Synthesis and anti-inflammatory evaluation of novel benzimidazole and imidazopyridine derivatives

Article abstract of DOI:10.1021/ml300282t

Sepsis, an acute inflammatory disease, remains the most common cause of death in intensive care units. A series of benzimidazole and imidazopyridine derivatives were synthesized and screened for anti-inflammatory activities, and the imidazopyridine series showed excellent inhibition of the expression of inflammatory cytokines in LPS-stimulated macrophages. Compounds X10, X12, X13, X14, and X15 inhibited TNF-α and IL-6 release in a dose-dependent manner, and X12 showed no cytotoxicity in hepatic cells. Furthermore, X12 exhibited a significant protection against LPS-induced septic death in mouse models. Together, these data present a series of new imidazopyridines with potential therapeutic effects in acute inflammatory diseases.

Full text of DOI:10.1021/ml300282t

Letter
pubs.acs.org/acsmedchemlett
Synthesis and Anti-inflammatory Evaluation of Novel Benzimidazole
and Imidazopyridine Derivatives
Gaozhi Chen,†,∥ Zhiguo Liu,†,∥ Yali Zhang,‡ Xiaoou Shan,§ Lili Jiang,§ Yunjie Zhao,† Wenfei He,†
Zhiguo Feng,† Shulin Yang,‡ and Guang Liang*,†
†School of Pharmacy, Wenzhou Medical College, 1210 University Town, Wenzhou, Zhejiang 325035, China
‡School of Environmental and Biological Engineering, Nanjing University of Science and Technology, 200 Xiaolingwei St., Nanjing,
Jiangsu 210094, China
§Department of Pediatrics, The Second Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang 325035, China
S
* Supporting Information
ABSTRACT: Sepsis, an acute inflammatory disease, remains the most common cause of death in intensive care units. A series of
benzimidazole and imidazopyridine derivatives were synthesized and screened for anti-inflammatory activities, and the
imidazopyridine series showed excellent inhibition of the expression of inflammatory cytokines in LPS-stimulated macrophages.
Compounds X10, X12, X13, X14, and X15 inhibited TNF-α and IL-6 release in a dose-dependent manner, and X12 showed no
cytotoxicity in hepatic cells. Furthermore, X12 exhibited a significant protection against LPS-induced septic death in mouse
models. Together, these data present a series of new imidazopyridines with potential therapeutic effects in acute inflammatory
diseases.
KEYWORDS: benzimidazoles, imidazopyridines, anti-inflammation, macrophages, sepsis
epsis, an acute inflammatory disease, is currently the 10th
leading cause of death overall and accounts for major
their expressions have attracted much attention in drug
development for inflammatory disease. For example, improved
survival following neutralization of these pro-inflammatory
mediators by antibodies or specific inhibitors has been recently
reported in animal models and clinical studies of sepsis.9,10
Small molecule inhibitors of cytokine expression are emerging
with a few having advanced into clinical trials.11 Our group has
previously designed and evaluated a lot of synthetic compounds
as anti-inflammatory agents, some of which are undergoing
preclinical assessment as antisepsis candidates.12,13
Recently, a series of benzimidazole derivatives were evaluated
for anti-inflammatory activity in carrageenan-induced rat paw
edema.14 In addition, studies also showed that imidazopyridines
possessed anti-inflammatory activity.15,16 More recently,
Ashwell et al.17 reported the discovery and optimization of a
series of imidazopyridines as selective and potent inhibitors of
protein kinase B (PKB or AKT), which functions as a key
signaling node in cell proliferation, survival, and inflammatory
S
healthcare expenditures in the developing world.1 It is widely
believed that the incidence of sepsis and sepsis-related mortality
will continue to rise. Research efforts in the field of sepsis have
largely focused on the innate immune system and have
conceptually viewed sepsis as a syndrome of hyperinflamma-
tion.2,3 Under this paradigm, overzealous activation of the host
inflammatory response, ostensibly intended for pathogen
eradication, becomes dysregulated and consequently causes
autoinjury to the host, leading to multiple organ dysfunction
and death.4 In this inflammatory storm, cytokines such as
tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and IL-
1β play important roles.5,6
The pro-inflammatory cytokines, including TNF-α and IL-6,
whose expression can be induced by endotoxin (lipopolysac-
charide, LPS), share many cellular and molecular features in
common and are deeply involved in sepsis and other
inflammation-mediated diseases, such as rheumatoid arthritis,
diabetic complications, cancer, atherosclerosis, and inflamma-
tory bowel disease, by amplifying inflammatory signals in
multiple pathways.7,8 Therefore, therapeutically relevant
candidates that target pro-inflammatory cytokines or inhibiting
Received: September 13, 2012
Accepted: November 21, 2012
© XXXX American Chemical Society
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Scheme 1. General Synthesis and Chemical Structures of Benzimidazole Derivatives (X1−X9)
Scheme 2. General Synthesis and Chemical Structures of Imidazopyridine Derivatives (X10−X32)
stress response. Thus, previous evidence prompted us to study
the cytokine-inhibitory and antisepsis activities of new
benzimidazole and imidazopyridine derivatives.
synthesis of imidazopyridines derivatives (X10−X32) pro-
ceeded in the similar route (Scheme 2). Generally, 2-amino-6-
chloro-3-nitropyridine (4a) and 2-amino-6-chloro-3-nitro-N-
propylpyridin (4b), which were derived from nucleophilic
substitution reactions of 2,6-dichloro-3-nitropyridine (3), were
treated with morpholine or 1-methylpiperazine to afford 6-
substituted derivatives (5a−d). Finally, the intermediates 5a−d
were cyclized with the various aldehydes in sodium hyposulfite
medium to afford the target imidazopyridines (X10−X32). All
As shown in Schemes 1 and 2, two novel series of
benzimidazoles (X1−X9) and imidazopyridines (X10−X32)
have been synthesized. Briefly, substituted ortho-nitroanilines
(2a and 2b) were prepared by direct displacement of 1,4-
dichloro-2-nitrobenzene (1) by amines at the reflux in
dimethylformamide (DMF). Subsequently, a cyclization of
ortho-nitroanilines by various aldehydes in sodium hyposulfite
medium at reflux provided target benzimidazoles (X1−X9)
with the yields ranging 30−70% (Scheme 1). In parallel, the
1
of the new compounds were characterized by H NMR and
ESI-MS, and details on their synthetic and structural
information are located in the Supporting Information. Before
B
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Figure 1. Benzimidazole and imidazopyridine derivatives inhibited LPS-induced TNF-α and IL-6 secretion in RAW 264.7 macrophages.
Macrophages were plated at a density of 4.0 × 105/plate for overnight in 37 °C and 5% CO2. Cells were pretreated with Ome or compounds (10
μM) for 2 h and then treated with LPS (0.5 μg/mL) for 22 h. TNF-α and IL-6 levels in the culture media were measured by ELISA and were
normalized by the total protein. The results were expressed as the percentage of LPS control. Statistical significance relative to LPS group is
indicated: *, p < 0.05; and **, p < 0.01.
Figure 2. Plots of predicted activity against the corresponding experimental activity on TNF-α (A) and IL-6 (B) inhibition. N, the number of
compounds taken into account in the regression; R2, the multiple correlation coefficient; Radj2, adjusted multiple correlation coefficient; s, residual
standard error; and the F value is related to the F-statistic analysis (Fischer test). The numbers in parentheses mean the standard deviation of the
coefficients.
they were used in biological experiments, compounds were
purified by silica gel column chromatography, and HPLC was
used to determine their purity (≥95.0%).
Although inflammatory cytokines can be produced by various
cells, macrophages are the prototypical cell source.18 The 32
synthetic derivatives were evaluated for their ability to decrease
the expression of TNF-α and IL-6 in mouse RAW 264.7
macrophages stimulated by LPS. Macrophages were pretreated
with 10 μM compounds for 2 h and then incubated with 0.5
μg/mL LPS for 22 h. The amount of TNF-α and IL-6 in media
was detected by enzyme-linked immunosorbent assays (ELISA)
and normalized by protein concentration of cells harvested in
homologous culture plates.
Figure 1 gives the cytokine-inhibitory activity of 32
derivatives with the benzimidazole-containing omeprazole
(Ome) as an anti-inflammatory comparison.19,20 The majority
of these compounds exhibited higher inhibition than
omeprazole against LPS-induced TNF-α and IL-6 expression.
Compounds X7, X10−X15, X17, X18, X20−X22, X26, X27,
and X31 showed inhibition of TNF-α production in a range of
C
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Figure 3. Cytokine-inhibitory activity and cytotoxicity of five active compounds. (A and B) Macrophages were plated at a density of 1.2 × 106/plate
overnight at 37 °C and 5% CO2. Cells were pretreated with specific compounds at indicated concentrations for 2 h, followed by LPS (0.5 μg/mL)
treatment for 22 h. TNF-α (A) and IL-6 (B) levels in the culture medium were measured by ELISA and were normalized to the total protein
amount. Statistical significance relative to the LPS group is indicated: *, p < 0.05; and **, p < 0.01. (C) Five imidazopyridines at 20 μM were tested
for their cytotoxicity in human normal liver HL-7702 Cells. The cell viability was determined by MTT method after a treatment period of 24, 48, and
72 h.
50−65%. With regard to IL-6, compounds X10 and X12−X15
exhibited more than 50% inhibitory effects against IL-6
expression as compared to LPS control. Among the tested
compounds, X10 is the strongest inhibitor on LPS-induced
TNF-α and IL-6 release with the inhibitory rates of 64.8 and
81.4%, respectively, as compared to the LPS control. According
the bioscreening, it is observed that imidazopyridine derivatives
showed stronger anti-inflammatory activity than benzimida-
zoles. Within the imidazopyridine series, compounds with n-
propyl group on the R3 and 1-methylpiperazine on the R4 were
the most active (X10−X15). Also, it should be noted that
various heterocycles at the R5 of the imidazopyridine have an
important impact on activity.
To further demonstrate the SAR of these compounds and to
evaluate the effects of various substituents on the activity, a
quantitative SAR (QSAR) was calculated. The concrete
description for QSAR study is shown in the Supporting
Information, and the descriptors studied here are listed in Table
S1 in the Supporting Information. The scatter plot of predicted
versus experimental values is illustrated in Figure 2. Using three
different variables, the statistically significant models, eq 1 and
eq 2, were obtained for anti-TNF-α and anti-IL-6 activities of
compounds, with relatively high regression coefficients (R2) of
0.83 and 0.70, respectively. As discussed in the Supporting
Information, the QSAR results indicate that the electro-
negativity and molecular polarizability may play an important
role in the anti-inflammatory activity of benzimidazole and
imidazopyridine derivatives.
Among tested compounds, the most promising compounds
X10, X12, X13, X14, and X15 were selected for further
assessment of their dose-dependent inhibitory effects against
LPS-induced TNF-α and IL-6 release, and their IC50 was
determined. RAW264.7 macrophages were pretreated with
compounds at a series of concentrations (1.0, 5.0, and 10 μM)
for 2 h and were subsequently incubated with LPS (0.5 μg/mL)
for 22 h. The release of TNF-α and IL-6 was determined by
ELISA. The results are shown in Figure 3A,B, indicating a dose-
dependent inhibition of LPS-induced TNF-α and IL-6 release
by these derivatives. Accordingly, their IC50 values were
determined, and all of them were under 10 μM except that
of X10 and X14 against TNF-α release. X13 exhibited the
lowest IC50 values against the expression of both TNF-α (1.99
μM) and IL-6 (1.21 μM), respectively. The fact that the
inhibition of TNF-α and IL-6 release by these compounds in a
D
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Figure 4. X12 improves survival of mice subjected to a lethal dose of LPS. Mice were treated with 15 mg/kg X12 (iv) either 15 min before or 15 min
after the injection of 20 mg/kg of LPS (iv). Survival (A) and body weight (B) were recorded for 7 days after the LPS injection at the interval of 1
day. Results from the summary of two different experiments are shown. n = 12 animals in each group (**p < 0.01 vs LPS group).
dose-dependent manner further suggests their potential as anti-
inflammatory agents.
In conclusion, we have synthesized and evaluated two series
of benzimidazole and imidazopyridine derivatives for their anti-
inflammatory activities in LPS-stimulated macrophages. It was
concluded that the imidazopyridine series showed better
activity than the benzimidazole series. Combined with the
QSAR analysis, an electron-donating substitution and low
molecular polarizability in imidazopyridine skeleton are
favorable to anti-inflammatory activities of imidazopyridine
derivatives. Compound X12 was selected for in vivo testing and
was found to markedly decrease LPS-induced lethality in septic
mice models. Thus, imidazopyridine derivatives may be
promising for drug discovery as anti-inflammatory agents.
Further studies should include testing of these new anti-
inflammatory compounds in more animal models and
examination of the underlying molecular mechanisms and
direct targets at the transcriptional or post-transcriptional level.
Before the in vivo evaluation, five active imidazopyridines
X10, X12, X13, X14, and X15 were tested for their cytotoxicity
and safety in human normal hepatic cell line HL-7702. Cell
viability was determined by MTT method after treatment with
compounds for 24, 48, and 72 h. As shown in Figure 3C, X13
shows a low toxicity, while the other four compounds at 20 μM,
particularly X12 and X14, are nontoxic in hepatic cells,
indicating that they are relatively safe. Thus, combining with
the anti-inflammatory activity and cytotoxicity of these
compounds, we chose the representative compound X12 for
the next in vivo evaluation.
As a major endotoxin, LPS from Gram-negative bacteria has
been implicated as a major cause of sepsis. A number of
different approaches have been investigated to treat and/or
prevent the septic shock associated with infections caused by
Gram-negative bacteria, including blockage of one or more of
the cytokines induced by LPS signaling.21 Our data have
demonstrated the inhibitory effects of these derivatives on LPS-
induced cytokine release. For the potential clinical application,
we further determine whether X12 are able to attenuate
endotoxin shock through inhibiting LPS-induced inflammatory
response. X12 was used in the form of X12-HCl for
intravenous (iv) administration in acute inflammatory models.
Mice were injected with LPS at the dosage of 20 mg/kg
intravenously in the presence or absence of X12 injection, and
their survival rates were monitored for 7 days, respectively. As
shown in Figure 4A, all animals treated with LPS alone died
within 3 days as a result of the septic shock. In animals
receiving X12 at 15 mg/kg either 15 min prior to LPS injection
(for preventive effect) or 15 min after LPS injection (for
therapeutic effect), the survival rates were significantly
increased as compared to that of the control group (50%
survivals in both the prevention group and the treatment group,
p < 0.01 in both groups v.s. LPS group). Meanwhile, the body
weight of mice in the prevention group decreased during 0−2
days but regained slowly 2−7 days after LPS injection (Figure
4B), while mice in the treatment group lost their body weight
till the third day after LPS injection. Thus, our data provide
evidence for the anti-inflammatory effects of these imidazopyr-
idine derivatives and X12's potent prevention and treatment
effects in sepsis. Although the anti-inflammatory mechanism
and underlying molecular targets are still unknown, the
beneficial effects of the imidazopyridines on LPS-induced
inflammation will become an objective in the continuing
research.
ASSOCIATED CONTENT
* Supporting Information
■
S
Full names, structures, and characterization of all compounds,
and details regarding biological assays and quantitative
structure−activity relationship (QSAR) study. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: (+86)577-86699524. Fax: (+86)577-86699527. E-mail:
cuiliang1234@163.com.
■
Author Contributions
∥These authors contributed equally to this work.
Funding
Financial support was provided by the National Natural Science
Funding of China (21272179 and 21202124), High-level
Inovative Talent Funding of Zhejiang Department of Health
(G.L.), Project of Wenzhou Sci&Tech Bureau (Y20120061 and
Y4090261), Zhejiang Natural Science Funding (Q12H300009),
Project of Zhejiang Provincial Key Constructive Subject
(Traditional Chinese Medicine, 2012-XK-A28), and Zhejiang
Key Group Project in Scientific Innovation (2010R50042).
Notes
The authors declare no competing financial interest.
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