Discovery of benzimidazole derivatives as modulators of mitochondrial function: A potential treatment for Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2016.11.017

In this study, we designed a library of compounds based on the structures of well-known ligands of the 18 kDa translocator protein (TSPO), one of the putative components of the mPTP. We performed diverse mitochondrial functional assays to assess their ability to restore cells from Aβ-induced toxicity in vitro and in vivo. Among tested compounds, compound 25 effectively improved cognitive function in animal models of AD. Given the excellent in vitro and in vivo activity and a favorable pharmacokinetic profile of compound 25, we believe that it can serve as a promising lead compound for a potential treatment option for AD.

Full text of DOI:10.1016/j.ejmech.2016.11.017

European Journal of Medicinal Chemistry 125 (2017) 1172e1192
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of benzimidazole derivatives as modulators of
mitochondrial function: A potential treatment for Alzheimer's disease
,
b
,
, d,
TaeHun Kim a 1, Ha Yun Yang a 1, Beoung Gun Park a, Seo Yun Jung a, Jong-Hyun Park a,
,
Ki Duk Park a b, Sun-Joon Min c, Jinsung Tae d, Hyejin Yang e, Suengmok Cho e,
Sung Jin Cho f, Hyundong Song g, Inhee Mook-Jung g, Jiyoun Lee h **, Ae Nim Pae a
,
, b, *
a Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology, PO Box 131, Cheongryang,
Seoul 130-650, Republic of Korea
b Biological Chemistry, Korea University of Science and Technology, Yuseong-Gu, Daejon 305-350, Republic of Korea
c Department of Applied Chemistry, Hanyang University, Ansan, Gyeonggi-do 15888, Republic of Korea
d Department of Chemistry, Yonsei University, Seodaemun-gu, Seoul 120-749, Republic of Korea
e Korea Food Research Institute, Sungnam 463-746, Republic of Korea
f New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, Republic of Korea
g Department of Biochemistry and Biomedical Sciences, College of Medicine, Seoul National University, 103 Daehak-ro, Seoul 110-799, Jongno-gu, Republic
of Korea
h Department of Global Medical Science, Sungshin University, Seoul 142-732, 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 study, we designed a library of compounds based on the structures of well-known ligands of the
18 kDa translocator protein (TSPO), one of the putative components of the mPTP. We performed diverse
mitochondrial functional assays to assess their ability to restore cells from Ab-induced toxicity in vitro
and in vivo. Among tested compounds, compound 25 effectively improved cognitive function in animal
models of AD. Given the excellent in vitro and in vivo activity and a favorable pharmacokinetic profile of
compound 25, we believe that it can serve as a promising lead compound for a potential treatment
option for AD.
Received 25 August 2016
Received in revised form
5 November 2016
Accepted 7 November 2016
Available online 9 November 2016
Keywords:
Translocator protein
TSPO
© 2016 Elsevier Masson SAS. All rights reserved.
Mitochondrial permeability transition pore
Alzheimer's disease
Mitochondrial dysfunction
A
b
1. Introduction
patients, but many recent studies have found that high levels of
cellular Ab cause mitochondrial dysfunction [4,5]. Since mito-
Alzheimer's disease (AD) is a neurodegenerative disorder,
known as the most common cause of dementia associated with
impairments of cognitive functions and memory [1]. The mecha-
nism of AD pathogenesis still remains largely unknown, however,
chondria are responsible for a diverse array of cellular processes
such as energy production, metabolism, and cell death, the after-
math of mitochondrial dysfunction in neuronal cells would be
particularly devastating. Moreover, given the protective functions
of mitochondria against oxidative stress and protein misfolding,
mitochondrial dysfunction further aggravates and even accelerates
the progression of AD [6]. It has been suggested that the accumu-
amyloid-b (Ab) is recognized as the major hallmark of AD [2,3]. Not
only A is the main component of the amyloid plaques found in AD
b
lation of A
b
disrupts intracellular Ca2þ homeostasis [7] and results
appears
in apoptosis of neurons [8]. Furthermore, mitochondrial A
b
* Corresponding author. Convergence Research Center for Diagnosis, Treatment
and Care System of Dementia, Korea Institute of Science and Technology, PO Box
131, Cheongryang, Seoul 130-650, Republic of Korea.
to interact with cyclophilin D (CypD) and promotes opening of the
mitochondrial permeability transition pore (mPTP), a multimeric
protein complex in the inner membrane of mitochondria [8,9].
Opening of the mPTP induces the depolarization of the
** Corresponding author.
E-mail addresses: jlee@sungshin.ac.kr (J. Lee), anpae@kist.re.kr (A.N. Pae).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2016.11.017
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
1173
mitochondrial membrane potentials (DJm), which in turn allows
for uncontrolled passage of cytosolic solutes, damaging mito-
chondrial structure. This structural damage leads to mitochondrial
dysfunction, the consequences of which include the impairment of
energy production, the initiation of cell death pathways, and the
accumulation of neurotoxic proteins [9].
TSPO
¼
2.68
nM),
N-(4-chloro-2-phenoxyphenyl)-N-(2-
isopropoxybenzyl)acetamide (DAA1097, IC50 for TSPO ¼ 0.92 nM),
N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acet-
amide (DAA1106, IC50 for TSPO ¼ 1.6 nM), and N-(sec-butyl)-1-(2-
chlorophenyl)-N-methylisoquinoline-3-carboxamide
(PK-11195,
IC50 for TSPO ¼ 1.1 nM) [25]. From this set of compounds, two
pharmacophore models (model 1 and 2) were generated by
differing in ring aromatic and hydrophobic feature options. Model 1
consists of one hydrogen bond acceptor, one hydrophobic aromatic
and four hydrophobic features, whereas model 2 includes six
different common features: one hydrogen bond acceptor, two ring
aromatic, and three hydrophobic features. Instead of the hydro-
phobic aromatic feature in model 1, the ring aromatic feature was
varied in model 2 to cover the hydrophobic property and to
introduce a planar or flexible ring aromatic substituent in the hit
compounds. Based on these two pharmacophore models, our in-
house library as well as commercial libraries from Asinex (Asi-
nexGold, 229,398 compounds; AsinexPlatinum, 125,231 com-
pounds, Asinex, Moscow, Russia, www.asinex.com) and ChemDiv
(693,042 compounds, ChemDiv, Inc. California, USA, www.
chemdiv.com) have been utilized for virtual screening. Through
the BEST flexible search of the databases, 278 compounds (model 1:
172; model 2: 106) were selected by fit values (3.50 out of 6.00)
from the two models. Among them, 22 compounds (VS001eVS022,
Table S1) were manually selected based on fit values, structural
diversity, and the presence of essential functionality. All the
selected compounds (VS001eVS022) share a common bicyclic core
ring: 3H-imidazo[4,5-c]pyridine (VS001eVS007), 1H-benzo[d]
Prolonged opening of the mPTP has been observed in many
diseases such as myocardial reperfusion injury [10], amyotrophic
lateral sclerosis (ALS) [11,12], traumatic brain injury [13], and AD
[14], therefore, many research efforts have focused on finding
regulators of the mPTP [13,15,16]. However, considering that the
complete structure of the mPTP has not been fully characterized,
identifying specific regulators of the mPTP is a challenging task.
According to several hypothetical models, the mPTP appears to
contain at least four proteins [16,17]; CypD, a voltage-dependent
anion channel (VDAC), the adenine nucleotide translocator (ANT),
and the 18 kDa translocator protein (TSPO) [18,19]. It has been
suggested that natural compounds such as cyclosporine A [20],
sangliferin A [21], and bongkrekic acid [22] appear to bind these
putative components of the mPTP, and regulate its opening. Several
recent studies have identified small molecule modulators of the
mPTP that can restore cellular viability from A
chondrial dysfunctions [23,24].
b-induced mito-
The 18 kDa TSPO has been studied extensively due to its
involvement in chronic inflammation and neurological disorders
[25,26]. Although it was first introduced as peripheral benzodiaz-
epine receptor (PBR), later it was found to be expressed throughout
the whole body including the brain, therefore renamed as TSPO.
Recent reports suggesting its regulatory roles in the mPTP opening
have drawn renewed attention to TSPO as a novel therapeutic
target for neurodegenerative diseases [25,27,28]. In spite of much
interest, small molecule ligands of TSPO remain relatively scarce in
literature, and only a few compounds with a benzodiazepine core
have been developed to date for diagnostic imaging and thera-
peutic applications [29]. We believe that structurally diverse sets of
compounds would facilitate the identification of novel ligands with
desirable physicochemical properties, therefore, set out to design
new compounds by employing ligand-based virtual screening.
Based on the virtual screening results, we designed and synthe-
sized a library of compounds containing a benzimidazole scaffold.
Biological activity of the synthesized library was evaluated by
determining the mitochondrial membrane potential, ATP produc-
imidazole
(VS011eVS022).
(VS008eVS010),
and 1H-imidazo[4,5-b]pyridine
All 22 compounds were initially screened by mitochondrial
functional assays including JC-1 assay and ATP production assay.
Interestingly,
(VS008eVS010) demonstrated excellent recovery of mitochondrial
membrane potential (over 50% at 5 M) in the JC-1 assay (see
supplementary material, Table S1). Moreover, compound VS008
showed suitable mapping with the built pharmacophore model
(model 2) which is illustrated in Fig. 1A. Compounds VS008eVS010
were further screened by the ATP production assay. VS008 again
three
1H-benzo[d]imidazole
compounds
m
showed moderate recovery of ATP production (20% at 5 mM) in Ab
treated cells, while VS009 and VS010 did not appear to affect ATP
production. Therefore, we decided to focus on VS008 as our lead
compound 1 (Fig. 1B).
tion, and ROS generation in cells suffering Ab induced mitochon-
drial dysfunction. In addition, we tested a few selected compounds
in both acute and transgenic (Tg) mice models of AD to assess their
effects on the cognitive impairment. We performed in vitro binding
assays of the most active compound for TSPO to confirm its target-
specific activity, and analyzed its binding interactions via molecular
docking studies.
2.2. Chemistry
Based on the structure of compound 1, we designed a library of
benzimidazole derivatives, compounds 10e32, which contained
various functional groups corresponding to the common feature
pharmacophore model (Fig. 1B). After performing preliminary
mitochondrial functional assays with compounds 1 and 10e32, we
modified the existing scaffold to have diverse hydrophobic groups
(36e44) as well as an additional hydrogen bond donor (51e53).
The benzimidazole derivatives 10e32 were synthesized by
following the pathway described in Scheme 1. Substituted N-(2-
iodophenyl)amide compounds 4aef were prepared via nucleo-
2. Results and discussion
2.1. Pharmacophore modeling and virtual screening
To design TSPO ligands with a novel scaffold, we first generated
a common feature pharmacophore model based on the structures
of the previously reported neuroprotective TSPO ligands. A ligand-
based pharmacophore model was generated by commercially
available pharmacophore generation program, Catalyst/HipHop. To
generate common feature pharmacophore models, five represen-
tative TSPO ligands were collected from the Integrity® database of
Prous, and were used as training set compounds: 2-(2-(4-
fluorophenyl)-1H-indol-3-yl)-N,N-dihexylacetamide (FGIN-1-27,
philic addition-elimination of the 2-iodoaniline
2 to aryl-
substituted acyl chlorides 3aef. Compounds 4aef were then con-
verted to the corresponding benzimidazole analogues 7aef via
Ullmann-type condensation reaction in moderate yields ranging 40
to 60%. On the other hand, compounds 7g and 7h were obtained via
an intramolecular cyclization of compounds 5aeb with 2-(2,6-
dichlorophenyl)acetic acid 6 in the presence of polyphosphoric
acid (PPA) [30]. The N-alkylation of compounds 7aeh with methyl
2-bromoacetate generated compounds 8aeh, and the subsequent
Ki for TSPO
chlorophenyl)imidazo[1,2-a]pyridin-3-yl)acetamide
¼
3.25 nM), N,N-dibutyl-2-(6,8-dichloro-2-(4-
(Ki for

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T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
A
B
Fig. 1. Design of novel benzimidazole derivatives from hit compound 1 (VS008). (A) Compound 1 was mapped to the common feature pharmacophore model generated from
representative TSPO ligands (Oxygen atom in red; nitrogen atom in blue; sulfur atom in yellow; fluoride atom in cyan; hydrophobic features in a cyan sphere; hydrophobic ring
features in a yellow sphere with a vector; and hydrogen bond feature in a light green sphere with a vector). (B) Newly designed scaffolds of TSPO ligands (HY; Hydrophobic group,
H1; H-bond donor group). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
hydrolysis followed by a peptide coupling reaction with substituted
anilines led to the desired compounds 10e32.
hydrolysis of 49, followed by a peptide coupling reaction with alkyl
amines led to the desired compounds 51e53.
Compounds 36e44 were prepared from compound 7g as shown
in Scheme 2. The N-alkylation of 7g by using methyl 4-(bromo-
methyl)benzoate 33 yielded compound 34. Compound 34 was then
hydrolyzed to carboxylic acid 35, which was subjected to a peptide
coupling reaction with N-substituted amines to generate 2-(2,6-
2.3. Effects on mitochondrial membrane potential
The electrochemical proton gradient across the mitochondrial
membrane is the driving force of electron transport chain and
essential for cellular respiration and energy production. This proton
gradient is tightly regulated by double layers of mitochondrial
membranes, and depolarization of the membrane potential is
considered to be one of the first signs of mPTP opening. Therefore,
dichlorobenzyl)-1H-benzo[d]imidazole
derivatives
36e44.
Notably, the R1 position of the N-substituted amines contains a
diverse set of cycloalkylamines, which correspond to a hydrophobic
feature in the pharmacophore model.
Compounds 51e53 were synthesized from commercially avail-
able 2-(1H-benzo[d]imidazol-2-yl)acetonitrile 45 as described in
Scheme 3. The cyano group of 45 was hydrolyzed by 4 M NaOH to
afford the carboxylic acid compound 46. The subsequent peptide
coupling reaction with 5-isopropyl-2-methylaniline 47 yielded
compound 48, which was further N-alkylated with methyl 4-
(bromomethyl)benzoate 33 to generate compound 49. Finally, the
to evaluate effects of the synthesized compounds on Ab-induced
mPTP opening, we used the ratiometric fluorescent dye JC-1
(5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenz-imidazolocarbo-
cyanine iodide), since the mitochondrial uptake of JC-1 is depen-
dent on mitochondrial membrane potential (DJm). JC-1 taken up
by mitochondria forms red fluorescent J-aggregates, whereas
cytoplasmic JC-1 emits green fluorescent light, hence,

T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
1175
Scheme 1. Synthesis of benzimidazole derivatives 10e32.a
a
Reagents and conditions: (a) THF, r.t., 12e16 h, 63e87%, (b) (i) 30% NH4OH, CuI, L-Proline, NaOH, DMSO, r.t., 1e2 h, (ii) AcOH, 80 ꢀC, 6e7 h, 40e77%, (c) PPA, 180 ꢀC, 4 h, 92%, (d)
methyl 2-bromoacetate, NaH, DMF, r.t., 16 h, 39e95%, (e) 1 N NaOH, MeOH, r.t., 1e2 h, 70e95%, (f) aniline, HATU, Et3N, MeCN, r.t., 16 h, 33e95%.
Scheme 2. Synthesis of 2-(2,6-dichlorobenzyl)-1H-benzo[d]imidazole Derivatives 36e44.a
a Reagents and conditions: (a) NaH, DMF, r.t., 16 h, 52%, (b) LiOH, THF/MeOH, H2O, r.t., 3 h, 53%, (c) (i) Oxalyl chloride, cat. DMF, CH2Cl2, 2 h, (ii) N-substituted amines, DIPEA, THF, r.t.,
16 h, 37e79%.
mitochondrial membrane depolarization can be determined by a
decrease in the red/green fluorescence intensity ratio. To measure
the effects of each compound on DJm, first, we treated a mouse
normal cells, the percent recovery value for this particular com-
pound should be 100%. We also included known neuroprotective
compounds, piracetam and cyclosporine A (CsA) for comparison,
since both compounds have been reported to exert their protective
effects by restoring mitochondrial function [31,32] or blocking
mPTP opening [17], respectively.
Initially, we measured the percent recovery values of DJm for
compounds 10e32, and these results are shown in Table 1. Com-
pounds in this series mostly retained DJm to some extent.
hippocampal cell line, HT22 cells, with 5
incubated with 5 M of each compound, and performed the JC-1
assay. Based on the changes of the red/green fluorescence in-
tensity ratio between normal cells and A -treated cells, we calcu-
mM of Ab, subsequently
m
b
lated percent recovery of DJm for each compound. For example, if
the measured ratio of a compound treated sample is identical with

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T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
Scheme 3. Synthesis of 2-(1H-benzo[d]imidazol-2-yl)-N-(5-isopropyl-2-methylphenyl) acetamide Derivatives 51e53.a
a Reagents and conditions: (a) 4 M NaOH, EtOH, Reflux, 8 h, 62%, (b) 5-isopropyl-2-methylaniline 47, HATU, Et3N, DMF, r.t., 4 h, 81%, (c) methyl 4-(bromomethyl)benzoate 33, DIPEA,
DMF, 80 ꢀC, 6 h, 90%, (d) LiOH, THF/MeOH, H2O, r.t., 3 h, 80%, (e) N-substituted amines, BOP, Et3N, DMF, r.t., 16 h, 37e79%.
Compounds 10e15 and 24e31 showed comparable or greater
percent recovery values compared to piracetam (60%) and CsA
(55%). Compounds without aryl-substituents on the benzimidazole
core (R1 ¼ H; 14 and 15) were shown to be more potent (91%) than
Cl-substituted derivatives (R1 ¼ Cl; 29e32). On the other hand,
compounds with a 2,5-dichlorophenyl group at the R2 position
(R2 ¼ 2,5-diCl; 14 and 15) demonstrated higher activity than
compounds without substituents (R2 ¼ H; 11 and 12). Compounds
with a 2-methyl 5-isopropyl group at the R3 position (compounds
11, 14, 25, 27, 29, and 30) demonstrated significantly higher percent
recovery compared to the rest of the compounds. Based on these
findings, we further modified the benzimidazole scaffold to contain
a dichlorobenzyl group (compounds 36e44), and a 2-methyl 5-iso-
propyl group at the 2-position (compounds 51e53). To focus on
determining structure-activity relationship of hydrophobic inter-
action mimicking groups, we decided to replace the N-phenyl-
acetamide group with various N-alkylbenzamide groups as shown
in Table 2.
effects of each compound, we treated HT22 cells with 5 mM of Ab
and selected compounds in the same manner as described for the
JC-1 assays, and measured the amount of ATP generated by a
luciferase-based assay. Based on the changes of luminescence in-
tensity between untreated control cells and Ab-treated cells, we
calculated percent recovery of ATP production for each compound
as shown in Table 3. We also calculated cell viability by measuring
luminescence intensity of compound-treated cells in the absence of
Ab. Interestingly, piracetam appeared to induce over-production of
ATP (127%), whereas CsA inhibited ATP production (ꢁ46%).
Although it is unclear whether there is a correlation between the
effects on DJm and ATP production of each compound, seven
compounds (13, 25, 28, 30, 36, 38, and 41) restored over 50% of ATP
production. In particular, compounds 25, 28, and 38 recovered ATP
production close to control cells (25, 95%; 28, 94%; and 38, 85%)
while maintaining excellent cell viability (25,109%; 28, 81%; and 38,
93%). On the basis of these findings, we decided to move forward
with eight compounds (13, 25, 26, 28, 30, 36, 38, and 41) for further
testing.
Interestingly, compounds in this series showed generally higher
percent recovery compared to piracetam (60% at 5 mM) as well as
compounds 10e32. Two notable structural changes in this series
are the removal of a hydrogen bond acceptor, and the addition of an
alkylamide group to the benzyl side chain of the benzimidazole
core, both of which appear to be beneficial for recovering mito-
chondrial membrane potential. The presence of a relatively bulky
alkylamide group at the R1 position seem to contribute to the
protective effects regardless of their chain length. While com-
pounds 36e44 share the same dichloro-substituted (2,6-diCl)
benzyl group at the 2 position of the benzimidazole core, com-
pounds 36, 41, and 42 demonstrated remarkably high percent re-
2.5. Effects on cell viability and reactive oxygen species (ROS)
generation
To determine the effects on cell viability, we tested eight
selected compounds (13, 25, 26, 28, 30, 36, 38, and 41) by using 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
(MTT). Again, we treated HT22 cells with 5 M of A
bromide
and each
m
b
compound in the same manner as described in the previous sec-
tion, and calculated percent inhibition values based on the changes
of the signal intensity between untreated cells and A
We also measured cell viability upon the treatment of each com-
pound in the absence of A via the MTT assay as described in
Table 4. Among tested compounds, compounds 25 and 26 inhibited
30% and 36% of cellular toxicity induced by the treatment of A
b-treated cells.
covery values, 89e90% recovery of DJm at 5
mM. Compounds
51e53 showed comparable potency (68e80%) at the same con-
centrations, although the addition of an extra amide bond did not
affect overall activity significantly.
b
b,
which is comparable to the effects of piracetam (29%). Several other
2.4. Effects on mitochondrial ATP production
compounds (13, 28, and 39) also showed noticeable protective ef-
fects against A
and 38 demonstrated high percent recovery values in both ATP
generation and DJm, however, they did not appear to inhibit A
induced cellular toxicity significantly, suggesting that these
b induced toxicity. Surprisingly, compounds 30, 36
Based on the JC-1 assay results, we selected compounds with
great percent recovery values (over 60%), and further evaluated
their effects on mitochondrial ATP production. To determine the
b

T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
1177
Table 1
In vitro activity of compounds 10e32 against Ab-induced dissipation of DJm.
Compds
n
R1
R2
R3
Recovery of DJm (%)a
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
57
86
63
69
65
91
91
41
0
41
29
24
0
38
42
57
74
54
81
68
60
87
77
18
55
60
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
0
0
0
0
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
5-Cl
6-Cl
5-Cl
6-Cl
H
H
H
H
3,5-di-t-Bu
2-Me-5-iPr
3,5-diCl
2-t-Bu-6-Me
2-Me-5-iPr
3,5-diCl
2-t-Bu-6-Me
5-(Biphenyl-4-yloxy)-2-F
2-Me-5-iPr
3,5-di-t-Bu
2-Me-5-iPr
5-(Biphenyl-4-yloxy)-2-F
2-t-Bu-6-Me
3,5-di-t-Bu
2-Me-5-iPr
2-Me-5-iPr
3,5-di-t-Bu
2-Me-5-iPr
3,5-diCl
2,5-diCl
2,5-diCl
2,5-diCl
2,5-diOMe
2,5-diOMe
H
H
H
H
3,4-diCl
3,4-diCl
2,6-diCl
2,5-diOMe
2,5-diOMe
2,5-diOMe
2,5-diCl
2,5-diCl
2,5-diCl
2,5-diCl
2-Me-5-iPr
2-Me-5-iPr
3,5-diCl
3,5-diCl
CsA
Piracetam
a
The increase of fluorescence ratio (green/red) after the treatment of each compound (5
m
M) and A
b (5 mM) in HT22 cell was normalized by dividing the difference of the
ratio between 0% (A
b
-induced damaged condition) and 100% (normal condition in the absence of Ab
).
compounds may act on different pathways.
mPTP opening, resulting in the increased intracellular Ca2þ
,
Next, we measured cellular ROS levels by using 20,7’-dichloro-
fluorescein diacetate (DCFDA), a fluorescent indicator of cellular
ROS. We measured fluorescence signal intensities and calculated
percent inhibition of ROS generation based on the changes of the
therefore, compounds that can inhibit mPTP opening maintain
intracellular Ca2þ level within a normal range. We tested this hy-
pothesis by measuring intracellular Ca2þ concentrations upon
treating cells with a well-known TSPO ligand, PK-11195 in the
signal intensity between untreated cells and A
Piracetam and compounds 25 and 30 effectively reduced cellular
ROS generated by the treatment of A (piracetam: 129%, 25: 85%,
and 30: 107%). Most of the tested compounds reduced cellular ROS
levels to some extent, which generally corresponded to the results
from MTT assays, except compounds 26 and 30. Compound 26
demonstrated a relatively high recovery percent in the MTT assay
(36%), however, it did not inhibit ROS generation significantly (10%).
On the other hand, compound 30 did not recover cell death during
the MTT assay (0%), while completely inhibited ROS generation
b
-treated cells.
presence of 5
mM of Ab. As shown in Fig. 2A, the treatment of Ab at
5
m
M increased intracellular Ca2þ concentrations up to 30%, while
b
the treatment of PK-11195 appeared to suppress opening of the
mPTP in dose-dependent manner, demonstrating significantly
reduced intracellular Ca2þ level compared to the A
b treated control.
Next, based on the results from in vitro testings, we selected
compound 25 (DJm recovery 74%, ATP recovery 95%, MTT assay
30%, and ROS inhibition 85%) to assess inhibitory effects against
mPTP opening, and measured intracellular Ca2þ level. As shown in
Fig. 2B, compound 25 suppressed mPTP opening in dose-
dependent manner, exhibiting the reduced Ca2þ level even at the
(100%), suggesting this compound might act as
antioxidant.
a cellular
lowest concentration (0.1 mM). At 5 mM or higher concentrations,
both PK-11195 and compound 25 maintained the intracellular Ca2þ
levels within a normal range.
2.6. Effects on intracellular Ca2þ level
Opening of the mPTP allows sudden exchange of substances
between mitochondria and cytosol, and also releases mitochondrial
Ca2þ into cytosol resulting in the increased levels of intracellular
2.7. CYP450 and hERG liability
Before we move forward to animal studies, we examined the
toxicity profile of several selected compounds by measuring the
Ca2þ [7]. It has been reported that the accumulation of A
b
induces

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T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
Table 2
In vitro activity of compounds 36e44 and 51e53 against Ab-induced dissipation of DJm.
Compds
R1
R2
H
Recovery of DJm (%)a
36
90
37
H
81
38
39
40
H
H
H
82
79
86
41
42
43
H
H
H
90
89
76
44
51
Me
Me
e
73
68
52
53
e
e
68
80
CsA
Piracetam
55
60
a
The increase of fluorescence ratio (green/red) after the treatment of each compound (5
mM) and A
b (5 mM) in HT22 cell was normalized by dividing the difference of the
ratio between 0% (A
b
-induced damaged condition) and 100% (normal condition in the absence of Ab
).
inhibition of human ether-a-go-go-related gene (hERG) potassium
channels and cytochrome P450 (CYP450) upon the treatment of
each compound (Table 5). While all tested compounds appear to
inhibit CYP450 to some extent, the treatment of compounds 25 and
38 affect overall activity of CYP450 less significantly, showing over
50% of remaining CYP450 activity for all five isoforms. Unfortu-
nately, however, compounds 26 and 30 inhibited all tested isoforms
of CYP450. As for the hERG channel related toxicity, compound 25
did not inhibit hERG channels, whereas compounds 26, 36 and 38
inhibited hERG channels at a low micromolar range. Although
compounds 26, 30, 36, and 41 are potent inhibitors of mPTP
opening in various cell-based assays, these compounds may exhibit
moderate to high toxicity in vivo based on the inhibitory effects of
CYP450 and hERG channels. Thus, we decided to continue further
in vivo studies with compounds 25 and 38 in acute AD model mice.
2.8. Y-maze spontaneous alternation test
Based on the results from mitochondrial functional assays, we
selected compounds 25 and 38 for further testings in acute AD
model mice, which were prepared by injecting a 500 pmol of
Ab1e42 i.c.v. by following the previously described method [33]. We
administered compounds 25 and 38 by intraperitoneal injections
for 6 days (30 mg/kg daily), and assessed spatial working memory
of each mouse by performing Y-maze spontaneous alternation tests
one day after the end of drug administration. We also included
piracetam treated mice in parallel for comparison. The Y-maze test
is a method to determine the willingness of rodents to explore new

T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
1179
Table 3
In vitro ATP production recovery Activities of the selected compounds.
Table 5
CYP450 and hERG liability profiles of selected compounds.
Compds CYP450 (% remaining Activity @ 10 M)
CYP1A2 CYP2D6 CYP2C9 CYP3A4 CYP2C19
Compds
ATP Production
Compds
ATP Production
m
hERG IC50 (m
M)a
Recoverya
Viabilityb
Recoverya
Viabilityb
10
11
13
14
15
25
26
27
28
29
30
4%
71%
67%
65%
93%
107%
109%
112%
96%
81%
78%
86%
36
37
38
39
41
42
43
52
51%
8%
85%
31%
64%
4%
62%
94%
93%
100%
86%
87%
98%
97%
100%
82%
88%
25
26
30
36
38
41
107.60
92.52
56.29
62.97
66.14
68.41
140.41
56.28
82.23
82.36
119.81
25.49
54.69
36.09
29.65
50.58
53.91
61.08
223.73
43.30
15.43
130.27
206.03
176.46
57.30
43.93
11.89
31.43
72.88
67.28
>100
19%
55%
0%
0.02 0.02
NDb
1.57 0.29
4.53 1.50
NDb
0%
95%
46%
0%
94%
3%
1%
a
IC50 values ( SD) were obtained from a dose-response curve.
Not determined.
14%
23%
ꢁ46%
127%
b
53
CsA
Piracetam
69%
memory. As shown in Fig. 3, both compounds 25 and 38 appear to
restore cognitive function compared to vehicle control and pira-
cetam administered mice. Compound 25 significantly improved
a
Recovery of ATP production at 5
m
M of each test compound against A
b-induced
mitochondrial ATP reduction.
b
HT22 cell viability after the treatment with 5
mM of each compound only.
Table 4
In vitro MTT assay and ROS assay results.
Compds
MTT
ROS
% Inhibitiona
Viabilityb
% Inhibitiona
13
25
26
28
30
36
38
39
16%
30%
36%
12%
0%
6%
7%
12%
3%
29%
95%
88%
99%
82%
106%
122%
111%
101%
118%
132%
0%
85%
10%
0%
107%
24%
9%
15%
27%
129%
41
Piracetam
a
Against A
b
-induced toxicity at 5
mM.
b
HT22 cell viability after the treatment with 5
m
M of each compound only.
Fig. 2. Alleviation of Ab
-induced increase of intracellular Ca2þ level by treating PK-11195 (A) or compound 25 (B). After the treatment of each compound in HT22 cell, intracellular
Ca2þ level (% of control) was measured by fluorescence of Fura-2 dye. (#) p < 0.05.
environments. Since rodents prefer to seek a new arm of the maze
rather than revisit the previously explored one, the number of arm
entries and their sequences are recorded to calculate the percent
alternation. Therefore, Y-maze tests can quantify cognitive func-
tion, and higher percent alternation indicates better spatial
learning and memory in acute AD model mice, reversing 72% of the
cognitive deficit induced by A , whereas a known nootropic agent,
piracetam only restored 20% (Fig. 3A). Compound 38 also reversed
62% of the A induced memory deficit, whereas piracetam reversed
32% (Fig. 3B). Overall, both compounds 25 and 38 successfully
b
b

1180
T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
improved spatial working memory function by ameliorating the
cognitive deficit induced by A in mice.
b
2.9. Contextual fear conditioning test in transgenic AD model mice
As observed in the Y-maze spontaneous alternation test, the
treatment of compound 25 alleviated learning and memory deficits
in acute AD mice to an extent similar to wild type mice. To further
assess neuroprotective effects of compound 25, we decided to
perform contextual fear conditioning tests by using a transgenic
mouse model of AD, APPswe/PSEN1dE9 2X (11 months, n ¼ 7). For
testing, we orally administered compound 25 for 1 month (30 mg/
kg daily), and then carried out contextual fear conditioning tests by
following the previously reported procedures [34]. We measured
the number of total freezing responses of the mice in response to
fear-relevant stimuli, and higher freezing percent indicates better
cognitive function. As demonstrated in Fig. 4, compound 25
partially restored fear-associated learning and memory, reversing
12% of cognitive deficits in transgenic mice, whereas piracetam did
not affect behavior of the tested mice. Based on these results, we
believe that compound 25 is capable of reversing learning and
memory deficits in acute and transgenic mice models of AD.
Fig. 4. Alleviation of emotion-associated learning memory in Tg AD model mice by
treating compounds 25. The therapeutic effect of compound 25 (30 mg/kg daily, 1
month) on emotional learning and memory in APPswe/PSEN1dE9 2x transgenic model
(12 month, n ¼ 7) was assessed by contextual fear conditioning test compared to
piracetam (30 mg/kg daily, 1 month). Data are mean
SEM (n ¼ 7 per group): (*)
p < 0.01, compared with WT, (#) p < 0.05, compared with vehicle-treated transgenic
AD model mice.
2.10. Pharmacokinetics of compound 25
To further validate in vivo activity of compound 25, we evalu-
ated its pharmacokinetic profile in Sprague-Dawley (SD) male rats
(Table 6). Compound 25 demonstrated relatively high brain-to-
plasma (B/P) ratio (2.88) after 2 h of its intravenous injection,
indicating that compound 25 is blood brain barrier (BBB) perme-
able. However, compound 25 exhibited unexpectedly low oral
bioavailability (3.7%) and low plasma concentration when admin-
istered orally. This low oral bioavailability may explain the rela-
tively lower percent efficacy of 25 in orally administrated Tg mice
(12%, Fig. 4) compared to the efficacy in intraperitoneally injected
acute AD model mice (72%, Fig. 3A). Therefore, our future efforts for
further optimization will be focused on the improvement of the
pharmacokinetic profiles, particularly oral bioavailability.
2.11. Interaction of compound 25 with the 18 kDa TSPO
Our in vitro and in vivo tests indicate that compound 25 exerts
neuroprotective effects by blocking Ab-induced mPTP opening, in
which the 18 kDa TSPO participates. To verify whether the neuro-
protective effects of compound 25 is derived from the binding of
TSPO, we measured the binding affinity of 25 for purified TSPO by
Fig. 3. Alleviation of learning and memory deficits in acute AD model mice by treating compounds 25 or 38. The effects of compounds 25 (A) or 38 (B) (30 mg/kg daily, 6 days, i.p.)
on learning and memory deficits in acute AD model mice was assessed by Y-maze spontaneous alternation test. Each compound was individually evaluated comparing with
piracetam (30 mg/kg daily, 6 days, i.p.). Data are mean SEM (n ¼ 7 per group): (*) p < 0.05.

T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
1181
Table 6
bond with the indole-NH group of Trp53, which was also observed
as a hydrogen bond donor for PK-11195. The nitrogen atom of the
benzimidazole core forms a hydrogen bond with the OH group of
Tyr57, which corresponds to the halogen bond interaction between
Tyr57 and Cl atom in PK-11195. The benzimidazole group of com-
pound 25 is stacked between the two Trp resides (Trp53 and
Mean ( SD) pharmacokinetic parametersa after intravenous (n ¼ 4) and oral (n ¼ 5)
administration (10 mg/kg) of compound 25 to SD male rats.
Plasma
Intravenous
Oral
AUC0e∞
(
m
m
g min/ml)
g min/ml)
Terminal half-life (min)
Cmax g/ml)
111.02 45.06
104.75 45.41
157.48 126.04
e
8.17 2.51
7.68 2.53
89.25 21.68
0.07 0.01
AUClast
(
Trp95), interacting through hydrophobic
p-p interactions. In
(
m
addition, the di-Cl substituted benzyl group attached to the benz-
imidazole of compound 25 interact with Arg24 through hydro-
phobic interactions. The 2-methyl 5-isopropyl phenyl group
exhibits additional hydrophobic interactions with Phe99. As a
result of these binding interactions, we believe that compound 25
competes with PK-11195 in the same binding site of the TSPO.
Tmax (min)
CL (mL/mim/kg)
MRT (min)
Vss (mL/kg)
e
33
e
54.55 31.38
67.14 28.87
6205.39 5089.22
0.03
e
e
Ae (%)
0.01
1.02
3.7
Brain-to-plasma ratio (B/P) at 2 h
F (%)
2.88
a
AUC0e∞, total area under the plasma concentrationetime curve from time zero
to time infinity; AUClast, total area under the plasma concentrationetime curve from
time zero to last measured time; Cmax, peak plasma concentration; Tmax, time to
3. Conclusion
In this study, we have developed novel benzimidazole de-
rivatives as an mPTP blocker to treat mitochondrial dysfunction in
AD. We generated a common feature pharmacophore model based
on the structures of the previously reported neuroprotective li-
gands of TSPO, one of the putative components of the mPTP. To
identify a lead compound, we screened our in-house library as well
as commercially available chemical libraries against our pharma-
cophore model, discovering compound 1 as a virtual hit. Based on
the structure of compound 1, we designed and synthesized a library
of benzimidazole derivatives, and evaluated biological effects of the
newly generated compounds. Among the tested compounds,
reach Cmax; CL, time-averaged total body clearance; MRT, mean residence time; Vss
apparent volume of distribution at steady state; Ae, Excreted amount; F,
bioavailability.
,
using [3H] PK-11195 as a competitive ligand. As shown in Table 7,
compound 25 appeared to be a potent ligand for TSPO, exhibiting Ki
and IC50 values in a low nanomolar range (Ki ¼ 22.8 10.6 nM,
IC50 ¼ 74.3 14.7 nM). Additionally, we performed surface plasmon
resonance (SPR) measurements to analyze the interaction of com-
pound 25 with recombinant human TSPO (Fig. 5). The SPR data
indicate that compound 25 interacts with the human TSPO
demonstrating the KD value of 108 nM. PK-11195 showed a lower
KD value (11.1 nM), which corresponds to the results from
competitive binding assays. Although the binding affinity of com-
pound 25 is not superior to that of PK-11195, these results support
our hypothesis that the neuroprotective effects of compound 25
come from its binding to the TSPO.
To investigate the binding mode of compound 25, we performed
molecular docking studies by using a homology model of human
TSPO (Fig. 6). Since crystal structure of human TSPO has not been
reported yet, the most reliable homology model of human TSPO
was generated by using recently reported crystal structure of the
bacterial TSPO (PDB ID: 4RYI). It has been suggested that PK-11195
fits in the central cavity of TSPO [35], therefore, we also docked
compound 25 in the same binding site. Both of PK-11195 and
compound 25 were docked into the hydrophobic central cavity
(indicated as grooves in Fig. 6) of the TSPO with additional
hydrogen bond interactions. The predicted binding mode showed
that the carbonyl oxygen of PK-11195 (Glide docking
score ¼ ꢁ10.80) interacts with the indole-NH groups of Trp53 and
Trp143 through two hydrogen bond interactions as shown in
Fig. 6A. Also, the Cl atom attached to the phenyl ring of PK-11195
forms a halogen bond with the oxygen atom of the OH group of
Tyr57. Furthermore, the isoquinoline group of PK-11195 interacts
compound 25 effectively alleviated Ab-induced mitochondrial
dysfunction in cells, recovering the mitochondrial membrane po-
tential, ATP production, cellular viability, and suppressing ROS
generation and intracellular calcium levels. More importantly,
compound 25 successfully recovered AD-associated cognitive def-
icits in preclinical AD model mice. Pharmacokinetic studies
revealed that compound 25 readily crosses the BBB, and bind to the
18 kDa TSPO located in mitochondrial outer membrane, although
its oral bioavailability is relatively low. Competitive binding assays
and SPR kinetics indicated that compound 25 binds toTSPO with KD
values of 108 nM, supporting that neuroprotective effects of com-
pound 25 comes from the direct interaction with TSPO. Binding
mode analysis also supports that compound 25 interacts with TSPO
in the similar manner as a known TSPO ligand, PK-11195. In
conclusion, compound 25 is a promising lead for the development
of modulators of mitochondrial function. We believe that further
optimization for more potent and orally bioavailable benzimidazole
derivatives may provide a possible therapeutic option for the
treatment of neurodegenerative diseases such as AD.
4. Experimental section
4.1. Chemistry
with the rings of Trp95 through hydrophobic
p-p interactions.
4.1.1. General methods
Next, compound 25 was docked into the same binding pocket of
PK-11195 as shown in Fig. 6B (Glide docking score ¼ ꢁ9.24). The
best fitted pose of compound 25 exhibited hydrogen bond in-
teractions. The amide group of compound 25 forms a hydrogen
All reagents were obtained from commercial sources and used
without further purification. All reactions were performed under a
nitrogen atmosphere in oven-dried glassware. Reactions were
monitored by analytical thin-layer chromatography (TLC) plates
(Merck, catalog no. 1.05715) with spots visualized by UV light
(
l
¼ 254 nm) or using a KMnO4 solution. Solvents were evaporated
Table 7
Inhibition of TSPO.
using a rotary evaporator under a reduced pressure of 50 mBar. The
reaction products were purified by flash column chromatography
using silica gel 60 (Merck, catalog nos. 1.07734). Melting points
were determined using an OptiMelt melting point apparatus
(Stanford Research System, Inc.) in open capillary tube without
correction. 1H (300 or 400 MHz) and 13C (75 or 100 MHz) NMR
spectra were recorded using tetramethylsilane (TMS) as the
Compds
IC50 (nM)a
Ki (nM)a
25
74.3 14.7
1.32 1.10
22.8 10.6
8.85 1.09
PK-11195
a
Values are expressed as the mean
SD from at least three independent
experiments.

1182
T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
Compound 25
KD = 108 ± 41.6 nM
PK-11195
KD = 11.1 ± 0.45 nM
A
B
10
10
5 μM
5 μM
1 μM
8
6
1 μM
8
6
0.5 μM
0.1 μM
0.01 μM
0.1 μM
0.01 μM
4
4
2
2
0
0
-2
-2
-50
0
50 100 150 200 250 300 350 400
-50
0
50 100 150 200 250 300 350 400
Time (s)
Time (s)
Fig. 5. Representative SPR sensorgrams for compound 25 (A) and PK-11195 (B). KD values are expressed as the mean SD from at least three independent experiments.
internal standard. Chemical shifts (
d
) are reported in parts per
(2C), 30.42 (6C); HRMS (ESIþ): m/z: calcd for C29H34N3O 440.2624
[MþH]þ; found: 440.2696.
million (ppm) values relative to TMS, and the coupling constants (J)
are reported in hertz (Hz). The purity (ꢂ95%) of the samples was
determined by analytical HPLC using a Waters E2695 system with
4.1.2.2. N-(5-Isopropyl-2-methylphenyl)-2-(2-phenyl-1H-benzo[d]
imidazol-1-yl)acetamide (11). Following the general procedure for
compounds 10e32, 2-(2-phenyl-1H-benzo[d]imidazol-1-yl)acetic
acid 9a (20 mg, 0.08 mmol), 5-iso-propyl-2-methylaniline (15 mg,
SunFire C18 column (4.6 mm ꢃ 150 mm; 5
mm). HPLC data were
recorded using parameters as follows: H2O/MeCN, 80/20 / 0/100
in 20 min, þ3 min isocratic, flow rate of 1.0 mL/min,
l
¼ 254 and
280 nm. Higheresolution mass spectra (HRMS) were recorded on a
LTQ Orbitrap (Thermo Electron Corporation) instrument. Liquid
chromatograph mass spectra (LC/MS) data were recorded on a
Shimadzu LCMS-2020 instrument equipped with Shimadzu's VP-
0.10 mmol), HATU (38 mg, 0.10 mmol), and Et3N (56 mL, 0.40 mmol)
in MeCN (3 mL) gave the title compound 11 (11 mg, 35%) as a white
solid; Rf ¼ 0.20 (n-hexane/EtOAc: 1/1); mp: 129.0e132.9 ꢀC; HPLC
purity: 16.84 min, 99.9%; 1H NMR (400 MHz, MeOD)
d 7.82e7.83
ODS column (4.6 mm ꢃ 150 mm; 4.6
mm). LC/MS data were
(m, 2H), 7.75 (d, J ¼ 7.6 Hz, 1H), 7.61e7.63 (m, 4H), 7.35e7.43 (m,
2H), 7.15e7.17 (m, 2H), 7.05 (d, J ¼ 7.2 Hz, 1H), 5.19 (s, 2H), 2.85
(septet, J ¼ 7.2 Hz, 1H), 2.18 (s, 3H), 1.23 (d, J ¼ 6.8 Hz, 6H); 13C NMR
recorded using parameters as follows: H2O/MeCN, 90/10 / 0/100
in 15 min, þ3 min isocratic, flow rate of 1.0 mL/min,
280 nm. Reaction yields are for purified products.
l
¼ 254 and
(101 MHz, MeOD)
d 166.69, 147.13, 141.87, 136.08, 134.59, 130.20,
130.15 (2C), 129.47, 129.24 (2C), 128.64 (2C), 124.45, 123.41, 123.21,
122.76, 118.48, 113.23, 110.07, 47.17 (overlapped with MeOD peaks),
33.48, 22.95 (2C), 16.33; HRMS (ESIþ): m/z: calcd for C25H26N3O
384.1998 [MþH]þ; found: 384.2069.
4.1.2. General procedure for the synthesis of compounds 10e32
To a stirred solution of compound 9 (1.0 equiv) and 1-[bis(di-
methylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-
oxid hexafluorophosphate (HATU, 1.2 equiv) in MeCN was added
triethylamine (Et3N, 5.0 equiv). After 1 h of stirring at room tem-
perature, the desired substituted amine (1.2 equiv) was added
dropwise. The reaction mixture was stirred at room temperature
for 12e16 h, concentrated in vacuo, and extracted with EtOAc
(3 ꢃ 30 mL). The organic layer was dried over anhydrous MgSO4,
filtered, concentrated, and purified by column chromatography on
SiO2 (n-hexane/EtOAc: 1/1). The resultant product was recrystal-
lized with diethyl ether to yield the desired compound.
4.1.2.3. N-(3,5-Dichlorophenyl)-2-(2-phenyl-1H-benzo[d]imidazol-1-
yl)acetamide (12). Following the general procedure for compounds
10e32, 2-(2-phenyl-1H-benzo[d]imidazol-1-yl)acetic acid 9a
(30 mg, 0.12 mmol), 3,5-dichloroaniline (25 mg, 0.15 mmol), HATU
(91 mg, 0.24 mmol), and Et3N (86 mL, 0.60 mmol) in MeCN (3 mL)
gave the title compound 12 (24 mg, 50%) as a white solid; Rf ¼ 0.30
(n-hexane/EtOAc: 1/1); mp: 236.6e238.8 ꢀC; HPLC purity:
19.44 min, 99.8%; 1H NMR (300 MHz, MeOD)
d
7.75 (m, 3H), 7.60 (m,
5H), 7.51e7.53 (m, 1H), 7.36e7.38 (m, 2H), 7.19 (s, 1H), 5.11 (s, 2H);
13C NMR (101 MHz, DMSO-d6)
166.93, 154.01, 142.92, 141.17,
d
4.1.2.1. N-(3,5-Di-tert-butylphenyl)-2-(2-phenyl-1H-benzo[d]imida-
zol-1-yl)acetamide (10). Following the general procedure for com-
pounds 10e32, 2-(2-phenyl-1H-benzo[d]imidazol-1-yl)acetic acid
9a (20 mg, 0.08 mmol), 3,5-di-tert-butylaniline (21 mg, 0.10 mmol),
136.91, 134.69 (2C), 130.45, 120.32, 129.58 (2C), 129.29 (2C), 123.53,
123.18, 122.70, 119.63, 118.00 (2C), 111.09, 48.20; HRMS (ESIþ): m/z:
calcd for C21H16Cl2N3O 396.0592 [MþH]þ; found: 396.0663.
HATU (38 mg, 0.10 mmol) and Et3N (56 mL, 0.40 mmol) in MeCN
(3 mL) gave the title compound 10 (21 mg, 60%) as a white solid;
4.1.2.4. N-(2-tert-Butyl-6-methylphenyl)-2-(2-phenyl-1H-benzo[d]
imidazol-1-yl)acetamide (13). Following the general procedure for
compounds 10e32, 2-(2-phenyl-1H-benzo[d]imidazol-1-yl)acetic
acid 9a (30 mg, 0.12 mmol), 2-tert-butyl-6-methylaniline (78 mg,
Rf ¼ 0.20 (n-hexane/EtOAc: 1/1); mp: 238.4e241.5 ꢀC; HPLC purity:
10.58 min, 97.3%; 1H NMR (300 MHz, MeOD)
d 7.78 (m, 2H),
7.71e7.74 (m, 1H), 7.57 (m, 3H), 7.51 (d, J ¼ 5.1 Hz, 1H), 7.45 (m, 2H),
7.32e7.34 (m, 2H), 7.24 (s, 1H), 5.07 (s, 2H), 1.30 (s, 18H); 13C NMR
0.48 mmol), HATU (91 mg, 0.24 mmol), and Et3N (84 mL, 0.48 mmol)
(75 MHz, MeOD)
d
165.86, 154.38, 151.41 (2C), 141.75, 137.27, 136.02,
in MeCN (3 mL) gave the title compound 13 (36 mg, 78%) as a white
130.12, 129.34 (2C), 129.20 (2C), 128.63 (2C), 123.22, 122.75, 118.41,
114.31 (2C), 110.09, 47.03 (overlapped with MeOD peaks), 34.39
solid; Rf ¼ 0.25 (n-hexane/EtOAc: 1/1); mp: 213.6e215.9 ꢀC; HPLC
purity: 15.39 min, 99.1%; 1H NMR (400 MHz, CDCl3)
d 7.90 (m, 3H),

T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
1183
Fig. 6. Binding modes of PK-11195 (A) and compound 25 (B) with the homology model of human TSPO built on a crystal structure of the bacterial TSPO (PDB ID: 4RYI). The predicted
binding mode demonstrated that PK-11195 and compound 25 form several hydrogen bond interactions (indicated in green lines) and hydrophobic interactions (indicated in pink
lines). (Carbon atoms in yellow, Oxygen atom in red, Nitrogen atom in blue, and Chlorine atom in green). (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
7.54e7.60 (m, 4H), 7.44e7.50 (m, 2H), 7.21 (d, J ¼ 7.6 Hz, 1H),
7.08e7.12 (m, 2H), 6.73 (br s, 1H), 5.22 (s, 2H), 2.05 (s, 3H), 1.10 (s,
J ¼ 6.9 Hz, 1H), 2.10 (s, 3H), 1.20 (d, J ¼ 7.2 Hz, 6H); 13C NMR
(75 MHz, MeOD)
d 166.00, 150.13, 147.09, 141.79, 135.32, 134.45,
9H); 13C NMR (75 MHz, CDCl3)
d
165.57, 153.67, 146.61, 143.32,
132.90, 132.48, 132.26, 131.90, 131.10, 130.58, 130.16, 130.10, 124.44,
123.90, 123.40, 122.89, 118.84, 110.28, 46.22, 33.45, 22.93 (2C),
16.25; HRMS (ESIþ): m/z: calcd for C25H24Cl2N3O 452.1218 [MþH]þ;
found: 452.1288.
137.61, 135.49, 132.32, 130.52, 129.56 (2C), 129.21 (2C), 129.13 (2C),
128.11, 124.79, 123.86, 123.71, 120.62, 110.07, 48.54, 34.90, 30.63
(3C), 18.95; HRMS (ESIþ): m/z: calcd for C26H28N3O 398.2227
[MþH]þ; found: 398.2218.
4.1.2.6. N-(3,5-Dichlorophenyl)-2-(2-(2,5-dichlorophenyl)-1H-benzo
[d]imidazol-1-yl)acetamide (15). Following the general procedure
for compounds 10e32, 2-(2-(2,5-dichlorophenyl)-1H-benzo[d]
imidazol-1-yl)acetic acid 9b (45 mg, 0.14 mmol), 3,5-
dichloroaniline (29 mg, 0.18 mmol), HATU (160 mg, 0.42 mmol),
4.1.2.5. N-(5-Isopropyl-2-methylphenyl)-2-(2-phenyl-1H-benzo[d]
imidazol-1-yl)acetamide (14). Following the general procedure for
compounds 10e32, 2-(2-(2,5-dichlorophenyl)-1H-benzo[d]imida-
zol-1-yl)acetic acid 9b (50 mg, 0.17 mmol), 5-iso-propyl-2-
methylaniline (35
m
L, 0.22 mmol), HATU (84 mg, 0.22 mmol), and
and Et3N (98 mL, 0.70 mmol) in MeCN (5 mL) gave the title com-
Et3N (119 L, 0.85 mmol) in MeCN (7 mL) gave the title compound
m
pound 15 (30 mg, 40%) as a white solid; Rf ¼ 0.25 (n-hexane/EtOAc:
14 (23 mg, 33%) as a white solid; Rf ¼ 0.25 (n-hexane/EtOAc: 2/1);
2/1); mp: 215.0e219.1 ꢀC; 19.37 min, 99.2%; 1H NMR (300 MHz,
mp: 185.1e188.2 ꢀC; HPLC purity: 19.38 min, 99.7%; 1H NMR
MeOD)
(m, 2H), 7.41e7.51 (m, 2H), 7.16e7.18 (m, 1H), 5.01 (s, 2H); 13C NMR
(75 MHz, MeOD) 165.62, 150.10, 141.73, 140.00, 135.24, 134.90
d 7.60 (m, 1H), 7.67e7.68 (m, 1H), 7.62e7.64 (m, 3H), 7.58
(300 MHz, MeOD)
d 7.74e7.75 (m,1H), 7.64e7.69 (m, 4H), 7.38e7.44
(m, 2H), 7.08e7.10 (m, 2H), 7.03 (m, 1H), 5.08 (s, 2H), 2.83 (septet,
d

1184
T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
(2C), 132.93, 132.44, 132.22, 131.91, 131.04, 130.42, 123.76, 123.60,
122.93, 118.83 (2C), 117.70, 110.26, 46.73 (overlapped with MeOD
peaks); HRMS (ESIþ): m/z: calcd for C21H14Cl4N3O 463.9885
[MþH]þ; found: 463.9872.
compounds 10e32, 2-(2-benzyl-1H-benzo[d]imidazol-1-yl)acetic
acid 9d (30 mg, 0.11 mmol), 3,5-di-tert-butylaniline (30 mg,
0.15 mmol), HATU (57 mg, 0.15 mmol), and Et3N (98 mL, 0.70 mmol)
in MeCN (3 mL) gave the title compound 19 (37 mg, 72%) as a white
solid; Rf ¼ 0.30 (n-hexane/EtOAc: 1/1); mp: 238.9e240.8 ꢀC; HPLC
4.1.2.7. N-(2-tert-Butyl-6-methylphenyl)-2-(2-(2,5-dichlorophenyl)-
1H-benzo[d]imidazol-1-yl)acetamide (16). Following the general
procedure for compounds 10e32, 2-(2-(2,5-dichlorophenyl)-1H-
benzo[d]imidazol-1-yl)acetic acid 9b (26 mg, 0.08 mmol), 2-
methyl-6-tert-butylaniline (16 mg, 0.10 mmol), HATU (38 mg,
purity: 17.03 min, 100%; 1H NMR (300 MHz, CDCl3)
d 7.84e7.86 (m,
1H), 7.25e7.36 (m, 7H), 7.17e7.20 (m, 2H), 7.05 (s, 2H), 7.00 (s, 1H),
4.80 (s, 2H), 4.32 (s, 2H), 1.27 (s, 18H); 13C NMR (75 MHz, CDCl3)
d
164.28, 153.49, 151.60 (2C), 142.63, 135.76, 135.48, 135.38, 129.23
(2C), 128.48 (2C), 127.50, 123.58, 123.12, 119.91, 119.35, 115.09 (2C),
109.34, 48.02, 34.92 (2C), 34.49, 31.34 (6C); HRMS (ESIþ): m/z: calcd
for C30H36N3O 454.2780 [MþH]þ; found: 454.2853.
0.10 mmol), and Et3N (56 mL, 0.40 mmol) in MeCN (3 mL) gave the
title compound 16 (21 mg, 57%) as a white solid; Rf ¼ 0.25 (n-
hexane/EtOAc: 2/1); mp: 193.2e194.6 ꢀC; HPLC purity: 17.74 min,
99.6%; 1H NMR (300 MHz, CDCl3)
d
7.92 (d, J ¼ 3.9 Hz, 1H), 7.71 (s,
4.1.2.11. 2-(2-Benzyl-1H-benzo[d]imidazol-1-yl)eN-(5-isopropyl-2-
methylphenyl)acetamide (20). Following the general procedure for
compounds 10e32, 2-(2-benzyl-1H-benzo[d]imidazol-1-yl)acetic
acid 9d (25 mg, 0.09 mmol), 5-iso-propyl-2-methylaniline (21 mg,
1H), 7.51e7.57 (m, 3H), 7.44e7.48 (m, 2H), 7.11e7.19 (m, 3H), 6.67 (s,
1H), 5.00 (s, 2H), 2.02 (s, 3H), 1.08 (s, 9H); 13C NMR (75 MHz,
Acetone-d6)
d 165.48, 150.16, 147.59, 143.29, 138.18, 135.83, 134.08,
132.83, 132.51, 132.30, 131.93, 131.50, 131.16, 128.39, 127.44, 124.44,
123.02, 122.24, 119.90, 110.26, 46.49, 34.79, 30.56 (3C), 17.98; HRMS
(ESIþ): m/z: calcd for C26H26Cl2N3O 466.1375 [MþH]þ; found:
466.1380.
0.12 mmol), HATU (46 mg, 0.12 mmol), and Et3N (63 mL, 0.45 mmol)
in MeCN (3 mL) gave the title compound 20 (27 mg, 73%) as a white
solid; Rf ¼ 0.30 (n-hexane/EtOAc: 1/1); mp: 213.1e214.3 ꢀC; HPLC
purity: 12.50 min, 95.7%; 1H NMR (300 MHz, CDCl3)
d 7.85 (d,
J ¼ 5.4 Hz, 1H), 7.17e7.36 (m, 10H), 6.89e6.96 (m, 2H), 6.56 (br s,
4.1.2.8. N-(5-(Biphenyl-4-yloxy)-2-fluorophenyl)-2-(2-(2,5-
1H), 4.86 (s, 2H), 4.38 (s, 2H), 2.83 (septet, J ¼ 7.2 Hz, 1H), 1.57 (s,
3H), 1.21 (d, J ¼ 6.9 Hz, 6H); 13C NMR (75 MHz, CDCl3)
d
164.11,
dimethoxyphenyl)-1H-benzo[d]imidazol-1-yl)acetamide
Following the general procedure for compounds 10e32, 2-(2-(2,5-
dimethoxyphenyl)-1H-benzo[d]imidazol-1-yl)acetic acid 9c
(72 mg, 0.23 mmol), 5-(biphenyl-4-yloxy)-2-fluoroaniline (82 mg,
0.29 mmol), HATU (110 mg, 0.29 mmol), and Et3N (130 L,
(17).
153.36, 147.68, 142.70, 135.25, 134.89, 133.95, 130.29, 129.19 (2C),
128.49 (2C), 127.48, 126.29, 123.83, 123.65, 123.31, 120.82, 120.21,
109.16, 47.82, 34.55, 33.76, 23.96 (2C), 16.53; HRMS (ESIþ): m/z:
calcd for C26H28N3O 398.2154 [MþH]þ; found: 398.2128.
m
0.92 mmol) in MeCN (3 mL) gave the title compound 17 (56 mg,
44%) as a white solid; Rf ¼ 0.20 (n-hexane/EtOAc: 2/1); mp:
116.5e119.2 ꢀC; HPLC purity: 12.29 min, 99.4%; 1H NMR (400 MHz,
4.1.2.12. 2-(2-Benzyl-1H-benzo[d]imidazol-1-yl)eN-(5-(biphenyl-4-
yloxy)-2-fluorophenyl)acetamide (21). Following the general pro-
cedure for compounds 10e32, 2-(2-benzyl-1H-benzo[d]imidazol-
1-yl)acetic acid 9d (30 mg, 0.11 mmol), 2-methyl-6-tert-butylani-
CDCl3)
d
8.41 (dd, J ¼ 10.4 Hz, 2.8 Hz, 1H), 8.16 (br s, 1H), 7.73 (d,
J ¼ 7.6 Hz, 1H), 7.51 (d, J ¼ 7.2 Hz, 2H), 7.46 (t, J ¼ 7.6 Hz, 2H),
7.37e7.38 (m, 1H), 7.29e7.32 (m, 2H with CDCl3 peak), 7.23 (d,
J ¼ 6.4 Hz, 1H), 7.19e7.22 (m, 2H), 7.05e7.12 (m, 2H), 6.93e6.96 (m,
2H), 6.82 (td, J ¼ 8.0 Hz, 3.2 Hz, 1H), 6.47 (d, J ¼ 8.4 Hz, 2H), 4.76 (s,
line (42 mg, 0.15 mmol), HATU (57 mg, 0.15 mmol), and Et3N (77 mL,
0.55 mmol) in MeCN (3 mL) gave the title compound 21 (26 mg,
44%) as a white solid; Rf ¼ 0.50 (n-hexane/EtOAc: 1/1); mp:
205.5e207.4 ꢀC; HPLC purity: 17.31 min, 94.9%; 1H NMR (300 MHz,
2H), 3.81 (s, 3H), 3.64 (s, 3H); 13C NMR (101 MHz, CDCl3)
d 165.82,
159.28 (d, 1J ¼ 241.4 Hz), 156.04, 154.03, 151.99, 151.07, 143.19,
140.27, 140.04, 136.24, 134.85, 130.92 (d, 3J ¼ 12.1 Hz), 128.77 (2C),
128.35 (2C), 127.09, 126.85 (2C), 123.70, 123.10, 121.02 (d,
3J ¼ 10.1 Hz),120.26,118.59,118.35,117.15,116.27 (2C),112.31,110.87
(d, 2J ¼ 24.2 Hz), 109.30, 107.92 (d, 2J ¼ 29.3 Hz), 55.92, 55.88, 46.50;
HRMS (ESIþ): m/z: calcd for C35H29FN3O4 574.2064 [MþH]þ; found:
574.2134.
CDCl3)
d
8.05 (d, J ¼ 5.1 Hz, 1H), 7.75 (d, J ¼ 3.9 Hz, 1H), 7.56 (d,
J ¼ 3.6 Hz, 2H), 7.47 (t, J ¼ 9.0 Hz, 2H), 7.37e7.39 (m, 3H), 7.36 (s,
1H), 7.17e7.20 (m, 5H), 7.09e7.10 (m, 3H), 6.83e6.85 (m, 2H), 6.47
(d, J ¼ 4.5 Hz, 2H), 4.76 (s, 2H), 4.26 (s, 2H); 13C NMR (101 MHz,
CDCl3)
d
164.31, 158.99 (d, 1J ¼ 242.5 Hz), 155.79, 153.09, 142.57,
140.24, 140.02, 136.43, 134.94 (d, 2J
¼
24.1 Hz), 130.11 (d,
3J ¼ 12.0 Hz), 129.08 (2C), 128.87 (2C), 128.45 (2C), 128.43 (2C),
127.26, 127.21, 126.86 (2C), 123.56, 123.07, 120.25 (d, 3J ¼ 9.0 Hz),
120.09, 116.44 (2C), 111.14(d, 2J ¼ 24.1 Hz), 108.57, 108.34, 108.05,
47.80, 34.60; HRMS (ESIþ): m/z: calcd for C34H27FN3O2 528.2009
[MþH]þ; found: 528.2078.
4.1.2.9. 2-(2-(2,5-Dimethoxyphenyl)-1H-benzo[d]imidazol-1-yl)eN-
(5-isopropyl-2-methylphenyl)acetamide (18). Following the general
procedure for compounds 10e32, 2-(2-(2,5-dimethoxyphenyl)-1H-
benzo[d]imidazol-1-yl)acetic acid 9c (50 mg, 0.13 mmol), 5-iso-
propyl-2-methylaniline (30
mL, 0.19 mmol), HATU (65 mg,
0.17 mmol), and Et3N (73 L, 0.52 mmol) in MeCN (3 mL) gave the
m
4.1.2.13. 2-(2-Benzyl-1H-benzo[d]imidazol-1-yl)eN-(2-tert-butyl-6-
methylphenyl)acetamide (22). Following the general procedure for
compounds 10e32, 2-(2-benzyl-1H-benzo[d]imidazol-1-yl)acetic
acid 9d (20 mg, 0.075 mmol), 2-methyl-6-tert-butylaniline (49 mg,
title compound 18 (24 mg, 44%) as a white solid; Rf ¼ 0.25 (n-
hexane/EtOAc: 2/1); mp: 205.4e209.1 ꢀC; HPLC purity: 9.90 min,
99.6%; 1H NMR (400 MHz, MeOD)
d
7.74 (d, J ¼ 7.6 Hz, 1H), 7.61 (d,
J ¼ 7.6 Hz, 1H), 7.35e7.40 (m, 2H), 7.11e7.19 (m, 5H), 7.01 (dd,
J ¼ 7.6 Hz, 1.6 Hz, 1H), 5.07 (s, 2H), 3.83 (s, 3H), 3.81 (s, 3H), 2.85
(septet, J ¼ 6.8 Hz, 1H), 2.07 (s, 3H), 1.22 (d, J ¼ 6.8 Hz, 6H); 13C NMR
0.30 mmol), HATU (114 mg, 0.30 mmol), and Et3N (21 mL,
0.15 mmol) in MeCN (2 mL) gave the title compound 22 (15 mg,
45%) as a white solid; Rf ¼ 0.55 (n-hexane/EtOAc: 1/1); mp:
213.2e214.6 ꢀC; HPLC purity: 16.35 min, 99.0%; 1H NMR (300 MHz,
(75 MHz, MeOD)
d 166.55, 153.93, 152.14, 151.52, 147.05, 142.03,
135.72, 134.68, 130.10, 129.66, 124.14, 123.02, 122.98, 122.37, 118.94,
118.35, 117.43, 117.13, 112.68, 110.11, 55.40, 54.90, 47.30 (overlapped
with MeOD peaks), 33.46, 22.91 (2C), 16.12; HRMS (ESIþ): m/z:
calcd for C27H30N3O3 444.2209 [MþH]þ; found: 444.2280.
CDCl3)
7.12e7.20 (m, 3H), 6.62 (br s, 1H), 4.87 (s, 2H), 4.46 (s, 2H), 2.17 (s,
3H), 1.14 (s, 9H); 13C NMR (75 MHz, CDCl3)
165.39, 152.94, 146.53,
d
7.85 (d, J ¼ 5.4 Hz, 1H), 7.27e7.39 (m, 7H with CDCl3 peak),
d
142.84, 137.38, 135.18, 134.90, 132.21, 129.23 (2C), 129.19 (2C),
128.46, 128.16, 127.53, 124.84, 123.53, 123.29, 120.32, 109.37, 47.48,
34.93, 34.58, 30.64 (3C), 19.07; HRMS (ESIþ): m/z: calcd for
4.1.2.10. 2-(2-Benzyl-1H-benzo[d]imidazol-1-yl)eN-(3,5-di-tert-
butylphenyl)acetamide (19). Following the general procedure for
C
27H30N3O 412.2311 [MþH]þ; found: 412.2313.

T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
1185
4.1.2.14. N-(3,5-Di-tert-butylphenyl)-2-(2-(3,4-dichlorobenzyl)-1H-
benzo[d]imidazol-1-yl)acetamide (23). Following the general pro-
cedure for compounds 10e32, 2-(2-(3,4-dichlorobenzyl)-1H-benzo
[d]imidazol-1-yl)acetic acid 9e (20 mg, 0.06 mmol), 3,5-di-tert-
butylaniline (18 mg, 0.09 mmol), HATU (46 mg, 0.12 mmol), and
(6C), 27.31; HRMS (ESIþ): m/z: calcd for C32H40N3O3 514.2991
[MþH]þ; found: 514.3063.
4.1.2.18. 2-(2-(2,5-Dimethoxybenzyl)-1H-benzo[d]imidazol-1-yl)eN-
(5-isopropyl-2-methylphenyl)acetamide (27). Following the general
procedure for compounds 10e32, 2-(2-(2,5-dimethoxybenzyl)-1H-
benzo[d]imidazol-1-yl)acetic acid 9f (20 mg, 0.06 mmol), 5-iso-
propyl-2-methylaniline (12 mg, 0.08 mmol), HATU (46 mg,
Et3N (25 mL, 0.18 mmol) in MeCN (3 mL) gave the title compound 23
(26 mg, 83%) as a white solid; Rf ¼ 0.35 (n-hexane/EtOAc: 1/2); mp:
221.4e224.1 ꢀC; HPLC purity: 19.15 min, 96.5%; 1H NMR (300 MHz,
CDCl3)
(m, 2H), 7.10 (s, 2H), 6.81 (br s, 1H), 4.83 (s, 2H), 4.32 (s, 2H), 1.29 (s,
18H); 13C NMR (101 MHz, DMSO-d6)
165.44, 153.82, 151.24 (2C),
142.66, 138.49, 138.46, 136.27, 131.48, 131.26, 130.82, 129.95, 129.69,
122.49, 122.05, 119.08, 117.79, 113.91 (2C), 110.55, 46.18, 35.01 (2C),
32.26, 31.65 (6C); HRMS (ESIþ): m/z: calcd for C30H34Cl2N3O
522.2001 [MþH]þ; found: 522.2069.
d
7.86e7.90 (m,1H), 7.43 (s,1H), 7.32e7.41 (m, 4H), 7.14e7.20
0.12 mmol), and Et3N (17 mL, 0.12 mmol) in MeCN (3 mL) gave the
title compound 27 (20 mg, 72%) as a white solid; Rf ¼ 0.50 (n-
d
hexane/EtOAc: 1/1); mp: 161.6e163.2 ꢀC; HPLC purity: 16.46 min,
95.8%; 1H NMR (300 MHz, MeOD)
d 7.66 (m, 1H), 7.47 (m, 1H),
7.28e7.31 (m, 2H), 7.16 (s, 1H), 7.11 (d, J ¼ 7.8 Hz, 1H), 7.01 (m, 1H),
6.92 (d, J ¼ 8.1 Hz, 1H), 6.74e6.80 (m, 2H), 5.13 (s, 2H), 4.32 (s, 2H),
3.81 (s, 3H), 3.64 (s, 3H), 2.83 (septet, J ¼ 7.2 Hz, 1H), 2.12 (s, 3H),
1.21 (dd, J ¼ 6.9 Hz, 4.5 Hz, 6H); 13C NMR (75 MHz, MeOD)
d 166.00,
4.1.2.15. 2-(2-(3,4-Dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)eN-(5-
isopropyl-2-methylphenyl)acetamide (24). Following the general
procedure for compounds 10e32, 2-(2-(3,4-dichlorobenzyl)-1H-
benzo[d]imidazol-1-yl)acetic acid 9e (20 mg, 0.06 mmol), 5-iso-
propyl-2-methylaniline (14 mg, 0.09 mmol), HATU (46 mg,
154.47, 153.88, 151.01, 147.05, 141.18, 135.43, 134.62, 130.10, 124.69,
124.11, 122.99, 122.73 (2C), 122.35, 117.82, 115.79, 112.85, 111.56,
109.55, 55.17, 54.60, 45.78, 33.52, 27.24, 22.96 (2C), 16.18; HRMS
(ESIþ): m/z: calcd for C28H32N3O3 458.2438 [MþH]þ; found:
458.2436.
0.12 mmol), and Et3N (25 mL, 0.18 mmol) in MeCN (3 mL) gave title
compound 24 (18 mg, 60%) as a white solid; Rf ¼ 0.35 (n-hexane/
4.1.2.19. N-(3,5-Dichlorophenyl)-2-(2-(2,5-dimethoxybenzyl)-1H-
benzo[d]imidazol-1-yl)acetamide (28). Following the general pro-
cedure for compounds 10e32, 2-(2-(2,5-dimethoxybenzyl)-1H-
benzo[d]imidazol-1-yl)acetic acid 9f (20 mg, 0.06 mmol), 3,5-
dichloroaniline (15 mg, 0.20 mmol), HATU (46 mg, 0.12 mmol),
EtOAc: 1/2); mp: 226.3e229.1 ꢀC; HPLC purity: 16.48 min, 95.0%; 1H
NMR (300 MHz, MeOD) d 7.66 (s, 1H), 7.44e7.52 (m, 3H), 7.25e7.31
(m, 3H), 7.13 (m, 2H), 7.02 (s, 1H), 5.15 (s, 2H), 4.39 (s, 2H), 2.87
(septet, J ¼ 7.2 Hz, 1H), 2.15 (s, 3H), 1.21 (d, J ¼ 4.2 Hz, 6H); 13C NMR
(75 MHz, MeOD with 2 drops of DMSO)
d
165.53, 147.04, 141.78,
and Et3N (17 mL, 0.12 mmol) in MeCN (3 mL) gave the title com-
137.32, 135.87, 134.05, 131.93, 130.97, 130.65 (2C), 130.41 (2C),
129.45, 129.03, 123.99, 123.12, 122.91, 122.48, 118.49, 110.20, 33.56,
32.14, 31.24, 23.43 (2C), 16.83; HRMS (ESIþ): m/z: calcd for
pound 28 (19 mg, 44%) as a white solid; Rf ¼ 0.50 (n-hexane/EtOAc:
1/1); mp: 223.9e225.1 ꢀC; HPLC purity: 10.11 min, 96.5%; 1H NMR
(300 MHz, CDCl3)
d 7.94 (s, 1H), 7.69e7.70 (m, 1H), 7.27e7.29 (m,
C
26H26Cl2N3O 466.1375 [MþH]þ; found: 466.1377.
2H), 7.22 (s, 2H), 7.02 (s, 1H), 6.80 (m, 1H), 6.70 (d, J ¼ 9.0 Hz, 1H),
6.58e6.61 (m, 1H), 4.92 (s, 2H), 4.24 (s, 2H), 3.76 (s, 3H), 3.60 (s,
4.1.2.16. 2-(2-(2,6-Dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)eN-(5-
isopropyl-2-methylphenyl)acetamide (25). Following the general
procedure for compounds 10e32, 2-(2-(2,6-dichlorobenzyl)-1H-
benzo[d]imidazol-1-yl)acetic acid 9g (20 mg, 0.06 mmol), 5-iso-
propyl-2-methylaniline (21 mg, 0.10 mmol), HATU (46 mg,
3H); 13C NMR (75 MHz, CDCl3)
d 165.05, 154.38, 153.82, 150.35,
138.83, 135.20, 134.84 (2C), 124.60, 124.49, 124.44, 123.56, 123.16,
119.32, 118.26 (2C), 116.58, 112.76, 111.98, 109.31, 56.17, 55.47, 47.59,
27.24; HRMS (ESIþ): m/z: calcd for C24H22Cl2N3O3 470.1032
[MþH]þ; found: 470.1029.
0.12 mmol), and Et3N (17 mL, 0.12 mmol) in MeCN (3 mL) gave the
title compound 25 (33 mg, 95%) as a white solid; Rf ¼ 0.40 (n-
4.1.2.20. 2-(5-Chloro-2-(2,5-dichlorophenyl)-1H-benzo[d]imidazol-
1-yl)eN-(5-iso-propyl-2-methyl phenyl)acetamide (29) and 2-(6-
chloro-2-(2,5-dichlorophenyl)-1H-benzo[d]imidazol-1-yl)eN-(5-iso-
propyl-2-methyl phenyl)acetamide (30). Following the general
procedure for compounds 10e32, an 1:1 mixture of 2-(5-chloro-2-
(2,5-dichlorophenyl)-1H-benzo[d]imidazol-1-yl)acetic acid and
methyl 2-(6-chloro-2-(2,5-dichlorophenyl)-1H-benzo[d]imidazol-
1-yl)acetic acid 9h (40 mg, 0.11 mmol), 5-iso-propyl-2-
methylaniline (35 mg, 0.17 mmol), HATU (84 mg, 0.22 mmol), and
hexane/EtOAc: 1/1); mp: 250.5e253.6 ꢀC; HPLC purity: 17.59 min,
95.2%; 1H NMR (300 MHz, DMSO-d6)
d 9.85 (br s, 1H), 7.48e7.58 (m,
4H), 7.37e7.42 (m, 1H), 7.32 (s, 1H), 7.20e7.25 (m, 1H), 7.12e7.16 (m,
2H), 6.99 (d, J ¼ 7.8 Hz, 1H), 5.32 (s, 2H), 4.54 (s, 2H), 2.82 (septet,
J ¼ 7.2 Hz, 1H), 2.22 (s, 3H), 1.15 (d, J ¼ 6.9 Hz, 6H); 13C NMR
(75 MHz, DMSO-d6) d 165.92, 152.29, 146.77, 142.52, 136.33, 136.01,
135.97 (2C), 133.65, 130.79, 129.91, 129.48, 128.80 (2C), 124.04,
123.15, 122.37, 121.87, 119.06, 110.19, 46.43, 33.43, 30.02, 24.32 (2C),
17.95; HRMS (ESIþ): m/z: calcd for C26H26Cl2N3O 466.1375 [MþH]þ;
found: 466.1361.
Et3N (46 mL, 0.33 mmol) in MeCN (4 mL) gave the mixture of title
compounds 29 and 30 (1:1). Two title compounds were separated
by column chromatography on SiO2 (diethyl ether/CH2Cl2: 1/40)
and confirmed by 2D NOE spectroscopy. Compound 29; white solid
(20 mg, 45%); mp: 213.2e214.2 ꢀC; HPLC purity: 16.35 min, 95.2%;
4.1.2.17. N-(3,5-Di-tert-butylphenyl)-2-(2-(2,5-dimethoxybenzyl)-
1H-benzo[d]imidazol-1-yl)acetamide (26). Following the general
procedure for compounds 10e32, 2-(2-(2,5-dimethoxybenzyl)-1H-
benzo[d]imidazol-1-yl)acetic acid 9f (20 mg, 0.06 mmol), 3,5-di-
tert-butylaniline (16 mg, 0.08 mmol), HATU (46 mg, 0.12 mmol),
1H NMR (300 MHz, MeOD)
d
7.76 (d, J ¼ 1.8 Hz, 1H), 7.67 (m, 3H),
7.64 (s, 1H), 7.42 (dd, J ¼ 8.7 Hz, 2.1 Hz, 1H), 7.09e7.13 (m, 2H), 7.01
(dd, J ¼ 7.8 Hz, 1.8 Hz, 1H), 5.09 (s, 2H), 2.83 (septet, J ¼ 7.2 Hz, 1H),
2.03 (s, 3H), 1.20 (d, J ¼ 6.9 Hz, 6H); 13C NMR (75 MHz, DMSO-d6)
and Et3N (17 mL, 0.12 mmol) in MeCN (3 mL) gave the title com-
pound 26 (27 mg, 86%) as a white solid; Rf ¼ 0.60 (n-hexane/EtOAc:
d 165.36, 162.34, 151.02, 146.69, 141.54, 137.01, 135.68, 132.55 (2C),
1/1); mp: 194.4e197.2 ꢀC; HPLC purity: 10.39 min, 95.1%; 1H NMR
132.40, 132.01, 131.20, 130.69, 129.65, 128.12, 124.13, 123.32, 123.05,
121.26, 111.74, 47.12, 33.38, 24.29 (2C), 17.67; HRMS (ESIþ): m/z:
calcd for C25H23Cl3N3O 486.0901 [MþH]þ; found: 486.0898. Com-
pound 30; white solid (20 mg, 45%); mp: 211.9e213.3 ꢀC; HPLC
(300 MHz, MeOD)
d 7.61 (m, 1H), 7.36e7.42 (m, 1H), 7.37 (s, 2H),
7.20e7.26 (m, 3H), 6.84 (d, J ¼ 8.4 Hz, 1H), 6.71e6.75 (m, 2H), 5.02
(s, 2H), 4.29 (s, 2H), 3.76 (s, 3H), 3.61 (s, 3H), 1.29 (s, 18H); 13C NMR
(75 MHz, MeOD)
d
165.29, 154.52, 153.81, 151.29 (2C), 151.06, 141.26,
purity: 16.67 min, 95.1%; 1H NMR (300 MHz, MeOD)
d 7.82 (br s,
137.28, 135.70, 124.69, 122.60, 122.15, 118.13, 117.76, 115.89, 114.32
(2C), 112.90, 111.46, 109.54, 55.15, 54.63, 45.95, 34.38 (2C), 30.43
1H), 7.74 (d, J ¼ 4.5 Hz, 1H), 7.71 (m, 3H), 7.39 (d, J ¼ 7.5 Hz, 1H), 7.12
(d, J ¼ 7.8 Hz,1H), 7.09 (s,1H), 7.02 (dd, J ¼ 7.8 Hz,1.5 Hz,1H), 5.09 (s,

1186
T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
2H), 2.83 (septet, J ¼ 7.2 Hz, 1H), 2.11 (s, 3H), 1.21 (d, J ¼ 6.9 Hz, 6H);
13C NMR (75 MHz, DMSO-d6)
165.31,151.43,146.68, 143.58,135.67,
135.06, 132.41 (2C), 132.03, 131.19, 130.69, 129.64, 127.18, 124.11,
123.71, 123.30, 119.36, 113.05, 47.12, 33.38, 24.29 (2C),17.67; HRMS
(ESIþ): m/z: calcd for C25H23Cl3N3O 486.0901 [MþH]þ; found:
486.0898.
48.15, 46.77, 40.89, 30.30, 28.30, 23.00, 14.16; HRMS (ESIþ): m/z:
calcd for C29H31Cl2N4O 521.1797 [MþH]þ; found: 521.1795.
d
4.1.3.2. 4-((2-(2,6-Dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)
methyl)eN-(1-methylpiperidin-4-yl)benzamide (37). Following the
general
dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)methyl)benzoic
35 (50 mg, 0.12 mmol), oxalyl chloride (20 L, 0.24 mmol), 1-
methylpiperidin-4-amine (15 L, 0.13 mmol), and DIPEA (43 L,
procedure
for compounds
36e44,
4-((2-(2,6-
acid
4.1.2.21. 2-(5-Chloro-2-(2,5-dichlorophenyl)-1H-benzo[d]imidazol-
1-yl)eN-(3,5-dichlorophenyl)acetamide (31) and 2-(6-chloro-2-(2,5-
dichlorophenyl)-1H-benzo[d]imidazol-1-yl)eN-(3,5-dichlorophenyl)
acetamide (32). Following the general procedure for compounds
10e32, an 1:1 mixture of 2-(5-chloro-2-(2,5-dichlorophenyl)-1H-
benzo[d]imidazol-1-yl)acetic acid and methyl 2-(6-chloro-2-(2,5-
dichlorophenyl)-1H-benzo[d]imidazol-1-yl)acetic acid 9h (47 mg,
0.13 mmol), 3,5-dichloroaniline (32 mg, 0.20 mmol), HATU (84 mg,
m
m
m
0.30 mmol) gave the title product 37 as a white solid (50 mg, 40%);
Rf ¼ 0.40 (CH2Cl2/MeOH: 10/1); mp: 175.2e177.4 ꢀC; HPLC purity:
10.09 min, 98.2%; 1H NMR (400 MHz, DMSO-d6)
d
8.23 (d, J ¼ 4.0 Hz,
1H),7.83 (d, J ¼ 8.4 Hz, 2H), 7.48e7.54 (m, 4H), 7.39 (t, J ¼ 8.4 Hz,
1H), 7.27 (d, J ¼ 8.0 Hz, 2H), 7.13e7.20 (m, 2H), 5.73 (s, 2H), 4.50 (s,
2H), 3.73 (quintet, J ¼ 3.6 Hz, 1H), 2.80 (d, J ¼ 11.6 Hz, 2H), 2.20 (s,
3H), 2.01 (t, J ¼ 11.2 Hz, 2H), 1.76 (d, J ¼ 10.8 Hz, 2H), 1.59 (qd,
0.22 mmol), and Et3N (46 mL, 0.33 mmol) in MeCN (4 mL) gave the
mixture of title compounds 31 and 32 (1:1). Two title compounds
were separated by column chromatography on SiO2 (diethyl ether/
CH2Cl2: 1/40) and confirmed by 2D NOE spectroscopy. Compound
31; white solid (20 mg, 66%); mp: 289.9e291.7 ꢀC; HPLC purity:
J ¼ 11.6 Hz, 2.4 Hz, 2H); 13C NMR (101 MHz, DMSO-d6)
d 165.72,
151.65, 142.65, 140.21, 135.89 (2C), 134.53, 133.47, 129.99, 128.82
(2C), 128.26 (2C), 126.95 (2C), 122.56, 122.03, 121.99, 119.25, 110.60,
54.80 (2C), 46.81, 46.42, 46.19, 31.69 (2C), 30.28; HRMS (ESIþ): m/z:
calcd for C28H29Cl2N4O 507.1640 [MþH]þ; found: 507.1642.
17.24 min, 98.7%; 1H NMR (400 MHz, DMSO-d6)
d
10.60 (br s, 1H),
7.87 (s, 1H), 7.69e7.78 (m, 3H), 7.66 (s, 1H), 7.53 (s, 2H), 7.32e7.36
(m, 2H), 5.04 (s, 2H); 13C NMR (101 MHz, DMSO-d6)
165.98,
d
4.1.3.3. 4-((2-(2,6-Dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)
150.85, 141.55, 140.66, 136.89, 134.68 (2C), 132.48 (2C), 132.41,
132.04, 130.94, 128.48, 123.58, 123.23, 121.30 (2C), 117.90, 111.82,
110.12, 47.61; HRMS (ESIþ): m/z: calcd for C21H13Cl5N3O 497.9496
[MþH]þ; found: 497.9498. Compound 32; white solid (20 mg, 66%);
mp: 288.6e290.6 ꢀC; HPLC purity: 17.30 min, 95.5%; 1H NMR
methyl)eN-(3-(dimethylamino)propyl)benzamide
(38).
Following the general procedure for compounds 36e44, 4-((2-(2,6-
dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)methyl)benzoic acid 35
(50 mg, 0.12 mmol), oxalyl chloride (20
dimethylpropane-1,3-diamine (16 L, 0.13 mmol), and DIPEA
m
L, 0.24 mmol), N1,N1-
m
(300 MHz, CDCl3)
d
7.74 (m, 2H), 7.65 (m, 3H), 7.51 (d, J ¼ 1.8 Hz,
(43 mL, 0.30 mmol) gave the title product 38 as a white solid (60 mg,
2H), 7.40 (d, J ¼ 10.5 Hz, 1H), 7.19 (m, 1H), 5.01 (s, 2H); 13C NMR
48%); Rf ¼ 0.20 (CH2Cl2/MeOH: 10/1); mp: 235.6e237.5 ꢀC; HPLC
(101 MHz, DMSO-d6)
d
165.99, 151.40, 143.59, 140.93, 134.89, 134.68
purity: 6.37 min, 98.9%; 1H NMR (400 MHz, MeOD)
d 7.84 (d,
(2C), 132.48 (2C), 132.40, 132.05, 128.23, 127.32, 123.86, 123.56,
J ¼ 8.0 Hz, 2H), 7.58 (dd, J ¼ 6.4 Hz, 2.8 Hz, 1H), 7.44e7.46 (m, 3H),
119.40, 117.91 (2C), 113.16, 111.90, 47.69; HRMS (ESIþ): m/z: calcd for
7.25e7.35 (m, 5H), 5.74 (s, 2H), 4.58 (s, 2H), 3.49 (t, J ¼ 6.4 Hz, 2H),
C
21H13Cl5N3O 497.9496 [MþH]þ; found: 497.9491.
3.18 (t, J ¼ 7.6 Hz, 2H), 2.92 (s, 6H), 2.03 (quintet, J ¼ 7.2 Hz, 2H); 13
C
NMR (75 MHz, MeOD)
d 168.38, 151.63, 141.66, 140.00, 135.99 (2C),
4.1.3. General procedure for the synthesis of compound 36e44
To a stirred solution of 4-((2-(2,6-dichlorobenzyl)-1H-benzo[d]
imidazol-1-yl)methyl)benzoic acid 35 (1.0 equiv) in CH2Cl2 was
added oxalyl chloride (2.0 equiv) dropwise at room temperature.
After catalytic amount of DMF (0.2 equiv) was added, the reaction
mixture was stirred for 2 h at room temperature. And then, the
reaction mixture was concentrated in vacuo. To the resulting res-
idue was added a mixture of the desired substituted amine
(1.2 equiv) and DIPEA (2.5 equiv) in THF. The reaction mixture was
stirred at room temperature for 16 h, concentrated in vacuo, and
extracted with CH2Cl2 (3 ꢃ 30 mL). The organic layer was dried over
anhydrous Na2SO4, filtered, concentrated, and purified by column
chromatography on SiO2 (CH2Cl2/MeOH: 10/1).
135.28, 133.54, 132.05, 129.22, 128.20 (2C), 127.56 (2C), 126.35 (2C),
122.72, 122.12, 118.14, 109.63, 56.09, 46.07, 43.09 (2C), 37.04, 29.60,
25.78; HRMS (ESIþ): m/z: calcd for C27H29Cl2N4O 495.1713 [MþH]þ;
found: 495.1694.
4.1.3.4. 4-((2-(2,6-Dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)
methyl)eN-(3-(pyrrolidin-1-yl)propyl)benzamide
(39).
Following the general procedure for compounds 36e44, 4-((2-(2,6-
dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)methyl)benzoic acid 35
(50 mg, 0.12 mmol), oxalyl chloride (20
mL, 0.24 mmol), 3-(pyrro-
lidin-1-yl)propan-1-amine (35 L, 0.27 mmol) and DIPEA (43
m
mL,
0.30 mmol) gave the title product 39 as a white solid (47 mg, 74%);
Rf ¼ 0.30 (CH2Cl2/MeOH: 10/1); mp: 218.0e220.2 ꢀC; HPLC purity:
7.48 min, 99.7%; 1H NMR (300 MHz, CDCl3 with MeOD)
d 7.80 (d,
4.1.3.1. 4-((2-(2,6-Dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)
J ¼ 8.4 Hz, 2H), 7.68e7.69 (m, 1H), 7.31e7.38 (m, 2H overlapped
with CDCl3 peak), 7.24e7.27 (m, 4H), 7.17e7.22 (m, 3H), 5.55 (s, 2H),
4.50 (s, 2H), 3.51 (t, J ¼ 6.3 Hz, 2H), 3.38e3.40 (m, 4H), 3.17 (t,
J ¼ 7.2 Hz, 2H), 2.03e2.11 (m, 6H); 13C NMR (75 MHz, DMSO-d6)
methyl)eN-((1-ethylpyrrolidin-2-yl)methyl)benzamide
(36).
Following the general procedure for compounds 36e44, 4-((2-(2,6-
dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)methyl)benzoic acid 35
(100 mg, 0.24 mmol), oxalyl chloride (42
mL, 0.48 mmol), (1-
d 166.55, 151.64, 142.66, 140.44, 135.88 (2C), 134.19, 133.14, 133.45,
ethylpyrrolidin-2-yl)methanamine (31 L, 0.26 mmol), and DIPEA
m
129.97, 128.80 (2C), 128.16 (2C), 127.06 (2C), 122.57, 122.00, 119.27,
110.57, 53.71 (2C), 52.57, 46.40, 37.00, 30.26, 26.44, 23.08 (2C);
HRMS (ESIþ): m/z: calcd for C29H31Cl2N4O 521.1869 [MþH]þ; found:
521.1865.
(86
mL, 0.60 mmol) gave the title product 36 as a white solid
(100 mg, 79%); Rf ¼ 0.40 (CH2Cl2/MeOH: 10/1); mp: 160.1e162.8 ꢀC;
HPLC purity: 7.87 min, 98.7%; 1H NMR (300 MHz, CDCl3)
d 7.76 (dd,
J ¼ 8.1 Hz, 4.5 Hz, 3H), 7.30e7.31 (m, 2H), 7.23e7.25 (m, 3H),
7.11e7.17 (m, 3H), 7.04 (br s,1H), 5.50 (s, 2H), 4.48 (s, 2H), 3.65e3.69
(m, 1H), 3.27e3.31 (m, 2H), 2.70e2.88 (m, 1H), 2.31 (s, 1H),
2.20e2.31 (m, 2H), 1.89e1.93 (m, 1H), 1.64e1.77 (m, 3H), 1.13 (t,
d 167.00, 151.14, 142.78,
139.02, 136.11 (2C), 135.57, 134.39, 132.38, 129.02, 128.40 (2C),
127.65 (2C), 126.26 (2C), 122.66, 122.12, 119.97, 109.19, 62.33, 53.58,
4.1.3.5. 4-((2-(2,6-Dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)
methyl)eN-(3-(4-methylpiperazin-1-yl)propyl)benzamide
(40).
Following the general procedure for compounds 36e44, 4-((2-(2,6-
dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)methyl)benzoic acid 35
J ¼ 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3)
(50 mg, 0.12 mmol), oxalyl chloride (20
methylpiperazin-1-yl)propan-1-amine (30
m
m
L, 0.24 mmol), 3-(4-
L, 0.27 mmol), and

T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
1187
DIPEA (43
m
L, 0.30 mmol) gave the title product 40 as a white solid
135.52, 134.46, 132.31, 129.07, 128.40 (2C), 127.72 (2C), 126.22 (2C),
122.72, 122.18, 119.88, 109.22, 67.00 (2C), 58.47, 53.81 (2C), 46.78,
40.42, 30.27, 24.40; HRMS (ESIþ): m/z: calcd for C29H31Cl2N4O2
537.1818 [MþH]þ; found: 537.1816.
(50 mg, 75%); Rf ¼ 0.25 (CH2Cl2/MeOH: 10/1); mp: 209.8e212.1 ꢀC;
HPLC purity: 6.99 min, 96.3%; 1H NMR (300 MHz, DMSO-d6)
d 8.49
(br t, J ¼ 4.8 Hz, 1H), 7.80 (d, J ¼ 8.1 Hz, 2H), 7.50 (d, J ¼ 8.1 Hz, 4H),
7.35e7.37 (m,1H), 7.24e7.28 (m, 2H), 7.14e7.16 (m, 2H), 5.72 (s, 2H),
4.48 (s, 2H), 3.39 (m, 2H), 3.10e3.21 (m, 8H overlapped with H2O
peak), 2.37e2.41 (m, 2H overlapped with DMSO-d6 peak), 2.29 (s,
4.1.3.9. 4-((2-(2,6-Dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)
methyl)-N,N-dimethylbenzamide (44). Following the general pro-
cedure for compounds 36e44, 4-((2-(2,6-dichlorobenzyl)-1H-
benzo[d]imidazol-1-yl)methyl)benzoic acid 35 (50 mg, 0.12 mmol),
3H), 1.66 (t, J ¼ 6.9 Hz, 2H); 13C NMR (101 MHz, DMSO-d6)
d 166.26,
151.65, 142.61, 140.24, 135.87 (2C), 134.39, 133.42, 129.95, 128.94,
128.78 (2C), 128.09 (2C), 127.02 (2C), 122.59, 122.00, 119.23, 110.56,
55.55, 54.12 (2C), 51.86 (2C), 46.36, 44.88, 38.11, 30.24, 26.30; HRMS
(ESIþ): m/z: calcd for C30H34Cl2N5O 550.2135 [MþH]þ; found:
550.2132.
oxalyl chloride (20
mL, 0.24 mmol), dimethylamine hydrochloride
(24 mg, 0.30 mmol), and DIPEA (43
mL, 0.30 mmol) gave the title
product 44 as a white solid (23 mg, 42%); Rf ¼ 0.30 (CH2Cl2/MeOH:
10/1); mp: 225.7e228.2 ꢀC; HPLC purity: 9.91 min, 99.7%; 1H NMR
(400 MHz, MeOD)
d
7.87 (dd, J ¼ 6.4 Hz, 3.2 Hz, 1H), 7.75e7.77 (m,
4.1.3.6. 4-((2-(2,6-Dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)
methyl)eN-(3-(piperidin-1-yl)propyl)benzamide (41). Following the
1H), 7.64e7.67 (m, 2H), 7.54 (d, J ¼ 8.4 Hz, 2H), 7.41e7.48 (m, 3H),
7.34 (d, J ¼ 8.4 Hz, 2H), 5.95 (s, 2H), 5.03 (s, 2H), 3.10 (s, 3H), 2.98 (s,
general
procedure
for compounds
36e44,
4-((2-(2,6-
acid
L, 0.24 mmol), 3-(4-
mL, 0.27 mmol), and
3H); 13C NMR (101 MHz, Acetone-d6)
d 169.75, 151.35, 137.42,
dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)methyl)benzoic
35 (50 mg, 0.12 mmol), oxalyl chloride (20
methylpiperazin-1-yl)propan-1-amine (34
136.30, 134.51, 132.87, 131.67, 130.46, 129.24 (2C), 127.97, 127.90
(2C), 127.09 (2C), 127.06 (2C), 126.93, 114.38, 113.08, 48.36 (2C),
38.59, 34.28; HRMS (ESIþ): m/z: calcd for C24H22Cl2N3O 438.1134
[MþH]þ; found: 438.1133.
m
DIPEA (43 mL, 0.30 mmol) gave the title product 41 as a white solid
(40 mg, 61%); Rf ¼ 0.35 (CH2Cl2/MeOH: 10/1); mp: 149.1e153.9 ꢀC;
HPLC purity: 11.92 min, 99.8%; 1H NMR (300 MHz, MeOD)
d
7.82
4.1.4. General procedure for the synthesis of compound 51e53
(dd, J ¼ 8.4 Hz, 1.8 Hz, 2H), 7.56e7.59 (m, 1H), 7.42e7.45 (m, 3H),
7.23e7.32 (m, 5H), 5.72 (s, 2H), 4.56 (s, 2H), 3.48 (t, J ¼ 6.6 Hz, 2H),
3.15 (m, 2H), 3.07 (t, J ¼ 7.8 Hz, 4H), 3.06 (q, J ¼ 7.8 Hz, 2H), 1.84 (t,
J ¼ 7.8 Hz, 4H) 1.31 (m, 2H); 13C NMR (101 MHz, Acetone-d6)
To
a
stirred
solution
of
4-((2-(2-(5-isopropyl-2-
methylphenylamino)-2-oxoethyl)-1H-benzo[d]imidazol-1-yl)
methyl)benzoic acid 50 (1.0 equiv), Et3N (1.2 equiv), and benzo-
triazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluoro-
phosphate (BOP; 2.0 equiv) in DMF was added the desired
substituted amine (1.2 equiv). After stirring for 12e16 h at room
temperature, the reaction mixture was concentrated in vacuo, and
extracted with EtOAc (3 ꢃ 30 mL). The organic layer was dried over
anhydrous MgSO4, filtered, concentrated, and purified by column
chromatography on SiO2 (CH2Cl2/MeOH: 10/1).
d
168.70, 151.22, 142.98, 141.04, 136.00 (2C), 135.83, 133.38, 132.85,
129.22, 128.22 (2C), 127.96 (2C), 126.75 (2C), 122.18, 121.62, 119.13,
109.70, 54.01, 53.14 (2C), 46.20, 36.11, 29.90, 24.60, 23.50 (2C),
21.76; HRMS (ESIþ): m/z: calcd for C30H33Cl2N4O 535.1953 [MþH]þ;
found: 535.1954.
4.1.3.7. 4-((2-(2,6-Dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)
methyl)eN-(3-(piperidin-1-yl)ethyl)benzamide (42). Following the
4.1.4.1. N-((1-Ethylpyrrolidin-2-yl)methyl)-4-((2-(2-(5-isopropyl-2-
methylphenylamino)-2-oxoethyl)-1H-benzo[d]imidazol-1-yl)methyl)
benzamide (51). Following the general procedure for compounds
51e53, 4-((2-(2-(5-isopropyl-2-methylphenylamino)-2-oxoethyl)-
1H-benzo[d]imidazol-1-yl)methyl)benzoic acid 50 (40 mg,
general
dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)methyl)benzoic
35 (50 mg, 0.12 mmol), oxalyl chloride (20 L, 0.24 mmol), 3-(4-
methylpiperazin-1-yl)ethan-1-amine (38 L, 0.27 mmol), and
procedure
for compounds
36e44,
4-((2-(2,6-
acid
m
m
DIPEA (43
m
L, 0.30 mmol) gave the title product 42 as a white solid
0.09 mmol), BOP (80 mg, 0.18 mmol), Et3N (25
mL, 0.18 mmol), and
(37 mg, 54%); Rf ¼ 0.30 (CH2Cl2/MeOH: 10/1); mp: 240.4e242.1 ꢀC;
(1-ethylpyrrolidin-2-yl)methanamine (14 L, 0.10 mmol) in DMF
m
HPLC purity: 11.92 min, 99.8%; 1H NMR (400 MHz, DMSO-d6)
d
8.68
(4 mL) gave the title product 51 as a white solid (40 mg, 80%)
Rf ¼ 0.15 (CH2Cl2/MeOH: 10/1); mp: 174.0e175.9 ꢀC; HPLC purity:
(br t, J ¼ 5.6 Hz, 1H), 7.85 (d, J ¼ 8.0 Hz, 2H), 7.49e7.54 (m, 4H),
7.38e7.42 (m, 1H), 7.31 (d, J ¼ 8.0 Hz, 2H), 7.13e7.21 (m, 2H), 5.75 (s,
2H), 4.50 (s, 2H), 3.60 (q, J ¼ 5.6 Hz, 2H), 3.52 (m, 2H), 3.21 (m, 2H),
2.93 (m, 2H), 1.81 (m, 2H), 1.67 (m, 3H), 1.41 (m, 2H); 13C NMR
16.17 min, 95.3%; 1H NMR (300 MHz, CDCl3)
d 10.53 (br s, 1H), 8.07
(s, 1H), 7.73e7.79 (m, 4H), 7.27e7.31 (m, 3H), 7.06e7.09 (m, 3H),
6.91 (d, J ¼ 7.5 Hz, 1H), 5.47 (s, 2H), 3.96 (s, 2H), 3.67e3.72 (m, 4H),
3.32e3.35 (m, 1H), 3.02e3.07 (m, 2H), 2.81 (m, 1H), 2.29 (s, 3H),
2.19e2.22 (m, 2H), 2.01e2.07 (m, 2H), 1.90 (s, 2H), 1.20 (s, 3H), 1.18
(101 MHz, DMSO-d6) d 167.03, 153.02, 151.64, 142.65, 140.82, 135.86
(2C), 133.42, 130.01, 128.82 (2C), 128.28 (2C), 127.14 (2C), 122.57,
122.14, 122.01, 119.29, 110.56, 52.79 (2C), 46.40 (2C), 30.42, 29.94,
23.00 (2C), 21.85; HRMS (ESIþ): m/z: calcd for C29H31Cl2N4O
521.1869 [MþH]þ; found: 521.1866.
(s, 3H); 13C NMR (75 MHz, CDCl3)
d 170.90, 164.78, 162.33, 149.25,
147.40, 141.71, 140.42, 135.77, 134.97, 131.35, 130.45, 128.46, 127.07,
126.71, 123.60, 123.20, 123.02, 121.05, 119.26, 109.94, 69.03, 53.59,
50.76, 46.64, 41.62, 35.24, 33.77, 26.38, 23.99 (2C), 23.57, 17.74,
11.34; HRMS (ESIþ): m/z: calcd for C34H42N5O2 552.3333 [MþH]þ;
found: 552.3333.
4.1.3.8. 4-((2-(2,6-Dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)
methyl)eN-(3-morpholinopropyl)benzamide (43). Following the
general
dichlorobenzyl)-1H-benzo[d]imidazol-1-yl)methyl)benzoic
35 (50 mg, 0.12 mmol), oxalyl chloride (20 L, 0.24 mmol), 3-
morpholinopropan-1-amine (39 L, 0.27 mmol), and DIPEA
procedure
for compounds
36e44,
4-((2-(2,6-
acid
4.1.4.2. N-(3-(Dimethylamino)propyl)-4-((2-(2-(5-isopropyl-2-
methylphenylamino)-2-oxoethyl)-1H-benzo[d]imidazol-1-yl)methyl)
benzamide (52). Following the general procedure for compounds
51e53, 4-((2-(2-(5-isopropyl-2-methylphenylamino)-2-oxoethyl)-
1H-benzo[d]imidazol-1-yl)methyl)benzoic acid 50 (40 mg,
m
m
(43 mL, 0.30 mmol) gave the title product 43 as a white solid (40 mg,
65%); Rf ¼ 0.30 (CH2Cl2/MeOH: 10/1); mp: 190.3e193.2 ꢀC; HPLC
purity: 8.29 min, 99.8%; 1H NMR (300 MHz, CDCl3)
d
8.09 (t,
0.09 mmol), BOP (80 mg, 0.18 mmol), Et3N (25
mL, 0.18 mmol), and
J ¼ 4.5 Hz, 1H), 7.72e7.78 (m, 3H), 7.20e7.31 (m, 5H), 7.12e7.18 (m,
3H), 5.51 (s, 2H), 4.47 (s, 2H), 3.68 (t, J ¼ 8.4 Hz, 4H), 3.55 (t,
J ¼ 8.4 Hz, 2H), 2.20e2.40 (m, 6H), 1.80 (q, J ¼ 8.4 Hz, 2H); 13C NMR
N1,N1-dimethylpropane-1,3-diamine (10
mL, 0.10 mmol) in DMF
(4 mL) gave the title product 52 as a white solid (15 mg, 50%)
Rf ¼ 0.20 (CH2Cl2/MeOH: 10/1); mp: 171.9e173.6 ꢀC; HPLC purity:
(75 MHz, CDCl3)
d
166.56, 162.57, 151.13, 142.69, 138.95, 136.07,
7.98 min, 99.1%; 1H NMR (300 MHz, DMSO-d6)
d 9.92 (s, 1H), 8.48 (t,

1188
T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
J ¼ 5.7 Hz, 1H), 7.77 (d, J ¼ 8.4 Hz, 2H), 7.63 (dd, J ¼ 7.5 Hz, 1.8 Hz,
1H), 7.36 (dd, J ¼ 6.0 Hz,1.5 Hz,1H), 7.21e7.26 (m, 3H), 7.15e7.18 (m,
2H), 7.12 (m, 1H), 6.95e6.97 (m, 1H), 5.64 (s, 2H), 4.14 (s, 2H),
3.23e3.28 (m, 2H), 2.75e2.82 (septet, J ¼ 6.9 Hz, 1H), 2.36 (m, 2H),
2.11e2.23 (m, 9H), 1.62e1.67 (quintet, J ¼ 6.9 Hz, 2H), 1.14 (d,
cells were treated with test compounds in a concentration of 5
per well and incubated at 37 ꢀC for 10 min. Then, for each test
compound, three wells were treated with 5 M of Ab1-42, and other
two wells were treated with 5 M of DMSO as vehicle control. The
fluorescence intensity was measured at every 1 h for 3 h by using a
microplate reader (Flexstation® 3, Molecular Devices, USA) reader
at ex/em 485 nm/535 nm (green) and ex/em 560 nm/595 nm (red).
The ratio of green to red fluorescence was calculated and normal-
ized by taking the percent changes using vehicle control as 100%.
mM
m
m
J ¼ 6.9 Hz, 6H); 13C NMR (75 MHz, DMSO-d6)
d 166.50, 166.26,
162.34, 150.52, 146.75, 142.37, 140.14, 136.12, 135.39, 134.08, 130.69,
129.26, 127.91 (2C), 127.10 (2C), 123.04, 122.89, 122.47, 119.12, 56.88,
47.02, 44.95 (2C), 38.00, 33.38, 26.81, 24.26 (2C), 17.75, 13.11; HRMS
(ESIþ): m/z: calcd for C32H40N5O2 526.3104 [MþH]þ; found:
526.3102.
4.2.4. Luciferase-based ATP assay
To a clear 96-well plate, HT-22 cells were seeded in a number of
7000 per well one day prior to the assay. After removing the me-
4.1.4.3. 4-((2-(2-(5-Isopropyl-2-methylphenylamino)-2-oxoethyl)-
1H-benzo[d]imidazol-1-yl)methyl)eN-(3-(piperidin-1-yl)propyl)ben-
zamide (53). Following the general procedure for compounds
51e53, 4-((2-(2-(5-isopropyl-2-methylphenylamino)-2-oxoethyl)-
1H-benzo[d]imidazol-1-yl)methyl)benzoic acid 50 (40 mg,
dium from the plate, four wells were treated with 25
each test compound (5 M). And the cells were incubated for
10 min at 37 ꢀC. For each test compound, two wells were treated
with 25 L of A solution (5 M, American peptide, 1e42) and the
other two wells were treated with 25 L of DMSO solution (5 M) as
mL/well of
m
m
b
m
0.09 mmol), BOP (80 mg, 0.18 mmol), Et3N (25
mL, 0.18 mmol), and
m
m
3-(piperidin-1-yl)propan-1-amine (14 L, 0.10 mmol) in DMF
m
vehicle controls. Then, the cells were incubated for 7 h at 37 ꢀC.
After the incubation, the test compound-treated wells were
washed twice with PBS, and lysed with 1% Triron-X 100 in TSBT
buffer solution. To measure the protein concentrations of each well,
BCA protein determination kit (Thermo scientific) was used. And
the same amount of cell lysate was taken from each well and plated
into a white 96-well plate (NUNC). And the ATP levels in each
sample were measured by ATP determination kit (Molecular
Probes, USA) containing D-luciferin and luciferase. The % inhibition
value was measured by luminescence from detecting ATP levels
from a microplate reader (Flexstation® 3, Molecular Devices, USA),
and the result was normalized by taking the vehicle control as 0%.
Cell viability (%) in each test compound was also determined by
detecting the ATP levels of each well containing only each test
(4 mL) gave the title product 53 as a white solid (48 mg, 90%)
Rf ¼ 0.20 (CH2Cl2/MeOH: 10/1); mp: 181.7e182.5 ꢀC; HPLC purity:
13.10 min, 95.9%; 1H NMR (300 MHz, MeOD)
d
7.83 (d, J ¼ 8.1 Hz,
2H), 7.76 (d, J ¼ 8.1 Hz, 1H), 7.35e7.43 (m, 5H), 7.20 (s, 1H), 7.14 (d,
J ¼ 8.1 Hz, 1H), 7.02 (d, J ¼ 8.1 Hz, 1H), 5.79 (s, 2H), 4.48 (s, 2H),
3.45e3.52 (m, 4H), 3.13 (t, J ¼ 8.1 Hz, 2H), 2.85e2.89 (m, 2H), 2.20
(s, 3H),1.98e2.03 (m, 4H),1.76e1.81 (m, 2H),1.43 (m,1H),1.26e1.28
(m, 1H), 1.21 (s, 3H), 1.19 (s, 3H); 13C NMR (75 MHz, MeOD)
d 168.79,
149.34, 147.05, 139.40, 134.91, 134.15, 133.35, 130.16 (2C), 129.77,
127.69 (2C), 126.70 (2C), 124.29 (2C), 123.86, 123.23, 117.19, 111.02,
54.31, 52.92 (2C), 48.16, 46.74 (overlapped with MeOD peaks),
36.31, 33.47, 24.14, 22.97 (3C), 22.94, 21.25, 16.24; HRMS (ESIþ): m/
z: calcd for C35H44N5O2 566.3417 [MþH]þ; found: 566.3415.
compound (5 mM) and the cells.
4.2. Cell-based assays
4.2.1. Cell culture
4.2.5. MTT assay
HT-22 (mouse hippocampal cells) cells were grown in Dulbec-
co's Modified Eagle's Medium (DMEM, GIBCO) supplemented with
10% (vol/vol) FBS and antibiotics (100 mg/mL penicillin/strepto-
mycin mix) in a humidified atmosphere at 37 ꢀC with 5% CO2.
To a clear 96-well plate, HT-22 cells were seeded in a number of
5000 per well as above described method for the ATP detection
assay. After incubating the cells for 24 h at 37 ꢀC, 10
m
L of MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide, Sigma-Aldrich) was directly added to each well. The
cells were incubated for 2 h at 37 ꢀC. And 135
L of MTT solubilizing
solution
4.2.2. Preparation of Ab1e42
m
A
b1e42 (American Peptide) was suspended in 1 mL of 1,1,1,3,3,3
solution (10% Triton-X 100 in isopropanol with 0.1 M HCl) was
added to each well followed by additional incubation of the cells for
2 h at 37 ꢀC. The % inhibition value was measured by the optical
density (OD) values of absorbance at 560 nm and normalized by
taking the vehicle control as 0%. Cell viability (%) was also deter-
mined by using OD values of each well containing only each test
hexafluoro-2-propanol (HFIP; SigmaeAldrich) using a gastight sy-
ringe. Peptide samples were vortexed to obtain a homogenous
solution, aliquoted into microcentrifuge tubes and lyophilized.
Protein amount for each batch was determined by BCA protein
assay. The dried peptide films were stored desiccated at ꢁ80 ꢀC. A
b
monomer solutions were prepared immediately before use to avoid
possible aggregation. For each biological experiment, peptide films
were resuspended to 5 mM in anhydrous dimethyl sulfoxide
(DMSO).
compound (5 mM) and the cells.
4.2.6. CM-H2DCFDA-fluorescent ROS assay
To a black 96-well plate with clear bottom, HT-22 cells were
4.2.3. JC-1 assay
seeded in a number of 10,000 per well and treated with 25
of each test compound (5 M) and 25 L/well of A solution (5
After incubating for 6 h at 37 ꢀC, compound-treated wells were
washed twice with HBSS. And 100 L of 1 M CM-H2DCFDA (Invi-
trogen, C6827) in HBSS solution was directly added to each well.
The cells were incubated for 30 min at 37 ꢀC and washed twice with
HBSS. The total fluorescent intensity from each well was measured
by using a high-content imaging system (Operetta, Perkin Elmer).
m
L/well
HT-22 (mouse hippocampal cells) cells were cultured in Dul-
becco's Modified Eagle's Medium (DMEM, GIBCO) supplemented
m
m
b
mM)
with 100
m
g/mL penicillin/streptomycin mixture and 10% (vol/vol)
m
m
fetal bovine serum (FBS) in a humidified atmosphere at 37 ꢀC with
5% CO2. The cells were seeded into a clear 96-well plate (FALCON) in
a number of 30,000 per well 24 h prior to the assay. JC-1 dye
(Stratagene, USA) was diluted with phenol red-free Opti-MEM
(GIBCO) medium to a concentration of 7.5
removed from the plate, and 100 L of JC-1 solution was added per
well. After incubation for 1 h and 15 min at 37 ꢀC, wells were
washed twice with 100 L of phosphate buffer solution (PBS). The
m
M. The medium was
The % inhibition of Ab-induced ROS production was determined by
calculating % ratio of the increased fluorescent intensity from the
test compound-treated wells to untreated wells in the presence of
the Ab solution.
m
m

T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
1189
4.2.7. Measurement of intracellular Ca2þ level
reached equilibrium by monitoring hERG tail currents. Whole-cell
recordings were analyzed using the Patchmaster/Fitmaster (HEKA
Elektronik, Lambrecht/Pfalz, Germany), IGOR Pro (WaveMetrics
Inc., Portland, OR, USA), and the GraphPad Prism 4.0 (GraphPad
Software, Inc., La Jolla, CA, USA) software.
To a black 96-well plate with clear bottom, HT-22 cells were
seeded in a number of 10,000 per well. And 25
lution (5 M), 25 L/well of each test compound, and 100
intracellular Ca2þ indicating dye (5
M), Fura-2 AM (Molecular
m
L/well of A
b so-
m
m
mL/well of
m
Probes), in phenol red-free medium was added. After incubating
the cells for 30 min at 37 ꢀC, each well was washed twice with
4.3. Pharmacokinetic study
100 mL/well of HBSS. And the plate was incubated for 30 min to
allow complete deesterification of intracellular AM esters. Then,
fluorescence was measured by a microplate reader (Flexstation® 3,
Molecular Devices, USA) at excitation and emission wavelengths
(ex/em) of 380 nm/340 nm. The intracellular Ca2þ level was
determined by calculating % ratio of the increased fluorescent in-
tensity from the test compound-treated wells to untreated wells in
4.3.1. Animals
Male SD rats (Nara Biotech, Korea), 8-week old, were used in the
experiments. Animals were housed under a 12-h light-dark cycle,
with food and water ad libitum. Rats were allowed to be accus-
tomed to the environment for at least 1 week prior to their usage in
the experiments. All animal handling was in accordance with
guidelines of the Animal Care and Use Committee in Korea Institute
of Science and Technology. The rats were studied after anesthesia
induced by intraperitoneal injection of isoflurane.
the presence of the Ab solution (control).
4.2.8. CYP inhibition assay
Inhibitions of CYP (CYP1A2, 2C9, 2D6, 2C19, and 3A4) activities
were measured using the Vivid® CYP450 screening kit (Invitrogen,
Madison, WI, USA) in a clear 96-well plate. Positive controls
4.3.2. Drug preparation and administration
Compounds were dissolved completely at 10 mg/2 mL of N-
Methyl-2-pyrrolidone, tween80, and D.W. (1:2:7 by volume) by
sonication for 5 min. This preparation was orally administered at
2 mL/kg with a blunt needle via the esophagus into the stomach.
Blood samples were collected from heart puncture by using a
syringe with heparinized needle at 30 m, 3 h (n ¼ 5) after drug
administration. The blood samples were centrifuged for 5 min at
10,000ꢃg, and the plasma samples were stored at ꢁ70 ꢀC until LC-
MS/MS analysis. After collection of blood samples, brain were
extracted (n ¼ 5, per sampling time). All extracted tissues were blot
dried, weighed and homogenized (Ultra-Turrax®, T25 basic, IKA
Labortechnic, Malasia) with 4 times its volume of distilled water.
Obtained tissue homogenates were kept at ꢁ70 ꢀC until LC-MS/MS
analysis.
including the
a-naphthoflavone (CYP1A2), sulfaphenazole
(CYP2C9), quinidine (CYP2D6), miconazol (CYP2C19), and ketoco-
nazole (CYP3A4) were prepared as 10 mM solution in MeCN. And
then, each sample (test compounds, positive inhibition control and
solvent control) and the Master Pre-Mix [CYP450 BACULOSOMES®
Reagent (recombinant human CYP450 isozyme and rabbit
NADPHP450 reductase) and Regeneration System (3.3 mM glucose-
6-phosphate and 0.3 U/ml glucose-6-phosphate dehydrogenase in
100 mM potassium phosphate, pH 8.0)] were added to each well.
After incubating the mixture for 5 min at 37 ꢀC, the Vivid® CYP
substrates and 0.1 mM NADPþ buffer was added to begin the
enzyme reaction. The remaining enzyme activity (% remaining ac-
tivity) was measured after 20 min by using a fluorescent plate
reader.
4.3.3. Sample preparation
4.2.9. hERG inhibition assay
To determine the drug concentration in the plasma, 50
plasma sample was mixed with 100 L carbamazepine (0.1
in acetonitrile, internal standard) and vortexed for 30 s. The
m
L of the
For automated patch-clamp NPC-16 Patchliner (Nanion Tech-
nologies, München, Germany) recordings, CHO-K1 Tet-On hERG
cells (IonGate Biosciences GmbH, Frankfurt, Germany) were plated
into the 100-mm culture dishes. Whole-cell currents were recorded
with the intracellular solution containing: 50 mM KCl, 60 mM kF,
10 mM NaCl, 2 mM MgCl2, 20 mM EGTA and 10 mM HEPES (pH 7.2),
and with the extracellular solution containing: 140 mM NaCl, 4 mM
KCl, 2 mM CaCl2,1 mM MgCl2, 5 mM Glucose and 10 HEPES (pH 7.4).
To assist stable seal formations, the seal enhancer containing:
80 mM NaCl, 3 mM KCl, 35 mM CaCl2, 10 mM MgCl2 and 10 mM
HEPES (pH 7.4) was used only at the seal formation step. Prior to the
whole-cell recordings, the external seal enhancing solution was
exchanged to the extracellular solution described above. hERG
channel currents were recorded using the parallel EPC-10 patch-
clamp amplifiers (HEKA Elektronik, Lambrecht/Pfalz, Germany),
and low-pass filtered (10 kHz) with a 4-pole Bessel filter. Cell sus-
pension and patch solutions were automatically added onto the
four recording wells in the microfabricated disposable chip (NPC-
16 Chip, Nanion Technologies, München, Germany). To obtain the
inhibitory constants, hERG tail currents were evoked by repolariz-
ing steps to ¡50 mV for 500 ms preceded by a 500-ms depolari-
zation potential of þ20 mV at a holding potential of ¡80 mV with a
20-s sweep interval. Whole-cell currents were acquired and digi-
tized at 5 kHz using Patchmaster (HEKA Elektronik, Lambrecht/
Pfalz, Germany). The extracellular solution was exchanged to the
extracellular solution containing each blocker via four pipette tips
m
mg/mL
mixture was centrifuged for 10 min at 10,000ꢃg and 100
mL su-
pernatant was analyzed by LC-MS/MS.
To determine the drug concentration in the brain, 100
brain sample was mixed with 200 L carbamazepine (0.1
acetonitrile, internal standard) and vortexed for 30 s. The mixture
mL of the
m
mg/mL in
was centrifuged for 10 min at 10,000ꢃg and 100
mL supernatant
was analyzed by LC-MS/MS.
4.3.4. LC-MS/MS analysis
The Agilent 1100Series (Agilent Technologies, Waldbronn, Ger-
many) was used. Mass spectrometer API 3200 (Applied Biosystems
Sciex, Rotterdam, The Netherlands) with electrospray-positive
ionization was used. The multiple reaction monitor was set at
274.3e185.0 m/z for each compound and 237.4e194.4 m/z for the
internal standard. The analytical column was Waters XTrerra® MS
C18 (3.5
consisted of (A) containing 0.1% formic acid and (B) 90% acetonitrile
containing 0.1% formic acid. 5 L aliquots were injected, and the
mm, 2.1 mm i.d. ꢃ 50 mm). The mobile phase of paclitaxel
m
flow rate was set at 0.35 mL/min. The initial composition was 10%
(B), programmed linearly to 90% (B) after 1 min, and held for 1 min.
The column was then re-equilibrated at 10% for 3 min.
4.4. In vivo studies
of NPC-16 Patchliner using a 4-fold volume of solution (40
a speed of 4 L/s, and the exchanged blocker solution was applied
for 100e200 s to the patch-clamped cells until blocker binding had
m
L) with
4.4.1. Animals
m
The acute AD mice model was prepared by administration of
A
b1e42 solution (10% DMSO/90% PBS) to male ICR mice (6 weeks

1190
T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
old, 30e33 g) via intracerebroventricular (ICV) injection (Ab1e42
dose: 500 pmol/mouse) as described previously [33]. The wild type
mice (B6C3F1) and double APP/PS1 transgenic mice (Tg AD mice
model; APPswe/PSEN1dE9) was purchased from Jackson Labora-
tory (Bar Harbor, Maine, USA). The Tg AD mice model was 11
months old at the beginning of the behavior test, and the mice were
housed for 1 month in a room under controlled temperature and
fed ad libitum. The behavior tests were conducted during daytime
in air-controlled and soundproof experiment room. The animal
experiments were abided by the guidelines of the Institutional
Animal Care and Use Committee of Korea Institute of Science and
Technology.
was incubated in a water bath (37 ꢀC) for 30 min. Next, the sus-
pension was centrifuged at 27,000ꢃg for 10 min. The final mem-
brane pellet was resuspended in 30 mL of Tris-HCl buffer and stored
in aliquots at ꢁ80 ꢀC until it was used in the binding assay. The
membrane preparation was thawed and washed with 20 mL of Tris-
citrate buffer (50 mM, pH 7.1, 0e4 ꢀC) 3 times. The pellet was
resuspended at a final concentration of 2.5
binding buffer, and the suspension was used for the binding assay.
A membrane suspension (180 L) was added to 10 L of a test so-
lution and 10
L of 1 nM (final concentration) [3H] PK-11,195 in a
mg protein in 100 mL
m
m
m
96-well plate. The solution was mixed and incubated for 60 min at
25 ꢀC. The binding reaction was terminated by rapid filtration onto
a Whatman GF/C glass fiber filter with ice-cold 30 mM Tris-HCl
buffer to remove any unbound [3H] PK-11195. The filters were
dried at 60 ꢀC for 30 min and suspended in Wallac microbeta plate
scintillation fluid. The amount of filter-bound radioactivity was
counted using a Wallac 1450 Microbeta liquid scintillation counter
(PerkinElmer Life and Analytical Sciences, Waltham, MA, USA).
Total binding and non-specific binding were determined using the
4.4.2. Y-maze spontaneous alternation test
To the acute AD mice model (n ¼ 7 per group), each test com-
pound (test compound in 20% cyclodextrin, 30 mg/kg) was intra-
venously injected daily for 6 days. The Y-maze spontaneous
alternation test was performed 1 day after the administration of
each test compound. The Y maze was made of three equally spaced
black plastic arms (40 L ꢃ 10 W ꢃ 12 H cm) positioned at an equal
angle. Each acute AD model mouse were placed at the end of one
arm and allowed to move freely through the maze for 8 min, and
the sequence of arm entries was recorded. The arm entry of the
mouse was counted when the all four limbs were within an arm. A
spontaneous alternation was considered as entry into all three
arms. The spontaneous alternation (%) was calculated by the
following equation:
binding buffer and non-labeled PK-11195 (1 mM, final concentra-
tion), respectively. The percent displacement of the radioligand
binding was determined by the following equation:
Binding displacement (%) ¼ 100 ꢃ {1 ꢁ (DPMsample ꢁ DPMNSB)/
(DPMTB ꢁ DPMNSB)}
where DPM, TB, and NSB denote disintegrations per minute, total
binding, and non-specific binding, respectively. IC50 values were
calculated from the binding displacement curve, which was fitted
to a one-site competition-binding model using the Prism 5.0
(GraphPad Software Inc., San Diego, CA, USA). Values of binding
affinity (Ki) were calculated by the following equation:
Spontaneous alternation (%) ¼ 100 ꢃ {number of alternations/(total
number of arm entries ꢁ 2)}
4.4.3. Contextual fear conditioning test
Ki ¼ IC50=ð1 þ ½Lꢄ=KdÞ
To the Tg AD mice model (n ¼ 7 per group), each test compound
(30 mg/kg) was orally administrated daily for 1 month. Each mouse
was placed in a fear-conditioning chamber (Courlbourn, USA) for
90 s before giving an electric shock. Trials of training were per-
formed using the chamber equipped with a fear conditioning sys-
tem (FreActimetrics, USA). The training was performed by giving a
conditional stimulus (CS) of 75 dB sound for 20 s followed by an
unconditional stimulus (US) of electric foot shock (0.5 mA) for the
last 2 s in CS. After an additional stay for 1 min, the mouse was
returned to its home cage. Fear conditioning test was conducted
24 h after the training. The mouse was placed in the same chamber
for 5 min without presentation of CS. Freezing behavior was
considered as the complete absence of any movement except for
respiration and heartbeat. Freezing response was measured by the
fear conditioning system without application of CS or US.
where [L] denotes the concentration of [3H] PK11195 used and Kd
denotes the competitor-ligand dissociation equilibrium constant
for [3H] PK11195.
4.6. Surface plasmon resonance (SPR) measurements
SPR measurements were performed with Biacore T200 optical
biosensor system equipped with CM5 sensor chips (GE Healthcare)
at 25 ꢀC. Recombinant protein of human TSPO (18 kDa, Origene
Technologies, Inc., No.: TP320107) was covalently immobilized on a
CM5 chip using standard amine-coupling protocols in 10 mM so-
dium acetate (pH 4.5) at a flow rate of 10 mL/min for 1500 s, to
4.5. TSPO binding assay
obtain densities of 4000e6000 response unit (RU). All of the tested
compounds were dissolved in 100% DMSO to obtain 10 mM solu-
tions, and serially diluted in PBS-P buffer (10 mM NaH2PO4,150 mM
NaCl, pH 7.4) to a final concentration of 1.0%. The filtered PBS-P
buffer containing 1.0% DMSO was prepared for the running buffer.
For each tested compounds, the SPR measurements were per-
formed using a five-point concentration series, 0.01e5 mM. In each
analysis, multiple blank samples of running buffer alone were
included. Typically, a series of different concentrations were
A radioligand [3H] PK11195 was purchased from PerkinElmer
Life and Analytical Sciences (Waltham, MA, USA). Non-labeled
PK11195 (Tocris, Bristol, UK) was used as reference materials. To
obtain a membrane preparation for the 18 kDa TSPO binding assay,
200e250 g male SD rats were purchased from Koatech Animal Inc.
(Pyeongtaek, Korea). All procedures involving animals were con-
ducted in accordance with the guidelines of the Korea Food
Research Institutional Animal Care and Use Committee (permission
No.: KFRI-M-09118). The cerebral cortex from 4 male SD rats was
homogenized for 10 s in 20 mL of Tris-HCl buffer (30 mM, pH 7.4).
The suspension was centrifuged at 27,000ꢃg for 10 min, and the
pellet was washed 3 times with Tris-HCl buffer. The washed pellet
was homogenized in 20 mL of Tris-HCl buffer, and the suspension
injected over the immobilized chip at a flow rate of 50
(contact time: 120 s, dissociation time: 300 s), followed by the
regeneration phase at a flow rate of 50 L/min (contact time: 120 s,
mL/min
m
stabilization time: 10 s). The KD values of each tested compounds
were determined by Biacore T200 evaluation software (GE
Healthcare) after the standard solvent correction process.

T. Kim et al. / European Journal of Medicinal Chemistry 125 (2017) 1172e1192
1191
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The homology model of three-dimensional (3D) human TSPO
was built on the atomic coordinates of the X-ray crystal structure of
bacterial TSPO (PDB ID: 4RYI) using Modeler9v7 program (Dis-
covery Studio 4.0, Accelrys, San Diego, CA, USA). Glide software
€
(Glide ver. 5.6; Schrodinger, LLC, NY, USA) was used to carry out
protein preparation, ligand preparation (in pH 7.4) and calculation,
and molecular docking studies. First, the glide, an enclosing box
centered at PK-11195, was generated from the built homology
model of human TSPO. Then, each docked ligand was prepared for
the glide docking. By using Glide software in extra precision (XP)
mode, the putative binding modes of compound 25 were gener-
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Acknowledgement
This work was supported by the National Research Council of
Science & Technology (NST) grant by the Korea government (MSIP)
(No. CRC-15-04-KIST) and KIST institutional program (2E26650). J.L.
was supported by the Sungshin Women's University Research
Grant of 2015-2-21-007.
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Appendix A. Supplementary data
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Abbreviations
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2385e2405.
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1309e1312.
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hibitor of the inner membrane permeability transition in liver mitochondria,
J. Biol. Chem. 264 (14) (1989) 7826e7830.
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A
AD
b
amyloid-b
Alzheimer's disease
mitochondrial permeability transition pore
translocator protein
cyclosphorin A
central nervous system
neurofibrillary tangles
endoplasmic reticulum
cyclophilin D
reactive oxygen species
amyotrophic lateral sclerosis
adenine nucleotide translocator
voltage-dependent anion channel
polyphosphoric acid
mPTP
TSPO
CsA
CNS
NFT
ER
Cyp D
ROS
ALS
ANT
VDAC
PPA
FBS
fetal bovine serum
PBS
BSA
ATP
phosphate buffered saline
bovine serum albumin
adenine triphosphate
CYP450 cytochrome P450
hERG
BBB
human ether-a-go-go-related gene
blood brain barrier
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]pyridinium3-oxidhexafluoro-phosphate
diisopropylamine
[25] R. Rupprecht, V. Papadopoulos, G. Rammes, T.C. Baghai, J. Fan, N. Akula,
G. Groyer, D. Adams, M. Schumacher, Translocator protein (18 kDa) (TSPO) as
a therapeutic target for neurological and psychiatric disorders, Nat. Rev. Drug
Discov. 9 (12) (2010) 971e988.
HATU
[26] M.K. Chen, T.R. Guilarte, Translocator protein 18 kDa (TSPO): molecular sensor
of brain injury and repair, Pharmacol. Ther. 118 (1) (2008) 1e17.
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R.C. Melcangi, C.J. Pike, Ligand for translocator protein reverses pathology in a
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M. Schumacher, E.M. Steidl, D. Maux, M. Delaage, C.E. Henderson, R.M. Pruss,
Identification and characterization of cholest-4-en-3-one, oxime (TRO19622),
a novel drug candidate for amyotrophic lateral sclerosis, J. Pharmacol. Exp.
Ther. 322 (2) (2007) 709e720.
DIPEA
TLC
thin layer chromatography
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