Design, synthesis and activity of benzothiazole-based inhibitors of NO production in LPS-activated macrophages

Article abstract of DOI:10.1016/j.bmcl.2010.08.112

Series of benzothiazoles were synthesized and evaluated their inhibitory activities for NO production in lipopolysaccharide-activated macrophages. The most potent compound was the indole-containing benzothiazole 3c with 4.18 μM of IC₅₀. The mec

Full text of DOI:10.1016/j.bmcl.2010.08.112

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6199–6202
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and activity of benzothiazole-based inhibitors
of NO production in LPS-activated macrophages
⇑
Guo Hua Jin , Hua Li, Semi An, Jae-Ha Ryu, Raok Jeon
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 benzothiazoles were synthesized and evaluated their inhibitory activities for NO production in
lipopolysaccharide-activated macrophages. The most potent compound was the indole-containing ben-
Received 24 May 2010
Revised 16 August 2010
Accepted 24 August 2010
Available online 16 September 2010
zothiazole 3c with 4.18
production by the suppression of iNOS protein and mRNA expression. They also suppressed the expres-
sion of COX-2 through the NF- B inactivation.
lM of IC50. The mechanistic study suggested that benzothiazoles inhibited NO
j
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Benzothiazole
Nitric oxide
NOS
Inhibitor
Nitric oxide (NO) is biosynthesized from
L
-arginine catalyzed by
which limited the application of them in vivo. Recent studies have
focused on design and synthesis of non-amino acid analogs such as
amino heterocycles,14 amidines,15 guanidines,16 isoquinolinam-
ines,17 and isothioureas.18,19 A number of these non-amino acid
analogs have been reported as selective iNOS inhibitors.20,21 Acti-
vation of iNOS enzyme requires homodimerization of NOS mono-
mers and binding of cofactors such as FMN, FAD, NADPH, and
tetrahydrobiopterin (BH4) to enzyme. Thus, inhibition of dimeriza-
tion would prevent overproduction of NO to attenuate NO-related
damages. Several groups have reported potent inhibitors of iNOS
dimerization.22–25 It was reported that BBS-4, potent imidazole-
based dimerization inhibitor disrupt the formation of NOS dimer
by direct coordination of the imidazole to the heme iron in the ac-
tive site of the enzyme.26,27 However, BBS-4 have been found to be
cross reactive to cytochrome P450.28 Diverse efforts have been
made to identify selective iNOS inhibitors with high safety profile
for effective attenuation of NO overproduction.
In order to identify new class of iNOS inhibitors, our study was
focused on the isosteric replacement of imidazole of the reported
dimerization inhibitor with another heterocycle. After careful con-
sideration of the structural features of several heterocycles, we
decided to investigate benzothiazole-based compounds as novel
inhibitors of dimerization and/or expression of iNOS. We initially
expected that benzothiazole could function as a metal coordinator
as imidazole did. It was supported by examination of the putative
binding mode of the benzothiazole-based analogs through molec-
ular docking analysis. We also anticipated the introduction of
appropriate lipophilic groups to the benzothiazole scaffold might
be beneficial for the activity and selectivity. Herein, we report
the design and synthesis of 4-alkoxy substituted benzothiazoles
three isoforms of nitric oxide synthases (NOSs).1 NO plays diverse
biological roles depending on which subtypes of NOS are involved
in its biosynthesis and which location is exposed to the released
NO. Constitutive isoforms found in neuronal tissues (nNOS) and
vascular endothelium (eNOS) are Ca2+-dependent and release
small amounts of NO required for homeostatic function.2 Mean-
while, inducible NOS (iNOS) induced by lipopolysaccharide (LPS)
and various cytokines such as IFN-a, IL-1b, and TNF-a
is Ca2+-inde-
pendent and produces high levels of NO.3 Overproduction of NO
has been implicated in several pathophysiological states, for exam-
ple, various inflammation,4 septic shock, vascular dysfunction in
diabetes, platelet aggregation, and cancer.5 Cyclooxgenase-2
(COX-2) is another inducible enzyme that plays a significant role
in the production of pro-inflammatory mediators,6 while COX-1
is the constitutively expressed form in normal conditions. NO acti-
vates COX-2 resulting in the increased release of inflammatory
prostaglandins (PGs). Therefore, the modulation of iNOS and
COX-2 can be a good strategy for the management of diseases
accompanying the overproduction of NO and PGs.
Early NOS inhibitors included the arginine derivatives such as
NG-methyl-
L
-arginine -ornitine
(
L
-NMA),7,8 N-iminoethyl-
L
(L-
NIO),9 NG-nitro- -NNA),10 and -thiocitrulline.11 These
L-arginine (L L
substrate analogs have been subjected to both animal models
and clinical trials to block overproduction of NO.12,13 However,
these compounds are not selective enough against NOS isoforms,
⇑
Corresponding author. Tel.: +82 2 710 9571; fax: +82 2 715 9571.
E-mail address: rjeon@sookmyung.ac.kr (R. Jeon).
This author is equally contributed to this work.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.112

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G. H. Jin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6199–6202
R1
R2
NO2
NH2
N
OH
N
H
d
b
a
N
S
O
O
O
O
N
S
N
S
N
S
N
S
1a
7, 2-NO2
8, 3-NO2
2a, 2-NH2
3a, 3-NH2
2d
Figure 1. Representative structure of benzothiazole derivatives.
c
e
f
(Fig. 1), and their inhibitory activities of the NO production in LPS-
activated macrophage. To understand the underlying mechanism
of active compounds, we examined the expression of iNOS/COX-2
H
O
N
R
R
N
H
N
R
O
O
O
O
protein and mRNA, the degradation of I-
j a, and the nuclear
B-
N
S
N
S
N
translocation of p65 subunit of NF-
and RT-PCR.
jB by Western blot analysis
S
2e-h
3b,c
2b, R = CH3CO
2c, R = CH3SO2
First, simply substituted benzothiazoles were prepared to figure
out the inhibitory activity of benzothiazole skeleton for NO pro-
duction (Scheme 1). Commercially available 2-amino-4-meth-
oxybenzothiazole (5) was converted to 4-methoxybenzothiazole
1b by treating isoamyl nitrite. Deprotection of 1b and 5 with
BBr3 gave corresponding 4-hydroxybenzothiazoles 1a and 1e, and
the following alkylation of the resulting 1a and 1e with appropri-
ate alkyl or benzyl halide provided 1b–d and 1f.
4a,b
Scheme 2. Preparation of 4-aminobenzyloxybenzothiazole derivatives. Reagents:
(a) 2- or 3-nitrobenzyl bromide, K2CO3, THF; (b) H2, 10% Pd/C, THF/MeOH; (c) AcCl
or CH3SO2Cl, TEA, DCM; (d) benzaldehyde, AcOH, NaBH3CN, CH3CN; (e) RCOOH,
BOP, TEA, DCM; (f) RCOCl, DCM.
Table 1
Next, amino-substituted 4-benzyloxybenzothiazole derivatives
were synthesized as depicted in Scheme 2. O-benzylation of 1a
by treatment of 2- or 3-nitrobenzyl bromide in the presence of
K2CO3 gave compounds 7 and 8, respectively. The followed reduc-
tion of nitro compounds 7 and 8 over 10% Pd/C under atmospheric
pressure of hydrogen gas provided amines 2a and 3a. Reductive
amination of compound 2a with benzaldehyde in the presence of
NaBH3CN and AcOH gave compound 2d. The amine 2a was also
acetylated and sulfonated to offer respective compounds 2b and
2c. Coupling reaction of the amine 2a and 3a with appropriate car-
boxylic acids using BOP as a coupling agent gave the amides 2e–h
and 3b, 3c. Amidation of secondary amine 2d with corresponding
acyl chlorides provided tertiary amides 4a, 4b.
Inhibitory activities of benzothiazole derivatives on the NO production in LPS-
activated macrophages
OR1
N
R2
S
Compounds
R1
R2
Inha (%)
1a
1b
1c
1d
1e
1f
H
CH3
H
H
H
H
NH2
NH2
8.6
18.6
12.3
10.4
11.4
14.4
Benzyl
4-Methylbenzyl
H
4-Methylbenzyl
a
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
Values mean the inhibition (%) of NO production at 20
compounds relative to the LPS control (n = 3).
l
M concentration of
with 1 lg/mL LPS in the presence of samples for 20 h. The amounts
of heterocycle-containing acyl group mostly enhanced activity ex-
cept compound 2f. The substituted positions of the compounds
also influenced on the activity. Regarding the position of the phe-
nyl substituents, meta-substituted amines 3a–c revealed higher
activity than the respective ortho-substituted amines 2a, 2e, and
2h. Effect of the second introduction of benzyl substituent at nitro-
gen of amide was not clear. The introduction of lipophilic and bulk-
ier tertiary amides increased the activity of 4a, but decreased that
of 4b, comparing with the corresponding secondary amides, 2e, 2g.
IC50 values of the representatively selected compounds 2d, 2e, 3b,
3c, 4a, and 4b were determined as 17.2, 72.3, 23.6, 4.2, 21.2, and
of NO released into culture media were determined by the Griess
method29 in the form of nitrite.30
The activities of the tested compounds were given in Tables 1
and 2. 4-Hydroxy or simple alkoxy substituted benzothiazoles re-
vealed little activities. Also, the existence of 2-amino group did
not show any difference on the activity. Activities of benzothiazole
derivatives were improved by introducing substituted amines on
the ortho- or meta-position of benzyloxy group as shown in Table 2.
Effect of the N-substitution (R1 and R2) of aniline on the activity
was investigated. Acetamide 2b and sulfonamide derivatives 2c
showed no activities. N-benzylated compound 2d showed the
highest activity among the N-substituted compounds. Introduction
20.9 lM, respectively. Among the tested benzothiazoles, indole-
containing compound 3c was the most potent.
To elucidate the possible mechanism of the active benzothia-
zole compounds for the inhibition of NO production, further bio-
logical study was carried out. Western blot analysis31 was
performed to examine the effects of compounds 2d, 3c, and 4b
on the expression of iNOS and COX-2 protein in LPS-activated
RAW 264.7 cells. A well known inhibitor of iNOS/COX-2 expres-
sion, curcumin was used as a positive control. The iNOS and
COX-2 protein expression was markedly induced by LPS-activa-
OR
OH
OCH3
OCH3
N
S
c
N
S
a
b
b
N
S
N
S
H2N
1a
1c-d
5
1b
OH
OR
c
N
S
N
S
H2N
H2N
tion. The treatment of 20 lV of compounds 2d, 3c, and 4b de-
creased the expression of iNOS and COX-2 in correlation with
their inhibitory potency of NO production. The most potent 3c sig-
nificantly inhibited the iNOS and COX-2 protein expression (Fig. 2).
At RT-PCR analysis,32 increased levels of iNOS and COX-2 mRNA
1e
1f
Scheme 1. Preparation of 4-alkoxybenzothiazole derivatives. Reagents: (a) isoamyl
nitrite, dioxane; (b) BBr3, DCM; (c) RBr, K2CO3, THF.

G. H. Jin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6199–6202
6201
Table 2
Inhibitory activities of benzothiazole derivatives on the NO production in LPS-
activated macrophages
R1
3
N
R2
2
O
N
S
La
R1
R2
Inhb (%)
IC50
(
lM)
c
Compounds
2a
2b
2c
2d
2
2
2
2
H
H
H
H
H
15.2
3.7
0.4
COCH3
SO2CH3
Bn
55.8
17.2
72.3
O
O
2e
2
H
32.5
11.6
O
O
O
O
O
2f
2
H
O
O
Figure 2. Effects of the prepared benzothiazoles on the expression of iNOS and
COX-2 proteins in LPS-activated macrophages. RAW 264.7 cells were treated for
20 h with compounds (20 lM) 2d, 3c, 4b, and curcumin during LPS (1 lg/mL)
2g
2h
2
2
H
H
44.7
36.4
activation. Cell lysates were prepared and the iNOS, COX-2, and actin protein levels
were determined by Western blotting. The relative iNOS, COX-2 protein levels were
normalized with the respective amounts of actin. Values represent mean SD of
OCH3
*
three independent densitometric analyses of bands. p <0.01 indicate significant
O
difference between LPS alone control and sample treatment.
N
H
3a
3b
3
3
H
H
H
32.9
58.9
O
23.6
4.2
O
O
O
O
OCH3
3c
3
H
75.9
N
H
O
O
4a
4b
2
2
Bn
Bn
54.9
39.8
21.2
20.9
O
O
O
a
L indicates the positions of amine substituents on the aromatic ring.
b
Values mean the inhibition (%) of NO production at 20
lM concentration of
compounds relative to the LPS control (n = 3).
c
Values are means SD of three experiments.
L indicates the position of
substituent.
were also decreased by treatment of compounds 2d, 3c, and 4b
(Fig. 3). These results suggested that the inhibition of NO produc-
tion by the active benzothiazole derivatives was caused by the sup-
pression of iNOS proteins and mRNA expression. When the
compounds were treated after the completion of iNOS induction
by LPS (post-treatment), they did not show significant inhibitory
activity. This result confirmed that benzothiazole derivatives
exhibited their activities mainly through the inhibition of iNOS
expression not the function of enzyme. The gene expressions of
Figure 3. Effects of the prepared benzothiazoles on the expression of iNOS and
COX-2 mRNAs in LPS-activated macrophages. RAW 264.7 cells were treated for 6 h
with compounds (20 lM) 2d, 3c, 4b, and curcumin during LPS (1 lg/mL) activation.
The mRNA levels of iNOS, COX-2 and b-actin were determined by RT-PCR from total
RNA extracts. The relative iNOS, COX-2 mRNA levels were normalized with the
respective amounts of b-actin. Values represent mean SD of three independent
*
densitometric analyses of bands. p <0.01 indicate significant difference between
LPS alone control and sample treatment.
iNOS and COX-2 are modulated by NF-
by the degradation of inhibitor- B (I- B) and the followed translo-
cation into nucleus. To verify further their inhibitory mechanism,
we examined the effects of benzothiazole derivatives on I- B-
degradation and nuclear translocation of p65 in LPS-activated mac-
rophages. Compounds 2d, 3c, and 4b decreased I- B- degradation
and resulted in the decrease of nuclear translocation of NF- B sub-
jB, which can be activated
In summary, a series of benzothiazole derivatives were identified
as novel inhibitors of NO production in LPS-activated macrophages.
Although they mostly revealed weak to moderate activity, several
compounds showed promising activity. The SAR study suggested
that amino-substituted 4-benzyloxy group on benzothiazole skele-
ton highly influenced on the activity. Among them, indole-contain-
j
j
j
a
j
a
j
ing compound 3c was the most potent with 4.18 lM of IC50.
unit, p65 (Fig. 4). Taking together the results from Western blot
analysis and RT-PCR, benzothiazoles 2d, 3c, and 4b decreased NO
production by the suppression of iNOS protein and mRNA through
Biological study indicated that possible mechanism of the active
benzothiazoles for inhibition of NO production was the suppression
of iNOS protein and mRNA expression through inhibition of I-
jB-a
the inhibition of NF-jB activation in LPS-activated macrophages.
degradation and NF- B activation. They also suppressed the expres-
j

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G. H. Jin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6199–6202
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j
a
sion of another inflammatory COX-2 through the same mechanism.
Further study of the other biological activities related with the over-
production of NO including in vivo experiments are in progress. Our
benzothiazole derivatives can be a promising lead for the develop-
ment of therapeutic agents for the treatment of NO-related diseases.
Acknowledgments
This work was supported by the SRC program of KOSEF (Re-
search Center for Women’s Diseases) and Basic Science Research
Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and Technology
(2010-0008340).
Supplementary data
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
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.08.112.
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.
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