Discovery of Novel 2-(piperidin-4-yl)-1H-benzo[d]imidazole Derivatives as Potential Anti-Inflammatory Agents

Article abstract of DOI:10.1111/cbdd.12513

A novel 2-(piperidin-4-yl)-1H-benzo[d]imidazole derivative 5 with good anti-inflammatory activity was identified from our in-house library. Based on hit compound 5, two series of 2-(piperidin-4-yl)-1H-benzo[d]imidazole derivative 6a-g and 7a-h were designed and synthesized as novel anti-inflammatory agents. Most of synthesized compounds exhibited good inhibitory activity on NO and TNF-α production in LPS-stimulated RAW 264.7 macrophages, in which the compound 6e showed most potent inhibitory activity on NO (IC₅₀ = 0.86 μm) and TNF-α (IC₅₀ = 1.87 μm) production. Further evaluation revealed that compound 6e displayed more potent in vivo anti-inflammatory activity than ibuprofen did on xylene-induced ear oedema in mice. Additionally, Western blot analysis revealed that compound 6e could restore phosphorylation level of IκBα and protein expression of p65 NF-κB in LPS-stimulated RAW 264.7 macrophages.

Full text of DOI:10.1111/cbdd.12513

Received Date : 11-Sep-2014
Revised Date : 04-Dec-2014
Accepted Date : 08-Jan-2015
Article type
: Research Article
Title page
Title:
Discovery of novel 2-(piperidin-4-yl)-1H-benzo[d]imidazole derivatives as potential
anti-inflammatory agents
Authors:
a#
b#
a
a
a
a
c
Qing Li , Qinghua Hu , Xinning Wang , Yang Zong , Leilei Zhao , Junhao Xing , Jinpei Zhou ,
Huibin Zhang a,b,∗
Affiliation and Address:
a
Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China
b
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR
China
c
Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing
210009, PR China
#
These two authors contributed equally to this work.
∗
Corresponding author: Huibin Zhang, Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR
China. Tel: +86-25-83271302. Fax: +86-25-83271480. Email: zhanghb80@cpu.edu.cn (H. Z.).
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
an 'Accepted Article', doi: 10.1111/cbdd.12513
This article is protected by copyright. All rights reserved.

*Corresponding author:
Huibin Zhang
Center of Drug Discovery, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China
Tel: +86-25-83271302; Fax: +86-25-83271480.
E-mail addresses: zhanghb80@cpu.edu.cn
This study was supported by the China National Key Hi-Tech Innovation Project for the R&D of Novel
Drugs (No. 2013ZX09301303-002), and supported by Natural Science Foundation of Jiangsu Province
(No. BK20141349).
Discovery of novel 2-(piperidin-4-yl)-1H-benzo[d]imidazole derivatives as potential
anti-inflammatory agents
a#
b#
a
a
a
a
c
Qing Li , Qinghua Hu , Xinning Wang , Yang Zong , Leilei Zhao , Junhao Xing , Jinpei Zhou ,
Huibin Zhang a,b,∗
a
Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China
b
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR
China
c
Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing
210009, PR China
#
These two authors contributed equally to this work.
Abstract: A novel 2-(piperidin-4-yl)-1H-benzo[d]imidazole derivative 5 with good anti-inflammatory
activity was identified from our in-house library. Based on hit compound 5, two series of
2-(piperidin-4-yl)-1H- benzo[d]imidazole derivative 6a-g and 7a-h were designed and synthesized as
novel anti-inflammatory agents. Most of synthesized compounds exhibited good inhibitory activity on
NO and TNF-α production in LPS-Stimulated RAW 264.7 Macrophages, in which the compound 6e
showed most potent inhibitory activity on NO (IC50 = 0.86 μM) and TNF-α (IC50 = 1.87 μM)
∗
Corresponding author: Huibin Zhang, Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR
China. Tel: +86-25-83271302. Fax: +86-25-83271480. Email: zhanghb80@cpu.edu.cn (H. Z.).
This article is protected by copyright. All rights reserved.

production. Further evaluation revealed that compound 6e displayed more potent in vivo
anti-inflammatory activity than ibuprofen did on xylene-induced ear edema in mice. Additionally,
western blot analysis revealed that compound 6e could restore phosphorylation level of IκBα and
protein expression of p65 NF-κB in LPS-stimulated RAW 264.7 macrophages.
Keywords: Inflammation; benzimidazole; 2-(piperidin-4-yl)-1H-benzo[d]imidazole derivatives;
anti-inflammatory activity.
Abbreviations:
NSAIDs
(non-steroidal
anti-inflammatory
drugs),
FLAP
(
(
(
(
(
(
5-lipoxygenase-activating protein), BK (Bradykinin), PPA (polyphosphoricacid), DIPEA
diisoproplethyl amine), DMF (dimethylformamide), PSA (polar surface area), LPS
lipopolysaccharides), TNF-α(Tumour necrosis factor-alpha), NO (Nitric oxide), ELISA
enzyme-linked immunosorbant assay), DMEM (Dulbecco’s modified Eagle medium ), DMSO
dimethyl sulfoxide), FBS (fetal bovine serum ), OD (optical density), MTT
3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide).
Inflammation is a protective immune response against harmful stimuli including damaged cells,
irritants, or pathogens (1). However, dysfunctional inflammation response will cause tissue damage,
leading to an emergence of chronic inflammation, which is a risk factor for a host of chronic diseases
such as autoimmune disorders, neurodegenerative disorders, and cancer (2, 3). Current treatment of
inflammation involves extensive use of non-steroidal anti-inflammatory drugs (NSAIDs) and
glucocorticoids (4). However, long term administration of NSAIDs is associated with serious
adverse effects, including cardiovascular toxicity, gastrointestinal ulcerations, platelet dysfunction,
and renal and hepatic toxicity (5, 6). Meanwhile, high doses and prolonged usage of glucocorticoids
cause serious side effects such as Cushing syndrome, osteoporosis, and reduced rate of bone growth in
children (7). Thus, there is clearly an unmet medical need for more effective and safe
anti-inflammatory agents.
Benzimidazole derivatives exhibit various biological activity, including antiparasitic(8), antiulcer(9),
antihypertensive(10, 11), antitumor(12, 13), antibacterial(14), etc, and have therefore attracted
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considerable pharmaceutical interest (15-18). Recently, benzimidazole scaffold has gained increasing
importance as a pharmacophore for designing anti-inflammatory and analgesic agents (Fig.1) (19, 20).
For example, benzoimidazole derivative 1 was found to be a highly selective CB2 agonist of Ki value
3
.3 nM in attenuating pain and neuroinflammation(21). Compound 2 was identified as a potent
-lipoxygenase-activating protein (FLAP) inhibitor (IC50 = 0.12 μM.), which is currently considered
5
as a promising and clinically relevant strategy for pharmacological intervention with inflammatory
diseases (22).
Also, 1-benzylbenzimidazole 3 was described as a Bradykinin (BK) B1 receptor antagonist with
excellent affinity (EC50 = 2 nM) and 3 was used as anti-inflammatory analgesic (23). In addition,
bilastine 4 has shown to possess anti-inflammatory activity (24). Based on above findings,
benzimidazole is a privileged structure that can be used in drug design as anti-inflammatory agents.
With promising anticipation on benzimidazole derivatives as stated above, we started a preliminary
study on investigating the anti-inflammatory effects of novel benzimidazole derivatives, and a novel
2-(piperidin-4-yl)-1H- benzo[d]imidazole derivative 5 with good anti-inflammatory activity was
identified from our in-house library. Compound 5 exhibited similar anti-inflammatory activity with
that of ibuprofen in xylene-induced ear swelling assay in mice. Further evaluation revealed that
compound 5 showed potent activity in inhibitions of NO and TNF-a production in LPS-Stimulated
RAW 264.7 macrophages with IC50 value of 10.35 μM and 7.70 μM, respectively. Physicochemical
properties calculation revealed that compound 5 with a moderate molecular weight of 393.48 and log
P value of 3.56 might display good drug-like properties. Therefore, compound 5 would serve as a
promising hit compound for further optimization. Considering short side chain of ethyl group at
1-position of compound 5, we firstly modified this site by introducing long side chains and aromatic
rings (compound 6a-g, Fig. 1). Since phenylacetic acid derivatives also exhibited excellent
anti-inflammatory activity (25), a series of phenylacetic acid derivatives (compound 7a-h, Fig. 1)
were designed. In this paper, by modifying this hit compound 5, two series of
2-(piperidin-4-yl)-1H-benzo[d]imidazole derivative 6a-g and 7a-h were designed and synthesized,
and their anti-inflammatory activity were evaluated.
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Methods and Materials
General chemistry
All chemicals (reagent grade) used were purchased from Sigma-Aldrich (USA) and Sinopharm
Chemical Reagent Co. Ltd (China). Melting points were measured on capillary tube and were
uncorrected. IR spectra (in KBr pellets) were taken using Shimadzu FT-IR-8400S spectrophotometer.
1
H NMR spectra were measured on Bruker AV-300 or ACF 500 spectrometer at 25 °C and referenced
to TMS. Chemical shifts were reported in ppm (δ) using the residual solvent line as internal standard.
Splitting patterns were designed as s, singlet; d, doublet; t, triplet; m, multiplet. Elemental analyses
were performed on CHN-O-Rapid instrument (Elementar, Hanau, Germany). Analytical thin-layer
chromatography (TLC) was performed on the glass-backed silica gel sheets (silica gel 60 Å GF 254).
Detailed synthetic procedures and characterization data for all new compounds 9-14, 5, 6a-g and 7a-h
are provided in the Supporting Information (Appendix S1) of this article.
Cell culture and treatment
Mouse RAW 264.7 macrophages were obtained from the Shanghai Institute of Cell Biology
(
Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Hyclone)
supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL of penicillin and 100 μg/mL of
streptomycin Amresco) at 37°C in 5% CO /95% air. LPS purchased from Sigma (St. Louis, MO) was
(
2
dissolved in FBS. Test compounds were dissolved in dimethyl sulfoxide (DMSO) for in vitro
experiments.
Cell viability test
Cell viability was assessed using a modified 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium
5
bromide (MTT) assay(26). Briefly, cells (3×10 cells/well) were seeded in a 96 well plate and treated
with test compounds at 80 μM with LPS (100 ng/mL) for 24 h. Then, 100 μL of MTT solution (5
mg/mL in phosphate buffered saline) was added to each well and further incubated for 4 h at 37 °C.
Subsequently, 100 μL of DMSO was added to each well to dissolve any deposited formazan. The
optical density (OD) of each well was measured at 540 nm with CORONA SH-1000 microplate
reader (Ibaraki-Ken, Japan). All experiments were carried out in triplicate.
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Measurement of nitrite (NO) levels
Measurement of nitrite (NO) levels was performed according to a described method (27). RAW 264.7
5
cells were seeded in 96-well plates at a density of 3.0 × 10 /well. Cells were pretreated with DMSO
and test compounds (0-80 μM dose range) for 2 h, and then incubated with LPS (100 ng/mL) for 24 h.
The culture media were collected to measure nitrite concentrations using Griess reaction by adding
100 μL Griess reagent (1% [w/v] sulfanilamide in 5% phosphoric acid and 0.1% [w/v]
N-1-naphthyl-ethylendiamide-dihydrochloride (NED) in H O)). The absorbance at 550 nm was
2
measured in CORONA SH-1000 microplate reader (Ibaraki-Ken, Japan) and a series of known
concentrations of sodium nitrite were used as the standards.
Measurement of TNF-α production using ELISA assay
Measurement of TNF-α production using ELISA assay was performed according to a described
method (28). RAW 264.7 Cells were pretreated with test compounds (0-80 μM) or vehicle control for
2
h, and then treated with LPS (100 ng/ml) for 24 h. After treatment, the culture medium was
collected separately and centrifuged at 1000 rpm for 10 min. After centrifugation, the supernatant was
separated and stored at -80 °C until further use. Levels of TNF-α were determined by enzyme-linked
immunosorbant assay (ELISA) using mouse TNF-α ELISA Kits (R&D, Minneapolis USA).
Experiment of xylene-induced ear edema in mice
Xylene-induced ear edema in mice was performed according to a previously described method (29).
Overnight fasted Male Kunming (KM) mice (20 ± 2 g) (12 h) were randomly divided into groups
(n=8/group). One group of mice, served as a control group, was given vehicle (0.5% CMC in water in
a volume of 20 mL/kg) only. The rest of groups, which were served as experimental groups, were
given two doses (4 and 12 mg/kg) of test compounds and ibuprofen suspended in vehicle (20 mL/kg)
orally (p.o.) for 5 days. 30 min after the last administration, 0.1 mL of xylene was applied to the
anterior and posterior surfaces of the right ear. The left ear was considered as control. One hour after
the application of xylene, mice were killed and both ears were removed. Circular sections were taken,
using a cork borer with a diameter of 9 mm, and measured. The degree of ear swelling was calculated
based on the weight of left ear without xylene. Ear edema (∆W) was calculated as follows: ∆W = W
R
−
W
L
. The weights of right (W ) and left (W ) ears were measured (mg) respectively of the same
R
L
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mouse. Edema inhibitory activity was calculated according to the following formula: Percentage
inhibition (%) = (Wcontrol − Wtreated) / Wcontrol × 100.
Measurement of protein expressions (p-IκB, IκB, p65 NF-κB) using Western Blot
RAW 264.7 Cells were pretreated with test compounds (10 μM) or vehicle control for 2 h, and then
treated with LPS (100 ng/ml) for 24 h. Protein extraction of cytosolic and nuclear fractions was
performed by using a cytosolic/nuclei isolation kit (KeyGEN, Nanjing, China), according to a
multiple centrifugation method. Protein concentration was determined using an enhanced BCA
protein assay (Beyotime, China). Equal quantities of cytosolic/nuclear proteins from each treatment
(
80 μg of total protein/lane) were resolved on 10% SDS-PAGE and then transferred to polyvinylidene
fluoride membranes (Millipore, MA, USA). Membranes were blocked in TBST (Tris-buffered saline
(TBS) containing 0.1% Tween-20) containing 5% skimmed milk power for 1 h at 37 °C. The
membranes were then treated individually with specific antibody diluted in TBST containing 5%
skimmed milk power, which includes p-IκB, IκB, p65 NF-κB (#9936, 1:1000) and α-tubulin (#2144,
1:1000) antibodies (CST, MA, USA). After incubating with horseradish peroxidase-conjugated
secondary antibody (1:5000, Bioworld, MN, USA), immunoreactive bands were visualized by TMB
reagent (KPL, Maryland, USA).
Statistical analysis
Data were expressed as means ± S.D. of at least three independent experiments performed in triplicate.
Statistical analysis was performed using a one-way analysis of variance (ANOVA). The difference
between the mean values of two groups was assessed by the Student–Newman–Keuls test. Multiple
group comparisons were performed using a Dunnett's test. SPSS version 12 was used for all statistical
analyses. Column charts were generated using Prism 5.0 software (GraphPad Software, INC, La Jolla,
CA). P-values of less than 0.05 were considered significant.
Results and discussion
Chemistry
The synthetic route adopted to obtain compound 5 and the target compounds 6a-g and 7a-h is
diagrammed in Scheme 1. Cyclization of commercially available benzene-1,2-diamine (8) with
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piperidine-4-carboxylic acid in polyphosphoricacid (PPA) afforded compound 9. Compound 9 was
protected with t-butyloxycarboryl (Boc) group at the piperidine nitrogen to give compound 10, which
was further alkylated with NaH and alkyl halides (R X) to give compounds 11a-h. Subsequently,
1
deprotection of Boc from 11a-h yielded compounds 12a-h. Compounds 14i-ii were obtained from
substitution of commercially available 4-hydroarmotic acid ester 13i-ii with 1,2-dibromoethane. The
final coupling of compounds 12a-h and compounds 14i-ii using diisoproplethyl amine (DIPEA) in
dimethylformamide (DMF) gave 15a-h and 16a-h, which were hydrolyzed with LiOH to afford
compound 5 and the target compounds 6a-g and 7a-h. The target compounds 6a-g and 7a-h are
presented in Table 1, and their structures were fully characterized by spectroscopic methods and the
elemental analysis.
Physicochemical properties.
Physicochemical properties of drugs strongly influenced the absorption, distribution, biological
availability, and pharmacological activity of drugs(25). Lipophylicity and polar surface area (PSA)
and Lipinski Rule of five parameters were important physicochemical parameters of target
compounds that were calculated and shown a result that all these new compounds except compounds
6g and 7h met the criteria of Lipinski’s “Rule of Five” and might have favorable physicochemical
properties. The results were presented in Table 1.
Inhibition of NO Production in LPS-Stimulated Macrophages.
Nitric oxide (NO) is an important pro-inflammatory mediator in the pathogenesis of inflammation(30).
Excessive production of NO was found to be detrimental to host tissues (31). Thus, pharmacological
inhibition of NO is considered to be a promising therapeutic strategy for the treatment of
inflammatory disorders(32). Therefore, the effect of target compounds 6a-g and 7a-h on NO
inhibition at 50 μM was firstly determined by using the lipopolysaccharides (LPS)-stimulated Raw
264.7 machrophages (27). Dexamethasone and ibuprofen were chosen as positive controls. These
compounds by which the inhibition ratio of NO production exceeded 40% were chosen for the IC50
value test. The results were listed in Table 2.
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In general, most compounds shown more potent or comparable activity as compared with ibuprofen.
Hit compound 5 with R
1
as ethyl group showed potent inhibitory activity (IC50 = 10.35 μM), being
5-fold more potent than positive control ibuprofen (IC50 = 56.34 μM). In the series of benzoimidazole
derived benzoic acid 6a-g, when ethyl group was replaced with long-chain groups such as butyl (6a),
methoxyethyl (6b), ethoxyethyl (6c) and phenoxyethyl (6d), the inhibitory activity decreased
dramatically. Compound 6e, containing a benzyl group, showed the most potent activity with IC50
values 0.86 μM, which was tenfold more potent than hit compound 5, and was 50-fold more potent
than ibuprofen. However, substituted benzyl groups 6h and 6g showed slightly decreased potency
compared to benzyl group 6e. In the case of benzoimidazole derived phenylacetic acid 7a-h, all of the
compounds exhibited lower inhibitory activity, of which compound 7b (IC50 = 25.39 μM) showed
twofold less potent activity than hit compound 5.
Comparing with dexamethasone (IC50= 10.57 μM), Hit compound 5 showed comparable inhibitory
activity (IC50 = 10.35 μM). Compounds 6a (IC50= 20.59 μM), 6f (IC50= 24.47 μM), 6g (IC50= 26.74
μM), 7b (IC = 25.39 μM) and 7e (IC = 28.67 μM) were two-fold less potent than positive control
5
0
50
dexamethasone. Compound 6e showed the most potent inhibitory activity with IC50 values 0.86 μM,
which was tenfold more potent than hit compound 5 and positive control dexamethasone, respectively.
In this regard, Compounds 6a, 6e, 6f, 6g, 7b and 7e were selected for further studies of
anti-inflammatory activity.
To exclude the possibility that the inhibition of inflammatory response were due to cytotoxicity
caused by treatment of these compounds, the cell viability was determined by MTT assay (26). RAW
264.7 macrophages were treated with individual compounds at 80 μM with LPS for 24 h. As shown in
Table 2, compared with the positive control dexamethasone and ibuprofen, all compounds did not
show significant cytotoxicity (the viability of Raw 264.7 cells: >95%) at the concentrations of 80 μM.
Inhibition of TNF-α production in in LPS-Stimulated Macrophages.
Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine that plays an important role in
many inflammatory processes. Over-production of TNF-α is associated with the pathogenesis of a
range of inflammatory diseases(33). Thus, further evaluation on effectiveness of compounds 6a, 6e, 6f,
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6g, 7b and 7e in inhibition of TNF-α production in RAW 264.7 macrophages was performed.
Dexamethasone and ibuprofen were served as positive controls. Macrophages were incubated with
LPS for 24 h in the presence of the compounds at a range of concentration (0-80 μM). The levels of
TNF-α in the culture medium supernatant were detected by enzyme-linked immunosorbant assay
(
ELISA) (28).
The result in Table 2 indicated that hit compound 5 shown strong inhibition on TNF-α production
with IC50 value of 7.70 μM. Its activity was more potent than ibuprofen (IC50 = 50.47 μM), but was
less potent than dexamethasone (IC50 = 2.45 μM). Compounds 6a, 6f, 6g, 7b and 7e exhibited
moderate inhibitory effect on TNF-α production. Compound 6e showed the most potent inhibitory
effects on TNF-α production with IC value of 1.87 μM, which was more potent than positive control
5
0
dexamethasone. Compound 6e showed most potent inhibitory activity on both NO (IC50 = 0.86 μM)
and TNF-α (IC50 = 1.87 μM) production. Next, the in vivo anti-inflammatory activity of compound 6e
was performed.
In vivo anti-inflammatory activity on xylene-induced ear edema in mice
In vivo anti-inflammatory activity of hit compound 5 and compound 6e was evaluated by the
xylene-induced ear edema in mice(29). Topical application of xylene induced cutaneous inflammation
at the ears of mice, which caused a significant increase in earplug weight of the right ear when
compared to that of vehicle-treated left ear. Treating the mice with ibuprofen in doses of 4 mg/kg and
12 mg/kg significantly gave rise to an inhibition of 26.77% and 39.34% in earplug weight (Fig. 2).
Moreover, oral administration of compounds 5 and 6e at 4 and 12 mg/kg significantly decreased
earplug weight of xylene-treated mice with a dose-dependent manner. Hit compound 5 displayed
similar inhibition rate (26.23% and 37.70%) with ibuprofen, while compound 6e showed the highest
anti-inflammatory inhibition rate with 33.30% and 50.60%, respectively. These findings indicated that
compound 6e contains reasonable anti-inflammatory activity and is more potent than ibuprofen.
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Inhibition of NF-κB pathways
Finally, in order to determine whether the inhibition of NF-κB pathways contributed to the
anti-inflammation, we evaluated the effects of hit compound 5 and the most active compound 6e on
phosphorylation levels of IκBα and protein expressions of p65 NF-κB in LPS-stimulated RAW264.7
macrophages by western blot analysis (Fig. 3). LPS stimulation significantly up-regulated the
phosphorylation level of IκB (p-IκB) and protein expression of p65 NF-κB, which could be restored
by compounds 5 (p-IκB: 36%, p65 NF-κB: 48%) and 6e (p-IκB: 68%, p65 NF-κB: 81%) at 10 μM, as
well as positive control dexamethasone (p-IκB: 56%, p65 NF-κB: 70%) at 10 μM. More importantly,
6e was more potential in regulating expressions of p-IκB, IκB and p65 NF-κB than dexamethasone,
nevertheless, the effect of hit compound 5 was weaker than that of dexamethasone. Thus, data suggest
that compound 6e could restore the phosphorylation of IκB (p-IκB) and protein expressions of p65
NF-κB in LPS-stimulated RAW264.7 macrophages.
Conclusion
In
this
study,
by
modifying
the
hit
compound
5,
two
series
of
2-(piperidin-4-yl)-1H-benzo[d]imidazole derivative 6a-g and 7a-h were designed, synthesized and
tested for anti-inflammatory activity. Firstly, physicochemical parameters calculation revealed that
most of new compounds met the criteria of Lipinski’s “Rule of Five” and might have favorable
physicochemical properties. Then, all compounds were tested for the effect on NO inhibition and parts
of them were tested their inhibition of TNF-a production by using the LPS-stimulated Raw 264.7
machrophages. Compound 6e displayed the most potent inhibitory activity on NO (IC50 = 0.86 μM)
and TNF-α (IC50 = 1.87 μM) production. Furthermore, in vitro anti-inflammatory evaluation on
xylene-induced ear edema in mice revealed that compound 6e was more potent than ibuprofen. Finally,
western blot analysis revealed that compound 6e could restore phosphorylation levels of IκBα and
protein expressions of p65 NF-κB in LPS-stimulated RAW 264.7 macrophages. Taking all data into
account, compound 6e could be served as a promising lead for novel anti-inflammatory agents
Acknowledgements
This study was supported by the China National Key Hi-Tech Innovation Project for the R&D of
Novel Drugs (No. 2013ZX09301303-002), and supported by Natural Science Foundation of Jiangsu
This article is protected by copyright. All rights reserved.

Province (No. BK20141349).
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Figures and schemes captions
Figure 1 Structure of reported anti-inflammatory benzimidazole compounds and hit compound
, and design of the target compounds.
5
Figure 2 The effects of compounds 5 and 6e on xylene-induced ear edema in mice. Compounds 5
and 6e, and ibuprofen (4, 12 mg/kg, p.o.) were administered 30 min before topical application of
*
xylene (0.1 ml) to the right ears of mice. Values are expressed as means ± S.D (n=8). P<0.05,
**
***
P<0.01, P<0.001 versus vehicle group.
Figure 3 The effects of compouds 5 and 6e on phosphorylation levels of IκBα and protein
expressions of p65 NF-κB in LPS-stimulated RAW264.7 macrophages. RAW264.7 macrophages
were preteated with 5, 6e and dexamethasone at 10 μM for 2 h, and stimulated by 100 ng/mL LPS for
another 24 h. Protein levels of p-IκB, IκB, p65 NF-κB and α-tubulin were detected by Western blot
analysis. The protein level of p-IκB was normalized to that of IκB while the protein level of p65
#
##
NF-κB was normalized to that of α-tubulin. Values are expressed as means ± S.D (n=4). P<0.001
*
**
***
versus normal group; P<0.05, P<0.01, P<0.001 versus LPS vehicle group.
Scheme 1. Synthesis of the compound 5 and the target compounds 6a-g and 7a-h. Reagents and
2
conditions: (a) piperidine-4-carboxylic acid, PPA, 150 °C, 5 h; (b) (Boc) O, NaOH, 0 °C, 16 h; (c)
R X, NaH, DMF, , 80 °C, 3 h; (d) HCl (gas), ethyl acetate/dichloromethane (3:1), 0 °C, 5 h; (e)
1
2 3 3
1,2-dibromoethane, K CO , KI, CH CN, 70°C, 18 h; (f) DIPEA, DMF, 80 °C, 16 h; (g) LiOH, MeOH/
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tetrahydrofuran/ H O(1: 2: 2), 70 °C, 16 h.
2
Table 1. Structures and calculated physicochemical parameters of target compounds 6a-g and
7a-h.
Table 2. Cell viability and inhibitory activity of target compounds 6a-g and 7a-h on NO and
TNF-α production in LPS-Stimulated RAW 264.7 Cells.
Table 1. Structures and calculated physicochemical parameters of target compounds 6a-g and
7a-h.
a
2
3
Compounds
5
R
1
MW
LogP
HBA HBD
RB
≤ 10
7
TPSA Å
≤ 140
65.37
65.37
74.6
MR[cm /mol]
≤
500
≤ 5
≤ 10
5
≤ 5
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
ethyl
393.479
421.532
423.505
437.531
485.574
455.548
485.574
489.993
407.505
435.559
437.531
451.558
499.601
469.575
499.601
504.02
3.56
4.46
3.07
3.41
4.89
4.96
4.83
5.51
3.56
4.46
3.07
3.41
4.83
4.96
4.77
5.46
111.41
120.6
6
6
a
1-butyl
5
9
b
2-methoxyethyl
2-ethoxyethyl
2-phenoxyethyl
benzyl
6
9
117.53
122.33
137.25
131.1
6
c
6
10
10
8
74.6
6
d
6
74.6
6
e
5
65.37
74.6
6f
4-methoxybenzyl
4-chlorobenzyl
ethyl
6
9
138.35
135.71
115.72
124.92
121.84
126.64
141.57
135.42
142.67
140.02
6g
7a
7b
5
8
65.37
65.37
65.37
74.6
5
8
1-butyl
5
10
10
11
11
9
7
c
2-methoxyethyl
2-ethoxyethyl
2-phenoxyethyl
benzyl
6
7
d
6
74.6
7
e
6
74.6
7f
5
65.37
74.6
7
7
g
4-methoxybenzyl
4-chlorobenzyl
6
10
9
h
5
65.37
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MW: Molecular weight (g/mol); LogP: n-octanolewater partition coefficient are calculated by Chemdraw 11.0;
HBD: number of hydrogen bond donors; HBA: number of hydrogen bond acceptors; RB: number of rotatable
bonds; TPSA: topological polar surface area; MR:
Table 2. Cell viability and inhibitory activity of target compounds 6a-g and 7a-h on NO and
TNF-α production in LPS-Stimulated RAW 264.7 Cells
Inhibition (%) at
0 μM
Inhibition of NO
Inhibition of TNF-α
Cell viability
compds
a
a
b
5
IC50 (μM)
IC50 (μM)
(%)
5
107.08±3.06
112.56±4.22
50.08±2.23
38.97±1.83
47.95±2.83
92.91±3.87
81.65±4.25
56.15±1.63
36.67±2.46
64.33±3.76
37.84±1.48
36.67±1.23
46.15±2.37
36.37±1.41
45.64±2.23
45.13±2.38
82.56±1.18
41.73±2.08
10.35±0.46
20.59±1.26
37.32±2.17
ND
7.70±1.06
18.03±1.52
ND
98.87±1.36
96.85±0.87
95.46±0.29
97.34±1.47
96.85±0.59
98.45±1.83
94.32±2.93
96.87±0.33
95.74±2.71
96.86±2.63
97.56±1.98
97.29±2.12
94.67±2.98
99.12±0.43
95.76±1.57
97.26±2.61
97.45±0.83
6a
6b
6
c
ND
6d
34.32±1.23
0.86±0.12
24.47±1.82
26.76±1.92
ND
ND
6
e
1.87±0.21
19.24±1.32
24.38±2.46
ND
6
f
6
g
a
7
7b
25.39±1.42
ND
13.50±1.52
ND
7
c
7d
ND
ND
7
e
28.67±2.25
ND
33.49±2.34
ND
7
f
7g
37.83±3.41
40.83±2.68
10.57±0.42
ND
7h
ND
dexamethasone
Ibuprofen
2.45±0.16
50.47±3.18
56.34±3.22
98.87±0.43
a
b
Values are the mean values of three experiments. IC50 values were not determined (ND). Cell
viability after treatment with 80 μM of each compound was expressed as a percentage (%) of the
value obtained from LPS alone group.
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.