Anti-neuroinflammatory activity of 1,5-benzodiazepine derivatives

Article abstract of DOI:10.1016/j.bmcl.2010.04.133

A series of 2,3-dihydro-1,5-benzodiazepines were synthesized and evaluated for anti-inflammatory effects in microglia cells. Among the 1,5-benzodiazepines tested, compound 3e strongly inhibited LPS-induced nitric oxide (NO) production, with an IC₅₀

Full text of DOI:10.1016/j.bmcl.2010.04.133

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3969–3971
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anti-neuroinflammatory activity of 1,5-benzodiazepine derivatives
Sang Keun Ha a, Donthabhaktuni Shobha b,c, Eunjung Moon a, Murugulla A. Chari b, Kagga Mukkanti c,
a,e,
Sung-Hoon Kim d, Kwang-Hyun Ahn b, , Sun Yeou Kim
*
*
a Graduate School of East-West Medical Science, Kyung Hee University, Global Campus, #1 Seocheon-dong, Giheung-gu, Yongin, Gyeonggi-do 446-701, Republic of Korea
b Department of Applied Chemistry, Kyung Hee University, #1 Seocheon-dong, Giheung-gu, Yongin, Gyeonggi-do 446-701, Republic of Korea
c Center for Pharmaceutical Sciences, IST, JNT University, Hyderabad 85, AP, India
d Cancer Preventive Material Development Research Center, Kyung Hee University, Seoul Campus, #1 Hoegi-dong, Dongdaemun-gu, Seoul, Republic of Korea
e East-West Integrated Medical Science Rearch center, Kyung Hee University Global Campus, #1 Seocheon-dong, Giheung-gu, Yongin-si, Gyeonggi-do 446-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2,3-dihydro-1,5-benzodiazepines were synthesized and evaluated for anti-inflammatory
Received 4 November 2009
Revised 21 April 2010
Accepted 28 April 2010
Available online 27 May 2010
effects in microglia cells. Among the 1,5-benzodiazepines tested, compound 3e strongly inhibited LPS-
induced nitric oxide (NO) production, with an IC50 value of 7.0 lM in the microglia cells. Also, compound
3e significantly inhibited the enzymatic activity of inducible NO synthase (iNOS) without changes in iNOS
protein expression or NO scavenging activity. This result suggests that compound 3e showed anti-neur-
oinflammatory effects by suppressing iNOS enzyme activity.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
2,3-Dihydro-1,5-benzodiazepines
Anti-neuroinflammation
Microglia
Inducible NO synthase
Microglia, resident macrophages, are the immune surveillance
cells of the central nervous system (CNS) and play an active role in
brain inflammatory, immune, and neurodegenerative processes.1
Upon neuronal injury or inflammatory stimulation, activated
microglia produce and release a variety of pro-inflammatory factors,
and neuronal plasticity.8 However, the elevated level of NO pro-
duced by the expression of inducible iNOS is an important media-
tor of inflammation and neuronal cell death.9
Benzodiazepines are important organic molecules with a wide
array of biological activities and therapeutic functions. Among
the benzodiazepines, the 1,4- and the 1,5-benzodiazepines are
commonly used as anxiolytic and anticonvulsive drugs. These ef-
fects are primarily mediated via the benzodiazepine receptors lo-
cated in the central nervous system.10 Also, peripheral-type
such as nitric oxide (NO), tumor necrosis factor-a(TNF-a), and inter-
leukin-1b (IL-1b), that can result in neuronal cell death in neurode-
generative diseases.2–5 Thus, microglial activation appears to play
a pivotal role in the initiation and progression of neurodegenerative
diseases. Therefore, modulation of activated microglia is important
to the protection of neuronal cells from exposure to inflammatory
substances and may have therapeutic potential in neurodegenera-
tive diseases that are accompanied by microglial activation.
benzodiazepine binding sites have been detected in microglia11
,
macrophages12, and other tissues.13 In addition to the anxiolytic
activity of the benzodiazepines, these molecules also have activity
as anti-cancer14 and anti-neuroinflammatory15 agents.
In this study we designed and synthesized 2,3-dihydro-1,5-ben-
zodiazepine derivatives to identify novel neuroprotective agents.
Evaluation of the ten synthesized 1,5-benzodiazepine derivatives
as potential anti-neuroinflammatory agents was performed using
BV-2 microglia cells.
NO can be produced by three isoforms of nitric oxide synthase
(NOS): neuronal NOS (nNOS, type I), inducible NOS (iNOS, type
II), and endothelial NOS (eNOS, type III). Generally, eNOS and nNOS
are Ca2+-dependent and constitutively release small amounts of
NO required for physiological function. In contrast, iNOS is Ca2+
-
independent and is not normally expressed but can be induced
by lipopolysaccharide (LPS) and various cytokines, such as inter-
The benzodiazepine derivatives were synthesized according to
the procedure shown in Scheme 1. Thus, a mixture of o-phenylene-
feron-c (IFN-c), TNF-a
, and IL-1b.6,7 NO, a neuromodulator in the
CNS, participates in brain development, pain perception, memory,
R
H
N
NH2
NH2
CH3
O
SiO2-H2SO4 Cat.
CH3OH, RmT
+
* Corresponding authors. Tel.: +82 031 201 2477; fax: +82 031 202 7337 (K.H.A.);
address: Graduate School of East-West Medical Science, Kyung Hee University,
Global Campus, #1 Seocheon-dong, Giheung-gu, Yongin-si, Gyeonggi-do 446-701,
Republic of Korea; tel.: 82 31 201 2177; fax: 82 31 205 8962 (S.Y.K.).
R
CH3
N
3(a-j)
R
2
1
E-mail addresses: khahn@khu.ac.kr (K.-H. Ahn), sunnykim@khu.ac.kr (S.Y. Kim).
Scheme 1. Synthesis of 2,3-dihydro-1,5-benzodiazepines.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.133

3970
S. K. Ha et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3969–3971
Table 1
Inhibitory activities of 1,5-benzodiazepine derivatives on NO production in LPS-activated microglia
R
H
N
CH3
N
3(a-j)
R
Compounds
R
Timea (min)
Yieldb (%)
Inhc (%)
IC50 (lM)
3a
3b
3c
3d
3e
3f
3g
3h
3i
Methyl
Ethyl
Phenyl
Isopropyl
2-Thiophenyl
3-Thiophenyl
4-Chlorophenyl
3-Chlorophenyl
4-Methylphenyl
2-Nitrophenyl
30
40
40
98
95
92
94
86
82
95
85
96
95
28.6
27.4
59.8
26.3
70.3
13.2
28.2d
21.2
34.6
24.9
14.3
15.2
9.4
16.6
7.0
35.2
—
23.2
14.0
23.2
60
120
120
50
80
50
3j
50
a
b
c
Reaction time for the preparation of 3a–j.
Isolated yield in the syntheses.
Values obtained through three experiments and the mean inhibition of NO production relative to the LPS control at 10
Cytotoxic effect was observed.
lM concentration of compounds.
d
18
10
16
14
12
9
8
7
*
10
8
6
5
4
3
2
1
0
*
6
*
4
2
0
LPS
-
+
+
1
+
5
+
10
+
1
+
5
+
SNP
Compound 3e
+
-
+
1
+
5
+
10 µM
10 µM
Compound 3e
L-NMMA
30
25
20
15
10
5
*
iNOS
*
*
Tubulin
LPS
Compound 3e
-
+
-
+
+
+
-
1
5
10 µM
0
LPS
-
+
+
1
+
5
+
10
+
100µM
Compound 3e
L-NMMA
Figure 1. The inhibitory effect of compound 3e on NO production in LPS-activated BV-2 cells. (A) The effect of compound 3e on LPS-induced NO production in BV-2 cells.
Nitrite was measured using the Griess reaction after 24 h treatment with LPS (100 ng/ml) in the presence or absence of compound 3e. (B) The effect of compound 3e on NO
scavenging activity after a mixture of SNP and compound 3e were incubated at 25 °C for 150 min. (C) The effect of compound 3e on iNOS enzyme activity in BV-2 cells. (D) The
effect of compound 3e on iNOS protein expression in BV-2 cells. iNOS protein was detected using Western blot analysis after 6 h of treatment with LPS (100 ng/ml) in the
presence or absence of compound 3e (1, 5, and 10
lM). L-NMMA was used as positive control. All data were presented as the mean S.D. *p <0.05 indicates statistically
significant differences from treatment with LPS alone.

S. K. Ha et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3969–3971
3971
diamine 1 (1 mmol), ketone 2 (2.5 mmol), and silica gel-supported
sulfuric acid catalyst (0.1 mmol) in methanol was stirred at room
temperature for the appropriate time16 (Table 1). Completion of
the reaction was monitored via TLC. After completion of the reac-
tion, ethyl acetate was added to the reaction mixture, and the cat-
alyst was recovered by filtration. The organic layer was
concentrated, and the crude product was purified by silica gel col-
umn chromatography using an ethyl acetate/n-hexane mixture
(1:9) as an eluent to afford the desired product 3. The compounds
were purified further by recrystallization with ethyl acetate and
hexane to obtain the pure form with an 82–98% yield.
apeutic potential for neuroinflammatory diseases through the inhi-
bition of microglial activation and is a suitable target for further
structure activity relationship studies. Further studies are required
to elucidate the precise mechanisms underlying the anti-inflam-
matory activity of compound 3e.
Acknowledgments
This work was supported by the Korea Science and Engineering
Foundation (KOSEF) Grant funded by the Korea Government
(MEST) (No. 2009-0063466).
The inhibitory activities of NO production by the 2,3-dihydro-
1,5-benzodiazepine derivatives were evaluated using LPS-acti-
vated microglia cells. A murine microglia cell line, BV-2, was stim-
ulated with 100 ng/ml LPS for 24 h in the presence or absence of
the samples. The presence of nitrite, a soluble oxidation product
of NO, in the culture media was determined by Griess reaction. Cell
viability was measured using a 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT) assay.
As shown in the Table 1, the inhibitory activities of the 1,5-ben-
zodiazepines varied according to the structure of R. The benzodiaz-
epine containing a phenyl ring at the R position showed good
inhibitory activity (3c), as compared with alkyl groups. However,
the activity declined as substituents were attached to the phenyl
ring (3g–j), indicating that the BV-2 cell is very sensitive towards
both steric and electronic differences in position R.
References and notes
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4. McGeer, P. L.; McGeer, E. G. Parkinsonism Relat. Disord. 2004, 10, S3.
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6. Mayer, B.; Andrew, P. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1998, 358, 127.
7. Forstermann, U.; Kleinert, H. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1995,
352, 351.
8. Chabrier, P. E.; Demerle-Pallardy, C.; Auguet, M. Cell. Mol. Life Sci. 1999, 55,
1029.
9. Iadecola, C. Trends Neurosci. 1997, 20, 132.
10. Tallman, J. F.; Paul, S. M.; Skolnick, P.; Gallager, D. W. Science 1980, 207, 274.
11. Park, C. H.; Carboni, E.; Wood, P. L.; Gee, K. W. Glia 1996, 16, 65.
12. Zavala, F.; Haumont, J.; Lenfant, M. Eur. J. Pharmacol. 1984, 106, 561.
13. Awad, M.; Gavish, M. Life Sci. 1991, 49, 1155.
14. Dourlat, J.; Liu, W. Q.; Gresh, N.; Garbay, C. Bioorg. Med. Chem. Lett. 2007, 17,
2527.
15. Wilms, H.; Claasen, J.; Rohl, C.; Sievers, J.; Deuschl, G.; Lucius, R. Neurobiol. Dis.
2003, 14, 417.
The highest NO-inhibitory effect on BV-2 cells among the com-
pounds tested in this study was obtained with the benzodiazepine
containing 2-thiophene ring (3e). Interestingly, the activity sharply
changed depending upon the attachment position of the thiophene
ring. The compound with the 3-thiophenyl group (3f) gave poor
inhibitory activity. Since the steric difference between 2-thio-
phenyl and 3-thiophenyl group is marginal and can be ignored in
this case, the activity difference might be due to the electronic
effects.
INOS expression is induced in activated microglia and mediates
NO synthesis. Thus, we investigated the effect of 3e on the activ-
ity17 and expression18 of iNOS protein in LPS-induced BV-2 cells.
Also, we evaluated the NO scavenging activity19 of compound 3e.
As shown in Figure 1, compound 3e did not have a significant
inhibitory effect on the expression of iNOS and NO scavenging
activity but did significantly inhibit iNOS enzyme activity. There-
fore, the inhibitory effect of compound 3e on NO production may
be partly due to inhibition of iNOS enzyme activity.
16. Catalyst preparation: To a slurry of silica gel (10 g) in dry diethyl ether (50 ml),
commercially available sulfuric acid (3 ml) was added with shaking for 5 min.
The solvent was evaporated under reduced pressure, resulting in free-flowing
silica gel-supported sulfuric acid (SiO2–H2SO4), which was then dried at 110 °C
for 3 h.
17. For the assay the iNOS enzyme activity in intact cells, BV-2 cells were plated in
100 mm tissue culture dishes (4 Â 106 cells) and incubated with LPS (100 ng/
ml) for 12 h. Then, the cells were washed twice with PBS, and cells were
harvested and plated in a 96-well plate (2 Â 106 cells/well), and incubated in
the presence or absence of different concentrations of compound 3e for a
further 12 h. The amount of NO in the supernatant was detected by Griess
reaction.
18. Western blot was performed to analyze iNOS 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 3e for 6 h. The protein sample (40
lg for each) from the
BV-2 cell extract was separated by 8% SDS–PAGE and transferred to
a
nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire,
UK). The membrane was blocked with 5% skim milk and incubated with
primary antibodies (rabbit anti-iNOS; Transduction Laboratories, San Diego,
CA, USA) and secondary antibodies (goat anti-rabbit IgG; Amersham Pharmacia
Biotech). The blots were developed using ECL Western Blotting Detection
Reagents (Amersham Pharmacia Biotech).
In summary, we have reported a new class of 1,5-benzodiaze-
pines that demonstrate anti-neuroinflammatory activity in
microglia cells. Compound 3e has shown the highest NO-inhibitory
effect on BV-2 cells. Compound 3e showed inhibitory effects on NO
production through the suppression of iNOS enzyme activity.
These results suggest that compound 3e may be of beneficial ther-
19. Sodium nitroprusside (SNP) was dissolved in phosphate buffered saline (PBS)
at 100 mM. This SNP solution (50
ll) was mixed with 950
ll PBS containing
various concentrations of compound 3e (1, 5 and 10
lM). These mixtures were
incubated at 25 °C for 2.5 h, and nitrite formed through the combination of
oxygen and NO released from the SNP, as measured by the Griess reaction.