A substituted 3,4-dihydropyrimidinone derivative (compound D22) prevents inflammation mediated neurotoxicity; Role in microglial activation in BV-2 cells

Article abstract of DOI:10.1016/j.bmcl.2012.06.082

A novel synthetic 3,4-dihydropyrimidinone derivative, compound D22 (ethyl 6-methyl-4-(3-phenoxyphenyl)-2-thioxo-3,4-dihydropyrimidine-5-carboxylate), was found to exert anti-inflammatory properties in lipopolysaccharide-stimulated microglial BV-2 cells. Compound D22 reduced the pro-inflammatory factors such as nitric oxide, prostaglandin E₂, tumor necrosis factor-α and interleukin-1β. Moreover, it suppressed the expressions of inducible NO synthase and cyclooxygenase-2. Compound D22 inhibited the activation of mitogen-activated protein kinases. When compound D22-conditioned media from BV-2 cells were applied to N2a cells, neuronal cell death was inhibited via suppression of caspase-3 activation and regulation of Bcl-2 and Bax proteins expression. These results suggest that compound D22 may be useful for treating neurodegenerative diseases related with neuroinflammation.

Full text of DOI:10.1016/j.bmcl.2012.06.082

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5199–5203
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A substituted 3,4-dihydropyrimidinone derivative (compound D22) prevents
inflammation mediated neurotoxicity; role in microglial activation in BV-2 cells
Oh Wook Kwon a, Eunjung Moon a, Murugulla A. Chari b, Tae Woo Kim a, Ae-jung Kim c, Pyeongjae Lee d,
Kwang-Hyun Ahn b, Sun Yeou Kim e,
⇑
a Graduate School of East-West Medical Science, Kyung Hee University Global Campus, #1732 Deogyeong-daero, Giheung-gu, Yongin, Gyeonggi-do 446-701, Republic of Korea
b Department of Applied Chemistry, Kyung Hee University Global Campus, #1732 Deogyeong-daero, Giheung-gu, Yongin, Gyeonggi-do 446-701, Republic of Korea
c Graduate School of Alternative Medicine, Kyonggi University, Seoul 120-837, Republic of Korea
d Department of Natural Medicine Resource, Semyung University, #21-1, Sinwoul-dong, Jecheon, Chungcheongbuk-do 390-711, Republic of Korea
e College of Pharmacy, Gachon University, #191, Hambakmoero, Yeonsu-gu, Incheon 406-799, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel synthetic 3,4-dihydropyrimidinone derivative, compound D22 (ethyl 6-methyl-4-(3-phenoxyphe-
nyl)-2-thioxo-3,4-dihydropyrimidine-5-carboxylate), was found to exert anti-inflammatory properties in
lipopolysaccharide-stimulated microglial BV-2 cells. Compound D22 reduced the pro-inflammatory
Received 17 January 2012
Revised 23 May 2012
Accepted 25 June 2012
Available online 3 July 2012
factors such as nitric oxide, prostaglandin E2, tumor necrosis factor-
a and interleukin-1b. Moreover, it
suppressed the expressions of inducible NO synthase and cyclooxygenase-2. Compound D22 inhibited
the activation of mitogen-activated protein kinases. When compound D22-conditioned media from BV-2
cells were applied to N2a cells, neuronal cell death was inhibited via suppression of caspase-3 activation
and regulation of Bcl-2 and Bax proteins expression. These results suggest that compound D22 may be
useful for treating neurodegenerative diseases related with neuroinflammation.
Keywords:
DHPM
Ethyl 6-methyl-4-(3-phenoxyphenyl)-2-
thioxo-3,4-dihydropyrimidine-5-
carboxylate
Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
Neuroinflammation
Neuroprotection
Microglia
Microglia, the immune cells of the central nervous system, play
roles in defense against injury and tissue repair.1 However, exces-
sive activation of microglia due to pathogenic bacterial infection or
injury can produce diverse pro-inflammatory factors including tu-
anti-inflammatory14 activities. However, little is known about their
neuroprotective effects via regulation of microglial cell activation.
In this study, we examined the effect of synthesized 3,4-
dihydropyrimidinone derivatives as candidate novel neuroprotec-
tive agents. First, the 30 synthesized 3,4-dihydropyrimidinone
derivatives examined in this study were investigated as potential
anti-neuroinflammatory substances using LPS-stimulated BV-2
microglial cells (data not shown). Among the 3,4-dihydropyrimid-
inone derivatives, compound D7 (ethyl 1,2,3,4-tetrahydro-6-
methyl-2-oxo-4-(3-phenoxyphenyl)pyrimidine-5-carboxylate),
compound D10 (ethyl 4-(5-chloro-2-hydroxyphenyl)-1,2,3,4-tetra-
hydro-6-methyl-2-oxopyrimidine-5-carboxylate), and compound
D22 (ethyl 6-methyl-4-(3-phenoxyphenyl)-2-thioxo-3,4-dihydro-
pyrimidine-5-carboxylate) exhibited inhibitory effects on NO pro-
duction in BV-2 cells. Compound D22 showed stronger inhibitory
activity on NO production than the others in LPS-stimulated BV-2
cells. Therefore, we investigated the effect of compound D22 on
regulation of microglial cell activation in activated BV-2 cells.
Compound D22 (Fig. 1) was obtained through the following syn-
thetic process. Solutions containing mixtures of aldehyde
(1.0 mmol), b-ketoester (1.2 mmol), and thiourea (1.2 mmol) in ace-
tonitrile (96%) were heated under reflux conditions in the presence
of rhodium(III) chloride (RhCl3) catalyst (5 mol %) for 4 h. Reaction
mor necrosis factor
a (TNF-a), nitric oxide (NO), interleukin-1b (IL-
1b) and prostaglandin E2 (PGE2),2,3 which may increase neuronal
damage and may results in neuronal cell death in the brain.4 These
processes exacerbate brain injury and cause neuroinflammation,
which has been shown to be a risk factor of neurodegenerative dis-
eases such as Alzheimer’s disease, Parkinson’s disease, and brain
ischemia.5 Therefore, much attention has focused on agents that
may regulate neuroinflammation via inhibition of microglial acti-
vation. Previously, we demonstrated that arctigenin,6 apigenin,7
wogonin8 and chrysin9 had anti-inflammatory activities in lipo-
polysaccharide (LPS)-activated microglial cells and in the brains
of ischemic animals.
3,4-Dihydropyrimidinone is associated with three structures:
ethyl acetoacetate, benzaldehyde and urea/thiourea.10 The
3,4-dihydropyrimidinone has many biological and therapeutic
effects, including anti-viral,11 anti-bacterial,12 anti-tumor,13 and
⇑
Corresponding author. Tel.: +82 32 899 6411; fax: +82 32 820 4829.
E-mail address: sunnykim@gachon.ac.kr (S.Y. Kim).
0960-894X/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.082

5200
O. W. Kwon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5199–5203
Figure 1. Structure of compounds D7, D10 and D22.
Figure 2. Effects of compound D22 on NO and PGE2 production, COX-2 and iNOS regulation in LPS-stimulated BV-2 microglial cells. BV-2 micriglial cells were pretreated with 5, 10
and 20 M of compound D22 for 30 min and stimulated with 100 ng/ml of LPS for 24 h. Measurement of NO production and PGE2 production (A and C). MTT assay (B). The
l
effects of compound D22 on the induction of iNOS and COX-2 expression in BV-2 cells after 6 h LPS treatment (D). Densitometry analysis of bands was performed as described
in the methods section. All data are presented as the mean S.E.M of three independent experiments. ⁄p<0.05 indicates significant differences compared to treatment with
LPS alone.
completion was monitored by thin-layer chromatography. The reac-
tion mixture was then poured onto crushed ice and the solid product
was filtered and recrystallized in methanol. All products were char-
acterized using nuclear magnetic resonance and infrared spectros-
copy by comparison with the spectra of authentic samples and
based on the melting points of the samples mixed with authentic
samples.10 The yields were in the range of 80–84%.
pro-inflammatory factors in BV-2 cells. Among these factors, NO
is released from activated microglia.16 Compounds D7, D10 and
D22 were compared with chrysin, which reduces NO production,
in BV-2 cells.9,17,18 Compounds D7 and D10 had little effect on
NO production in BV-2 cells. But, compound D22 showed 8.5%
stronger inhibitory activity on NO production than chrysin at a
concentration of 10 lM in LPS-stimulated BV-2 cells (Fig. 2A).
Compound D22 did not affect cell viability at this concentration
(Fig. 2B).
LPS is widely used as an inflammatory inducer in the brain.15
The presence of LPS leads to the production of several

O. W. Kwon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5199–5203
5201
Figure 3. Effects of compound D22 on production of IL-1b and TNF-
stimulated with LPS (100 ng/ml) for 24 h. The culture medium was subsequently collected to measure the quantity of IL-1b (A) and TNF-
are presented as the mean S.E.M of three independent experiments. ⁄p<0.05 indicates significant differences compared to treatment with LPS alone.
a
in LPS-stimulated BV-2 microglial cells. BV-2 cells were pretreated with compound D22 for 30 min and then
a
(B) using ELISA analysis. ALL data
Together with NO, PGE2 is also an important inflammatory
mediator.19 Therefore, we investigated whether compound D22
had inhibitory activity against PGE2 production in LPS-treated
BV-2 cells.18,20 As shown in Figure 2C, compound D22 suppressed
LPS-induced PGE2 production in BV-2 cells.
NO and PGE2 production were catalyzed by inducible NO syn-
thase (iNOS) and cyclooxygenase-2 (COX-2), which are key neuro-
inflammatory enzymes in the brain.21,22 Thus, we investigated the
inhibitory effect of compound D22 on iNOS and COX-2 expres-
sion.23 As shown in Figure 2D, compound D22 inhibited expression
of iNOS and COX-2 proteins.
D22-conditioned media (CM) from BV-2 cells were applied to
N2a cells. N2a cells have been widely used as a model of neuronal
cell death.26 We measured neuronal cell survival,17 and confirmed
DNA fragmentation27 and the expression of apoptosis-related fac-
tors such as Bcl-2, Bax and cleaved caspase-3.23 As shown in Fig-
ure 5A and B, compound D22-CM prevented neuronal cell death,
and DNA fragmentation in a dose-dependent manner. Moreover,
the expression of anti-apoptotic protein Bcl-2 was recovered, of
pro-apoptotic protein Bax was inhibited, and caspase-3 activation
was blocked by compound D22-CM from LPS-stimulated BV-2 cells
in N2a cells (Fig. 5C).
We also investigated the effects of compound D22 on the
3,4-Dihydropyrimidinone derivative exert anti-inflammatory
effects on carrageenan induced animal paw edema in animal mod-
els.14 Also, 3,4-dihydropyrimidinone regulates gamma-aminobuty-
tic acid (GABA) receptor in brain.28 However, their inhibitory
effects on inflammation in the brain have not been reported. Thus,
we investigated the anti-inflammatory effects of 3,4-dihydropyri-
midinone derivatives via regulation of microglial cell activation.
Among the 3,4-dihydropyrimidinone derivative, compounds D22
and D7 have inhibitory effect on NO production in BV-2 cells and
have nearly the same chemical structures. But, compound D22
had much stronger inhibitory activity on NO production than com-
pound D7 in LPS-stimulated BV-2 cells. Compound D22 is a 3,4-
dihydropyrimidinone derivative containing thiourea skeleton,
and compound D7 is an urea counterpart of compound D22. The
replacement strategy of oxygen atom in urea by sulfur atom
production of pro-inflammatory cytokines such as TNF-
a and IL-
1b.18,20 As shown Figure 3A and B, compound D22 reduced TNF-
a
and IL-1b levels in the medium in a dose-dependent manner.
The mitogen-activated protein kinases (MAPKs) signaling path-
way plays an important role in controlling the synthesis and
releases of pro-inflammatory factors in LPS-stimulated BV-2
cells.24,25 Thus, we investigated the effects of compound D22 on
MAPKs activation in BV-2 cells.23 Compound D22 inhibited LPS-
induced phosphorylation of p38, extracellular signal-regulated ki-
nase (ERK) and c-Jun N-terminal kinase (JNK) in a dose-dependent
manner (Fig. 4).
Activated microglia secretes many inflammatory factors that
induce apoptotic neuronal cell death. To investigate the inhibitory
effects of compound D22 on neuronal cell death, compound
Figure 4. Effects of compound D22 on LPS-induced MAPKs activation in BV-2 cells. BV-2 cells were treated with LPS and with or without 5–20 lM compound D22 for 30 min. Cell
lysates were prepared to evaluate the protein levels of MAPKs. Phophorylation of p38, JNK and ERK was measured by Western blotting. Densitometry analysis of bands was
performed as described in the methods section.

5202
O. W. Kwon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5199–5203
Figure 5. Effects of compound D22-conditioned media from LPS-stimulated BV-2 cells on apoptotic neuronal cell death in N2a cells. BV-2 micriglial cells were pretreated with 5, 10
and 20 M compound D22 for 30 min and stimulated with 100 ng/ml of LPS for 24 h. After 24 h LPS treatment, the conditioned media (CM) were collected and treated in N2a
cells. (A) MTT assay and (B) Gel electrophoresis of DNA fragmentation. Illustrated lanes represent: molecular markers (M); vehicle (CM) (lane 1); LPS with vehicle (CM) (lane
l
2); LPS with compound D22 5, 10, and 20 lM (CM) (lanes 3, 4, 5). (C) Western blot analysis for expression of cleaved caspase-3, Bax, and Bcl-2 proteins. Densitometry analysis
of bands was performed as described in the methods section. ALL data are presented as the mean S.E.M of three independent experiments. ⁄p<0.05 indicates significant
differences compared to control group.
has been successfully used in many studies for drug develop-
ment.29–33 Several studies have demonstrated that some com-
pounds containing sulfur instead of oxygen significantly
enhanced the pharmacological activities of urea.32,33 In the writh-
ing test, the percentage of maximal inhibition of 1-phenyl-3-{4-
[(2E)-3-phenylprop-2-enoyl]phenyl}-thiourea derivatives showed
higher values than that of 1-phenyl-3-{4-[(2E)-3-phenylprop-2-
enoyl]phenyl}-urea derivatives in mice induced nociceptive re-
sponses by many stimuli such as acetic acid, formalin, capsaicin,
glutamate and high temperature.32 Moreover, isoxyl (1,3-bis[4-
(3-methylbutoxy)phenyl]) thiourea, a thiourea drug used in the
treatment of tuberculosis, shows the 80 times more potent mini-
mum inhibitory concentration (MIC) activity against Mycobacte-
rium tuberculosis, compared with isoxyl’s urea counterpart (1,3-
bis[4-(3-methylbutoxy)phenyl]urea).33 We think that the anti-
nociceptive and the anti-tuberculosis effects of thioureas are not
enough to support the anti-neuroinflammatory effect of compound
D22. However, the potent activity of thiourea-containing com-
pound D22 may be associated with enhancement of the pharmaco-
logical activities when an oxygen replaced by a sulfur in urea. We
suggest that it may be mainly due to the different physical proper-
ties of urea and thiourea in spite of their structural similarity: size
(C@O bond distance of urea 1.256 0.007 Å and C@S of thiourea
1.681 0.020 Å),34 polarity (dipole moment of urea 4.56 D and
thiourea 4.89 D),35 and pKa (urea 26.9 and thiourea 21.0).36 In
LPS-stimulated BV-2 microglial cells, the NO production was sig-
nificantly down-regulated by the small molecular change from
compound D7 (9.8%) to compound D22 (51.0%) (Fig. 2A). Based
on structure-activity relationship, 3,4-dihydropyrimidin-2-thione
can be a putative pharmacophore for preventing inflammation-
mediated neurotoxicity.
In this study, compound D22 inhibited NO and PGE2 production
in BV-2 cells. In addition, compound D22 inhibited the expression
of iNOS and COX-2 in BV-2 cells. These results suggest that com-
pound D22 inhibits NO and PGE2 production via inhibition of iNOS
and COX-2 expression.
Overproduction of pro-inflammatory cytokines has harmful ef-
fects on neuronal cells.37,38 In this study, compound D22 reduced
levels of secreted TNF-
that compound D22 may regulate neuroinflammation via inhibi-
tion of TNF- and IL-1b production.
a and IL-1b in BV-2 cells. Our results suggest
a
These pro-inflammatory factors were regulated by the MAPKs
pathway. In this study, compound D22 inhibited the phosphoryla-
tion of ERK, p38, and JNK in BV-2 cells. These results suggest that
compound D22 inhibits pro-inflammatory factors via inhibition
of MAPKs activation.
Neuroinflammation responses induce apoptotic neuronal cell
death. Apoptosis is a gene-regulated phenomenon which is charac-
terized by obvious morphological features, such as chromatin con-
densation, cell and nuclear shrinkage, membrane blebbing, and

O. W. Kwon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5199–5203
5203
10 and 20
l
M) for 24 h. Nitrite, a soluble oxidation product of NO, in the
l)
oligonucleosomal DNA fragmentation. Numerous investigations
have shown that anti- or pro- apoptosis signaling pathways in-
volve the activation of caspase-3, Bax and Bcl-2.39–41 Caspase-3 is
a family of cysteine proteases that execute apoptosis induced by
many stimuli. Bcl-2 plays a major role in the permeability of mito-
chondrial outer membrane. Apoptosis progresses when the pro-
apoptotic protein Bax binds to the mitochondrial outer membrane,
where it triggers changes in membrane permeability. Finally, these
results induce apoptotic neuronal cell death. Thus, we investigated
neuronal cell death by neurotoxicity mediated compound D22-CM
from LPS-stimulated BV-2 cells in N2a cells. Our results indicate
that compound D22 prevents neuronal cell death and DNA frag-
mentation by LPS-induced microglial cell activation mediated
neurotoxicity, and regulates the expression of apoptosis related
factors such as Bcl-2, Bax and cleaved caspase-3. These results sug-
gest that compound D22-CM from LPS-stimulated BV-2 cells inhib-
its apoptotic neuronal cell death via regulation of caspase-3
activity and expression of Bcl-2 and Bax proteins in N2a cells.
In conclusion, compound D22 suppressed pro-inflammatory
factors in LPS-activated microglial BV-2 cells. In addition, com-
pound D22 prevented LPS-induced microglial activation mediated
neurotoxicity in N2a cells. Thus, our results suggest that compound
D22 has potential neuroprotective effects in inflammatory-related
brain damage induced by microglial cell activation.
culture media was detected using the Griess reaction. The supernatant (50
l
was harvested and mixed with an equal volume of Griess reagent (1%
sulfanilamide, 0.1% N-1-napthylethylenediamine dihydrochloride in 5%
phosphoric acid). After 10 min, the absorbance at 570 nm was measured
using a microplate reader. Sodium nitrite was used as a standard to calculate
NO2 concentration. Cell viability was measured using a 3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. To measure neuronal cell
viability, BV-2 cells were treated with 100 ng/ml of LPS in the presence or
absence of compound D22. After 24 h, CM were collected and treated in N2a
cells for 24 h. Viabilities of N2a cells were measured using MTT assay.
18. The data were analyzed using Statistical Analysis System (SAS) software
(PRISM). All data are expressed as mean S.E.M. Statistical comparisons
between different treatments were performed using one-way ANOVA with
Tukey’s multiple comparison post test. ⁄P-values <0.05 were considered to be
statistically significant.
19. Tilley, S. L.; Coffman, T. M.; Koller, B. H. J. Clin. Invest. 2001, 108, 15.
20. Measurement of cytokines (IL-1b, PGE2 and TNF-a) — BV-2 cells were plated in
a 6-well plate at a density of 1.5 Â 106 cells/well. Then, BV-2 cells were treated
with LPS (100 ng/ml) in the presence or absence of compound D22 for 24 h.
PGE2, IL-1b and TNF-
kits (R&D Systems, Minneapolis, MN, U.S.A) according to the manufacture’s
protocol.
a were measured by competitive enzyme immunoassay
21. Iadecola, C. Trends Neurosci. 1997, 20, 132.
22. Mitchell, J. A.; Larkin, S.; Williams, T. J. Biochem. Pharmacol. 1995, 50, 1535.
23. Western blot analysis of various proteins (iNOS, COX, MAPKs, a-tubulin, Bcl-2,
caspase-3 and Bax) expression — BV-2 cells were seeded in a 6-well plate and
exposed to LPS (100 ng/ml) in the presence or absence of compound D22 for
various time periods (for detection of iNOS and COX-2 expression, 6 h; for
MAPKs expression, 30 min). Also, to investigate the effect of compound D22 on
apoptosis of neuronal cells, we treated CM from LPS-stimulated BV-2 cells for
24 h in N2a cells, and performed Western blot analysis using primary
antibodies (Cell Signaling) against cleaved caspase-3, Bax, and Bcl-2. Proteins
from cell lysates were separated by 8–12% sodium dodecyl sulfate
Acknowledgments
polyacrylamide gel electrophoresis and transferred to
a nitrocellulose
membrane. After transfer, membranes were incubated for 12 h with primary
antibodies (Cell Signaling, Beverly, MA, U.S.A), followed by incubation for 1 h
with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling)
at room temperature. Blots were developed using ECL Western Blotting
Detection Reagents (Amersham Pharmacia Biotech, Buckinghamshire, U.K).
Densitometry analysis of the bands was performed using ImageMaster™ 2D
Elite software (version 3.1, Amersham, Pharmacia Biotech).
This research was supported by Korea Institute of Planning and
Evaluation for Technology in Food, Agriculture, Forestry and Fish-
eries (iPET, 810006-03-1-SB120), Republic of Korea.
References and Notes
24. Choi, M. S.; Kwon, K. J.; Jeon, S. J.; Go, H. S.; Kim, K. C.; Ryu, J. R.; Lee, J.; Han, S.;
Cheong, J. H.; Ryu, J. H.; Bae, K.; Shin, C. Y.; Ko, K. H. Biomol. Ther. 2009, 17, 47.
25. Kaminska, B. Biochim. Biophys. Acta 2005, 1754, 253.
1. Perry, V. H.; Gordon, S. Trends Neurosci. 1988, 11, 273.
2. Hanisch, U. K. Glia 2002, 40, 140.
26. Yang, L.; Zhou, X.; Yang, J.; Yin, X.; Han, L.; Zhao, D. J. Neuroimmunol. 2008, 199,
10.
27. DNA laddering analysis — N2a cells were seeded in 6-well plates, and cells
3. Minghetti, L.; Levi, G. Prog. Neurobiol. 1998, 54, 99.
4. Pan, X. D.; Chen, X. C.; Zhu, Y. G.; Zhang, J.; Huang, T. W.; Chen, L. M.; Ye, Q. Y.;
Huang, H. P. Biochem. Pharmacol. 2008, 76, 362.
5. Dickson, D. W.; Lee, S. C.; Mattiace, L. A.; Yen, S. H.; Brosnan, C. Glia 1993, 7, 75.
6. Park, J. H.; Hong, Y. J.; Moon, E.; Kim, S.; Kim, S. Y. Biomol. Ther. 2011, 19, 425.
7. Ha, S. K.; Lee, P.; Park, J. A.; Oh, H. R.; Lee, S. Y.; Park, J. H.; Lee, E. H.; Ryu, J. H.;
Lee, K. R.; Kim, S. Y. Neurochem. Int. 2008, 52, 878.
8. Son, D.; Lee, P.; Lee, J.; Kim, H.; Kim, S. Y. Eur. J. Pharmacol. 2004, 493, 99.
9. Ha, S. K.; Moon, E.; Kim, S. Y. Neurosci. Lett. 2010, 485, 143.
10. Kappe, C. O. Acc. Chem. Res. 2000, 33, 879.
11. Dougherty, A. M.; Guo, H.; Westby, G.; Liu, Y.; Simsek, E.; Guo, J. T.; Mehta, A.;
Norton, P.; Gu, B.; Block, T.; Cuconati, A. Chemotherapy 2007, 51, 4427.
12. Muravyova, E. A.; Desenko, S. M.; Musatov, V. I.; Knyazeva, I. V.; Shishkina, S.
V.; Shishkin, O. V.; Chebanov, V. A. J. Comb. Chem. 2007, 9, 797.
13. Barrow, J. C.; Nantermet, P. G.; Selnick, H. G.; Glass, K. L.; Rittle, K. E.; Gilbert, K.
F.; Steele, T. G.; Homnick, C. F.; Freidinger, R. M.; Ransom, R. W.; Kling, P.; Reiss,
D.; Broten, T. P.; Schorn, T. W.; Chang, R. S.; O’Malley, S. S.; Olah, T. V.; Ellis, J.
D.; Barrish, A.; Kassahun, K.; Leppert, P.; Nagarathnam, D.; Forray, C. J. Med.
Chem. 2000, 43, 2703.
were exposed to CM from BV-2 cells treated with 5, 10 and 20 lM of
compound D22 plus 100 ng/ml of LPS. After 24 h incubation, the cells were
scraped from the dishes using a rubber policeman, centrifuged at 7500 rpm for
5 min. DNA fragmentation was analyzed using a 2% agarose gel containing
ethidium bromide according to the manufacturer’s protocol (Apoptotic DNA
Ladder Detection Kit, Abcam, Cambridge, U.K).
28. Lewis, R. W.; Mabry, J.; Polisar, J. G.; Eagen, K. P.; Ganem, B.; Hess, G. P.
Biochemistry 2010, 49, 4841.
29. Aaramadaka, S. K.; Guha, M. K.; Prabhu, G.; Kini, S. G.; Vijayan, M. Chem. Pharm.
Bull. 2007, 55, 236.
30. Venketaraman, V. Global View of HIV Infection InTech; Croatia, 2011. Chapter 7.
31. Khan, S. A.; Saleem, Z.; Khan, Z. Eur. J. Med. Chem. 2007, 42, 103.
32. Santos, L. D.; Lima, L. A.; Cechinel-Filho, V.; Corrêa, R.; De Campos Buzzi, F.;
Nunes, R. J. Bioorg. Med. Chem. 2008, 16, 8526.
33. Brown, J. R.; North, E. J.; Hurdle, J. G.; Morisseau, C.; Scarborough, J. S.; Sun, D.;
Kordulakova, J.; Scherman, M. S.; Jones, V.; Grzegorzewicz, A.; Crew, R. M.;
Jackson, M.; McNeil, M. R.; Lee, R. E. Bioorg. Med. Chem. 2011, 19, 5585.
34. Haynes, W. M. CRC Handbook of Chemistry and Physics, 92nd Edition; Taylor &
Francis Group: London, 2011. Chapter 9-1.
14. Tozkoparan, B.; Ertan, M.; Kelicen, P.; Demirdamar, R. Farmaco 1999, 54, 588.
15. Lehnardt, S.; Massillon, L.; Follett, P.; Jensen, F. E.; Ratan, R.; Rosenberg, P. A.;
Volpe, J. J.; Vartanian, T. Natl. Acad. Sci. U.S.A. 2003, 100, 8514.
35. Kumler, W. D.; Fohlen, G. M. J. Am. Chem. Soc. 1944, 1942, 64.
36. Bordwell, F. G.; Algrim, D. J., Jr; Harrelson, J. A. J. Am. Chem. Soc. 1988, 110, 5903.
37. McGuire, S. O.; Ling, Z. D.; Lipton, J. W.; Sortwell, C. E.; Collier, T. J.; Carvey, P. M.
Exp. Neurol. 2001, 169, 219.
38. McGeer, P. L.; Yasojima, K.; McGeer, E. G. Neurosci. Lett. 2002, 326, 67.
39. Lavrik, I. N.; Golks, A.; Krammer, P. H. J. Clin. Invest. 2005, 115, 2665.
40. Fadeel, B.; Oreenius, S. J. Intern. Med. 2005, 258, 479.
16. Chao, C. C.; Hu, S.; Molitor, T. W.; Shaskan, E. G.; Peterson, P. K. J. Immunol.
1992, 149, 2736.
17. Cell culture, nitrite oxide assay and MTT assay — BV-2 cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 5% fetal
bovine serum (FBS) and 1% penicillin–streptomycin (PS). N2a cells were
maintained in DMEM supplemented with 10% FBS and 1% PS. The cells were
cultured in a humidified atmosphere of 5% CO2 at 37 °C. In order to measure NO
production, BV-2 cells were plated into 96-well plate (3 Â 104 cells/well) and
treated with 100 ng/ml of LPS in the presence or absence of compound D22 (5,
41. Sharifi, A. M.; Mousavi, S. H.; Farhadi, M.; Larijani, B. J. Pharmacol. Sci. 2007, 104,
258.