Design and synthesis of carbamate and thiocarbamate derivatives and their inhibitory activities of NO production in LPS activated macrophages

Article abstract of DOI:10.1016/j.bmcl.2012.03.010

Series of carbamate and thiocarbamate derivatives were designed and synthesized and their inhibitory activities of NO production in lipopolysaccharide-activated macrophages were evaluated. Several thoicarbamate derivatives revealed promising inhibitory ac

Full text of DOI:10.1016/j.bmcl.2012.03.010

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3301–3304
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of carbamate and thiocarbamate derivatives
and their inhibitory activities of NO production in LPS activated macrophages
⇑
Guo Hua Jin, Hwa Jin Lee, Hyo Jin Gim, Jae-Ha Ryu, Raok Jeon
Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women’s University, 52 Hyochangwon-Gil, Yongsan-Ku, Seoul 140-742, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Series of carbamate and thiocarbamate derivatives were designed and synthesized and their inhibitory
activities of NO production in lipopolysaccharide-activated macrophages were evaluated. Several thoic-
arbamate derivatives revealed promising inhibitory activity. The structure–activity relationship study of
these compounds is also reported. Among these compounds, compound 12b was the most potent with
Received 25 January 2012
Revised 22 February 2012
Accepted 3 March 2012
Available online 16 March 2012
6.5
lM of IC50. They inhibited NO production through the suppression of iNOS protein and mRNA expres-
sion and nuclear translocation of p65.
Keywords:
Carbamate
Thiocarbamate
Nitric oxide
iNOS
Ó 2012 Elsevier Ltd. All rights reserved.
Nitric oxide (NO) plays critical roles in various physiological
processes,1,2 including neurotransmission, smooth muscle contrac-
tility, platelet reactivity, and the cytotoxic activity of immune cells.
NO is produced by neuronal nitric oxide synthase (nNOS) or mam-
malian endothelial NOS (eNOS) and serves as a second messenger
molecule for homeostatic function in neuronal tissues and vascular
endothelium, respectively.3–6 Meanwhile, the overproduction of
NO caused by inducible NOS (iNOS) has been linked to the patho-
genesis of a number of disease states, including septic shock, neu-
rodegenerative disorders, and various inflammatory processes.7 It
suggests the rationale for therapeutic agents that modulate over-
production of NO. Among several strategies for controlling the
NO concentrations, main efforts have been directed to develop
selective inhibitors of iNOS.
on this finding, we investigated carbazole-linked carbamate and
thiocarbamate derivatives depicted in Figure 1 as new inhibitors
of NO production. Here we report the design and synthesis of a ser-
ies of carbamates and thiocarbamates and their effects on the NO
production and iNOS expression.
The preparation of the carbazole-linked carbamate and thiocar-
bamate derivatives is outlined in Schemes 1–3. N-alkylation of car-
bazole gave alcohol 2 which was mesylated to obtain compound 3.
Alkylation of 4-nitrophenol by treatment of compound 3 in the
presence of NaH gave compound 4. Following reduction of nitro
compound 4 over 10% Pd/C under atmospheric pressure of hydro-
gen gas provided amine 5 which was reacted with thiophosgene to
give isothiocyanate 6. Addition of appropriate alcohol to isothiocy-
anate gave the target compounds 7. Also, condensation of amine 5
with 4-nitrophenyl chloroformate offered carbamate 8, which was
converted to the desired thiocarbamates 9 by treating sodium
thioalkoxide.
Other carbamates and thiocarbamates were obtained by substi-
tution reaction of hydroquinone with mesylate 3 and following
addition of the resulted alcohol 10 to the appropriate isocyanates
or isothiocyanates offered the target compounds 11 and 12.
The activities of the prepared compounds were evaluated for
the inhibition of NO production in LPS-activated macrophages.
Murine macrophage cell line, RAW 264.7 cells were stimulated
Early NOS inhibitors were mainly the arginine analogs such as
N-monomethyl-
L
-arginine (
L
-NMMA), N-(iminoethyl)-L-ornithine
(
L
-NIO), and N-nitro-
L
-arginine (L
-NNA), which were potent but
little selective against NOS isoforms.8 Recently, many classes of
non-arginine type iNOS inhibitors such as amino heterocycles,9
amidines,10 isoquinolinamines,11 and isothiourea12,13 have been
reported but still limited their application in clinical due to their
insufficient potency and/or subtype selectivity.
In our previous Letters, we reported urea, thiourea, and isot-
hiourea derivatives as inhibitors for the NO production in LPS-acti-
vated macrophage.14,15 It was suggested that introduction of
appropriate lipophilic groups such as carbazole and phenoxazine
to urea or thiourea moiety was necessary for the activity. Based
with 1 lg/mL of LPS in the presence of samples for 20 h. The
amounts of NO released into culture media were determined by
the Griess method in the form of nitrite.16
The inhibitory activities of the tested compounds on the NO
production are given in Table 1. Aminoguanidine, a well known
⇑
Corresponding author.
E-mail address: rjeon@sookmyung.ac.kr (R. Jeon).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.010

3302
G. H. Jin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3301–3304
corresponding compound 7a. Methyl substituted 9a showed a little
bit higher activity than ethyl substituted compound 9b. Regarding
N-substituents of compounds 11 and 12, the activities of alkoxy-
carbonylmethyl substituted derivatives (11b, 11c, and 12b) were
slightly higher than ethyl substituted derivatives 11a and 12a.
Among these compounds, thiocarbamate 12b was the most potent
X
Z
R
X, Y, Z = N, O, S
N
Y
O
Figure 1. Representative structure of carbamate and thiocarbamate.
showing 6.5 lM of IC50 value.
specific inhibitor of iNOS was used as positive control that showed
74% inhibition of NO production at 100 M.
For the further biological study of our compounds, we examined
the effects of 7a and 12b on the expression of iNOS protein and
mRNA in LPS-activated RAW 264.7 cells. The amounts of iNOS
protein was analyzed in Western blot analysis after 20 h incuba-
l
Effect of O-alkyl substituent of thiocarbamates 7a–c on the
inhibitory activity was evaluated. When methyl substituent (7a)
was replaced with isopropyl (7b) or benzyl (7c) group, the inhibi-
tory activity was decreased. This result suggested that bulky
substituents were inappropriate for the activity. The activity of
thiocarbamate 9a, which has the reverse position of S and O
comparing with thiocarbamate 7a, was lower than those of the
tion with compounds during LPS (1
phages.17 Compounds 7a and 12b significantly reduced the
amounts of iNOS at 20
M ( Fig. 2). At RT-PCR analysis,18 the
lg/mL) activation of macro-
l
expression level of iNOS mRNA was increased by LPS-activation
for 6 h. Both compounds 7a and 12b suppressed the induction of
a
b
N
H
N
N
OH
OMs
3
1
2
d
NO2
NH2
c
N
N
O
O
4
5
H
N
e
NCS
OR
f
N
N
S
O
O
7a, R = Me
6
7b, R = isopropyl
7c, R = benzyl
Scheme 1. Preparation of carbazole-linked thiocarbamate derivatives. Reagents: (a) 2-iodoethanol, NaH, DMF; (b) MsCl, TEA, DMF; (c) 4-nitrophenol, NaH, DMF; (d) H2, 10%
Pd/C, THF/MeOH; (e) thiophosgene, K2CO3, CHCl3; (f) ROH, NaH, DMF.
H
N
O
NH2
a
N
O
N
O
NO2
O
5
8
H
N
b
SR
9a, R = Me
9b, R = Et
N
O
O
Scheme 2. Preparation of carbazole-linked thiocarbamate derivatives. Reagents: (a) 4-nitrophenyl chloroformate, MC; (b) NaSR, MC.
O
NHR
OH
a
b
N
Y
N
N
O
O
OMs
3
11a, Y= O, R= Et
10
11b, Y= O, R= CH2CO2Me
11c, Y= O, R= CH2CO2Et
12a, Y= S, R= Et
12b, Y= S, R= CH2CO2Et
Scheme 3. Preparation of carbazole-linked carbamate and thiocarbamate derivatives. Reagents and conditions: (a) hydroquinone, NaH, DMF; (b) (i) RNCO, TEA, toluene or
RNCO, K2CO3, THF, (ii) RNCS, TEA, toluene or RNCS, 18-crown-6, KH, THF.

G. H. Jin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3301–3304
3303
Table 1
Inhibitory activities of carbazole-linked carbamates and thiocarbamates on the NO
production in LPS-activated macrophages
X
Z
R
X, Y, Z = N, O, S
N
Y
O
Inha (%)
IC50 (lM)
b
Compds
X
Y
Z
R
7a
7b
7c
8
9a
9b
11a
11b
11c
12a
12b
NH
NH
NH
NH
NH
NH
O
O
O
O
O
S
S
S
O
O
O
O
O
O
S
O
O
O
O
S
S
NH
NH
NH
NH
NH
Me
i-Pro
Bn
4-NO2–C6H4
Me
Et
Et
CH2CO2Me
CH2CO2Et
Et
60
20
18
22
44
26
10
33
35
10
73
74
12.0 0.6
Figure 4. Effects of the tested compounds on the nuclear translocation of p65 in
LPS-activated macrophages. Cells were pretreated with compounds 7a and 12b (5,
20 lM) for 30 min and followed by further incubation with LPS (1 lg/mL) for
15 min. The protein levels of nuclear p65 were determined by Western blotting.
PARP was used as an internal control. Images are the representative of three
independent experiments that shows similar results.
of NF-jB to the nucleus. The nuclear NF-jB activates the transcrip-
tion of iNOS, leading to the induction of its mRNA expression.
S
CH2CO2Et
6.5 0.5
Aminoguanidine
To examine whether compounds 7a and 12b prevented the
nuclear translocation of the p65 subunit of NF-jB, the nuclear lev-
a
Values mean the inhibition (%) of NO production at 20
lM of compounds rel-
els of p65 were determined. Treatment with compounds 7a and
12b decreased the nuclear translocation of LPS-induced p65 sub-
unit. Compound 12b treatment showed markedly decreased the
ative to the LPS control (n = 3).
b
Values are means SD of three experiments.
nuclear level of p65 at 5 and 20 lM as shown in Figure 4. PARP
was used as the internal control of nuclear extract. These observa-
tions suggest that compounds 7a and 12b inhibit the LPS-induced
activation of NF-jB by suppressing the translocation to the nucleus
in macrophage cells.
In conclusion, we prepared a series of carbamate and thiocarba-
mate derivatives and evaluated their inhibitory activities of NO pro-
duction in LPS-activated macrophages. Some of the prepared
compounds revealed promising inhibitory activity. They sup-
pressed the release of NO into culture media through the suppres-
sion of iNOS protein and mRNA expression and also inhibited the
Figure 2. Effects of the tested compounds on the expression of iNOS protein in LPS-
activated macrophages. RAW 264.7 cells were treated for 20 h with compounds 7a
and 12b (5, 20 lM) during LPS (1 lg/mL) activation. Cell lysates were prepared and
nuclear translocation of p65 subunit of NF-jB. Further study of
the other biological activities related with the overproduction of
NO, and the detailed mechanism for the activities of the representa-
tive compounds are in progress. Our carbamate and thiocarbamate
derivatives might serve as useful lead compounds for the develop-
ment of therapeutic agents for the management of NO-related
diseases.
the iNOS and b-actin protein levels were determined by Western blotting. b-Actin
was used as an internal control. The results shown are the representative of three
independent experiments.
Acknowledgments
This work was supported by the National Research Foundation
of Korea Grant funded by the Ministry of Education, Science and
Technology (Project No 2011-0030699) and by the Sookmyung
Women’s University Research Grants (2010).
Figure 3. Effects of the tested compounds on the expression of iNOS mRNA in LPS-
activated macrophages. RAW 264.7 cells were treated for 6 h with compounds 7a
and 12b (5, 20 lM) during LPS (1 lg/mL) activation. The mRNA levels of iNOS and b-
Supplementary data
actin were determined by RT-PCR from total RNA extracts. b-Actin was used as an
internal control. The results shown are the representative of three independent
experiments.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.03.010.
References and notes
iNOS mRNA at 20
lM. Compound 12b revealed remarkable inhibi-
tion even at 5 M (Fig. 3). These results indicated that the inhibi-
tion of NO production by thiocarbamate derivatives was resulted
from the suppression of iNOS protein and mRNA.
To elucidate the action mechanism of compounds 7a and 12b
for the inhibitory consequence of iNOS expression in activated
macrophages, we analyzed whether compounds 7a and 12b affect
l
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16. Cell culture and nitrite assay in LPS-activated RAW 264.7 cells—Cells in 10%
fetal bovine serum (FBS)-DMEM medium, were plated in 48-well plates
(1 ꢀ 105 cells/mL), and then incubated for 24 h. The cells were replaced with
fresh media with 1% FBS, and then incubated for 20 h in the presence or
18. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of iNOS
mRNA expression—RAW 264.7 cells (8 ꢀ 105 cells/60 mm dish) were
stimulated for 6 h with LPS (1 lg/mL) in the presence or absence of test
compounds. After washing twice with phosphate buffered saline, total RNA
was isolated from cell pellet, using an RNA isolation reagent (Trizol, Invitrogen,
Carlsbad, CA). Two microgram of RNA was reverse transcribed into cDNA using
reverse transcriptase and random hexamer. The PCR samples, contained in the
reaction mixture, were comprised of mixture buffer, dNTP, Taq DNA
polymerase (Promega, Madison, WI) and primers (sense and antisense). The
sense and antisense primers for iNOS were 50-ATGTCCGAAGCAAACATCAC-30
and 50-TAATGTCCAGGAAGTAGGTG-30, respectively. The sense and antisense
primers for b-actin were 50-TGTGATGGTGGGAATGGGTCAG-30 and 50-
TTTGATGTCACGCACGATTTCC-30, respectively. The PCR amplification was
performed under following conditions; 25 cycles of denaturation at 94 °C for
30 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s, using thermal
cycler (Gene Amp PCR system 2400, Applied Biosystems, Foster City, CA). The
amplified PCR products were separated on a 2% agarose gel.
absence of test compounds with LPS (1
was assessed by measuring the accumulated nitrite in culture supernatant.
Samples (100 L) of media were incubated with Griess reagent (150 L) for
lg/mL). NO production in each well
l
l
10 min at room temperature in 96 well microplate. Absorbance at 570 nm was
read using an ELISA plate reader. A standard calibration curve was prepared
using sodium nitrite as a standard. A dose–response curve was prepared, and
the results were typically expressed as IC50 values.
17. Western blot analysis of iNOS protein expression—RAW 264.7 cells (8 ꢀ 105
cells/60 mm dish) were stimulated with LPS (1 lg/mL) in the presence or