Design and synthesis of urea and thiourea derivatives and their inhibitory activities on lipopolysaccharide-induced NO production

Article abstract of DOI:10.1016/j.bmcl.2007.04.005

Series of ureas and thioureas were designed and synthesized, and their inhibitory activities of NO production in lipopolysaccharide-activated macrophages were evaluated. We found several essential moieties in the structure of the prepared compounds for th

Full text of DOI:10.1016/j.bmcl.2007.04.005

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3317–3321
Design and synthesis of urea and thiourea derivatives and
their inhibitory activities on lipopolysaccharide-induced
NO production
Yoon Jung Kim, Jae-Ha Ryu, Ye Jin Cheon, Hyo Jin Lim and Raok Jeon*
College of Pharmacy, Sookmyung Women’s University, 52 Hyochangwon-Gil, Yongsan-Ku, Seoul 140-742, Republic of Korea
Received 17 November 2006; revised 20 March 2007; accepted 2 April 2007
Available online 6 April 2007
Abstract—Series of ureas and thioureas were designed and synthesized, and their inhibitory activities of NO production in lipopoly-
saccharide-activated macrophages were evaluated. We found several essential moieties in the structure of the prepared compounds
for the activity. Thiourea derivatives revealed higher inhibitory activity than the corresponding urea derivatives. Among these com-
pounds, 7e having carboxymethyl group at N3 position of thiourea was the most potent in the inhibition of NO production. They
inhibited NO production through the suppression of iNOS protein and mRNA expression.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The critical role of nitric oxide (NO) in various patho-
logical conditions has led to the discovery of new inhib-
itors of NO production as potential therapeutic agents.
NO, a gaseous free radical, is produced through the oxi-
dation of L-arginine by three isoforms of nitric oxide
synthase (NOS).1 The constitutive NOS (cNOS) found
in neuronal tissues (nNOS, type I) and vascular endo-
can be applied for the treatment of diseases accompany-
ing high levels of NO. Regulation of iNOS can be
achieved by the control of expression level and/or enzy-
matic activity of iNOS. Many classes of iNOS inhibitors
can be classified according to their structural features,
such as, amino acid analogues,7 amino heterocycles,8
amidines,9 guanidines,10 isoquinolinamines,11 and isot-
hiourea.12,13 But most of the inhibitors are neither po-
tent nor selective enough against NOS isoforms, that
limited the application of them in vivo. Only one class
of iNOS inhibitors, L-lysine analogue, was reported to
enter the clinical trial in human.14
thelium (eNOS, type III) is Ca2+-dependent and releases
small amounts of NO required for homeostatic func-
tion.2 Meanwhile, inducible NOS (iNOS, type II), which
can be induced by lipopolysaccharide (LPS) and various
cytokines such as IFN-a, IL-1b, and TNF-a, is Ca2+
-
independent and produces micromolar levels of NO.3
Low concentrations of NO produced by iNOS possess
beneficial roles in antimicrobial activity of macrophages
against pathogens,4 while the overproduction of NO and
its derivatives, such as peroxynitrite and nitrogen diox-
ide, has been suggested to be mutagenic in vivo and to
provoke the pathogenesis of septic shock and various
inflammatory processes.5 Furthermore, NO and its oxi-
dized forms have also been known to be carcinogenic.6
Urea was known to inhibit not only the activity of iNOS
in macrophages during the uremia,15 but also the expres-
sion of iNOS in LPS-activated macrophages.16 Urea was
suggested as an important modulator for renal function
through the fine tuning of NO production.17 Several
groups have reported that urea and thiourea18 or iso-
thiourea12,13,19 inhibit iNOS expression and/or NO pro-
duction. Based on these reports, we tried to investigate
urea and thiourea derivatives as novel inhibitors having
mechanism for enzymatic inhibition and/or downregula-
tion of iNOS expression.
Among the many strategies for providing rational con-
trol of NO levels, the efforts have been mainly directed
toward development of selective inhibitor of iNOS that
Herein, we report the design and synthesis of urea and
thiourea derivatives as depicted in Figure 1. Their effects
on the NO production and expression of iNOS were
evaluated in LPS-activated macrophage cell culture
system.
Keywords: Urea; Thiourea; Carbazole; Nitric oxide synthase; Nitric
oxide; Inhibitor.
*
Corresponding author. Tel.: + 82 2 710 9571; fax: + 82 2 715 9571;
e-mail: rjeon@sookmyung.ac.kr
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.005

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Y. J. Kim et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3317–3321
R1
N
as positive control that showed 85% inhibition of NO
R1, R2 = H, alkyl
X = O, S
Y = H, CH3, N-carbazolylethyl
H
N
production at 0.1 lM. Most of the thiourea derivatives
revealed higher activity than the corresponding urea
derivatives. For example, thioureas 7a, 7c, 7e, and 7f
showed significantly higher activities than ureas 6a, 6b,
6g, and 6i, respectively.
R2
Y
X
O
Figure 1. Representative structure of urea and thiourea.
The preparation of the carbazole-linked urea and thio-
urea derivatives is outlined in Schemes 1 and 2. Hydroxy
ethyl group was introduced at nitrogen of carbazole and
the resulting alcohol 2 was mesylated to obtain com-
pound 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 hydrogen gas pro-
vided amine 5. Condensation of amine 5 with the appro-
priate isocyanates or isothiocyanates offered the desired
compounds 6 and 7.
Effects of the alkyl substituents at the N1 and N3 posi-
tion of urea 6a and thiourea 7a were investigated. Intro-
duction of methyl group 6h at the N1 position of urea 6a
lowered the activity, while introduction of bulkier sub-
stituent retrieved the activity. Compound 6i with ethyl
group showed the similar activity as 6a. Meanwhile,
activity of compound 6j with cyclopropylmethyl at N1
became 2.5-fold higher than that of 6a. On the other
hand, thiourea derivatives 7f substituted with ethyl
and 7g with cyclopropylmethyl at N1 of 7a revealed
2-fold lower activity than 7a.
The activities of the prepared compounds were evalu-
ated for the inhibition of NO production in LPS-acti-
vated macrophages. Murine macrophage cell line,
RAW 264.7 cells, was stimulated 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 method20 in the form of nitrite.21
The effects of substituents at N3 position were consider-
ably different between urea and thiourea derivatives.
While the substitution at N3 of urea derivatives 6b–6g
showed no significant effects on the activity, the alkyl
substitution of thiourea greatly enhanced the activity.
Compounds 7b, 7c, 7e with methyl, ethyl, or carboxym-
ethyl group at N3 revealed similar activity ranging from
80% to 90% inhibition of NO production at 5lM con-
centration. In both cases of ureas and thioureas, the
carboxymethyl was the best substituent at N3 for the
improvement of activity. IC50 values of 6j, 7a–7c, and
The inhibitory activities of the prepared compounds on
the NO production are given in Table 1. Aminoguani-
dine, a well-known specific inhibitor of iNOS, was used
a
b
N
H
N
N
OH
OMs
3
1
2
d
c
NO
NH
2
2
N
N
O
O
4
5
Scheme 1. Preparation of 4-(2-carbazol-9-ylethoxy)phenylamine. Reagents: (a) ICH2CH2OH, NaH, DMF; (b) MsCl, TEA, DMF; (c) 4-nitrophenol,
NaH, DMF; (d) H2, 10% Pd/C.
H
N
a
b
c
NH2
6a, X = O
7a, X = S
N
N
X
X
O
O
6b-e, X = O
6f, X = O
NH2
H
N
H
N
d
d
6g, X = O
7b-ef, X = S
7d, X = S
7e, X = S
R2
N
O
5
R1
N
NH2
6h-j, X = O
7f,g, X = S
N
X
O
Scheme 2. Preparation of carbazole-linked urea and thiourea derivatives. Reagents: (a) NaCNX (X = O, S), AcOH, H2O; (b) R2NCX (X = O, S),
0
0
K2CO3, THF; (c) i—R1 CHO (R1 = corresponding alkyl-CH2), NaCNBH3, CH3CN; ii—NaCNX (X = O, S), AcOH, H2O; (d) Ba(OH)2, THF/
MeOH/H2O.

Y. J. Kim et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3317–3321
3319
Table 1. Inhibitory activities of carbazole-linked phenylureas and phenylthioureas on the NO production in LPS-induced NO production
R1
H
N
N
R1, R2 = H, alkyl
X = O, S
R2
N
X
O
R1
R2
X
Inhibitiona (%)
b
IC50 (nM)
Compound
6a
6b
6c
6d
6e
6f
H
H
Et
O
O
O
O
O
O
O
O
O
O
S
24
30
14
15
28
38
43
8
H
H
Pro
i-Pro
Ph
H
H
H
CH2CO2Et
CH2CO2H
6g
6h
6i
H
Me
H
Et
Cyclopropylmethyl
H
25
61
55
80
90
41
87
33
34
6j
H
3120 250
5310 702
730 180
901 211
7a
7b
7c
7d
7e
7f
H
H
Me
H
S
H
Et
CH2CO2Et
CH2CO2H
S
H
S
H
Et
S
153 83
H
H
S
7g
Cyclopropylmethyl
S
a Values mean the inhibition (%) of NO production at 5 lM concentration of compounds relative to the LPS control (n = 3).
b Values are means SD of three experiments.
7e, which showed more than 50% inhibitory responses at
5 lM, were determined as 3.12, 5.31, 0.73, 0.90, and
0.15 lM, respectively.
120
100
80
60
40
20
0
In order to find the influence of lipophilic tail on the
activity, carbazolylmethyl group of thiourea derivatives
7b–7c was eliminated. These compounds showed lower
activities, less than 15% inhibition of NO production
at 5 lM, than the corresponding carbazole-linked thiou-
reas. It has been reported that a carbazole derivative
inhibited iNOS expression in the LPS-activated macro-
phage.22 Our results also demonstrated that both thio-
urea and carbazole moieties might play an important
role for their activities although carbazole moiety devoid
of thiourea group revealed no activity. We expect to
potentiate the activity by the structural modification of
thiourea and lipophilic segment.
iNOS
actin
LPS
Sample
-
+
+
6j
+
7a
+
7b
+
7c
+
7e
Figure 2. Effects of the prepared compounds on the expression of
iNOS protein in LPS-activated macrophages. RAW 264.7 cells were
treated for 20 h with compounds (10 lM) during LPS (1 lg/mL)
activation. Cell lysates were prepared and the iNOS and actin protein
levels were determined by Western blotting. The relative iNOS protein
levels were normalized with the respective amounts of actin. Values
represent means SD of three independent densitometric analyses of
bands.
For the further biological study of our derivatives, we
examined the effects of 6j, 7a–7c, and 7e on the expres-
sion of iNOS protein and mRNA in LPS-activated
RAW 264.7 cells. The amounts of iNOS protein were
analyzed in Western blot analysis after 20-h incubation
with compounds during LPS (1 lg/mL) activation of
macrophages.23 Compounds 7b and 7e significantly
reduced the amounts of iNOS at 10 lM (Fig. 2). At
RT-PCR analysis,24 the expression level of iNOS
mRNA was increased markedly by LPS-activation for
6 h. Compounds 7b and 7e suppressed the induction of
iNOS mRNA at 10 lM (Fig. 3). These results indicated
that the inhibition of NO production by thiourea deriv-
atives resulted from the suppression of iNOS protein
and mRNA.
weak activity compared with the results of the co-treat-
ment of compounds with LPS. Even the most potent
compound 7e showed 12% inhibition at 10 lM by
post-treatment. These results suggested that thiourea
derivatives exhibited their activities mainly through
the inhibition of iNOS expression with marginal inhibi-
tion against enzymatic activity. It has been reported
that urea itself inhibited the activity of iNOS by transcrip-
tional15 and post-transcriptional16 mechanisms. Many
of thioureas and isothioureas were reported12,25 as
inhibitors of iNOS enzyme devoid of controlling the
expression step. In addition, a carbazole compound,
When we treated the compounds after the completion of
iNOS induction by LPS (post-treatment), they showed

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120
References and notes
100
80
60
40
20
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LPS
Sample
-
+
+
+
+
+
+
6j
7a
7b
7c
7e
Figure 3. Effects of the prepared compounds on the expression of
iNOS mRNA in LPS-activated macrophages. RAW 264.7 cells were
treated for 6 h with compounds (10 lM) during LPS (1 lg/mL)
activation. The mRNA levels of iNOS and b-actin were determined
by RT-PCR from total RNA extracts. The relative iNOS mRNA levels
were normalized with the respective amounts of b-actin. Values
represent means SD of three independent densitometric analyses of
bands.
9-(2-chlorobenzyl)-9H-carbazole-3-carbaldehyde, was
reported as an inhibitor of iNOS mRNA expression
through a signaling pathway that does not involve NF-
jB pathway.22 The exact difference between the mecha-
nism of our compounds and that of the reported thioureas
and carbazole derivative was not explained in this report.
The study of the mechanism for the iNOS inhibition by
our compounds might be worthy to pursue further.
14. Hansel, T. T.; Kharitonov, S. A.; Donnelly, L. E.; Erin, E.
M.; Currie, M. G.; Moore, W. M.; Manning, P. T.;
Recker, D. P.; Barnes, P. J. FASEB J. 2003, 17, 1298.
15. Moeslinger, T.; Friedl, R.; Volf, I.; Brunner, M.; Baran,
H.; Koller, E.; Spieckermann, P. G. Kidney Int. 1999, 56,
581.
In conclusion, we prepared a series of urea and thiourea
derivatives and evaluated their inhibitory activities of
NO production in LPS-activated macrophages. They
suppressed the release of NO into culture media through
the suppression of iNOS protein and mRNA expression.
The SAR studies demonstrated that thiourea is superior
to urea and N3 substitution of thiourea with alkyl group
is highly beneficial for their activity. Further study of the
other biological activities related with the overproduc-
tion of NO, and the detailed mechanism for the activities
of these derivatives, is in progress. Our thiourea deriva-
tives that can control the expression of iNOS can be
good leads for the development of therapeutic agents
for the management of NO-related diseases.
16. Prabhakar, S. S.; Zeballos, G. A.; Montoya-Zavala, M.;
Leonard, C. Am. J. Physiol. 1997, 273, C1882.
17. Wang, W.; Jittikanont, S.; Falk, S. A.; Li, P.; Feng, L.;
Gengaro, P. E.; Poole, B. D.; Bowler, R. P.; Day, B. J.;
Crapo, J. D.; Schrier, R. W. Am. J. Physiol. Renal Physiol.
2003, 284, F532.
18. Goodyer, C. L. M.; Chinje, E. C.; Jaffar, M.; Stratford, I.
J.; Threadgill, M. D. Bioorg. Med. Chem. 2003, 11, 4189.
19. Shearer, B. G.; Lee, S.; Oplinger, J. A.; Frick, L. W.;
Garvey, E. P.; Furfine, E. S. J. Med. Chem. 1997, 40, 1901.
20. Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P.
L.; Wishnok, J. S.; Tannenbaum, S. R. Anal. Biochem.
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21. Cell culture and nitrite assay in LPS-activated RAW 264.7
cells—cells in 10% fetal bovine serum (FBS)–DMEM,
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 absence of test compounds with LPS (1 lg/
mL). NO production in each well was assessed by
measuring the accumulated nitrite in culture supernatant.
Samples (100 lL) of media were incubated with Griess
reagent (150 lL) for 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.
Acknowledgment
This work was supported by the SRC program of
MOST/KOSEF (R11-2005-017, RESEARCH CEN-
TER FOR WOMEN’S DISEASES).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2007.04.005.
22. Tsao, L. T.; Lee, C. Y.; Huang, L. J.; Kuo, S. C.; Wang, J.
P. Biochem. Pharmacol. 2002, 63, 1961.

Y. J. Kim et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3317–3321
3321
23. Western blot analysis of iNOS protein expression—RAW
264.7 cells (1.5 · 106 cells/60-mm dish) were stimulated
with LPS (1 lg/mL) in the presence or absence of test
compounds. After incubation for 20 h, the cells were
washed and lysed with lysis buffer. Twenty lg protein of
cell lysates was applied on 8% SDS–polyacrylamide gels
and transferred to PVDF membrane by a standard
method. The membrane was probed with antibody for
anti-mouse iNOS (Transduction Laboratories, Lexington,
KY) and anti-actin (Sigma, St. Louis, MO). The bands
were visualized using an enhanced chemiluminescence
(ECL) detection kit (Amersham Bioscience, Piscataway,
NJ) according to the manufacturer’s instruction.
24. Reverse transcription-polymerase chain reaction (RT-
PCR) analysis of iNOS mRNA expression—RAW 264.7
cells (1.8 · 106 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 micrograms 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-TGT
GATGGTGGGAATGGGTCAG-30 and 50-TTTGATGT
CACGCACGATTTCC-30, respectively. The PCR ampli-
fication 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.
25. Garvey, E. P.; Oplinger, J. A.; Tanoury, G. J.; Sherman,
P. A.; Fowler, M.; Marshall, S.; Harmon, M. F.; Paith, J.
E.; Furfine, E. S. J. Biol. Chem. 1994, 269, 26669.