid sulfatase
(STS) inhibitor to enter diverse clinical trials for patients with ad-
vanced hormone-dependent cancer is closely related to the struc-
tures of tricyclic coumarin sulfonates in this manuscript, we
investigated whether STX-64 has inhibitory effects on the LPS-in-
duced NO and PGE2 production in RAW 264.7 macrophages. As
shown in Figure 5A and B, STX-64 inhibited LPS-induced NO and
PGE2 production in a dose-dependent manner. However, STX-64
more potently inhibited LPS-induced PGE2 production (IC50
:
81.04 nM) than NO production (IC50: 85.76 M). These results sug-
l
gest that STX-64 showed similar inhibitory effects of four most ac-
tive target compounds (Table 3) via a selective PGE2 inhibition in
LPS-stimulated macrophages.
The bioavailability of compounds 1g, 1j, 1m, and 1n with prom-
ising biological results was assessed using ADME (absorption, dis-
tribution, metabolism, and excretion) prediction methods. In
particular, we calculated the compliance of compounds to the

H.-L. Jang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 571–575
575
12. Li, C.-J.; Chen, D.-L.; Lu, Y.-Q.; Haberman, J. X.; Mague, J. T. Tetrahedron 1998, 54,
2347.
compound for further development of potential antiinflammatory
coumarin compounds.
13. Woo, L. W. L.; Ganeshapillai, D.; Thomas, M. P.; Sutcliffe, O. B.; Malini, B.;
Mahon, M. F.; Purohit, A.; Potter, B. V. L. ChemMedChem 2011, 6, 2019.
14. Cell culture and sample treatment: the RAW 264.7 macrophage cell line was
obtained from the Korea Cell Line Bank (Seoul, Republic of Korea). Cells were
grown at 37 °C in Dulbecco’s Modified Eagle Medium (DMEM) supplemented
with 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin
Acknowledgment
This work was supported by Korea Institute of Science and
Technology (KIST), KIST Project (2E22360).
sulfate (100
lg/mL) in a humidified 5% CO2 atmosphere. Cells were incubated
with various concentrations of tested samples and then stimulated with LPS
(1 lg/mL) for the indicated time.
15. Nitrite determination: RAW 264.7 macrophages were plated at 4 ꢀ 105 cells/
Supplementary data
well in 24-well plates and then incubated with or without LPS (1 lg/mL) in the
absence or presence of various concentrations (1.60, 3.15, 6.25, 12.5, 25, 50 and
100 M) of tested samples for 24 h. Nitrite levels in culture media were
determined using the Griess reaction and presumed to reflect NO levels.
Briefly, 100 L of cell culture medium was mixed with 100 L of Griess reagent
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.12.
018.
l
l
l
[equal volumes of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid and 0.1%
(w/v) naphtylethylenediamine.HCl], and incubated at room temperature for
10 min. Absorbance was then measured at 540 nm using a microplate reader
(Perkin Elmer Cetus, Foster City, CA, USA). Fresh culture media were used as
blanks in all experiments. The amount of nitrite in the samples was measured
with the sodium nitrite serial dilution standard curve and nitrite production
was measured.
References and notes
1. Coussens, L. M.; Werb, Z. Nature 2002, 420, 860.
2. Qui, H.; Johansson, A.-S.; Sjostrom, M.; Wan, M.; Schroder, O.; Palmblad, J.;
Haeggstrom, J. Z. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 6913.
3. Lee, I.-A.; Bae, E.-A.; Hyun, Y.-J.; Kim, D.-H. J. Inflamm. 2010, 7, 7.
4. Hochberg, M. C. Semin. Arthritis Rheum. 1990, 19, 294.
5. Sung, B.; Prasad, S.; Yadav, V. R.; Lavasanifar, A.; Aggarwal, B. B. Free Radical Res.
2011, 45, 864.
16. PGE2 assay: RAW 264.7 macrophages were pretreated with tested samples for
1 h and then stimulated with LPS (1 lg/mL) for 24 h. The levels of PGE2 in the
culture media was quantified using EIA kits (R&D Systems, Minneapolis, MN,
USA) according to the manufacturer’s instructions.
6. Sastre, M.; Richardson, J. C.; Gentleman, S. M.; Brooks, D. J. Curr. Alzheimer Res.
2011, 8, 132.
17. MTT assay for cell viability: RAW 264.7 macrophages were plated at a density
of 105 cells/well in 96-well plates. To determine the appropriate concentration
not toxic to cells, cytotoxicity studies were performed 24 h after treating cells
with various concentrations of tested compounds. Cell viabilities were
determined using colorimetric MTT assays, as described previously.18
18. Kim, J. Y.; Park, S. J.; Yun, K. J.; Cho, Y. W.; Park, H. J.; Lee, K.-T. Eur. J. Pharmacol.
2008, 584, 175.
7. Yun, H. Y.; Dawson, V. L.; Dawson, T. M. Crit. Rev. Neurobiol. 1996, 10, 291.
8. Hinz, B.; Brune, K. J. Pharmacol. Exp. Ther. 2002, 300, 367.
9. Bansal, Y.; Sethi, P.; Bansal, G. Med. Chem. Res. 2012, 22, 3049.
10. Li, Z.; Hu, J.; Sun, M.; Ji, H.; Chu, S.; Liu, G.; Chen, N. Int. Immunopharmacol.
2012, 14, 145.
11. Hemshekhar, M.; Sunithaa, K.; Thusharaa, R. M.; Sebastin Santhosha, M.;
Shanmuga Sundarama, M.; Kemparajua, K.; Girisha, K. S. Biochimie 2013, 95,
1326.
19. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev.
2001, 46, 3.
20. Clark, D. E.; Pickett, S. D. Drug Discovery Today 2000, 5, 49.