Discovery of 2-iminobenzimidazoles as potent hepatitis C virus inhibitors with a novel mechanism of action

Article abstract of DOI:10.1016/j.ejmech.2014.03.030

In this report we describe 2-iminobenzimidazole (IBI) analogs, identified during the course of a phenotypic high-throughput screening campaign, as novel hepatitis C virus (HCV) inhibitors. A series of IBI derivatives was synthesized and evaluated for their inhibitory activity against infectious HCV. Among the IBIs derivatives studied in this work, we identified promising compounds with high antiviral efficacy, high selectivity index and good microsomal stability. Noteworthy, the IBI series exhibited inhibitory activity on early and late steps of the viral cycle, but not in the HCV replicon system demonstrating a mechanism of action distinct from clinical-stage and approved anti-HCV drugs. Overall, our results suggest that IBIs are predestinated for further exploration as lead compounds for novel HCV interventions.

Full text of DOI:10.1016/j.ejmech.2014.03.030

European Journal of Medicinal Chemistry 78 (2014) 35e42
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Discovery of 2-iminobenzimidazoles as potent hepatitis C virus
inhibitors with a novel mechanism of action
, ,
,
Marc Peter Windisch a 3, Suyeon Jo b 3, Hee-Young Kim a, Soo-Hyun Kim a,
*
Keumhyun Kim a, Sunju Kong b, Hyangsuk Jeong c, Sujin Ahn c, Zaesung No d
,
,
1
, ,
2
Jong Yeon Hwang b
*
a Applied Molecular Virology, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Republic of Korea
b Medicinal Chemistry Group, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Republic of Korea
c DMPK Group, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Republic of Korea
d Late Discovery Program, Institut Pasteur Korea (IP-K), Seongnam-si, Gyeonggi-do, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In this report we describe 2-iminobenzimidazole (IBI) analogs, identified during the course of a
phenotypic high-throughput screening campaign, as novel hepatitis C virus (HCV) inhibitors. A series of
IBI derivatives was synthesized and evaluated for their inhibitory activity against infectious HCV. Among
the IBIs derivatives studied in this work, we identified promising compounds with high antiviral efficacy,
high selectivity index and good microsomal stability. Noteworthy, the IBI series exhibited inhibitory
activity on early and late steps of the viral cycle, but not in the HCV replicon system demonstrating a
mechanism of action distinct from clinical-stage and approved anti-HCV drugs. Overall, our results
suggest that IBIs are predestinated for further exploration as lead compounds for novel HCV
interventions.
Received 24 January 2014
Received in revised form
11 March 2014
Accepted 11 March 2014
Available online 13 March 2014
Keywords:
Hepatitis C virus
Phenotypic screening
Iminobenzimidazole
Entry
Ó 2014 Elsevier Masson SAS. All rights reserved.
Release
1. Introduction
content assay designed to identify compounds that can inhibit all
stages of the viral life cycle [6]. In order to enrich for inhibitors with
More than 170 million people worldwide are chronically infected
with the hepatitis C virus (HCV) and are at risk of developing life-
threatening liver diseases [1]. Despite the recent approval of two
direct-acting antiviral agents (DAAs), the standard of care (SOC) still
relies on a combination therapy of pegylated interferon-alpha
a novel mechanism of action, we counter-screened the active hits in
the HCV replicon system, thereby reducing competition with
replication inhibitors in clinical development. The inhibitors were
finally tested in a series of secondary assays to elucidate their pu-
tative MoA. Thereby, we observed that IBI has a novel MoA by
interfering with early and late steps in the HCV life cycle. These
results prompted us to explore further the potential of the IBIs series
as a new anti-HCV lead series. Initial structureeactivity relationship
(SAR) studies revealed the functional groups responsible for anti-
HCV activity and identified a lead candidate suitable for further
lead optimization studies.
(PEG-IFN
sustained virologic response rate (SVR) of only 70e80% [2e5]. As for
the DAAs, PEG-IFN therapy is accompanied by serious side effects
a) and ribavirin (RBV), associated with an unsatisfactory
a
responsible for low adherence to the therapy. This obvious unmet
medical need encouraged us to screen small molecule compound
libraries for new antiviral agents with a novel mechanism of action
(MoA). We screened more than 200,000 small-molecules in a high-
Here we report the results of our SAR and virological studies.
2. Results and discussion
* Corresponding authors.
E-mail addresses: mpwindisch@ip-korea.org (M.P. Windisch), jyhwang@krict.re.
kr, jongyeonhwang@gmail.com (J.Y. Hwang).
2.1. High throughput screening and hit selection
1
Current address: Gyeonggi Bio-Center, Suwon 443-270, Republic of Korea.
Current address: Korea Research Institute of Chemical Technology, Daejeon
200,000 compounds were screened against the entire HCV life
cycle [7]. Briefly, naïve Huh-7 target cells were plated in 384-well
microplates, incubated with 6 mM of the test compounds in
2
305-600, Republic of Korea.
3
These authors equally contributed to this work.
http://dx.doi.org/10.1016/j.ejmech.2014.03.030
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

36
M.P. Windisch et al. / European Journal of Medicinal Chemistry 78 (2014) 35e42
Fig. 1. Structures of IBI (1a-c) and a known HCV IRES inhibitor (2).
duplicate, and inoculated with cell culture derived HCV (HCVcc). At
72 h post-infection experiments were analyzed and the half
maximal effective concentration (EC50) to inhibit viral replication
was calculated. In addition, the cytotoxic concentration (CC50) was
determined to rule out whether the observed antiviral effects were
due to toxicity induced by compounds. Active hits were cheery-
picked and confirmed for activity by 10-point dose response
curve (DRC) analysis in the primary assay as described previously
[7]. Confirmed active hits were triaged in HCV pseudoparticle
(HCVpp) and replicon systems to determine inhibitory effects on
viral entry and viral RNA replication, respectively.
treated with methylamine to give intermediates 4, which were
then reduced to methylaminoanilines 5. Cyclization of 5 with
1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea 6 gave
Boc-protected 2-aminobenzimidazoles 7 in good yields [10]. Af-
ter deprotection of Boc on compound 7 by treatment of 4 N
hydrogen chloride, N-alkylations with three types of reagents
including benzyl bromides, phenoxyethyl bromides, and 2-
bromoacetophenones were conducted to afford the correspond-
ing acyclic IBI analogs 9, 10, and 11, respectively. Several IBI
analogs such as compound 16, 20, 23, and 25 were synthesized
as shown in Scheme 2. 2-Chlorobenzimidazole 12 was subjected
to alkylation with phenoxyethyl bromide to afford intermediate
13, which was condensed with ethanolamine to give interme-
diate 14. Chlorination by treatment with POCl3 and subsequent
cyclization under thermal heating provided the desired cyclic
IBI 16 [11]. Compound 20, imidazole-IBI, was synthesized from
2-aminobenzimidazole 17. Alkylation of compound 17 with
4-chlorophenoxyethylbromide gave compound 18, which was
then treated with 1-bromobutan-2-one to give intermediate 19.
Cyclization of compound 19 under heating led to imidazolo-IBI
20 [12]. Compound 23 bearing cyano in imino group of IBI
was obtained from compound 21 by treatment of diphenyl cya-
Following this rational, we identified
a
series of 2-
iminobenzimidazoles (IBIs, 1aec) that exhibited submicromolar
inhibitory activity in the infectious HCVcc assay (Fig. 1). Interest-
ingly, 2-aminobenzimidazole (2), which has a chemical structure
resembling IBI 1, has been reported as an in vitro inhibitor of HCV
translation by interaction with the viral internal ribosome entry
side (IRES) [8,9]. However, the IBIs series was inactive in an HCV
genotype 1 and 2 replicon systems, suggesting that IBIs 1 have a
MoA distinct from the 2-aminobenzimidazole series (2) which
encouraged us to initiate a SAR study.
nocarbonimidate
[10],
followed
by
alkylation
with
2.2. Chemistry
4-chlorophenoxyethylbromide. Benzimidazolone 25 was synthe-
sized through cyclization of compound 21 with carbonyl diimi-
dazole (CDI), followed by alkylation.
A
general scheme for synthesis of IBIs is depicted in
Scheme 1. Commercially available chloronitrobenzenes 3 were
Scheme 1. Reagents and conditions: (a) Methylamine, 5 mol% Cu, H2O, air, 100 ꢀC, 8 h; (b) Raney Ni, H2, EtOH, rt, 3 h; (c) p-TsOH, iPrOH, 65 ꢀC, 16 h; (d) 4 N HCl, 1,4-dioxane, 100 ꢀC,
30 min; (e) BnBr, DMF, rt-150 ꢀC; (f) Bromoacetophenone, DMF, rt-150 ꢀC; (g) Phenoxyethyl bromide, DMF, rt-150 ꢀC.

M.P. Windisch et al. / European Journal of Medicinal Chemistry 78 (2014) 35e42
37
Scheme 2. Reagents and conditions: (a) 4-ClPhOEtBr, K2CO3, MeCN, 80 ꢀC; (b) ethanolamine, microwave, 200 ꢀC, 30 min; (c) POCl3, 110 ꢀC; (d) TEA, toluene, reflux;
(e) 1-bromobutan-2-one, TEA, EtOH, reflux, 2 h; (f) 2-aminoethan-1-ol, microwave, 200 ꢀC, 30 min; (g) diphenyl cyanocarbonimidate, MeCN, 100 ꢀC, 8 h; (h) CDI, pyridine, THF,
65 ꢀC, 1 h.
2.3. Structureeactivity relationship (SAR) study
is depicted in Scheme 2, and their anti-HCV activities are summa-
rized in Table 2. Cyclic-IBI 16 showed similar anti-HCV activity
All IBIs were evaluated for their anti-HCV activity and cytotox-
icity (Tables 1e3) as described in Section 2.1. First we examined R2
substitution effect (Table 1). IBIs could be divided in three groups
(benzyl 9, acetophenonyl 10, and phenoxyethyl 11), depending on
the functional group (Fig. 1). IBIs 9 bearing a benzyl moiety
(EC50 ¼ 0.050
IBI 20, bearing an aromatic characteristic, had strongly reduced
M). These results indicate that cyclic
structure is tolerated but aromatic structure is not favorable for
anti-HCV activity. 2-Aminobenzimidazole 18 and IBI 19 with ketone
moiety showed at least 100-fold reduced potency compared to IBI
11a. Compound 23 bearing cyano moiety in imine group of IBI was
inactive. In addition, replacement of imine moiety in IBI to ketone
mM) compared to acyclic IBI 11a, whereas imidazole-
anti-HCV activity (EC50 ¼ 6.0
m
exhibited good anti-HCV activities (EC50 ¼ 0.17e0.46
mM), but had a
narrow selectivity index (SI ¼ 11.8e34.7). In case of acetophenone
substituted IBIs (10), compound 10a and 10c displayed good anti-
HCV activity (EC50 ¼ 0.073 and 0.079
cytotoxicity value (CC50 ¼ 20 and 8.9
m
m
M, respectively) with low
M, respectively), thereby
(25) led to a loss of activity (>20 mM). This result strongly suggested
that the imine group in IBI is crucial for anti-HCV activity.
providing good selectivity index value (SI ¼ 274 and 113, respec-
We further explored the effect of R1 substituent on anti-HCV
activity (Table 3). Four compounds (26ae26d) with chloro
substituent in position 4, 5, 6, and 7 were examined. Among chloro-
substituted compounds, 4-Cl substituted IBI (26a) exhibited four-
fold improved anti-HCV potency (EC50 ¼ 0.0033) compared to
compound 11a, while 5-Cl (26b) and 7-Cl (26d) substituted IBI
retained antiviral activities. 6-Chloro substituted IBI (26c) displayed
slightly reduced anti-HCV activity. In case of methyl substitution,
5-Me substituted IBI (26f) exhibited highly potent anti-HCV activity
tively). Compound 10d also showed good antiviral activity
(EC50 ¼ 0.046
m
M), but provided low selectivity index value
(SI ¼ 59) due to increased cytotoxicity (CC50 ¼ 2.7
mM) in com-
parison with compound 10a. 4-Et substituted IBI 10b and naph-
thalene IBI 10e had a weaker antiviral activities compared to other
substitutions. Phenoxyethyl substituted IBIs (11), had excellent
anti-HCV potency (EC50 ¼ 0.016e0.050
mM). Among them the most
promising compound was 4-chloro substituted IBI 11a with EC50
value of 0.016
high SI of 475.
m
M and CC50 value of 7.6
m
M, thereby providing a
with EC50 value of 0.001
m
M, while 4-Me and 6-Me substituted IBI
(26e and 26g) were less active (EC50 ¼ 0.13 and 0.090
mM,
Next, we synthesized several IBI analogs bearing various func-
tional groups to determine essential chemical features of IBIs
associated with potency. The scheme for synthesis of these analogs
respectively). Among all IBIs tested in this study, compound 26f
exhibited the highest selectivity index value (SI ¼ 2900) in the
HCVcc system.

38
M.P. Windisch et al. / European Journal of Medicinal Chemistry 78 (2014) 35e42
Table 1
Anti-HCV activity of IBIs 9e11.
No.
R2
EC50
(m
M)
CC50
(
mM)
SI (CC50/EC50)
9a
9b
9c
10a
10b
10c
10d
4-Et
4-tertBu
4-Ph
4-Cl
4-Et
0.46
0.17
0.22
0.073
0.38
5.8
5.9
2.6
20
9.5
8.9
2.7
12.6
34.7
11.8
274
25
4-tertBu
4-Ph
0.079
0.046
113
59
10e
0.34
4.5
13
11a
11b
11c
11d
4-Cl
4-Et
4-tertBu
4-Ph
0.016
0.050
0.045
0.20
7.6
8.9
2.8
3.4
475
178
62
17
EC50 and CC50 values were determined by 10-point DRC analysis in duplicates with quadruplicate measurements.
Table 2
Anti-HCV activity of IBI 16, 18, 19, 20, 23, and 25.
In order to determine metabolic stability, all IBI derivatives lis-
ted in Table 3 were tested in human and rat liver microsomes.
Chloro-substituted IBIs (26ae26d) displayed an excellent micro-
somal stability in rat microsomes (t1/2 ¼ 37 or >60 min), whereas
compound 11a (t1/2 ¼ 13.5 min) and 26ee26g (t1/2 ¼ 8.7e24.6 min)
had a less favorable stability.
2.4. Mechanism of action studies
Our primary screening campaign was conducted using the in-
fectious HCV cell culture system, which in principle allows to target
any step of the entire viral life cycle. Consequently, the MoA and
ultimately the molecular target of active hit compounds remain to
be elucidated. In order to determine which step is inhibited by the
IBI series, we analyzed the activity of selected derivatives in HCVpp,
HCV replicon and HCVcc late assays enabling to monitor viral entry,
RNA replication and virus secretion, respectively. Using a viral su-
pernatant transfer assay employing transiently transfected cells
(producer cells) secreting infectious viral particle, we monitored
inhibition of late stages in the viral life cycle by IBI, as previously
No.
Q
EC50
(
m
M)
CC50
20
(mM)
16
0.050
4.0
18
19
8.9
8.5
2.3
6.0
Table 3
Anti-HCV activity and microsomal stability of IBIs 26.
20
20
No.
R1
EC50
(
m
M)
CC50
(m
M)
SI
Metabolic stability
Human/rat
(CC50/EC50
)
(t1/2, min)
11a
26a
26b
26c
26d
26e
26f
H
0.016
7.6
1.5
4.1
9.84
4.3
15
2.9
10.62
475
455
373
246
443
115
2900
118
>60/13.5
>60/>60
>60/>60
>60/>60
>60/37.4
>60/24.6
>60/8.7
23
25
>20
>20
>20
>20
4-Cl
5-Cl
6-Cl
7-Cl
4-Me
5-Me
6-Me
0.0033
0.011
0.04
0.0097
0.13
0.0010
0.09
26g
>60/18.0
EC50 and CC50 values were determined by 10-point DRC analysis in duplicates with
quadruplicate measurements.
EC50 and CC50 values were determined by 10-point DRC analysis in duplicates with
quadruplicate measurements.

M.P. Windisch et al. / European Journal of Medicinal Chemistry 78 (2014) 35e42
39
described by Wichroski et al. [13]. No HCV RNA replication inhibi-
4.1.1. The representative procedure for the preparation of N-methyl-
tion of producer cells was observed (black diamonds), corrobo-
rating the finding that the IBI series is inactive in HCV replicon
assays (Fig. 2A). Transfer cell culture supernatant of producer cells
to naïve target cells revealed a strong dose dependent reduction of
secreted infectious HCV particles (blue squares), suggesting that
IBIs interfere with a late step of the viral life cycle that affects the
production of infectious virions. In addition, we monitored inhibi-
tion of viral particle attachment and/or internalization by using
HCVpps. In order to demonstrate HCV-specificity of IBIs, we used
vesicular stomatitis virus glycoprotein expressing pseudoparticles
(VSVGpps) which were capable of transducing Huh-7 target cells in
the presence of IBIs. Nonetheless, transduction of HCVpps was
inhibited indicating that IBIs interfere with HCV attachment and/or
internalization (Fig. 2B). Taken together, our data demonstrate that
IBIs exhibit anti-HCV activity by inhibiting early and late steps of
the viral cycle, whereas viral RNA replication was not affected.
2-nitroaniline (4, R1 ¼ H)
A solution of 1-chloro-2-nitrobenzene (1.57 g, 10 mmol), 40%
aqueous methylamine (4.3 mL, 50 mmol), and copper powder
(31 mg, 0.5 mmol) was sealed in a 30 mL screw-cap tube and stirred
at 100 ꢀC for 8 h. The reaction mixture was then cooled-down to
room temperature and extracted with ethyl acetate. The combined
organic layers were dried over anhydrous sodium sulfate and
concentrated in vacuo to give crude product 4 (1.4 g, yield 95%) as
oil. The crude product was used directly in next reaction step. 1H
NMR (400 MHz, CDCl3)
d
8.18 (d, J ¼ 8.0 Hz, 1H), 8.16 (NH, 1H), 7.45
(t, J ¼ 7.8 Hz, 1H), 6.84 (d, J ¼ 8.0 Hz, 1H), 6.65 (t, J ¼ 7.8 Hz, 1H), 3.02
(d, J ¼ 5.2 Hz, 3H); LC/MS (electrospray) m/z (M þ H)þ 153.05.
4.1.2. Representative procedure for the preparation of N1-methyl-
benzene-1,2-diamine (5, R1 ¼ H)
A solution of compound 4 (R1 ¼ H, 760 mg, 5 mmol) and Raney
Nickel (3 g) in ethanol was hydrogenated under 1 atm at room
temperature for 3 h. The mixture was filtered over a pad of Celite.
The filtrate was concentrated in vacuo and purified by silica gel
column chromatography (eluent: n-Hex/EA ¼ 5:1) to give the
product 5 (366 mg, yield 60%) as oil. 1H NMR (400 MHz, CDCl3)
3. Conclusion
In conclusion, a series of iminobenzimidazoles was identified by
a target-free phenotypic HTS assay using the infectious HCV cell
culture system. Through our preliminary SAR study, we identified a
series of promising IBI lead compounds that show excellent po-
tency and safety margin, and an acceptable stability in human and
rat liver microsomes. In addition, we demonstrated that the IBIs
series inhibits early and late steps of the HCV life cycle. Although
further intensive lead optimization and MoA studies are required,
the IBI scaffold is not only a new chemical class for anti-HCV drug
development, but also an interesting tool compound to study some
basic aspects of the HCV life cycle.
d
6.86 (t, J ¼ 8.4 Hz,1H), 6.69 (m, 3H), 3.32 (NH, 2H), 2.87 (s, 3H); LC/
MS (electrospray) m/z (M þ H)þ 123.98.
4.1.3. Representative procedure for the preparation of tert-butyl
(1-methyl-1H-benzo[d]imidazol-2-yl)carbamate (7, R1 ¼ H)
A solution of compound 5 (R1 ¼ H, 100 mg, 0.657 mmol), 1,3-
bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea 6 (209 mg,
0.722 mmol), and p-toluene sulfonic acid (12 mg, 0.065 mmol) in
isopropanol was heated at 65 ꢀC for 16 h. The reaction mixture was
concentrated in vacuo and diluted with ethyl acetate. The organic
layer was successively washed with 1N sodium hydroxide and
brine, and dried over sodium sulfate. After concentration in vacuo,
the crude product was purified by silica gel column chromatog-
raphy (eluent : n-Hex/EA ¼ 5 : 1) to give the compound 7 (R1 ¼ H,
4. Experimental section
4.1. Chemistry
All materials were obtained from commercial suppliers and
used without further purification. Solvents in this study were dried
using an aluminum oxide column. Thin-layer chromatography was
performed on pre-coated silica gel 60 F254 plates. Purification of
intermediates was carried out by normal phase column chroma-
tography (MPLC, Silica gel 230e400 mesh). NMR spectra were
recorded on a Varian 400 MHz. LC/MS data were obtained using a
Waters 2695 LC and Micromass ZQ spectrometer. Identity of final
compounds was confirmed by proton NMR, carbon NMR, and mass
spectrometry.
121 mg, yield 75%) as solid. 1H NMR (400 MHz, DMSO-d6)
d 11.9
(NH, 1H), 7.38 (m, 2H), 7.14 (m, 2H), 3.65 (s, 3H), 1.45 (s, 9H); LC/MS
(electrospray) m/z (M þ H)þ 248.03.
4.1.4. Representative procedure for the preparation of 1-methyl-1H-
benzo[d]imidazol-2-amine (8, R1 ¼ H)
A solution of compound 7 (R1 ¼ H, 75 mg, 0.303 mmol) and 4 N
HCl in 1,4-dioxane solution (1.5 mL, 6 mmol) was heated at 100 ꢀC
for 30 min. The reaction mixture was then cooled-down to room
temperature and the precipitant was collected and dissolved in
Fig. 2. Evaluation of HCV entry and secretion in the presence of IBIs. (A) Transiently transfected Huh-7.5 producer cells secreting HCV particle were treated with increasing IBI 11a
concentration [ M] (X-axis). 48 h after treatment, viral cell culture supernatants were transferred to naïve target cells. Viral RNA replication (black diamonds) and cytotoxicity (red
m
diamonds) of transiently transfected producer cells was determined at 72 h post compound treatment. Viral RNA replication of inoculated target cells was determined at 72 h post
infection (blue squares). Data were normalized to the 1% DMSO control that was set to 0% inhibition. (B) HCVpp expressing E1/E2 viral glycoproteins of HCV and VSVGpp were
incubated with 3.3 mM of IBI 11a. Inhibition of pseudoparticle transduction (%) and cell viability (%) is shown by black bars (left Y-axis) and red bars (right Y-axis), respectively. Data
were normalized to the 1% DMSO control that was set to 0% inhibition. Experiments were conducted in triplicates with quadruple measurements (n ¼ 12). Representative ex-
periments are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

40
M.P. Windisch et al. / European Journal of Medicinal Chemistry 78 (2014) 35e42
dichloromethane. The organic solution was neutralized with 1 N
NaOH solution and washed with brine and dried over sodium
sulfate. After concentration in vacuo, the crude solid product was
washed with ether to give the pure product 8 (R1 ¼ H, 42 mg, yield
mp. 271.5 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d
8.18 (d, J ¼ 8.4 Hz,
2H), 7.96 (d, J ¼ 8.4 Hz, 2H), 7.81 (d, J ¼ 7.6 Hz, 2H), 7.61 (d,
J ¼ 8.0 Hz, 2H), 7.54 (t, J ¼ 7.6 Hz, 2H), 7.46 (t, J ¼ 7.2 Hz, 1H), 7.36 (t,
J ¼ 7.6 Hz, 1H), 7.29 (t, J ¼ 7.6 Hz, 1H), 6.09 (s, 2H), 3.76 (s, 3H); LC/
MS (electrospray) m/z (M þ H)þ 342.06.
95%) as solid. 1H NMR (400 MHz, CDCl3)
d 7.13 (m, 2H), 6.89 (m, 2H),
6.37 (NH, 2H), 3.47 (s, 3H); LC/MS (electrospray) m/z (M þ H)þ
148.07.
4.1.5.8. 2-(2-imino-3-methyl-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)-1-(naphthalen-2-yl)ethanone hydrobromide (10e). Yield 48%;
mp. 258.0 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d 8.88 (NH2, 2H), 8.81
4.1.5. General procedure for the preparation of compounds 9, 10, 11
A solution of compound 8 (0.203 mmol, 1 eq) and the corre-
sponding halides (0.244 mmol, 1.2 eq) in DMF (2 mL) was stirred at
room temperature for 12 h. After the reaction was completed, ethyl
acetate was added to the reaction mixture. The precipitate was
filtered and washed with ether to give the corresponding product 9,
10, 11 as a salt form.
(s, 1H), 8.21 (d, J ¼ 8.0 Hz, 1H), 8.13 (d, J ¼ 8.8 Hz, 1H), 8.06 (t,
J ¼ 9.2 Hz, 2H), 7.75 (m, 2H), 7.68 (t, J ¼ 9.2 Hz, 2H), 7.38 (t,
J ¼ 8.2 Hz, 1H), 7.31 (t, J ¼ 8.2 Hz, 1H), 6.13 (s, 2H), 3.76 (s, 3H); LC/
MS (electrospray) m/z (M þ H)þ 316.14.
4.1.5.9. 1-(2-(4-chlorophenoxy)ethyl)-3-methyl-1H-benzo[d]imida-
zol-2(3H)-imine hydrobromide (11a). Yield 41%; mp. 300.8 ꢀC; 1H
4.1.5.1. 1-(4-ethylbenzyl)-3-methyl-1H-benzo[d]imidazol-2(3H)-
NMR (400 MHz, DMSO-d6)
d
8.84 (NH2, 2H), 7.66 (d, J ¼ 2.1 Hz, 1H),
imine (9a). Yield 43%; mp. 281.4 ꢀC; 1H NMR (400 MHz, DMSO-d6)
7.55 (d, J ¼ 2.1 Hz, 1H), 7.35 (m, 2H), 7.30 (d, J ¼ 8.8 Hz, 2H), 6.85 (d,
d
8.99 (NH2, 2H), 7.53 (d, J ¼ 7.6 Hz, 1H), 7.42 (d, J ¼ 7.6 Hz, 1H), 7.31
J ¼ 8.8 Hz, 2H), 4.60 (t, J ¼ 5.0 Hz, 2H), 4.29 (t, J ¼ 5.0 Hz, 2H), 3.65 (s,
3H); 13C NMR (100 MHz, DMSO-d6)
d 157.2, 150.8, 132.5, 131.8,
(t, J ¼ 3.8 Hz, 1H), 7.25 (m, 5H), 5.42 (s, 2H), 3.68 (s, 3H), 2.55 (q,
J ¼ 4.9 Hz, 2H),1.12 (t, J ¼ 7.8 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
130.5, 129.9, 125.4, 124.1, 116.8, 111.5, 110.8, 66.2, 43.0, 30.2; LC/MS
(electrospray) m/z (M þ H)þ 302.05.
d
150.9, 144.2, 132.8, 131.2, 130.1, 128.8, 128.0, 124.0, 111.1, 110.8,
45.9, 30.3, 28.4, 16.2; LC/MS (electrospray) m/z (M þ H)þ 266.13.
4.1.5.10. 1-(2-(4-ethylphenoxy)ethyl)-3-methyl-1H-benzo[d]imida-
4.1.5.2. 1-(4-(tert-butyl)benzyl)-3-methyl-1H-benzo[d]imidazol-
zol-2(3H)-imine hydrobromide (11b). Yield 42%; mp. 276.0 ꢀC; 1H
2(3H)-imine hydrochloride (9b). Yield 40%; mp. 286.2 ꢀC; 1H NMR
NMR (400 MHz, DMSO-d6)
d
8.84 (NH2, 2H), 7.65 (d, J ¼ 2.1 Hz, 1H),
(400 MHz, DMSO-d6)
d
7.55 (d, J ¼ 7.6 Hz, 1H), 7.47 (d, J ¼ 7.6 Hz,
7.54 (d, J ¼ 2.1 Hz, 1H), 7.34 (m, 2H), 7.06 (d, J ¼ 8.8 Hz, 2H), 6.72 (d,
J ¼ 8.8 Hz, 2H), 4.59 (t, J ¼ 5.0 Hz, 2H), 4.26 (t, J ¼ 5.0 Hz, 2H), 3.64 (s,
3H), 2.50 (q, J ¼ 4.9 Hz, 2H), 1.09 (t, J ¼ 7.8 Hz, 3H); 13C NMR
1H), 7.37 (d, J ¼ 8.0 Hz, 2H), 7.28 (m, 2H), 7.24 (t, J ¼ 8.0 Hz, 2H), 5.44
(s, 2H), 3.72 (s, 3H), 1.25 (s, 9H); LC/MS (electrospray) m/z (M þ H)þ
294.16.
(100 MHz, DMSO-d6) d 156.5, 150.7, 136.9, 130.8, 130.5, 129.3, 124.1,
114.8, 111.6, 110.8, 65.9, 43.2, 30.3, 27.9, 16.5; LC/MS (electrospray)
4.1.5.3. 1-([1,1’-biphenyl]-4-ylmethyl)-3-methyl-1H-benzo[d]imida-
zol-2(3H)-imine (9c). Yield 35%; mp. 315.4 ꢀC; 1H NMR (400 MHz,
m/z (M þ H)þ 296.13.
DMSO-d6)
d
9.04 (NH, 1H), 7.64 (m, 4H), 7.57 (d, J ¼ 7.6 Hz, 1H), 7.31
4.1.5.11. 1-(2-(4-(tert-butyl)phenoxy)ethyl)-3-methyl-1H-benzo[d]
(m, 8H), 5.52 (s, 2H), 3.70 (s, 3H); LC/MS (electrospray) m/z
imidazol-2(3H)-imine hydrobromide (11c). Yield 48%; mp. 270.4 ꢀC;
(M þ H)þ 314.16.
1H NMR (400 MHz, DMSO-d6)
d
8.88 (NH2, 2H), 7.66 (d, J ¼ 2.1 Hz,
1H), 7.55 (d, J ¼ 2.1 Hz,1H), 7.33 (m, 2H), 7.23 (d, J ¼ 8.8 Hz, 2H), 6.73
(d, J ¼ 8.8 Hz, 2H), 4.61 (t, J ¼ 5.0 Hz, 2H), 4.27 (t, J ¼ 5.0 Hz, 2H), 3.65
4.1.5.4. 1-(4-chlorophenyl)-2-(2-imino-3-methyl-2,3-dihydro-1H-
benzo[d]imidazol-1-yl)ethanone hydrobromide (10a). Yield 45%;
(s, 3H), 1.20 (s, 9H); 13C NMR (100 MHz, DMSO-d6)
d 156.2, 150.7,
mp. 257.2 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d 8.91 (NH2, 2H), 8.11
143.8, 130.8, 130.5, 126.7, 124.0, 114.4, 111.6, 110.8, 65.9, 43.2, 34.3,
(d, J ¼ 6.8 Hz, 2H), 7.73 (d, J ¼ 6.8 Hz, 2H), 7.62 (d, J ¼ 8.0 Hz, 2H),
31.9, 30.4; LC/MS (electrospray) m/z (M þ H)þ 324.17.
7.36 (t, J ¼ 8.2 Hz, 1H), 7.29 (t, J ¼ 8.2 Hz, 1H), 6.01 (s, 2H), 3.74 (s,
3H); 13C NMR (100 MHz, DMSO-d6)
d 191.1, 151.3, 139.8, 133.4, 131.1,
4.1.5.12. 1-(2-([1,1’-biphenyl]-4-yloxy)ethyl)-3-methyl-1H-benzo[d]
imidazol-2(3H)-imine hydrobromide (11d). Yield 46%; mp. 291.9 ꢀC;
130.8, 129.6, 124.3, 111.2, 111.0, 50.7, 30.4; LC/MS (electrospray) m/z
(M þ H)þ 300.01.
1H NMR (400 MHz, DMSO-d6)
d
8.86 (NH2, 2H), 7.69 (d, J ¼ 2.0 Hz,
1H), 7.56 (m, 5H), 7.41 (t, J ¼ 7.8 Hz, 2H), 7.36 (m, 2H), 7.29 (t,
J ¼ 7.4 Hz,1H), 6.91 (d, J ¼ 8.8 Hz, 2H), 4.63 (t, J ¼ 5.0 Hz, 2H), 4.35 (t,
J ¼ 5.0 Hz, 2H), 3.66 (s, 3H); LC/MS (electrospray) m/z (M þ H)þ
344.10.
4.1.5.5. 1-(4-ethylphenyl)-2-(2-imino-3-methyl-2,3-dihydro-1H-
benzo[d]imidazol-1-yl)ethanone hydrobromide (10b). Yield 55%;
mp. 254.7 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d 8.94 (NH2, 2H), 8.03
(d, J ¼ 8.0 Hz, 2H), 7.60 (t, J ¼ 7.6 Hz, 2H), 7.48 (d, J ¼ 8.4 Hz, 2H), 7.35
(t, J ¼ 7.8 Hz, 1H), 7.28 (t, J ¼ 7.8 Hz, 1H), 6.02 (s, 2H), 3.75 (s, 3H),
2.73 (q, J ¼ 4.9 Hz, 2H), 1.22 (t, J ¼ 7.8 Hz, 3H); 13C NMR (100 MHz,
4.1.6. Synthesis of 2-chloro-1-(2-(4-chlorophenoxy)ethyl)-1H-
benzo[d]imidazole (13)
DMSO-d6) d 191.4, 151.7, 151.3, 132.5, 130.8, 129.4, 128.9, 124.3, 111.1,
1H NMR (400 MHz, Acetone-d6)
d
7.65 (d, J ¼ 8.4 Hz, 1H), 7.58 (d,
111.0, 50.6, 30.4, 29.0, 16.0 LC/MS (electrospray) m/z (M þ H)þ
J ¼ 8.4 Hz, 1H), 7.29 (m, 2H), 7.23 (d, J ¼ 6.8 Hz, 2H), 6.89 (d,
J ¼ 6.8 Hz, 2H), 4.43 (t, J ¼ 5.2 Hz, 2H), 4.74 (t, J ¼ 5.2 Hz, 2H); LC/MS
(electrospray) m/z (M þ H)þ 309.
294.16.
4.1.5.6. 1-(4-(tert-butyl)phenyl)-2-(2-imino-3-methyl-2,3-dihydro-
1H-benzo[d]imidazol-1-yl)ethanone hydrobromide (10c). Yield 47%;
mp. 265.3 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d
8.83 (NH2, 2H), 8.03
4.1.7. 2-((1-(2-(4-chlorophenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)
(d, J ¼ 7.2 Hz, 2H), 7.67 (d, J ¼ 7.2 Hz, 2H), 7.59 (t, J ¼ 9.2 Hz, 2H), 7.36
(t, J ¼ 7.8 Hz, 1H), 7.29 (t, J ¼ 7.8 Hz, 1H), 5.95 (s, 2H), 3.73 (s, 3H),
1.34 (s, 9H); LC/MS (electrospray) m/z (M þ H)þ 322.13.
amino)ethan-1-ol (14)
1H NMR (400 MHz, CD3OD)
d
7.29 (d, J ¼ 8.4 Hz, 1H), 7.23 (d,
J ¼ 8.4 Hz, 1H), 7.18 (m, 2H), 7.03 (d, J ¼ 8.8 Hz, 2H), 6.83 (d,
J ¼ 8.8 Hz, 2H), 4.38 (t, J ¼ 5.2 Hz, 2H), 4.23 (t, J ¼ 5.2 Hz, 2H), 3.57 (t,
J ¼ 5.4 Hz, 2H), 3.06 (t, J ¼ 5.4 Hz, 2H); LC/MS (electrospray) m/z
(M þ H)þ 332.
4.1.5.7. 1-([1,10-biphenyl]-4-yl)-2-(2-imino-3-methyl-2,3-dihydro-
1H-benzo[d]imidazol-1-yl)ethanone hydrobromide (10d). Yield 46%;

M.P. Windisch et al. / European Journal of Medicinal Chemistry 78 (2014) 35e42
41
4.1.8. Synthesis of N-(2-chloroethyl)-1-(2-(4-chlorophenoxy)ethyl)-
1H-benzo[d]imidazol-2-amine (15)
1H NMR (400 MHz, CD3OD)
3H), 7.21 (d, J ¼ 8.8 Hz, 2H), 6.84 (d, J ¼ 8.8 Hz, 2H), 4.62 (t,
J ¼ 4.8 Hz, 2H), 4.37 (t, J ¼ 4.8 Hz, 2H), 3.89 (m, 4H); LC/MS (elec-
trospray) m/z (M þ H)þ 352.
4.1.15. Synthesis of 6,7-dichloro-1-methyl-1,3-dihydro-2H-benzo[d]
imidazol-2-one (24)
d
7.60 (d, J ¼ 8.4 Hz, 1H), 7.42 (m,
A solution of compound 21 (1.57 mmol, 1.0 equiv), pyridine
(3.14 mmol, 2.0 equiv) and CDI (3.14 mmol, 2.0 equiv) in THF (5 mL)
was heated at 65 ꢀC for 1 h. After reaction was completed, the re-
action mixture was poured into EtOAc. The precipitant was
collected and washed with EtOAc/Hexane, and dried to give com-
pound 24 (Yield 55%).
4.1.9. Synthesis of 9-(2-(4-chlorophenoxy)ethyl)-2,9-dihydro-3H-
benzo[d]imidazo[1,2-a]imidazole (16)
Yield 22%; Yellow oil; 1H NMR (400 MHz, CD3OD)
d 7.60 (m, 1H),
4.1.16. Synthesis of 4,5-dichloro-1-(2-(4-chlorophenoxy)ethyl)-3-
methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (25)
7.36 (m, 3H), 7.21 (d, J ¼ 8.8 Hz, 2H), 6.86 (d, J ¼ 8.8 Hz, 2H), 4.54 (t,
White solid; Yield 78%; mp. 167.3 ꢀC; 1H NMR (400 MHz, CDCl3)
J ¼ 4.8 Hz, 2H), 4.42 (m, 4H), 4.36 (t, J ¼ 4.8 Hz, 2H); 13C NMR
d
7.31 (m, 2H), 7.26 (d, J ¼ 8.4 Hz, 2H), 6.87 (d, J ¼ 8.4 Hz, 2H), 4.22
(100 MHz, CD3OD)
d 156.9, 156.5, 134.9, 129.2, 128.3, 126.2, 124.5,
(m, 4H), 3.61 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
d
157.4, 154.3,
123.5, 115.8, 111.5, 110.2, 65.6, 49.6, 43.9, 43.4; LC/MS (electrospray)
m/z (M þ H)þ 314.
130.5, 129.8, 127.7, 125.2, 124.9, 122.8, 116.8, 112.8, 109.0, 66.5, 41.2,
30.2; LC/MS (electrospray) m/z (M þ H)þ 372.
4.1.10. Synthesis of 1-(2-(4-chlorophenoxy)ethyl)-1H-benzo[d]
4.1.16.1. Synthesis of 4-chloro-1-(2-(4-chlorophenoxy)ethyl)-3-
imidazol-2-amine (18)
Yield 48%; mp. 180.6 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d 7.29 (d,
methyl-1H-benzo[d]imidazol-2(3H)-imine
hydrobromide
(26a).
Yield 43%; mp. 246.0 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d
8.98 (NH2,
J ¼ 6.8 Hz, 2H), 7.20 (d, J ¼ 7.2 Hz, 1H), 7.10 (d, J ¼ 7.2 Hz, 1H), 6.93 (t,
J ¼ 3.6 Hz, 1H), 6.90 (m, 3H), 6.39 (NH2, 2H), 4.37 (t, J ¼ 5.0 Hz, 2H),
4.20 (t, J ¼ 5.0 Hz, 2H); LC/MS (electrospray) m/z (M þ H)þ 288.97.
2H), 7.66 (d, J ¼ 8.8 Hz, 1H), 7.32 (m, 2H), 7.30 (d, J ¼ 8.8 Hz, 2H),
6.84 (d, J ¼ 8.8 Hz, 2H), 4.60 (t, J ¼ 5.0 Hz, 2H), 4.27 (t, J ¼ 5.0 Hz,
2H), 3.88 (s, 3H); LC/MS (electrospray) m/z (M þ H)þ 337.01.
4.1.11. Synthesis of 1-(3-(2-(4-chlorophenoxy)ethyl)-2-imino-2,3-
4.1.16.2. Synthesis of 5-chloro-1-(2-(4-chlorophenoxy)ethyl)-3-
dihydro-1H-benzo[d]imidazol-1-yl)butan-2-one (19)
methyl-1H-benzo[d]imidazol-2(3H)-imine
hydrobromide
(26b).
Yield 79%; mp. 218.8 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d 8.83
Yield 46%; mp. 314.2 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d
8.98 (NH2,
(NH2, 2H), 7.67 (d, J ¼ 8.4 Hz, 1H), 7.50 (d, J ¼ 8.4 Hz, 1H), 7.33 (m,
4H), 6.87 (d, J ¼ 6.8 Hz, 2H), 5.23 (s, 2H), 4.63 (t, J ¼ 5.0 Hz, 2H), 4.31
(t, J ¼ 5.0 Hz, 2H), 2.61 (q, J ¼ 20.2 Hz, 2H),1.01 (t, J ¼ 7.4 Hz, 3H); LC/
MS (electrospray) m/z (M þ H)þ 358.98.
2H), 7.77 (s, 1H), 7.67 (d, J ¼ 8.8 Hz, 1H), 7.39 (d, J ¼ 8.8 Hz, 1H), 7.29
(d, J ¼ 8.8 Hz, 2H), 6.85 (d, J ¼ 8.8 Hz, 2H), 4.59 (t, J ¼ 5.0 Hz, 2H),
4.27 (t, J ¼ 5.0 Hz, 2H), 3.60 (s, 3H); LC/MS (electrospray) m/z
(M þ H)þ 337.01.
4.1.12. Synthesis of 9-(2-(4-chlorophenoxy)ethyl)-2-ethyl-9H-
benzo[d]imidazo[1,2-a]imidazole (20)
4.1.16.3. Synthesis of 5-chloro-3-(2-(4-chlorophenoxy)ethyl)-1-
methyl-1H-benzo[d]imidazol-2(3H)-imine
hydrobromide
(26c).
Yield 70%; 1H NMR (400 MHz, CDCl3)
d
7.44 (d, J ¼ 8.0 Hz, 2H),
Yield 43%; mp. 281.3 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d
9.05 (NH2,
7.27 (t, J ¼ 3.6 Hz, 1H), 7.17 (m, 3H), 7.08 (s, 1H), 6.72 (d, J ¼ 8.0 Hz,
2H), 4.56 (t, J ¼ 5.0 Hz, 2H), 4.37 (t, J ¼ 5.0 Hz, 2H), 2.74 (q,
J ¼ 20.2 Hz, 2H), 1.32 (t, J ¼ 7.4 Hz, 3H); LC/MS (electrospray) m/z
(M þ H)þ 340.02.
2H), 7.80 (s, 1H), 7.57 (d, J ¼ 8.4 Hz, 1H), 7.38 (d, J ¼ 8.4 Hz, 1H), 7.30
(d, J ¼ 8.8 Hz, 2H), 6.83 (d, J ¼ 8.8 Hz, 2H), 4.63 (t, J ¼ 5.0 Hz, 2H),
4.29 (t, J ¼ 5.0 Hz, 2H), 3.65 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
d
157.2, 151.4, 131.6, 130.0, 128.3, 125.4, 124.0, 116.7, 112.2, 111.9, 66.1,
43.1, 30.5; LC/MS (electrospray) m/z (M þ H)þ 337.01.
4.1.13. Synthesis of N-(6,7-dichloro-1-methyl-1,3-dihydro-2H-
benzo[d]imidazol-2-ylidene)cyanamide (22)
4.1.16.4. Synthesis of 4-chloro-3-(2-(4-chlorophenoxy)ethyl)-1-
A solution of compound 21 (600 mg, 3.140 mmol) and diphenyl
cyanocarboimidate (748 mg, 3.140 mmol) in MeCN was heated at
100 ꢀC for 8 h. The reaction mixture was then cooled-down to room
temperature to precipitate the product. The precipitate was
collected and dried to afford the pure compound 22 (227 mg, yield
methyl-1H-benzo[d]imidazol-2(3H)-imine
hydrobromide
(26d).
Yield 30%; mp. 287.8 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d
9.02 (NH2,
2H), 7.57 (d, J ¼ 6.0 Hz,1H), 7.37 (m, 2H), 7.31 (d, J ¼ 4.8 Hz, 2H), 6.87
(d, J ¼ 4.8 Hz, 2H), 4.85 (t, J ¼ 5.0 Hz, 2H), 4.33 (t, J ¼ 5.0 Hz, 2H),
3.66 (s, 3H); LC/MS (electrospray) m/z (M þ H)þ 337.01.
30%). 1H NMR (400 MHz, DMSO-d6)
d
13.06 (NH, 1H), 7.38 (d, J ¼ 8.4
Hz, 2H), 7.14 (d, J ¼ 8.4 Hz, 2H), 3.68 (s, 3H); LC/MS (electrospray) m/
4.1.16.5. Synthesis of 1-(2-(4-chlorophenoxy)ethyl)-3,4-dimethyl-1H-
z (M þ H)þ 242.02.
benzo[d]imidazol-2(3H)-imine hydrobromide (26e). Yield 40%; mp.
242.2 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d 8.71 (NH2, 2H), 7.48 (d,
4.1.14. Synthesis of N-(4,5-dichloro-1-(2-(4-chlorophenoxy)ethyl)-
3-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-ylidene)cyanamide
(23)
A solution of compound 22 (50 mg, 0.207 mmol), 1-(2-
bromoethoxy)-4-chlorobenzene (73 mg, 0.311 mmol), K2CO3
(86 mg, 0.622 mmol) in DMF (2 mL) was heated at 80 ꢀC for 4 h. The
reaction mixture was then cooled-down to room temperature and
poured into water. The precipitate was filtered and washed with EA
to give the compound 23 (32 mg, Yield 40%). mp. 197.5 ꢀC; 1H NMR
J ¼ 8.4 Hz, 1H), 7.30 (d, J ¼ 4.8 Hz, 2H), 7.22 (t, J ¼ 7.8 Hz, 1H), 7.09 (t,
J ¼ 7.8 Hz,1H), 6.84 (d, J ¼ 4.8 Hz, 2H), 4.58 (t, J ¼ 5.0 Hz, 2H), 4.27 (t,
J ¼ 5.0 Hz, 2H), 3.84 (s, 3H), 2.66 (s, 3H); LC/MS (electrospray) m/z
(M þ H)þ 316.07.
4.1.16.6. Synthesis of 1-(2-(4-chlorophenoxy)ethyl)-3,5-dimethyl-1H-
benzo[d]imidazol-2(3H)-imine hydrobromide (26f). Yield 55%; mp.
319.8 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d 8.75 (NH2, 2H), 7.52 (d,
J ¼ 8.4 Hz, 1H), 7.37 (s, 1H), 7.29 (d, J ¼ 4.4 Hz, 2H), 7.16 (d, J ¼ 8.0 Hz,
1H), 6.85 (d, J ¼ 4.4 Hz, 2H), 4.56 (t, J ¼ 5.0 Hz, 2H), 4.27 (t,
J ¼ 5.0 Hz, 2H), 3.61 (s, 3H), 2.41 (s, 3H); 13C NMR (100 MHz, DMSO-
(400 MHz, DMSO-d6)
J ¼ 8.8 Hz, 2H), 4.66 (t, J ¼ 5.0 Hz, 2H), 4.31 (t, J ¼ 5.0 Hz, 2H), 3.87 (s,
3H); 13C NMR (100 MHz, DMSO-d6)
157.3,151.0,131.8,129.9,128.8,
d
7.54 (m, 2H), 7.26 (d, J ¼ 8.8 Hz, 2H), 6.84 (d,
d
d6) d 157.3, 150.6, 133.8, 130.9, 129.9, 128.5, 125.4, 124.8, 116.8, 111.2,
127.1, 125.3, 124.7, 116.8, 115.7, 114.2, 110.7, 66.7, 43.3, 32.5; LC/MS
111.0, 66.2, 43.0, 30.2, 21.6; LC/MS (electrospray) m/z (M þ H)þ
(electrospray) m/z (M þ H)þ 396.97.
316.07.

42
M.P. Windisch et al. / European Journal of Medicinal Chemistry 78 (2014) 35e42
4.1.16.7. Synthesis of 3-(2-(4-chlorophenoxy)ethyl)-1,5-dimethyl-1H-
50 mm ꢁ 2.1 mm, 1.7
mm, Waters, Milford, MA) followed by an
benzo[d]imidazol-2(3H)-imine hydrobromide (26g). White solid;
analytical column. The mobile phases were (A) water with 0.1% of
formic acid and (B) acetonitrile with 0.1% of formic acid at a flow
rate of 0.4 mL/min. The LC conditions were 5% B at 0 min, a linear
gradient from 5 to 50% B over 0.5 min, held at 50% for 0.25 min, then
ramped from 50 to 95% over 0.25 min, followed by 95% B for
0.75 min and back to 5% B over 0.25 min, then held at 5% B for the
remaining 0.75 min. The percentage of remaining compound was
calculated by comparing with the initial quantity at 0 min. Half-life
was then calculated based on first order reaction kinetics.
Yield 27%; mp. 303.9 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d 8.70 (NH2,
2H), 7.41 (m, 2H), 7.30 (d, J ¼ 2.0 Hz, 2H), 7.14 (d, J ¼ 8.0 Hz,1H), 6.85
(d, J ¼ 2.0 Hz, 2H), 4.54 (t, J ¼ 5.0 Hz, 2H), 4.29 (t, J ¼ 5.0 Hz, 2H),
3.60 (s, 3H), 2.41 (s, 3H); LC/MS (electrospray) m/z (M þ H)þ 316.14.
4.2. Biological evaluation
4.2.1. HCVcc assay
Naïve Huh-7 cells were plated in 384-well plates, incubated
with serially diluted compounds and inoculated with cell culture
adapted HCVcc 2 h after compound treatment. At 72 h post-
infection, HCV infection rates and cytotoxicity were determined [7].
Acknowledgments
This work was supported by the National Research Foundation
of Korea (NRF) grant funded by the Korea Government (MEST) (No.
2012-00011), Gyeonggi-do and KISTI.
4.2.2. HCVpp assay
HCV E1/E2-pseudotyped (or VSV-G pseudotyped as a control)
lentiviral particles expressing a GFP reporter gene were produced
as described previously [14]. Comparable amounts of HCVpp and
VSVGpp were used in every experiment. Briefly, Huh-7 target cells
were plated in 384-well plates, treated with compounds for 1 h
followed by transduction with HCVpp or VSVGpp. Cell culture su-
pernatants were replaced 12 h later and at 72 h post inoculation
cytotoxicity and transduction efficiency were evaluated by deter-
mining number of cell nuclei and GFP reporter gene expression,
respectively, by confocal microscopy at 20ꢁ magnification
(ImageXpress ULTRA, Molecular Device). Relative transduction
values were obtained using DMSO-treated cells as control.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.030.
References
[1] Hepatitis C, WHO, (2012).
[2] S. Chevaliez, R. Brillet, E. Lazaro, C. Hezode, J.M. Pawlotsky, Analysis of riba-
virin mutagenicity in human hepatitis C virus infection, Journal of Virology 81
(2007) 7732e7741.
[3] N. Hayashi, T. Takehara, Antiviral therapy for chronic hepatitis C: past, pre-
sent, and future, Journal of Gastroenterology 41 (2006) 17e27.
[4] M.P. Manns, H. Wedemeyer, M. Cornberg, Treating viral hepatitis C: efficacy,
side effects, and complications, Gut 55 (2006) 1350e1359.
[5] J. Shepherd, J. Jones, D. Hartwell, P. Davidson, A. Price, N. Waugh, Interferon
alpha (pegylated and non-pegylated) and ribavirin for the treatment of mild
chronic hepatitis C: a systematic review and economic evaluation, Health
Technology Assessment 11 (2007) 1e205 (iii).
[6] J.Y. Hwang, M.P. Windisch, S. Jo, K. Kim, S. Kong, H.C. Kim, S. Kim, H. Kim,
M.E. Lee, Y. Kim, J. Choi, D.S. Park, E. Park, J. Kwon, J. Nam, S. Ahn, J. Cechetto,
J. Kim, M. Liuzzi, Z. No, J. Lee, Discovery and characterization of a novel 7-
aminopyrazolo[1,5-a]pyrimidine analog as a potent hepatitis C virus inhibi-
tor, Bioorganic & Medicinal Chemistry Letters 22 (2012) 7297e7301.
[7] J.Y. Hwang, H.Y. Kim, S. Jo, E. Park, J. Choi, S. Kong, D.S. Park, J.M. Heo, J.S. Lee,
Y. Ko, I. Choi, J. Cechetto, J. Kim, J. Lee, Z. No, M.P. Windisch, Synthesis and
evaluation of hexahydropyrimidines and diamines as novel hepatitis C virus
inhibitors, European Journal of Medicinal Chemistry 70C (2013) 315e325.
[8] S. Zhou, K.D. Rynearson, K. Ding, N.D. Brunn, T. Hermann, Screening for in-
hibitors of the hepatitis C virus internal ribosome entry site RNA, Bioorganic &
Medicinal Chemistry 21 (2013) 6139e6144.
[9] J. Parsons, M.P. Castaldi, S. Dutta, S.M. Dibrov, D.L. Wyles, T. Hermann,
Conformational inhibition of the hepatitis C virus internal ribosome entry site
RNA, Nature Chemical Biology 5 (2009) 823e825.
[10] J.Y. Hwang, H.Y. Kim, D.S. Park, J. Choi, S.M. Baek, K. Kim, S. Kim, S. Seong,
I. Choi, H.G. Lee, M.P. Windisch, J. Lee, Identification of a series of 1,3,4-
4.2.3. HCVcc late assay
The production of HCV was determined by a cell culture su-
pernatant transfer assay as described previously [13]. Briefly, Huh-
7 cells transiently transfected with full length HCV RNA genomes
(producer cells) secreting infectious virions, were plated in 384-
well plates and incubated overnight at 37 ꢀC. The next day cells
were treated with serially diluted compounds at 37 ꢀC for 48 h
followed by a wash step. The viral cell culture supernatant was
clarified by centrifugation 24 h after the washing step and trans-
ferred to naïve Huh-7 target cells in 384-well plates. HCV RNA
replication in producer cells and in target cells was measured at
72
h post compound treatment and 72 h post inoculation
respectively.
4.2.4. HCV replication assay
Huh-7 replicon cells stably replicating subgenomic HCV ge-
nomes were plated in 384-well plates and incubated at 37 ꢀC with
serially diluted compounds. 72 h after compound treatment, the
EC50 and CC50 values were calculated by non-linear regression us-
ing GraphPad Prism (GraphPad software) [7].
trisubstituted pyrazoles as novel hepatitis
C virus entry inhibitors, Bio-
organic & Medicinal Chemistry Letters 23 (2013) 6467e6473.
[11] V.A. Anisimova, M.V. Levchendo, A.F. Pozharskii, Research on imidazo[1,2-a]
benzimidazole derivatives. 22. Synthesis of 2,3-dihydroimidazo[1,2-a]benz-
imidazoles starting from 2-imino-3-(2-hydroxyethyl)benzimidazolines,
Chemistry of Heterocyclic Compounds 22 (1986) 732e739.
4.3. Microsomal metabolic stability assay
[12] N. Abe, T. Nishiwaki, H. Yamamoto, Syntheses and regioselective cycloaddi-
Compounds (2
mM final concentration in 0.02% DMSO) were
tion
reactions
of
cyclohept[d]imidazo[1,2-a]imidazoles
(1,3a,9-
incubated with 0.5 mg/mL human liver (pool of 200, mixed gender,
Xenotech) and rat male (BD Gentest) microsomes in potassium
phosphate buffer. The reaction was initiated by the addition of
NADPH and stopped either immediately or at 10, 20, 30 and 60 min
for a precise estimate of clearance. A triple quadrupole Quattro
PremierÔ mass spectrometer (Waters, Milford, MA) with electro-
spray ionization (ESI) was employed for sample analysis. Samples
were passed through trapping cartridges (Acquity BEH RP18
triazacyclopent[a]azulenes): formation of 2,3-diazacyclohepta[ef]cycl[3.2.2]
azine system, Chemistry Letters 11 (1982) 805e806.
[13] M.J. Wichroski, J. Fang, B.J. Eggers, R.E. Rose, C.E. Mazzucco, K.A. Pokornowski,
C.J. Baldick, M.N. Anthony, C.J. Dowling, L.E. Barber, J.E. Leet, B.R. Beno,
S.W. Gerritz, M.L. Agler, M.I. Cockett, D.J. Tenney, High-throughput screening
and rapid inhibitor triage using an infectious chimeric Hepatitis C virus, PLoS
One 7 (2012) e42609.
[14] B. Bartosch, J. Dubuisson, F.L. Cosset, Infectious hepatitis C virus pseudo-
particles containing functional E1-E2 envelope protein complexes, Journal
of Experimental Medicine 197 (2003) 633e642.