Syntheses and biological evaluation of 1-heteroaryl-2-aryl-1H-benzimidazole derivatives as c-Jun N-terminal kinase inhibitors with neuroprotective effects

Article abstract of DOI:10.1016/j.bmc.2013.02.021

1-Heteroaryl-2-aryl-1H-benzimidazole derivatives were synthesized as inhibitors of c-Jun N-terminal kinases, JNK3. Their activities were evaluated through measurement of K_d using SPR, JNK3 kinase assay, and cell-viability of human neuroblastoma

Full text of DOI:10.1016/j.bmc.2013.02.021

Bioorganic & Medicinal Chemistry 21 (2013) 2271–2285
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Syntheses and biological evaluation of 1-heteroaryl-2-aryl-
1H-benzimidazole derivatives as c-Jun N-terminal kinase inhibitors
with neuroprotective effects
Mi-hyun Kim a, Junghun Lee a, Kyungjin Jung a, Minjung Kim a, Yun-Jin Park b, Heechul Ahn c,
Young Hye Kwon b, Jung-Mi Hah a,
⇑
a Department of Pharmacy, College of Pharmacy, Hanyang University, 1271 Sa 3-Dong, Sangnok-gu, Ansan-si, Gyunggi-do 426-791, Republic of Korea
b Department of Food and Nutrition, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
c Department of Pharmacy, Dongguk University, 32 Dongguk-ro, Ilsandong-gu, Goyang, Geonggi 410-820, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
1-Heteroaryl-2-aryl-1H-benzimidazole derivatives were synthesized as inhibitors of c-Jun N-terminal
kinases, JNK3. Their activities were evaluated through measurement of Kd using SPR, JNK3 kinase assay,
Received 4 January 2013
Revised 6 February 2013
Accepted 9 February 2013
Available online 21 February 2013
and cell-viability of human neuroblastoma cells. Most tested compounds showed high affinity (10
46 nM) to JNK3. Among them, compound 16f exhibited potent activities (Kd = 46 nM). Especially, 16f
was also found to present a potent cell protective effect (IC50 = 1.09 M) against toxicity induced by
anisomycin, showing a possibility as protective therapeutics in neuronal cell apoptosis.
Ó 2013 Elsevier Ltd. All rights reserved.
lM–
l
Keywords:
Benzimidazole
Neuroprotective effect
Neurodegenerative disease
Neuroblastoma cell line
Kinase inhibitor
JNK
1. Introduction
b-amyloid is postulated to contribute to the neuronal dysfunction
and loss observed in Alzheimer’s disease. b-Amyloid-induced cell
death is attenuated in cortical neurons from jnk3-null mice, and
JNK3 mediates this cell death through the activation of c-Jun and
the enhanced expression of Fas ligand.3 In addition, post-mortem
brain sections from Alzheimer’s disease patients revealed an al-
tered distribution and activation of JNKs,4,5 indicating either a cau-
sal role in, or a response to, the pathology. There are many reports
that JNK mediates phosphorylation of Tau, the major constituent of
NFTs,6 which is another hallmark of Alzheimer’s disease. Jnk2, Jnk3,
and especially Jnk2/3 knockout mice showed significantly less loss
of dopaminergic neurons and also increased levels of striatal
dopamine compared to wild type mice treatedwith1-methyl-4-
phenyl-1,2,4,6-tetrahydropyridine (MPTP),7 providing strong evi-
dence of JNK activation in Parkinson’s disease. Moreover, mice
deficient in JNK3 or expressing mutations in the phosphorylation
site of c-Jun were resistant to acute MPTP intoxication, which typ-
ically produces a pattern of neuropathology nearly identical to that
of post-mortem human Parkinson’s disease specimens, and also to
the hippocampal neurotoxic events associated with administration
of the glutamate receptor agonist, kainic acid.8
The stress activated protein kinases/c-Jun N-terminal kinases
(SAPKs/JNKs) are serine/threonine kinases and one member of
the three identified families of mitogen-activated protein(MAP) ki-
nases. Activated JNK leads to the phosphorylation of a number of
transcription factors, most notably the c-Jun component of AP-1,
and cellular proteins, particularly those associated with apoptosis
(e.g., Bcl2, p53). There are ten different JNK isoforms, which are
formed by alternate splicing of three JNK genes (JNK1, 2, and 3).1
We were intrigued by the fact that JNK3 is predominantly localized
to the brain andtestes5 whereas JNKs 1 and 2 exhibit a broad tissue
distribution,2 which suggests a potentially unique role for JNK3 in
central nervous system (CNS) disorders.
JNKs are known to be activated in response to various stimuli
such as cytokines, mitogens, osmotic stress and ultraviolet irradia-
tion. In pathological conditions, JNK3 are considered degenerative
signal transducers. Indeed, during the last decade a number of re-
ports have supported JNK as good therapeutic target for neurode-
generative diseases such as Alzheimer’s and Parkinsonian
diseases in addition to ischemic injury. The direct toxicity of
With these previous findings in mind, we set up a research pro-
gram focusing on the development of a JNK3 selective inhibitor
and began screening our in-house kinase-focused library toward
⇑
Corresponding author. Tel.: +82 31 400 5803; fax: +82 31 400 5958.
E-mail address: jhah@hanyang.ac.kr (J.-M. Hah).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.02.021

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M.-h. Kim et al. / Bioorg. Med. Chem. 21 (2013) 2271–2285
3,4-dichloro substituent; and (3) introduction of substituents into
the 5-/6-position on the benzimidazole scaffold (Fig. 2).
HN
N
OH
N
H
N
2. Chemistry
N
The synthetic routes for the1-heteroaryl-2-aryl-1H-benzimid-
azole derivatives are shown in Schemes 1 and 2. The N-alkyl-4-
(2-aryl-1H-benzoimidazol-1-yl)pyridin-2-amine derivatives in Ta-
ble 1 were synthesized as shown in Scheme 1. Secondary amines
were coupled with 2-chloro-4-nitropyridine 1-oxide (1) via ArSN
(nucleophilic aromatic substitution) using a modified version of
a known method9 to produce the N-alkylamino-4-nitropyridine-
1-oxidederivatives (2a–2d) and then reduced using Raney-Ni.
Compounds (3a–3d) were continuously coupled with 1-bromo-
2-nitrobenzene (1.2 equiv) under modified Buchwald conditions
(palladium acetate (0.1 equiv), BINAP ligand (0.1 equiv), and
phosphate base (2 equiv)). The coupling products (4a–4g) were
reduced and then oxidatively cyclized10 using ceric ammonium
nitrate and hydrogen peroxide to produce N-alkyl-4-(2-aryl-1H-
benzoimidazol-1-yl) pyridin-2-amine derivatives (6a–6j). In addi-
tion, demethylation in the condition of Lewis acid (boron tribro-
mide) or introduction of an N-acyl group after Boc deprotection
was conducted when necessary.
In the case of N-alkyl-4-(2-aryl-1H-benzoimidazol-1-yl)pyrimi-
din-2-amine derivatives, either oxidative cyclization from ben-
zene-1,2-diamine (9a) or 4-methoxybenzene-1,2-diamine (9b)
was accomplished using ceric ammonium nitrate and hydrogen
peroxide to produce a benzimidazole core as shown in Scheme 2.11
Then, the benzimidazole compounds (10a–10c) were coupled with
4-chloro-2-(methylthio)pyrimidine under modified Buchwald con-
ditions (Pd2(dba)3 (0.1 equiv), BINAP ligand (0.1 equiv), sodium
tert-butoxide (2 equiv)),12 and followed by oxidation of methyl sul-
fide (11a–11d) into methyl sulfoxide (12a–12d) using mCPBA and
the substitution of methylsulfoxide (12a–12d) into 1-pyrimidine-
benzimidazole derivatives (13a–13l). Demethylation or introduc-
tion of an N-acyl group was performed when necessary.
1
Figure 1. In-house screening hit (JNK2: 2.18
l
M, JNK3: 1.86 lM).
various kinds of JNK3 PDB. We identified several hit compounds
from the screening process and tested those toward JNK2/3. Com-
pound 1 (Fig. 1) was identified as an ATP-competitive inhibitor
with mild potency (IC50: JNK2 2.18 lM, JNK3 1.86 lM). Interest-
ingly, 1 showed almost no inhibitory activity toward 50 kinases
other than JNK2 and JNK3, which is encouraging with regard to fur-
ther development of JNK selective inhibitors.
We subsequently examined the docking pose of in-house hit (1)
with JNK3 (PDB: 3OY1) as shown in Fig. 2. The 2-amino pyridine
moiety of in-house hit (1) played a role as hinge binder against
the Met 149 residue, and the benzimidazole scaffold-occupied
hydrophobic pocket region was surrounded by Ile70, Val78,
Ile124, Val196, and Leu206 residues in the activation loop and gly-
cine rich loop. The 2-phenyl substituent of benzimidazole ap-
proached the gate keeper, a Met146 residue. In addition, the N-
piperidine ring linked to pyridine was located in the solvent expos-
able region. This plausible docking pose was used to modify hit (1)
to acquire a more potent JNK3 inhibitor (Fig. 2).
Herein we report the syntheses, characterization, and biological
evaluation of 1-heteroaryl-2-aryl-1H-benzimidazole derivatives as
selective JNK3 inhibitors. The SAR from the hit compound was pro-
cessed in three steps: (1) modification of the 2-substituted pyridyl
group with a variety of substituent pyridine and pyrimidine
groups; (2) change of the 30-substituted phenol group to other
substituted aromatic rings, especially bulkier 2-naphthyl and a
Figure 2. (A) Docking structure of in-house screening hit (bold, orange) in JNK3 (3OY1) (B) 2D map of 1 in the active site of JNK3: Red; negative charged, violet; positive
charged, cyan; polar, pale green; hydrophobic, pink-line; hydrogen bonding, yellow in ligand; ligand exposure.

M. Kim et al. / Bioorg. Med. Chem. 21 (2013) 2271–2285
2273
H
N
N
O
N
O
N
R
H
N
H
N
N
Cl
R
R
NH
i
ii
iii
iv
NH2
NO2
NO2
X
NO2
2a, 76%
2b, 51%
2c, 67%
2d, 99%
3a, 88%
3b, 83%
3c, 96%
3d, 91%
4a, 66%
4b, 50%
4c, 74%
4d, 60%
X= H
X= OMe 4e, 58%
4f, 40%
4g, 43%
5a ~ 5c,
5e ~ 5g
vii
8a X= OH
55%
6a X= H; 6e X= OMe
50%
R =
for 6e
38%
H
N
v
N
N
vii
R
8b X= OH
47%
R
6b X= H; 6f X= OMe
20% 33%
N
H
N
N
for 6f
O
NH
R =
vii
6c X= H; 6g X= OMe
8c X= OH,
NBoc
O
for 6g
vii
NH
38%
44%
67%
vi
X
NH2
X
8d X= OH
37%
7a X= H; 7b X= OMe
6a ~ 6d
6e ~ 6g
X= H
N
X= H
5a, 66%
5b, 70%
5c, 90%
5d, 89%
for 7b
R =
60%
48%
X= OMe
OH
6d X= H
60%
X= OMe 5e, 99%
5f, 96%
Y
5c, 5d
v
5g, 90%
O
N
vii
vi
NBoc
7c
6h
8e
R
Y=OMe,
vii
Y= OMe
Y= OH
55%
N
N
H
N
N
57%
53%
OH
6i Y= OMe
41%
8f Y= OH
64%
6h, 6i
Cl
N
5a
v
Cl
N
vii
8g X= OH
54%
N
N
H
6j X=OMe
38%
X
Scheme 1. Syntheses of N-alkyl-4-(2-aryl-1H-benzoimidazol-1-yl)pyridin-2-amine derivatives. Reagents and reaction conditions: (i) RNH2, EtOH, 80 °C, 13 h; (ii) Raney Ni,
H2, MeOH, rt; (iii) 1-Br-2-NO2C6H4 or 1-Br-4-MeO-2-NO2C6H4, Pd(OAC)2, BINAP, K2PO4, Tol, 130 °C; (iv) Pd-C, H2, MeOH, rt; (v) 2-naphthaldehyde, 3-methoxybenzaldehyde or
3,4-dichloroaldehyde, CAN, H2O2, EtOH, 50 °C, 8 h; (vi) (1) 4N-HCl in 1,4-dioxane, rt, 20 min, (2) cyclopropanecarbonyl chloride, DIPEA, CH2Cl2, 0 °C to rt, 2 h; (vii) BBr3,
CH2Cl2, ꢀ78 °C to rt, 2 h.
an acyl piperidine group presented a dramatic increase of affinity
in 16f and similar activity with other 6-ring alkyl substituents in
8d, 15a and 16e. This result implies that the binding conformation
of N-alkyl groups like acyl piperidine could be a significant contrib-
utor to the binding of inhibitors in the active site of JNK3.
3. Results and discussion
3.1. Structure–activity relationships studies for measuring JNK3
activities
Another modification was introducing a hydroxy or methoxy
substitution in the 5-/6-position of the benzimidazole scaffold (R2,
R3 in Tables 1 and 2). Despite a slight decrease of affinity in 8d and
16b, it was expected that insertion of a hydroxy group instead of
hydrogen as R2 and R3 substituents would in general increase affin-
ity. In contrast, a methoxy group in the R2 and R3 position resulted in
an increased pKd value in some case (13a and 13d) and a dramati-
cally decreased pKd value in other cases (6a and 6e), suggesting that
it is also related to the 1-alkyl group of benzimidazole.
Finally, three kinds of aryl groups were introduced at the 2-po-
sition of the benzimidazole core instead of a phenyl group since we
determined this would yield a bigger hydrophobic space in the
docking studies. Replacement of the naphthyl group with a 3-
hydroxylphenyl group (8e and 8f) or a 3,4-dichlorophenyl group
(8g and 15d) gave similar pKd values in all cases except for a severe
Tables 1 and 2 shows the structures for 28 compounds and pre-
sents the biochemical Kd values against JNK3. The first SAR element
that improved potency for JNK3 was replacement of the pyrimidine
ring at the 1-position of benzimidazole (6a–8g vs 13a–16f) of hit 1.
Generally, 1-pyrimidyl substituted compounds showed better
affinity for JNK3. For both the pyridine and pyrimidine series, we
introduced various amino groups bearing hydrophobic substitu-
ents (R1) such as cyclohexanamine, tetrahydro-2H-pyran-4-amine,
(S)-1-aminopropan-2-ol, and (R)-cyclopropanecarbonyl pyridin-3-
amine. When the effect of an R1 substituent was compared in the
set of 6a, 6b, 6d and 7a, it seemed that a simple alkyl group such
as a cyclohexyl or pyranyl group in the N-alkyl position of R1
was superior to polar functionalized groups like 2-propanol or acyl
piperidine. However, when comparing R1 composition in other sets
(8a, 8b, 8c and 8d, 13a, 13b, 15a, 16a, 16c, 16e and 16b, 16d, 16f),

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M.-h. Kim et al. / Bioorg. Med. Chem. 21 (2013) 2271–2285
Table 1
decrease in pKd value for 13j and 13k and a dramatically increased
pKd value for 6j. Out of all these derivatives of 1-heteroaryl-2-aryl-
1H-benzimidazole, the best compound from the biochemical assay
was 16f, which had a biochemical potency of 46 nM.
Structure of N-alkyl-4-(2-aryl-1H-benzoimidazol-1-yl)pyridin-2-amine derivatives
and affinity (Kd) against JNK3
N
Ar
R1
N
N
3.2. Cell-based JNK activity measurement by immunoblotting
Based on biochemical Kd values, we performed series of cell-
based assays to confirm the JNK inhibitor(s) with good potency
(13d, 15c, 16f) having neuroprotective effects in cell. Human neu-
roblastoma SH-SY5Y cells were selected to assess the protective
potency of JNK3 inhibitors in an apoptotic environment, and the
well-known pan-JNK inhibitor SP600125 was used as the reference
compound to compare the phosphorylation of c-Jun, the substrate
of JNK, at the cellular level. Immunoblotting against p-c-Jun (p-
Ser73) revealed an increase in c-Jun phosphorylation in cells trea-
ted with anisomycin. Treatment of cells with the JNK inhibitor alle-
viated c-Jun phosphorylation (Fig. 3A). Compound 16f almost
R2
R3
No.
Substituents
Activity
Kd M)
R1
R2
R3
Ar
(l
NH
NH
6a
6b
6d
H
H
5.26
4.65
12.7
H
H
H
H
O
OH
NH
NH
completely blocked the phosphorylation of c-Jun at 10 lM.
In addition, cell morphology was observed under a light micro-
scope (Fig. 3B). Changes in cell morphology are an indicator of the
cytotoxic effect of JNK activation by anisomycin. Neuronal loss
and reduced neurite outgrowth were observed in response to aniso-
mycin. Treatment of cells with various JNK inhibitors alleviated cell
death induced by anisomycin. These results suggest that the inhibi-
tion of JNK is involved in the protective effect against cytotoxicity
supporting inhibition of JNK may provide clinical benefit in neuro-
degenerative disorders, including Alzheimer’s disease.
Cl
Cl
6j
H
H
OMe
H
18.1
10.7
O
NH
7a
N
NH
8a
8b
8c
H
H
H
OH
OH
OH
24.5
10
NH
The most potent compound 16f was further investigated to
determine cellular IC50 value (Fig. 3C). JNK signaling pathway has
been shown to be critical in the development of neuronal cytotox-
O
NH
HN
6.05
icity and a previous study reported that TNF
dose-dependently inhibited by SP600125 with IC50 of 10
PBMCs stimulated with LPS.14 In the present study, we observed
the dose-dependent decrease in TNF -mRNA levels by a compound
16f and the IC50 was determined as 1.09 M, which is much better
a
-mRNA levels were
O
O
lM in
NH
NH
8d
8e
H
H
OH
H
27.7
2.64
N
N
a
l
HO
HO
than the reference compound, SP600125.
4. Conclusions
OH
8f
H
H
H
19.1
13.8
NH
NH
In summary, a potent series of 1-heteroaryl-2-aryl-1H-benz-
imidazole derivatives has been developed. Optimization of this
scaffold by modification of three substituents (R1, R2 and R3) affor-
ded a series of potent JNK3 inhibitors. Many compounds in this
scaffold showed potent biochemical activities toward JNK3
in vitro, and furthermore, one of the best compound 16f has been
confirmed as a potent and selective JNK3 inhibitor in cell, dramat-
ically reducing phosphorylation of c-Jun. In the present study, we
Cl
Cl
8g
OH
ducted with a Waters Auto Purification™ System with a PDA detec-
tor and an atmospheric pressure single quadrupole mass
spectrometer. High-resolution MS (HR/MS) experiments were per-
formed to assess JNK activity using a 6500 Series Accurate-Mass
Quadrupole Time-of-Flight (Q-TOF) LC/MS mass spectrometer
(Agilent Technologies) operated in positive-ion electrospray mode.
Experiments assessing Kd value through SPR measurement were
conducted with a ProteOn XPR36 (Bio-Rad). Sybyl X-1.3 (Tripos)
was used as a 3D-QSAR tool for molecular modeling and Glide
and Macro model (Schrodinger) were used to conduct receptor-
guided alignment.
observed dose-dependent decrease in TNFa-mRNA levels by a
compound 16f with IC50 = 1.09 M. Which suggests that it 1-het-
l
eroaryl-2-aryl-1H-benzimidazolederivativescould serve as protec-
tive therapeutics in neuronal cell apoptosis.
5. Instrumentation and chemicals
All chemicals (reagent grade) used were purchased from Aldrich
(USA). Separation of the compounds by column chromatography
was carried out with silica gel 60 (200–300 mesh ASTM, E. Merck,
Germany). The quantity of silica gel used was 50–100 times the
weight charged on the column. Thin layer chromatography (TLC)
was run on silica gel-coated aluminum sheets (silica gel 60
GF254, E. Merck, Germany) and visualized under ultraviolet (UV)
light (254 nm). 1H NMR spectra were recorded on Varian 300 and
500 MHz and Bruker Avance 400 MHz spectrometers at 25 °C using
tetramethylsilane (TMS) as the internal standard. Low-resolution
MS (LR/MS) experiments on intermediate compounds were con-
6. Synthesis
6.1. Synthesis of cyclohexyl-[4-(2-naphthalen-2-yl-
benzoimidazol-1-yl)-pyridin-2-yl]-amine (6a)
The mixture of 2-chloro-4-nitropyridine 1-oxide (1) (325 mg,
1.86 mmol) and cyclohexylamine (0.38 mL, 3.35 mmol) in EtOH
(9 ml) was heated at 80 °C until compound 1 disappeared in TLC.

M. Kim et al. / Bioorg. Med. Chem. 21 (2013) 2271–2285
2275
N
N
Ar
N
Ar
N
Ar
O
S
O
NH2
NH2
N
Cl
S
HN
X
N
N
X
S
N
N
X
N
N
ii
iii
X
i
9a X= H
9b X= OMe
Ar =
11a X= H
11b X= 5-OMe
11c X= 6-OMe
12a X= H,
12b X= 5-OMe, 37%
12c X= 6-OMe, 36%
52%
10a X= H,
10b X= OMe, 33%
48%
Cl
10c X= H, 44%
11d X= H
12d X= H, 61%
Cl
vi
16a X= 5-OH, 16b X= 6-OH
13a X= H; 13d X= 5-OMe; 13g X= 6-OMe
52%
for 13d, 13g
13%
73%
80%
94%
12a~12c
N
vi
R =
13b X= H; 13e X= 5-OMe; 13h X= 6-OMe
16c X= 5-OH, 16d X= 6-OH
R
O
iv
N
H
N
N
23%
33%
N
for 13e, 13h
(1) of v
71%
86%
71%
R =
NBoc
O
14 X= H,
63%
13c X= H; 13f X= 5-OMe; 13i X= 6-OMe
41% 35%
NH
for 13c
X
35%
v
vi
16e X= 5-OH, 16f X= 6-OH
15a X= H; 15b X= 5-OMe; 15c X= 6-OMe
N
for 15b, 15c
72%
74%
59%
55%
62%
Cl
Cl
O
N
12d
iv
v
NBoc
R
N
R =
N
H
N
N
O
N
13j
13k
40%
15d
60%
50%
Scheme 2. Syntheses of N-alkyl-4-(2-aryl-1H-benzoimidazol-1-yl)pyrimidin-2-amine derivatives. Reagents and reaction conditions:(i) 2-naphthaldehyde or 3,4-dichloro-
aldehyde, H2O2, MeOH, 50 °C, 8 h; (ii) Pd2(dba)3, BINAP, NaOt-Bu, toluene, 150 °C,3 h; (iii) m-CPBA, CH2Cl2, rt, 8 h, (iv) RNH2,THF, 60 °C, 5 h;(v) (1) 4N-HCl in 1,4-dioxane, rt,
20 min, (2) cyclopropanecarbonyl chloride, DIPEA, CH2Cl2, 0 °C to rt, 2 h; (vi) BBr3, CH2Cl2, ꢀ78 °C to rt, 2 h.
After reaction termination, the mixture was cooled to ambient
temperature and solvent was removed in vacuo. The concentrated
crude product was purified by flash column chromatography with
EA/Hex (1:1) as the eluent to produce cyclohexyl-(4-nitro-1-oxy-
solvent was removed in vacuo and the crude product was purified
by flash column chromatography on silica gel using EA/Hex (2:1–
1:1) as the eluent to produce N2-cyclohexyl-N4-(2-nitro-phenyl)-
pyridine-2,4-diamine (4a) as
a
yellow solid (66%); 1H NMR
pyridin-2-yl)-amine (2a) as
a
yellow solid (76%); 1H NMR
(500 MHz, CDCl3) d 9.20 (1H, s), 8.18 (1H, d, J = 8.5 Hz), 7.89 (1H,
d, J = 5.5 Hz), 7.47–7.56 (2H, m), 6.94–7.00 (1H, m), 6.41 (1H, dd,
J = 5.5 Hz), 6.14 (1H, s), 5.57 (1H, br s), 3.40 (1H, br s), 2.00–2.03
(2H, m), 1.75–1.77 (2H, m), 1.61–1.64 (1H, m), 1.32–1.40 (2H,
m), 1.22–1.29 (m, 3H).
(400 MHz, CDCl3) d 8.23 (1H, br, s), 7.39 (1H, s), 7.36 (1H, br, s),
6.92 (1H, br, s), 3.44 (1H, br, s), 2.04–2.06 (2H, m), 1.72–1.90
(2H, m), 1.56–1.60 (1H, m), 1.42–1.47 (3H, m), 1.26–1.30 (2H, m).
Cyclohexyl-(4-nitro-1-oxy-pyridin-2-yl)-amine (2a) (294 mg,
1.24 mmol) and Raney Ni (excess) were suspended in MeOH
(12 ml). The mixture was stirred under hydrogen gas at ambient
temperature for 9 h. After completion of the reaction, the catalyst
was removed by celite pad filtration and the residue washed with
MeOH. The solvent was evaporated under reduced pressure. N2-
Cyclohexyl-pyridine-2,4-diamine (3a) as a crude dark brown solid
(88%) was used for the next step without additional purification;
1H NMR (400 MHz, CDCl3) d 7.46 (1H, d, J = 6.4 Hz), 7.15 (–NH, br
s), 5.90 (1H, dd, J = 6.4 Hz, J = 2.0 Hz),5.59 (1H, d, J = 2.0 Hz),4.57
(2H, –NH, br, s), 3.22–3.29 (1H, m), 1.93–1.97 (2H, m), 1.74–1.77
(2H, m), 1.57–1.61 (1H, m), 1.25–1.37 (5H, m).
N2-Cyclohexyl-N4-(2-nitro-phenyl)-pyridine-2,4-diamine(4a)
(206 mg, 0.66 mmol) and palladium on activated carbon (excess)
were suspended in MeOH (7 ml). The mixture was stirred under
hydrogen gas at ambient temperature for 30 min. After the yellow
color of the reaction mixture disappeared, the catalyst was re-
moved by celite pad filtration and the residue was washed with
MeOH. The solvent was evaporated under reduced pressure. N4-
(2-Amino-phenyl)-N2-cyclohexyl-pyridine-2,4-diamine (5a) as a
crude yellow solid (66%) was used for the next step without addi-
tional purification; 1H NMR (500 MHz, CD3OD) d 7.41–7.44 (2H,
m), 7.00–7.50 (m, 2H), 6.64–6.66 (1H, m), 6.20–6.22 (1H, m),
5.69–5.72 (1H, m), 3.58–3.69 (3H, m), 1.93–1.97 (2H, m), 1.69–
1.78 (2H, m), 1.57–1.60 (1H, m), 1.19–1.38 (5H, m).
The mixture of compound 3a (159 mg, 0.83 mmol), 1-bromo-2-
nitrobenzene
(167 mg,
0.83 mmol),
Pd(OAC)2
(18.6 mg,
0.083 mmol), BINAP (51.5 mg, 0.083 mmol), and K3PO4 (351 mg,
1.6 mmol) was purged with N2, and then toluene (3.8 mL) was
added to the mixture. After sonication under N2 for 5 min, the mix-
ture was heated up to 130 °C under N2 and was stirred without N2
at 130 °C for 3 h. After cooling down to ambient temperature, the
The mixture of compound 5a (73 mg, 0.26 mmol), 2-naphthal-
dehyde (41 mg, 0.26 mmol), 30% w/w H2O2 (118 mg, 1.04 mmol),
NH4Ce(NO3)6 (14 mg, 0.026 mmol) in EtOH (0.2 ml) was heated
at 50 °C for 8 h. After completion of the reaction, the reaction mix-
ture was cooled and extracted with CH2Cl2. The organic layer was

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M.-h. Kim et al. / Bioorg. Med. Chem. 21 (2013) 2271–2285
Table 2
Structure of N-alkyl-4-(2-aryl-1H-benzoimidazol-1-yl)pyrimidin-2-amine derivatives and affinity (Kd) against JNK3
N
Ar
R1
N
N
N
R2
Substituents
R3
No.
Activity
Kd M)
R1
R2
H
R3
H
Ar
(l
NH
NH
NH
NH
NH
13a
13b
13d
13g
1.75
2.33
H
H
O
H
OMe
H
0.743
1.77
OMe
Cl
Cl
13j
14
H
H
H
H
21.8
2.98
O
NH
HN
O
O
O
O
NH
NH
NH
NH
15a
15b
15c
15d
H
H
1.93
2.91
1.17
7.69
N
N
N
H
OMe
H
OMe
H
Cl
Cl
H
N
NH
16a
16b
16c
16d
H
OH
H
1.89
31.7
1.66
6.00
NH
NH
NH
OH
H
OH
H
O
O
OH
O
O
NH
NH
16e
16f
H
OH
H
2.65
N
N
OH
0.0461
washed with saturated NaHCO3 solution, water and brine. After
drying over anhydrous MgSO4 and evaporation of solvent, the
crude product was purified by flash column chromatography with
EA/Hex (1:1) as the eluent to produce compound 6a as a white so-
lid (38%); 1H NMR (500 MHz, CDCl3) d 8.24 (1H, s), 8.15 (1H, d,
J = 5.0 Hz), 7.93 (1H, d, J = 6.4 Hz), 7.81–7.85 (3H, m), 7.60 (1H, d,
J = 8.5 Hz), 7.49–7.55 (2H, m), 7.45–7.47 (1H, m), 7.33–7.41 (2H,
m), 6.63 (1H, d, J = 5.5 Hz), 6.18 (1H, s), 5.18 (1H, br, s), 3.10–3.18
(1H, m), 1.65–1.70 (2H, m), 1.57–1.61 (2H, m), 1.03–1.10 (4H,
m), 0.87–0.89 (2H, m); HRMS (ESI) calcd for C28H27N4 [M+H]+:
419.2230, found 419.2229.
6.2. Synthesis of [4-(2-naphthalen-2-yl-benzoimidazol-1-yl)-
pyridin-2-yl]-(tetrahydro-pyran-4-yl)-amine (6b)
A mixture of compound 1 (348 mg, 1.99 mmol) and tetrahydro-
pyran-4-ylamine (300 mg, 3.00 mmol) in EtOH (9 ml) was treated
by identical method with compound 2a. The crude product was

M. Kim et al. / Bioorg. Med. Chem. 21 (2013) 2271–2285
2277
100
80
60
40
20
0
Figure 3A. Inhibition of c-Jun phosphorylation by the JNK inhibitor in SH-SY5Y
cells treated with anisomycin. Cells were pre-treated with 10 M of various JNK
inhibitor candidate compounds for 1 h prior to treatment with 10 g/ml anisomy-
cin for 30 min. Cell extracts were prepared, and relative protein levels of p-c-Jun,
total-c-Jun, and b-actin were determined by immunoblotting analysis. SP600125
was used as a positive control.
l
l
0
2
4
6
8
10
16f ( M)
Figure 3C. Inhibition of TNF-
a
mRNA expression by a JNK inhibitor 16f in SH-SY5Y
cells treated with anisomycin. Cells were pre-treated with various concentrations of
a compound 16f for 1 h prior to the treatment of 10 g/ml anisomycin for 30 min.
l
purified by flash column chromatography with EA/Hex (1:1) ?
CH2Cl2/MeOH = 10:1 as the eluent to produce (4-nitro-1-oxy-pyri-
din-2-yl)-(tetrahydro-pyran-4-yl)-amine (2b) as yellow solid
(51%); 1H NMR (400 MHz, DMSO-d6) d 8.37 (1H, d, J = 7.2 Hz),
7.67 (1H, d, J = 3.2 Hz), 7.45 (1H, d, J = 9.2 Hz), 7.39 (1H, dd,
J = 7.2 Hz, J = 3.2 Hz), 3.43–3.49 (2H, m), 3.28–3.30 (1H, m), 3.16–
3.24 (2H, m), 1.49–1.60 (4H, m).
Total RNA was extracted and mRNA levels of TNF-
quantitative real-time PCR. Results are expressed as
treatment only) and the means SEM (n = 4).
a
%
were determined by
control (anisomycin
N4-(2-Amino-phenyl)-N2-(tetrahydro-pyran-4-yl)-pyridine-2,4-
diamine (5b) as crude dark yellow solid (70%) was synthesized by
identical method with compound 5a except for use of compound
4b (31 mg, 0.1 mmol) instead of compound 4a; 1H NMR
(400 MHz, DMSO-d6) d 7.97 (1H, s), 7.56 (1H, d, J = 6.00 Hz),
6.91–6.97 (2H, m), 6.76 (1H, dd, J = 8.0 Hz, J = 1.2 Hz), 6.53–6.58
(2H, m), 6.05 (1H, dd, J = 6.4 Hz, J = 1.6 Hz), 5.62 (1H, d,
J = 1.6 Hz), 4.87 (2H, s), 3.81–3.83 (3H, m), 3.31–3.37 (2H, m),
1.77–1.80 (2H, m), 1.33–1.39 (2H, m).
N2-(Tetrahydro-pyran-4-yl)-pyridine-2,4-diamine (3b) as crude
pale pink solid (83%)was synthesized by identical method with
compound 3a except for use of compound 2b (239 mg, 1.00 mmol)
instead of compound 2a; 1H NMR (500 MHz, CDCl3) d 7.47 (1H, d,
J = 6.0 Hz),5.74–5.77 (2H, m),5.55 (1H, s),5.46 (2H, –NH, br, s),
3.82–3.84 (2H, m), 3.73–3.75 (1H, m), 3.32–3.37 (2H, m), 1.78–
1.81 (2H, m), 1.32–1.37 (2H, m).
N4-(2-Nitro-phenyl)-N2-(tetrahydro-pyran-4-yl)-pyridine-2,4-
diamine (4b) was synthesized by identical method with compound
4a except for use of compound 3b (38 mg, 0.20 mmol) instead of
compound 3a. Purification by flash column chromatography with
EA/Hex (1:1) as the eluent produced compound 4b as yellow oil
(50%); 1H NMR (400 MHz, CDCl3) d 9.20 (1H, s), 8.19 (1H, dd,
J = 8.8 Hz, J = 1.6 Hz), 7.95 (1H, d, J = 5.6 Hz), 7.48–7.56 (2H, m),
6.96–7.00 (1H, m), 6.47 (1H, dd, J = 5.6 Hz, J = 1.6 Hz), 6.18 (1H, d,
J = 1.6 Hz), 4.96 (1H, br s), 3.96–4.02 (2H, m), 3.77–3.84 (1H, m),
3.50–3.56 (2H, m), 2.02 (2H, br d), 1.50–1.60 (2H, m).
[4-(2-Naphthalen-2-yl-benzoimidazol-1-yl)-pyridin-2-yl]-(tet-
rahydro-pyran-4-yl)-amine (6b) was synthesized by identical
method with compound 6a except for use of compound 5b (scale
25 mg, 0.088 mmol). Purification by flash column chromatography
with EA only as the eluent produced compound 6b as pale pink so-
lid (20%); 1H NMR (500 MHz, CDCl3) d 8.23 (1H, s), 8.21 (1H, d,
J = 5.0 Hz), 7.92 (1H, d, J = 8.0 Hz), 7.80–7.84 (3H, m), 7.50–7.59
(3H, m), 7.45 (1H, d, J = 8.0 Hz), 7.39 (1H, t, J = 7.5 Hz), 7.33 (1H,
t, J = 7.5 Hz), 6.70 (1H, d, J = 5.0 Hz), 6.15 (1H, s), 4.73 (1H, br,
s),3.74 (2H, d, J = 11.5 Hz), 3.46–3.58 (1H, m), 3.24 (2H, t,
Figure 3B. Protective effect of the JNK inhibitor against cytotoxicity in SH-SY5Y cells treated with anisomycin. Cells were pre-treated with 10
lM of various JNK inhibitors for
1 h prior to the treatment of 10 g/ml anisomycin for 30 min. Cell morphology was observed under a light microscope. SP600125 was used as a positive control.
l

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M.-h. Kim et al. / Bioorg. Med. Chem. 21 (2013) 2271–2285
J = 11.5 Hz), 1.67 (1H, d, J = 13.0 Hz), 1.29–1.36 (2H, m); HRMS (ESI)
J = 6.80 Hz), 5.96 (1H, d, J = 2.00 Hz), 3.83 (3H, s), 3.56–3.64 (1H,
m), 1.84–1.87 (2H, m), 1.67–1.71 (2H, m), 1.55–1.58 (1H, m),
1.23–1.33 (2H, m), 1.09–1.81 (3H, m).
calcd for C27H25N4O [M+H]+: 421.2023, found 421.2029.
6.3. Synthesis of (S)-1-[4-(2-naphthalen-2-yl-benzoimidazol-1-
yl)-pyridin-2-ylamino]-propan-2-ol (6d)
N4-(2-Amino-4-methoxy-phenyl)-N2-cyclohexyl-pyridine-2,4-
diamine (5e) as crude dark red solid (99%)was synthesized by iden-
tical method with compound 5a except for use of compound 4e
(114 mg, 0.33 mmol) instead of compound 4a; 1H NMR
(400 MHz, CDCl3) d 7.71 (1H, d, J = 6.0 Hz), 6.97 (2H, d, J = 8.4 Hz),
6.28–6.33 (2H, m), 5.88 (1H, dd, J = 6.0 Hz, J = 2.0 Hz), 5.46 (1H, d,
J = 2.0 Hz), 5.32 (1H, s), 4.38 (1H, d, J = 6.4 Hz), 3.82 (2H, s), 3.75
(3H, s), 3.34–3.44 (1H, m), 1.93–1.97 (2H, m), 1.66–1.72 (2H, m),
1.55–1.60 (1H, m), 1.31–1.38 (2H, m), 1.13–1.20 (3H, m).
Cyclohexyl-{4-[2-(3,4-dichloro-phenyl)-5-methoxy-ben-
zoimidazol-1-yl]-pyridin-2-yl}-amine (6j) was synthesized by
identical method with compound 6a except for use of 3,4-dichloro-
aldehyde and compound 5e (20 mg, 0.064 mmol). Purification by
flash column chromatography with Hex/acetone (8:1) as eluent
produced compound 6j as white solid (38%); 1H NMR (400 MHz,
CDCl3) d 8.15 (1H, d, J = 5.2 Hz), 7.84 (1H, d, J = 2.4 Hz), 7.40 (1H,
d, J = 8.4 Hz), 7.28–7.32 (3H, m), 6.95 (1H, dd, J = 8.8 Hz,
J = 2.4 Hz), 6.51 (1H, dd, J = 5.2 Hz, J = 2.0 Hz), 6.12 (1H, d,
J = 2.0 Hz), 5.08 (1H, br, s),3.88 (3H, s), 3.30–3.32 (1H, m), 1.83–
1.86 (2H, m), 1.69–1.74 (2H, m), 1.59–1.62 (1H, m),1.12–1.34
(5H, m); HRMS (ESI) calcd for C25H25Cl2N4O [M+H]+: 467.1400,
found 467.1401.
(S)-1-(4-Nitro-1-oxy-pyridin-2-ylamino)-propan-2-ol (2d) was
synthesized by identical method with compound 2a except for
use of 1-amino-propan-2-ol (300 mg, 1.719 mmol) instead of (R)-
tert-butyl-3-aminopiperidine-1-carboxylate. Purification by flash
column chromatography with EA/Hex (1:1) as the eluent produced
compound 2d as yellow solid (99%); 1H NMR (400 MHz, CDCl3) d
8.22 (1H, d, J = 6.03 Hz), 7.52 (1H, s), 7.41 (2H, m), 4.20 (1H, s),
3.38 (2H, m), 3.30 (1H, m), 1.34 (3H, d, J = 6.13 Hz).
(S)-1-(4-Aminopyridin-2-ylamino)propan-2-ol (3d) as crude
yellow solid (91%) was synthesized by identical method with com-
pound 3a except for use of compound 2d (420 mg, 1.97 mmol) in-
stead of compound 2a; 1H NMR (400 MHz, DMSO-d6) d 7.45 (1H, d,
J = 5.6 Hz), 6.03 (1H, s), 5.81 (1H, dd, d, J = 5.6 Hz, J = 1.6 Hz),5.64
(2H, s), 5.58 (1H, d, J = 1.6 Hz), 3.67–3.74 (2H, m), 3.02–3.07 (2H,
m), 1.03 (3H, d, J = 6.0 Hz).
(S)-1-[4-(2-Nitro-phenylamino)-pyridin-2-ylamino]-propan-2-
ol (4d) was synthesized by identical method with compound 4a
except for use of compound 3d (200 mg, 1.2 mmol) instead of com-
pound 3a. Purification by flash column chromatography with EA/
Hex (1:1) as the eluent produced compound 4d as a pale yellow so-
lid (60%); 1H NMR (400 MHz, CDCl3) d 9.16 (1H, s), 8.17 (1H, dd,
J = 1.45 Hz, J = 1.48 Hz), 7.91 (1H, d, J = 5.76 Hz), 7.50 (2H, m),
6.94 (1H, td, J = 1.40 Hz), 6.44 (1H, dd, J = 1.86 Hz, J = 1.86 Hz),
6.24 (1H, s), 5.12 (1H, s), 3.99 (1H, m), 3.54 (1H, br), 3.42 (1H, d,
J = 13.7 Hz), 3.24 (1H, m), 1.21 (3H, d, J = 6.28 Hz).
(S)-1-[4-(2-Amino-phenylamino)-pyridin-2-ylamino]-propan-
2-ol (5d) as a crude dark red solid (89%) was synthesized by iden-
tical method with compound 5a except for use of compound 4d
(75 mg, 0.26 mmol) instead of compound 4a; 1H NMR (400 MHz,
DMSO-d6) d 7.55 (1H, d, J = 6.0 Hz), 7.49 (1H, s), 6.96 (1H, dd,
J = 8.0 Hz, J = 1.2 Hz), 6.90 (1H, td, J = 7.6 Hz, J = 1.2 Hz), 6.74 (1H,
dd, J = 8.0 Hz, J = 1.26 Hz), 6.55 (1H, td, J = 7.6 Hz, J = 1.2 Hz), 6.06
(1H, t, J = 5.6 Hz), 5.95 (1H, dd, J = 6.0 Hz, J = 1.6 Hz), 5.63 (1H, d,
J = 1.6 Hz), 4.78 (2H, s), 3.67–3.71 (1H, m), 3.17 (1H, s), 3.04–3.08
(2H, m), 1.00 (3H, J = 6.4 Hz).
6.5. Synthesis of (R)-cyclopropyl-{3-[4-(2-naphthalen-2-yl-
benzoimidazol-1-yl)-pyridin-2-ylamino]-piperidin-1-yl}-
methanone (7a)
The mixture of compound 1 (325 mg, 1.86 mmol)and (R)-tert-
butyl-3-aminopiperidine-1-carboxylate (671 mg, 3.35 mmol) in
EtOH (9 ml) was treated by identical method with compound 2a.
The concentrated crude product was purified by flash column chro-
matography with EA/Hex (1:1) as the eluent to produce (R)-3-(4-
nitro-1-oxy-pyridin-2-ylamino)-piperidine-1-carboxylic acid tert-
butyl ester (2c) as yellow solid (67%); 1H NMR (400 MHz, CDCl3)
d 8.23 (1H, d, J = 7.08 Hz), 7.46 (1H, s), 7.41 (1H, dd, J = 4.33 Hz,
2.71 Hz), 7.00 (1H, d, J = 7.65 Hz), 3.96 (1H, d, J = 13.07 Hz), 3.71
(1H, br, s), 3.54 (1H, br, s), 3.13 (2H, m), 2.12 (1H, m), 1.81 (1H,
m), 1.71 (2H, m), 1.47 (9H, s).
(S)-1-[4-(2-Naphthalen-2-yl-benzoimidazol-1-yl)-pyridin-2-
ylamino]-propan-2-ol (6d) was synthesized by identical method
with compound 6a except for use of compound 5d (scale 15 mg,
0.06 mmol). Purification by flash column chromatography on silica
gel using EA/Hex (1:1) as the eluent produced compound 6d as
white solid (60%); 1H NMR (400 MHz, CDCl3) d 8.20 (1H, s), 7.90–
7.92 (2H, m), 7.81–7.85 (3H, m), 7.51–7.56 (3H, m), 7.45–7.47
(1H, m), 7.39 (1H, t, J = 7.2 Hz), 7.33 (1H, t, J = 7.2 Hz), 6.95 (1H,
br, s), 6.67 (1H, s), 6.49 (1H, d, J = 4.8 Hz), 3.90–3.93 (1H, m),
3.33–3.36 (1H, m), 3.16–3.23 (1H, m), 1.13 (3H, d, J = 6.4 Hz);
(R)-3-(4-Amino-pyridin-2-ylamino)-piperidine-1-carboxylic
acid tert-butyl ester (3c) pale yellow solid (96%) was synthesized
by identical method with compound 3a except for use of com-
pound 2c (420 mg, 1.24 mmol) instead of compound 2a; 1H NMR
(400 MHz, CDCl3) d 7.96 (1H, s), 5,78 (1H, d, J = 4.5 Hz), 5.69 (1H,
d, J = 7.20 Hz), 5.57 (1H, s), 5.47 (1H, s), 3.75 (1H, br, s) 3.51 (1H,
br, s), 2.84 (1H, br, s), 1.83 (2H, m), 1.73 (1H, m), 1.81 (1H, m),
1.67 (2H, m), 1.35 (9H, s).
(R)-3-[4-(2-Nitro-phenylamino)-pyridin-2-ylamino]-piperi-
dine-1-carboxylicacid tert-butyl ester (4c) was synthesized by
identical method with compound 4a except for use of compound
3c (242 mg, 0.83 mmol) instead of compound 3a. Purification by
flash column chromatography with EA/Hex (2:1) as the eluent pro-
duced compound 4b as yellow oil (74%);1H NMR (400 MHz, DMSO-
d6) d 9.00 (1H, s), 8.06 (1H, d, J = 1.58 Hz), 7.81 (1H, d, J = 5.66 Hz),
7.61 (3H, m), 7.10 (1H, t, J = 1.01 Hz), 6.30 (2H, m), 5.62 (1H, s), 3.79
(1H, m), 3.54 (2H, m), 3.15 (2H, m), 2.13 (1H, m), 1.96 (1H, m), 1.67
(2H, m), 1.21 (9H, s).
HRMS (ESI) calcd for
395.1866.
C
25H23N4O [M+H]+: 395.1866, found
6.4. Synthesis of cyclohexyl-{4-[2-(3,4-dichloro-phenyl)-5-
methoxy-benzoimidazol-1-yl]-pyridin-2-yl}-amine (6j)
N 2-Cyclohexyl-N4-(4-methoxy-2-nitro-phenyl)-pyridine-2,4-
diamine (4e) was synthesized by identical method with compound
3a except for use of 1-bromo-4-methoxy-2-nitro-benzeneand
compound 3a (113 mg, 0.59 mmol). Purification by flash column
chromatography with EA/Hex (2:1) as the eluent produced com-
pound 4e as pale yellow solid (58%); 1H NMR (400 MHz, DMSO-
(R)-3-[4-(2-Amino-phenylamino)-pyridin-2-ylamino]-piperi-
dine-1-carboxylic acid tert-butyl ester (5c) as crude dark yellow
solid (90%)was synthesized by identical method with compound
5a except for use of compound 4c (252 mg, 0.66 mmol) instead
of compound 4a; 1H NMR (400 MHz, CDCl3) d 7.61 (1H, br, s),
7.10 (2H, d, J = 4.0 Hz), 6.82 (1H, d, J = 7.2 Hz), 6.76 (1H, t,
J = 7.2 Hz), 6.38 (1H, br, s), 6.02 (1H, d, J = 5.8 Hz), 5.62 (1H, s),
d6)
d 8.46 (1H, s), 7.70 (1H, d, J = 6.00 Hz), 7.54 (1H, d,
J = 3.20 Hz), 7.47 (1H, d, J = 9.00 Hz), 7.30 (1H, dd, J = 9.00 Hz,
J = 6.00 Hz), 6.12 (1H, dd, J = 6.80 Hz, J = 2.00 Hz), 6.03 (1H, d,

M. Kim et al. / Bioorg. Med. Chem. 21 (2013) 2271–2285
2279
3.66–3.92 (2H, m), 3.38–3.49 (1H, m), 2.89–3.13 (1H, m), 2.46–2.62
(4H, with –NH, m), 1.70–1.73 (1H, m), 1.42–1.54 (3H, m), 1.42 (9H,
s).
in vacuo and the concentrated crude product was purified by flash
column chromatography with CH2Cl2/MeOH (gradient from 40:1
to 5:1)as the eluent to afford pale yellow solid (55%); 1H NMR
(400 MHz, DMSO-d6) d 9.31 (1H, s), 8.21 (1H, s), 8.07 (1H, d,
J = 5.2 Hz), 7.89–7.95 (3H, m), 7.53–7.60 (3H, m), 7.22 (1H, d,
J = 8.8 Hz), 7.11 (1H, d, J = 2.0 Hz), 6.82 (1H, dd, J = 8.8 Hz,
J = 2.0 Hz), 6.61 (1H, d, J = 7.6 Hz), 6.45 (1H, dd, J = 5.2 Hz,
J = 2.0 Hz), 6.38 (1H, d, J = 2.0 Hz), 3.62 (1H, br, s),1.78–1.80 (2H,
m), 1.51–1.62 (3H, m), 1.04–1.23 (5H, m); HRMS (ESI) calcd for
(R)-3-[4-(2-Naphthalen-2-yl-benzoimidazol-1-yl)-pyridin-2-
ylamino]-piperidine-1-carboxylic acid tert-butyl ester (6c) was
synthesized by identical method with compound 6a except for
compound 5c (scale 25 mg, 0.064 mmol). Purification by flash col-
umn chromatography with EA/Hex (1:1) as the eluent produced
compound 6c as pale yellow solid (38%); 1H NMR (500 MHz, CDCl3)
d 8.24 (1H, s), 8.18 (1H, d, J = 4.5 Hz), 7.92 (1H, d, J = 8.0 Hz), 7.99–
7.85 (3H, m), 7.61–7.63 (1H, m), 7.49–7.55 (2H, m), 7.44 (1H, d,
J = 8.0 Hz), 7.38 (1H, t, J = 8.0 Hz), 7.33 (1H, t, J = 8.0 Hz), 6.58 (1H,
br, s), 6.33 (1H, br, s), 4.89 (1H, br, s), 3.76–3.78 (1H, m), 3.60
(1H, br, s), 3.46–3.50 (1H, m), 3.12–3.18 (2H, m), 1.75–1.78 (1H,
m), 1.57–1.61 (1H, m), 1.33–1.43 (2H, m, shield with impurity),
1.28 (9H, s).
Compound 6c (0.148 mmol) was dissolved in 1.4 ml of 1,4-diox-
ane and treated with 0.7 ml of anhydrous 4 M HCl in dioxane at
ambient temperature and stirred for 30 min. The mixture was then
diluted with ether and stirred until the product separated as a so-
lid. The solid product was filtered and washed with ether to pro-
duce the crude salt. The crude salt (0.048 mmol) in methylene
chloride (0.9 ml) was cooled to 0 °C and treated with DIPEA
C
28H27N4O [M+H]+: 435.2179, found 435.2172.
6.7. Synthesis of 2-naphthalen-2-yl-1-[2-(tetrahydro-pyran-4-
ylamino)-pyridin-4-yl]-1H-benzoimidazol-5-ol (8b)
N4-(4-Methoxy-2-nitro-phenyl)-N2-(tetrahydro-pyran-4-yl)-
pyridine-2,4-diamine (4f) was synthesized by identical method
with compound 4e except for use of compound 3b (77 mg,
0.4 mmol). Purification by flash column chromatography with
EA/Hex (1:1) ? EA only as gradient eluent produced compound
4f as pale yellow solid (40%); 1H NMR (400 MHz, CDCl3) d 8.67
(1H, s), 7.72 (1H, d, J = 7.2 Hz), 7.67 (1H, d, J = 3.2 Hz), 7.46–7.55
(2H, m), 7.24 (1H, dd, J = 9.2 Hz, J = 2.2 Hz), 6.38 (1H, dd,
J = 6.4 Hz, J = 2.0 Hz), 6.07 (1H, d, J = 2.0 Hz), 3.99–4.05 (2H, m),
3.90 (3H, s), 3.87–3.93 (1H, m), 3.52–3.58 (2H, m), 1.98–2.06
(2H, m), 1.68–1.72 (2H, m).
(32 lL, 0.18 mmol). The mixture was kept at 0 °C for 30 min, then
cyclopropanecarbonyl chloride (18 mg, 0.051 mmol) was added
and the mixture was warmed up to ambient temperature and stir-
red for 2 h. The reaction mixture was diluted with methylene chlo-
ride, and washed with water and saturated aqueous sodium
chloride solution. The organic phase was dried over sodium sulfate
and concentrated in vacuo. The crude product was purified by flash
column chromatography with EA/Hex (1:1) as the eluent to pro-
duce 7a as white solid (48%); 1H NMR (400 MHz, CDCl3) d 8.22
(1H, d, J = 1.2 Hz), 8.02–8.14 (1H, m), 7.91 (1H, d, J = 8.0 Hz),
7.78–7.83 (3H, m), 7.59 (1H, d, J = 8.0 Hz), 7.47–7.54 (2H, m),
7.28–7.39 (3H, m), 6.32–6.60 (2H, m), 5.26 (1H, br, s), 3.99 (1H,
br, s), 3.70–3.76 (1H, m), 3.15–3.46 (3H, m), 1.78–1.86 (1H, m),
1.54–1.69 (3H, m), 1.37–1.45 (1H, m), 0.81–1.11 (4H, m); HRMS
(ESI) calcd for C31H30N5O [M+H]+:488.2445, found 488.2447.
N4-(2-Amino-4-methoxy-phenyl)-N2-(tetrahydro-pyran-4-yl)-
pyridine-2,4-diamine (5f) was synthesized by identical method
with compound 5e except for use of compound 4f (43 mg,
0.13 mmol) instead of compound 4e. The compound 5f was given
as crude dark brown solid (96%); 1H NMR (400 MHz, CDCl3) d
7.56 (–NH, br s), 7.31–7.35 (2H, m), 6.94 (1H, d, J = 8.4 Hz), 6.28–
6.32 (1H, m), 5.96 (1H, d, J = 5.6 Hz), 5.48 (1H, s), 3.90–3.95 (2H,
m), 3.84 (2H, br s), 3.76 (3H, s), 3.58–3.64 (1H, m), 3.43–3.49
(2H, m), 1.90–1.93 (2H, m), 1.40–1.52 (2H, m).
[4-(5-Methoxy-2-naphthalen-2-yl-benzoimidazol-1-yl)-pyri-
din-2-yl]-(tetrahydro-pyran-4-yl)-amine (6f) was synthesized by
identical method with compound 6e except for use of compound
5f (35 mg, 0.11 mmol). Purification by flash column chromatogra-
phy with Hex/acetone (2:1) as eluent produced compound 6f as
pale pink solid (33%); 1H NMR (400 MHz, CDCl3) d 8.16–8.18 (2H,
m), 7.76–7.82 (3H, m), 7.46–7.55 (3H, m), 7.36 (1H, d, J = 2.4 Hz),
7.31 (1H, d, J = 8.8 Hz), 6.95 (1H, dd, J = 8.8 Hz, J = 2.4 Hz), 6.65
(1H, dd, J = 5.6 Hz, J = 1.6 Hz), 6.10 (1H, d, J = 1.6 Hz), 4.77 (1H, d,
J = 7.6 Hz), 3.89 (3H, s), 3.68–3.72 (2H, m), 3.44–3.51 (1H, m),
3.18–3.24 (2H, m), 1.62–1.66 (2H, m), 1.239–1.34 (2H, m, mixed
with impurity peak).
6.6. Synthesis of 1-(2-cyclohexylamino-pyridin-4-yl)-2-
naphthalen-2-yl-1H-benzoimidazol-5-ol (8a)
Cyclohexyl-[4-(5-methoxy-2-naphthalen-2-yl-benzoimidazol-
1-yl)-pyridin-2-yl]-amine (6e) was synthesized by identical meth-
od with compound 6a except for use of compound 5e (30 mg,
0.1 mmol). Purification by flash column chromatography with
Hex/acetone (5:1) as eluent produced compound 6e as pale yellow
solid (50%); 1H NMR (400 MHz, CDCl3) d 8.18 (1H, s), 8.13 (1H, d,
J = 5.2 Hz), 7.76–7.81 (3H, m), 7.58 (1H, d, J = 8.4 Hz), 7.45–7.51
(2H, m), 7.37 (1H, br, s), 7.31 (1H, d, J = 8.4 Hz), 6.95 (1H, dd,
J = 8.8 Hz, J = 1.6 Hz), 6.57 (1H, d, J = 8.4 Hz), 6.12 (1H, s), 4.78
(1H, d, J = 7.2 Hz), 3.89 (3H, s), 3.16–3.19 (1H, m), 1.68–1.71 (2H,
m), 1.49 (3H, br, s), 0.99–1.09 (5H, m); HRMS(ESI) calcd for
2-Naphthalen-2-yl-1-[2-(tetrahydro-pyran-4-ylamino)-pyri-
din-4-yl]-1Hbenzoimidazol-5-ol (8b) was synthesized by identical
method with compound 8a except for using compound 6f (15 mg,
0.033 mmol) instead of compound 8b as substrate. Purification by
flash column chromatography with CH2Cl2/MeOH (gradient from
40:1 to 5:1)as eluent produced compound 8b a spale yellow solid
(47%); 1H NMR (400 MHz, DMSO-d6) d 9.28 (1H, s), 8.21 (1H, s),
8.09 (1H, d, J = 5.2 Hz), 7.90–7.95 (3H, m), 7.54–7.61 (3H, m),
7.23 (1H, d, J = 8.8 Hz), 7.12 (1H, d, J = 2.0 Hz), 6.83 (1H, dd,
J = 8.8 Hz, J = 2.0 Hz), 6.74 (1H, d, J = 7.6 Hz), 6.49 (1H, dd,
J = 5.2 Hz, J = 1.6 Hz), 6.42 (1H, d, J = 1.6 Hz), 3.74–3.81 (3H,
m),1.74–1.77 (2H, m), 1.31–1.38 (4H, m); HRMS (ESI) calcd for
C
29H29N4O [M+H]+: 449.2336, found 449.2332.
1-(2-Cyclohexylamino-pyridin-4-yl)-2-naphthalen-2-yl-1H-
benzoimidazol-5-ol (8a) (22 mg, 0.048 mmol) in methylene chlo-
ride (0.34 ml) was cooled to ꢀ78 °C and slowly treated with a
1 M solution of boron tribromide (150 lM, 0.15 mmol). The reac-
tion mixture was kept at ꢀ78 °C for 1 h and was then allowed to
warm to room temperature for 2 h. The mixture was quenched
by the addition of methanol (0.2 ml) at 0 °C and was stirred at
room temperature for an additional hour. The mixture was diluted
with methylene chloride (5 ml) and washed with three portions of
saturated sodium bicarbonate solution (3 ml), two 5 ml portions of
water and two 5 ml portions of saturated sodium chloride solution.
The organic phase was dried over sodium sulfate and concentrated
C
27H25N4O2 [M+H]+: 437.1972, found 439.1971.
6.8. Synthesis of (R)-2-naphthalen-2-yl-1-[2-(piperidin-3-
ylamino)-pyridin-4-yl]-1H-benzoimidazol-5-ol (8c)
(R)-3-[4-(4-Methoxy-2-nitro-phenylamino)-pyridin-2-ylami-
no]-piperidine-1-carboxylicacid tert-butyl ester (4g) was synthe-
sized by identical method with compound 4e except for use of of

2280
M.-h. Kim et al. / Bioorg. Med. Chem. 21 (2013) 2271–2285
the compound 3c (117 mg, 0.40 mmol). Purification by flash col-
umn chromatography with EA/Hex (1:1) as the eluent produced
compound 4g as pale yellow solid (75 mg, 43%); 1H NMR
(400 MHz, CDCl3) d 8.77 (1H, s), 7.98–8.00 (1H, m), 7.59–7.72
(4H, m), 6.26–6.35 (2H, m), 3.90 (3H, s), 3.87–3.91 (2H, m), 3.56–
3.64 (2H, m), 2.95–3.10 (1H, m), 1.98–2.06 (2H, m), 1.68–1.72
(2H, m), 1.44 (9H, s).
(R)-3-{4-[2-(3-Methoxy-phenyl)-benzoimidazol-1-yl]-pyridin-
2-ylamino}-piperidine-1-carboxylic acid tert-butyl ester (5g) as
crude dark brown solid (90%) was synthesized by identical method
with compound 5e except for use of compound 4g (75 mg,
0.17 mmol) instead of compound 4e; 1H NMR (400 MHz, CDCl3) d
7.46–7.65 (2H, m), 6.98 (2H, d, J = 8.4 Hz), 6.36 (1H, d, J = 6.4 Hz),
6.33 (1H, dd, J = 8.4 Hz, J = 2.8 Hz),6.00 (1H, dd, J = 6.4 Hz,
J = 1.6 Hz),5.53 (1H, br, s), 3.91–3.96 (2H, m), 3.78 (3H, s), 3.72–
3.80 (2H, m), 3.26–3.39 (1H, m), 1.98–2.00 (2H, m), 1.74–1.75
(2H, m), 1.44 (9H, s).
(R)-3-[4-(5-Methoxy-2-naphthalen-2-yl-benzoimidazol-1-yl)-
pyridin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester
(6g) was synthesized by identical method with compound 6e ex-
cept for use of compound 5g (60 mg, 0.14 mmol). Purification by
flash column chromatography with Hex/acetone (3:1) as eluent
produced compound 6g as white solid (44%); 1H NMR (400 MHz,
CDCl3) d 8.20 (1H, d, J = 0.8 Hz), 8.16 (1H, d, J = 5.2 Hz), 7.78–7.84
(3H, m), 7.60 (1H, dd, J = 8.4 Hz, J = 2.0 Hz), 7.49–7.53 (2H, m),
7.39 (1H, d, J = 4.8 Hz), 7.31 (1H, d, J = 4.8 Hz), 6.96 (1H, dd,
J = 8.8 Hz, J = 2.4 Hz), 6.55 (1H, d, J = 4.8 Hz), 6.30 (1H, s), 4.83
(1H, br, s), 3.91 (3H, s), 3.75–3.79 (1H, m), 3.61 (1H, br, s), 3.45–
3.50 (1H, m), 3.08–3.19 (2H, m), 1.72–1.79 (1H, m), 1.56–1.62
(1H, m), 1.36 (9H, s), 1.25–1.46 (2H, m).
(R)-2-Naphthalen-2-yl-1-[2-(piperidin-3-ylamino)-pyridin-4-
yl]-1H-benzoimidazol-5-ol (8c) was synthesized by identical
method with compound 8a except for using compound 6g
(16 mg, 0.029 mmol) instead of compound 6e as substrate and
5 equiv of boron tribromide. Purification by flash column chroma-
tography with CH2Cl2/MeOH (5:1) as eluent produced compound
8c as pale yellow solid (67%); 1H NMR (400 MHz, DMSO-d6) d
8.21 (1H, s), 8.06 (1H, d, J = 5.2 Hz), 7.89–7.95 (3H, m), 7.54–7.61
(3H, m), 7.21 (1H, d, J = 8.8 Hz), 7.12 (1H, d, J = 2.4 Hz), 6.83 (1H,
dd, J = 8.8 Hz, J = 2.4 Hz), 6.70 (1H, d, J = 7.6 Hz), 6.47 (1H, d,
J = 1.6 Hz), 6.44 (1H, dd, J = 5.2 Hz, J = 1.6 Hz), 3.76 (1H, br, s),
3.07–3.09 (1H, m), 2.80–2.83 (1H, m), 2.33–2.38 (1H, m), 1.94–
1.97 (1H, m), 1.81–1.83 (1H, m), 1.54–1.62 (1H, m), 1.30–1.44
(2H, m); HRMS(ESI) calcd for C27H26N5O [M+H]+: 436.2132, found
436.2130.
6.10. Synthesis of (R)-cyclopropyl(3-((4-(2-(3-hydroxyphenyl)-
1H-benzo[d]imidazol-1-yl)pyridin-2-yl)amino)piperidin-1-yl)
methanone
(R)-3-{4-[2-(3-Methoxy-phenyl)-benzoimidazol-1-yl]-pyridin-
2-ylamino}-piperidine-1-carboxylic acid tert-butyl ester (6h) was
synthesized by identical method with compound 6c except for
using 3-methoxybenzaldehyde (36 mg, 0.26 mmol, 1 equiv). Purifi-
cation by flash column chromatography with EA/Hex (1:1) as the
eluent produced compound 6h as pale pink solid (57%); 1H NMR
(400 MHz, CDCl3)
d 8.18 (1H, d, J = 6.40 Hz), 7.87 (1H, d,
J = 7.52 Hz), 7.31(6H, m), 7.10 (1H, d, J = 7.89 Hz), 6.95 (1H, dd,
J = 2.62 Hz, J = 2.80 Hz), 6.54 (1H, d, J = 4.80 Hz), 6.30 (1H, s), 3.77
(3H, s), 3.53 (2H, m), 3.16 (2H, m), 1.95 (1H, m), 1.60 (4H, m),
1.36 (9H, s).
(R)-Cyclopropyl-(3-{4-[2-(3-methoxy-phenyl)-benzoimidazol-
1-yl]-pyridin-2-ylamino}-piperidin-1-yl)-methanone (7c) was
synthesized by identical method with compound 7a except for
use of compound 6h (74 mg, 0.15 mmol) instead of compound
6c. Purification by flash column chromatography with EA/Hex
(2:1) as eluent produced compound 7c as pale yellow solid
(53%); 1H NMR (400 MHz, CDCl3) d 8.85 (1H, s), 8.19 (1H, s), 7.92
(1H, d, J = 8.00 Hz), 7.88 (1H, d, J = 7.60 Hz), 7.56 (1H, m), 7.35
(3H, m), 6.95 (2H, m), 6.54 (1H, br), 6.28 (1H, s), 3.78 (3H, s),
3.73 (1H, m), 3.48 (2H, m), 2.33 (2H, m), 1.68 (4H, m), 0.87 (5H, m).
(R)-Cyclopropyl-(3-{4-[2-(3-hydroxy-phenyl)-benzoimidazol-
1-yl]-pyridin-2-ylamino}-piperidin-1-yl)-methanone (8e) was syn-
thesized by identical method with compound 8a except for using
compound 7c (36 mg, 0.077 mmol) instead of compound 6e as sub-
strate. Purification by flash column chromatography with CH2Cl2/
MeOH (5:1)as eluent produced compound 8e as pale yellow solid
(55%); 1H NMR (400 MHz, CDCl3) d 8.92 (1H, s), 8.03 (1H, d,
J = 5.65 Hz), 7.88 (1H, d, J = 7.60 Hz), 7.52 (1H, d, J = 4.40 Hz), 7.33
(4H, m), 6.88 (1H, d, J = 7.60 Hz), 6.81 (1H, s), 6.73 (1H, s), 6.32
(1H, d, J = 4.80 Hz), 4.72 (1H, m), 4.09–4.15 (1H, m, mixed with
EtOAc), 3.78 (1H, m), 3.39 (1H, m), 2.99 (1H, m), 2.06 (1H, m, mixed
with EtOAc), 1.72–1.85 (3H, m), 0.86–1.05 (5H, m); HRMS (ESI)
calcd for C27H28N5O2 [M+H]+: 454.2238, found 454.2238.
6.11. Synthesis of (S)-3-{1-[2-(2-hydroxy-propylamino)-pyridin-
4-yl]-1H-benzoimidazol-2-yl}-phenol (8f)
(S)-1-{4-[2-(3-Methoxy-phenyl)-benzoimidazol-1-yl]-pyridin-
2-ylamino}-propan-2-ol (6i) was synthesized by identical method
with compound 6h except for using compound 5d (20 mg,
0.08 mmol, 1 equiv). Purification by flash column chromatography
with EA/Hex (1:1) as the eluent produced compound 6i as pale
pink solid (41%); 1H NMR (400 MHz, CDCl3) d 8.12 (1H, d,
J = 5.60 Hz), 7.91 (2H, m), 7.36 (3H, m), 7.24 (2H, d, J = 8.21 Hz),
7.08 (1H, d, J = 7.76 Hz), 6.96 (1H, dd, J = 2.58 Hz, J = 2.58 Hz),
6.55 (1H, dd, J = 1.69 Hz, J = 1.70 Hz), 6.40 (1H, d, J = 1.22 Hz),
5.43 (1H, s), 3.97 (1H, m), 3.79 (3H, s), 3.44 (1H, m), 3.26 (1H,
m), 1.22 (3H, d, J = 6.29 Hz).
6.9. Synthesis of (R)-cyclopropyl-{3-[4-(5-hydroxy-2-
naphthalen-2-yl-benzoimidazol-1-yl)-pyridin-2-ylamino]-
piperidin-1-yl}-methanone (8d)
(R)-Cyclopropyl-{3-[4-(5-methoxy-2-naphthalen-2-yl-ben-
zoimidazol-1-yl)-pyridin-2-ylamino]-piperidin-1-yl}-methanone
(7b) was synthesized by identical method with compound 7a ex-
cept for use of compound 6g (19 mg, 0.035 mmol) instead of com-
pound 6c. Purification by flash column chromatography with EA/
Hex (2:1) as eluent produced compound 7b as pale yellow solid
(60%);
(R)-Cyclopropyl-{3-[4-(5-hydroxy-2-naphthalen-2-yl-ben-
zoimidazol-1-yl)-pyridin-2-ylamino]-piperidin-1-yl}-methanone
(8d) was synthesized by identical method with compound 8a ex-
cept for using compound 7b (11 mg, 0.021 mmol) instead of
compound 6e as substrate. Purification by flash column chroma-
tography with EA/Hex (2:1) as eluent produced compound 8d as
white solid (37%).
Compound 8f was synthesized by identical method with com-
pound 8a except for using compound 6i (10 mg, 0.026 mmol) in-
stead of compound 6e as substrate. Purification by flash column
chromatography with CH2Cl2/MeOH (5:1) as eluent produced
compound 8e as pale yellow solid (64%); 1H NMR (300 MHz,
CD3OD) d 8.07 (1H, d, J = 2.60 Hz), 7.77 (1H, d, J = 8.86 Hz),
7.38 (1H, m), 7.38 (2H, m), 7.24 (1H, t, J = 7.56 Hz), 7.03 (1H,
m), 6.91 (1H, d, J = 6.00 Hz) 6.68 (1H, s), 6.57 (1H, m), 5.57
(1H, s), 3.54 (1H, m), 3.43 (1H, m), 2.73 (1H, m), 1.01 (3H, d,
J = 4.02 Hz); HRMS (ESI) calcd for
C
21H21N4O2 [M+H]+:
361.1659, found 361.1660.

M. Kim et al. / Bioorg. Med. Chem. 21 (2013) 2271–2285
2281
6.12. Synthesis of 1-(2-cyclohexylamino-pyridin-4-yl)-2-(3,4-
dichloro-phenyl)-1H-benzoimidazol-5-ol (8g)
mixture was cooled up to ambient temperature and concentrated
in vacuo. The crude product was purified by flash column chroma-
tography with EA/Hex (1:1) as the eluent to produce cyclohexyl-
[4-(2-naphthalen-2-yl-benzoimidazol-1-yl)-pyrimidin-2-yl]-ami-
ne(13a) as white solid (73%); 1H NMR (400 MHz, CDCl3) d 8.20–
8.23 (2H, m), 7.80–7.90 (5H, m), 7.48–7.56 (3H, m), 7.32–7.40
(2H, m), 6.37 (1H, br, s), 5.31 (1H, br, s), 3.23–3.47 (1H, m), 2.02–
2.15 (1H, m), 1.46–1.76 (5H, m), 0.91–1.18 (4H, m);HRMS (ESI)
calcd for C27H26N5 [M+H]+: 420.2183, found 420.2179.
Compound 8g was synthesized by identical method with com-
pound 8a except for using compound 6j (9 mg, 0.019 mmol) in-
stead of compound 6e as substrate. Purification by flash column
chromatography with Hex/EtOAc (1:1)as eluent produced com-
pound 8g as white solid (54%); 1H NMR (400 MHz, DMSO-d6) d
9.34 (1H, s),8.10 (1H, d, J = 5.6 Hz), 7.82 (1H, d, J = 2.0 Hz), 7.70
(1H, d, J = 8.4 Hz), 7.44 (1H, dd, J = 8.4 Hz, J = 2.4 Hz), 7.19 (1H, d,
J = 8.8 Hz), 7.09 (1H, d, J = 2.4 Hz), 6.83 (1H, dd, J = 8.8 Hz,
J = 2.4 Hz), 6.71 (1H, d, J = 7.6 Hz), 6.45 (1H, dd, J = 5.6 Hz,
J = 1.6 Hz), 6.38 (1H, d, J = 1.6 Hz), 3.61 (2H, s), 1.83–1.85 (2H, m),
1.66–1.70 (2H, m), 1.55–1.59 (1H, m),1.23–1.32 (3H, m), 1.10–
6.14. Synthesis of [4-(2-naphthalen-2-yl-benzoimidazol-1-yl)-
pyrimidin-2-yl]-(tetrahydro-pyran-4-yl)-amine (13b)
Compound 13b was synthesized by identical method with com-
pound 13a except for use of tetrahydro-pyran-4-ylamine instead of
cyclohexylamine. The reaction time was 12 h. Purification by flash
column chromatography with Hex/EA (1:1) as eluent produced
compound 13b as white solid (71%); 1H NMR (400 MHz, CDCl3) d
8.29 (1H, br, s), 8.24 (1H, s), 7.89–7.99 (1H, m), 7.73–7.87 (4H,
m), 7.48–7.55 (3H, m), 7.33–7.41 (2H, m), 6.59 (1H, br, s), 5.40
(1H, s), 3.53–3.66 (2H, m), 3.20–3.39 (1H, m), 2.75–3.16 (2H, m),
1.14–1.63 (4H, m, mixed with H2O); HRMS (ESI) calcd for
C26H24N5O [M+H]+: 422.1975, found 422.1973.
1.19 (3H, m); HRMS (ESI) calcd for
453.1243, found 453.1239.
C
24H23Cl2N4O [M+H]+:
6.13. Synthesis of cyclohexyl-[4-(2-naphthalen-2-yl-
benzoimidazol-1-yl)-pyrimidin-2-yl]-amine (13a)
A mixture of benzene-1,2-diamine (9a) (540 mg, 5 mmol), 2-
naphthaldehyde (781 mg, 5 mmol) in MeOH (25 ml) was stirred,
and after the addition of 30% w/w H2O2 (1.2 g, 10 mmol) was
heated at 50 °C for 3 h. After completion of the reaction, the reac-
tion mixture was cooled and quenched with NaHSO3 (10 mmol).
Methanol in the reaction mixture was removed up to 1/10 of the
initial volume in vacuo. The concentrated mixture was filtered
off and washed with ethyl acetate. The combined organic layer
was concentrated in vacuo to produce crude product (10a) as a
crude brown solid (48%). Without additional purification, 2-naph-
thalen-2-yl-1H-benzoimidazole (10a) was identified by LCMS and
used in the next step; 1H NMR (500 MHz, CD3OD) d 8.57 (1H, s),
8.20 (1H, dd, J = 8.5 Hz, J = 1.5 Hz),7.70–7.74 (2H, m), 7.64–7.66
(2H, m), 7.59–7.61 (1H, m), 7.40–7.43 (1H, m), 7.33–7.36 (1H,
m), 7.21–7.24 (2H, m), 5.70 (NH, s).
6.15. Synthesis of cyclohexyl-[4-(5-methoxy-2-naphthalen-2-yl-
benzoimidazol-1-yl)-pyrimidin-2-yl]-amine (13d)
A mixture of 4-methoxy-benzene-1,2-diamine dihydrochloride
(9b) (1.0 g, 5 mmol), 2-naphthaldehyde(781 mg, 5 mmol) in
MeOH(50 ml) was stirred at 0 °C, and Et3N (1.4 mL, 10 mmol)
was subsequently added. After 30 min, H2O2 (1.2 g, 10 mmol)
was added to the reaction mixture and then the mixture was
heated at 50 °C for 3 h. After completion of the reaction, the reac-
tion mixture was cooled and quenched with NaHSO3 (10 mmol).
Methanol in the reaction mixture was removed up to 0.1 times
the initial volume in vacuo. The concentrated mixture was filtered
off and washed with ethyl acetate. The combined organic layer was
concentrated in vacuo to produce crude product, which was puri-
fied by flash column chromatography on silica gel using acetone/
Hex = 2:1 ? EA only as the eluent to produce 5-methoxy-2-naph-
thalen-2-yl-1H-benzoimidazole (10b) as brown solid (ratio of
two tautomer 10:1 in DMSO, 33%); 1H NMR (400 MHz, DMSO-d6)
d 12.92 (1H, s), 10.20 (1H, s),8.68 (1H, s), 8.28 (1H, dd, J = 8.8 Hz,
J = 1.6 Hz), 8.06 (1H, d, J = 8.8 Hz), 8.02–8.04 (1H, m), 7.97–7.99
(1H, m), 7.56–7.62 (2H, m), 6.86 (1H, dd, J = 8.8 Hz, J = 2.0 Hz),
7.52–7.55 (1H, m), 7.10 (1H, br s), 3.83 (3H, s).
5-Methoxy-1-(2-methylsulfanyl-pyrimidin-4-yl)-2-naphtha-
len-2-yl-1H-benzoimidazole (11b) and 6-methoxy-1-(2-meth-
ylsulfanyl-pyrimidin-4-yl)-2-naphthalen-2-yl-1H-benzoimidazole
(11c) were synthesized by identical method with compound 11a
except for use of compound 10b (288 mg, 1.0 mmol) instead of
compound10a. Purification by flash column chromatography with
Hex/acetone (3:1) as eluent produced the mixture with compound
11b and 11c (ratio 1:1) as white solid (80%); 1H NMR (400 MHz,
CDCl3) d 8.68 (1H, d, J = 5.6 Hz), 8.65 (1H,d, J = 5.6 Hz), 8.22 (1H,
d, J = 1.2 Hz), 8.19 (1H, d, J = 1.2 Hz), 7.95–8.00 (6H, m), 7.80 (1H,
d, J = 8.8 Hz), 7.75 (1H, d, J = 8.8 Hz), 7.54–7.64 (6H, m), 7.41 (1H,
d, J = 2.4 Hz), 7.39 (1H, d, J = 2.4 Hz), 7.11 (1H, d, J = 5.6 Hz), 7.03–
7.06 (3H, m), 3.86 (3H, s), 3.84 (3H, s), 2.28 (3H, s), 2.27 (3H, s).
1-(2-Methanesulfonyl-pyrimidin-4-yl)-5-methoxy-2-naphtha-
len-2-yl-1H-benzoimidazole (12b) and 1-(2-methane sulfonyl-
pyrimidin-4-yl)-6-methoxy-2-naphthalen-2-yl-1H-benzoimidaz-
ole (12c) were synthesized by identical method with compound
12a except for use of the mixture with compound 11b and 11c (ra-
tio 1:1, 200 mg, 0.51 mmol) instead of compound 11a. Purification
by flash column chromatography on silica gel using EA/Hex (1:1)
The mixture of compound 10a (317 mg, 1.3 mmol), 4-iodo-2-
(methylthio)pyrimidine (320 mg, 1.3 mmol), Pd2(dba)3 (140 mg,
0.13 mmol), BINAP (81 mg, 0.13 mmol) and NaOtBu (242 mg,
2.5 mmol) was purged with N2, and then toluene (13 mL) was
added to the mixture. After sonication under N2 for 5 min, the mix-
ture was heated up to 130 °C under N2 and was stirred without N2
at 130 °C for 3 h. After cooling down to ambient temperature, the
reaction mixture was filtered through a celite pad, removed in va-
cuo, and purified by flash column chromatography on silica gel
using EA/Hex (1:1) as the eluent. This produced a mixture of 1-
(2-methylsulfanyl-pyrimidin-4-yl)-2-naphthalen-2-yl-1H-benzo-
imidazole (11a) and 10a (ratio 2:1 in 1H NMR) as a pale yellow so-
lid (66%); 1H NMR (400 MHz, CDCl3) of 11a mixed with 10a d 8.40
(H, d, J = 5.6 Hz), 8.22–8.23 (1H,m), 7.86–7.96 (6H, m), 7.50–7.59
(2H, m), 7.41–7.44 (2H, m), 6.60 (1H, d, J = 5.6 Hz), 2.49 (3H, s).
The mixture of compound 11a (mixture 170 mg, 0.51 mmol)
and 70% m-CPBA (360 mg, 1.5 mmol) in methylene chloride
(5 ml) was stirred at ambient temperature for 8 h. After compound
11a disappeared in LCMS, the reaction mixture was extracted with
ethyl acetate and washed with saturated NaHCO3 (aq). The ex-
tracted organic layer was dried usingMgSO4and filtered. The fil-
trate was concentrated in vacuo and then the crude product was
purified by flash column chromatography with EA/Hex 1:1 to pro-
duce1-(2-methanesulfonyl-pyrimidin-4-yl)-2-naphthalen-2-yl-
1H-benzoimidazole (12a)as
a
white solid (52%); 1H NMR
(400 MHz, CDCl3) d 8.65 (1H, d, J = 5.6 Hz), 8.19 (1H, s), 8.12–8.14
(1H, m), 7.84–7.89 (4H, m), 7.52–7.60 (2H, m), 7.41–7.50 (3H,
m), 7.03 (1H, d, J = 5.6 Hz), 3.25 (3H, s).
The mixture of compound 12a (13 mg, 0.033 mmol) and cyclo-
hexylamine (8
ll, 0.066 mmol) in THF (0.25 ml) was stirred at60 °C
for 5 h. After compound 12a disappeared completely, the reaction

2282
M.-h. Kim et al. / Bioorg. Med. Chem. 21 (2013) 2271–2285
as eluent and repurification by ptlc (the scale of 30 mg) produced
compounds 12b and 12c separated by as white solid (ca. 80 mg
of 12b and ca. 75 mg of 12c, 72%);1H NMR (400 MHz, CDCl3) of
12b d 8.60 (1H, d, J = 1.6 Hz), 8.18 (1H, s), 8.08 (1H, d, J = 8.8 Hz),
7.85–7.90 (3H, m), 7.50–7.61 (3H, m), 7.35 (1H, d, J = 2.4 Hz),
7.05 (1H, dd, J = 8.8 Hz, J = 2.4 Hz), 6.96 (1H, d, J = 5.6 Hz), 3.90
(3H, s), 3.28 (3H, s); 1H NMR (400 MHz, DMSO-d6) of 12c d 9.01
(1H, d, J = 5.6 Hz), 8.26 (1H, d, J = 1.6 Hz), 8.01 (1H, d, J = 8.4 Hz),
7.77 (1H, d, J = 8.4 Hz), 7.73 (1H, d, J = 2.4 Hz), 7.57–7.65 (3H, m),
7.47 (1H, d, J = 5.6 Hz), 7.08 (1H, dd, J = 8.4 Hz, J = 2.4 Hz), 3.85
(3H, s), 3.30 (3H, s).
170 mg, 0.43 mmol) instead of compound11a. Purification by flash
column chromatography with EA/Hex (1:1)as eluent produced
compound 12d as white solid (61%); 1H NMR (400 MHz, CDCl3) d
8.87 (1H, d, J = 5.6 Hz), 8.07–8.11 (1H, m), 7.88–7.91 (1H, m),
7.83 (1H, d, J = 2.0 Hz), 7.54 (1H, d, J = 8.4 Hz), 7.45–7.49 (2H, m),
7.34 (1H, dd, J = 8.4 Hz, J = 2.0 Hz), 7.13 (1H, d, J = 5.6 Hz), 3.36
(3H, s).
{4-[2-(3,4-Dichloro-phenyl)-benzoimidazol-1-yl]-pyrimidin-2-
yl}-(tetrahydro-pyran-4-yl)-amine (13j) was synthesized by the
same method with compound 13a except for use of compound
12d (15 mg, 0.036 mmol). Purification by flash column chromatog-
raphy with Hex/acetone (2:1) as eluent produced compound 13j as
a white solid (80%);1H NMR (400 MHz, DMSO-d6) d 8.53 (1H, s),
7.82–7.84 (2H, m), 7.74 (1H, d, J = 8.0 Hz), 7.67–7.76 (1H, m), 7.60
(1H, d, J = 8.0 Hz), 7.46 (1H, d, J = 8.0 Hz), 7.37–7.42 (2H, m), 6.92
(1H, s), 3.73 (3H, br, s), 3.09–3.24 (2H, m), 1.23–1.39 (4H, m); HRMS
(ESI) calcd for C22H20Cl2N5O [M+H]+: 440.1039, found 440.1036.
Compound 12d as a white solid (61%);1H NMR (400 MHz,
CDCl3) d 8.87 (1H, d, J = 5.6 Hz), 8.07–8.11 (1H, m), 7.88–7.91
(1H, m), 7.83 (1H, d, J = 2.0 Hz), 7.54 (1H, d, J = 8.4 Hz), 7.45–7.49
(2H, m), 7.34 (1H, dd, J = 8.4 Hz, J = 2.0 Hz), 7.13 (1H, d,
J = 5.6 Hz), 3.36 (3H, s).
Cyclohexyl-[4-(5-methoxy-2-naphthalen-2-yl-benzoimidazol-
1-yl)-pyrimidin-2-yl]-amine (13d) was synthesized by identical
method with compound 13a except for use of compound 12b
(28 mg, 0.066 mmol). The reaction terminated after 12 h. Purifica-
tion by flash column chromatography with Hex/acetone
(1:1) ? EA only as eluent produced compound 13a as white solid
(80%); 1H NMR (400 MHz, CDCl3) d 8.21 (1H, s), 8.18 (1H, d,
J = 5.2 Hz), 7.78–7.86 (3H, m), 7.71 (1H, d, J = 8.8 Hz), 7.49–7.55
(3H, m), 7.36 (1H, d, J = 2.4 Hz), 6.98 (1H, dd, J = 8.8 Hz,
J = 2.4 Hz), 6.31 (1H, br, s), 5.24 (1H, d, J = 7.6 Hz), 3.89 (3H, s),
3.29–3.52 (1H, m), 1.40–1.80 (5H, m), 1.04–1.16 (5H, m); HRMS
(ESI) calcd for C28H28N5O [M+H]+: 450.2288, found 450.2285.
6.18. Synthesis of (R)-[4-(2-Naphthalen-2-yl-benzoimidazol-1-
yl)-pyrimidin-2-yl]-piperidin-3-yl-amine
(R)-3-[4-(2-Naphthalen-2-yl-benzoimidazol-1-yl)-pyrimidin-2-
ylamino]-piperidine-1-carboxylic acid tert-butyl ester (13c) was
synthesized by identical method with compound 13a except for
use of (R)-tert-butyl-3-aminopiperidine-1-carboxylate instead of
cyclohexylamine. The reaction time was 12 h. Purification by flash
column chromatography with Hex/acetone (2:1) as eluent pro-
duced compound 13c as white solid (41%) and recovered substrate
12a (50%); 1H NMR (400 MHz, CDCl3) d 8.32 (1H, br, s),8.17 (1H, br,
s), 7.85–8.00 (5H, m), 7.46–7.60 (5H, m), 6.38 (1H, br, s), 4.61 (1H,
br, s), 3.73 (1H, m), 3.62 (1H, m), 3.44–4.46 (1H, m), 3.35 (1H, m).
3.20 (1H, m), 1.79–1.84 (2H, m), 1.60–1.64 (2H, m), 1.45 (9H, s).
Compound 13c (20 mg, 0.038 mmol) was dissolved in 0.38 ml of
1,4-dioxane and treated with 0.19 ml of 4 M-HCl in 1,4-dioxane at
ambient temperature. The reaction mixture was stirred at that
temperature for 20 min. The mixture was then diluted with ether
and stirred until the product separated as a solid. The solid product
was filtered and washed off with ether followed by hexane. The
crude product was subsequently crystallized as a pale yellow solid
(63%);1H NMR (400 MHz, DMSO-d6) d 9.03–9.38 (1H, m), 8.48–8.68
(1H, m), 8.27 (1H, s), 7.98–8.01 (3H, m), 7.85–7.87 (1H, m), 7.79
(1H, d,, J = 7.2 Hz), 7.58–7.65 (3H, m), 7.40–7.45 (2H, m), 6.41–
6.77 (1H, m), 4.24 (1H, br s), 2.87–3.19 (2H, m), 2.67–2.78 (3H,
6.16. Synthesis of cyclohexyl-[4-(6-methoxy-2-naphthalen-2-yl-
benzoimidazol-1-yl)-pyrimidin-2-yl]-amine (13g)
Compound 13g as white solid (94%) was synthesized by identi-
cal method with compound 13a except for use of compound 12c;
1H NMR (400 MHz, CDCl3) d 8.20 (1H, s), 8.19 (1H, d, J = 5.2 Hz),
7.85 (2H, dd, J = 8.8 H, J = 6.4 H), 7.81 (1H, d, J = 8.8 Hz), 7.57 (1H,
d, J = 8.8 Hz), 7.48–7.57 (3H, m), 7.35 (1H, d, J = 2.4 Hz), 7.02 (1H,
dd, J = 8.8 Hz, J = 2.4 Hz), 6.28 (1H, s), 5.34 (1H, s), 3.88 (3H, s),
3.46–3.60 (1H, m), 1.69–1.88 (2H, m), 1.45–1.64 (3H, m), 1.07–
1.16 (5H, m); HRMS (ESI) calcd for C28H28N5O [M+H]+: 450.2288,
found 450.2282.
6.17. Synthesis of {4-[2-(3,4-dichloro-phenyl)-benzoimidazol-1-
yl]-pyrimidin-2-yl}-(tetrahydro-pyran-4-yl)-amine (13j)
m), 1.60–1.98 (4H, m); HRMS (ESI) calcd for
C26H25N6
[M+H]+:421.2135, found 421.2141.
2-(3,4-Dichloro-phenyl)-1H-benzoimidazole (10c) was synthe-
sized by identical method with compound 10a except for use of
3,4-dichlorobenzylaldehyde. Purification by flash column chroma-
tography with acetone/Hex = 2:1 ? EA only as the eluent produced
the compound 10c (tautomer ratio 1:0.18) as pale yellow solid
(44%); 1H NMR (400 MHz, DMSO-d6) d 8.41 (1H, d, J = 2.0 Hz),
8.16 (1H, dd, J = 8.8 Hz, J = 2.4 Hz), 7.85 (1H, d, J = 8.8 Hz), 7.61–
7.66 (2H, m), 7.24–7.28 (2H, m), 5.75 (1H, s).
2-(3,4-Dichloro-phenyl)-1-(2-methylsulfanyl-pyrimidin-4-yl)-
1H-benzoimidazole (11d) was synthesized by identical method
with compound 11a except for use of compound 10c (103 mg,
0.39 mmol) instead of compound 10a. Purification by flash column
chromatography with acetone/Hex (1:3) as eluent produced the
mixture with compound 11d and 10c as pale yellow solid (4:1
11d and 10c ratio in CDCl3, 80%); 1H NMR (400 MHz, CDCl3) d
8.54 (1H, d, J = 5.6 Hz), 7.85–7.87 (1H, m), 7.79–7.82 (2H, m),
7.46 (1H, d, J = 8.8 Hz), 7.38–7.41 (2H, m), 7.30 (1H, dd, J = 8.8 Hz,
J = 2.0 Hz), 6.70 (1H, d, J = 5.6 Hz), 2.46 (3H, s).
6.19. Synthesis of Cyclopropyl-{3-[4-(2-naphthalen-2-yl-
benzoimidazol-1-yl)-pyrimidin-2-ylamino]-piperidin-1-yl}-
methanone
Compound 15a were synthesized by the same method with
compound 7a except for use of compound 13c (21 mg, 0.04 mmol).
Purification by flash column chromatography with EA/Hex = 1:1 as
the eluent produced compound 15a as white solid (72%); 1H NMR
(400 MHz, CDCl3) d 8.25 (2H, br, s), 7.82–7.94 (5H, m), 7.50–7.57
(3H, m), 7.39–7.43 (2H, m), 6.35–6.49 (1H, m), 5.33–5.81 (1H,
m), 3.42–3.96 (5H, m), 1.98–2.12 (2H, m), 1.64–1.73 (2H, m),
1.56–1.62 (1H, m), 0.81–1.04 (1H, m), 0.78–0.96 (4H, m); HRMS
(ESI) calcd for C30H29N6O [M+H]+: 489.2397, found 489.2396.
6.20. Synthesis of (R)-cyclopropyl-{3-[4-(5-methoxy-2-
naphthalen-2-yl-benzoimidazol-1-yl)-pyrimidin-2-ylamino]-
piperidin-1-yl}-methanone (15b)
2-(3,4-Dichloro-phenyl)-1-(2-methanesulfonyl-pyrimidin-4-
yl)-1H-benzoimidazole (12d) was synthesized by identical method
with compound 12a except for use of compound 11d (mixture
(R)-3-[4-(5-Methoxy-2-naphthalen-2-yl-benzoimidazol-1-yl)-
pyridin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester

M. Kim et al. / Bioorg. Med. Chem. 21 (2013) 2271–2285
2283
(13f) was synthesized by identical method with compound 13c ex-
cept for use of compound 12b (43 mg, 0.1 mmol). The reaction
time up to limit of substrate consumption was 12 h. Purification
by flash column chromatography on silica gel using Hex/EtOAc
(1:1) as eluent produced compound 13f as white solid (35%) and
recovered substrate 12b (65%); 1H NMR (400 MHz, CDCl3) d 8.20
(1H, s), 8.16 (1H, d, J = 5.2 Hz), 7.78–7.84 (3H, m), 7.73–7.74 (1H,
m), 7.49–7.55 (3H, m), 7.36 (1H, d, J = 2.4 Hz), 7.05 (1H, dd,
J = 8.8 Hz, J = 2.4 Hz), 6.24 (1H, br, s), 5.42 (1H, br s), 3.87 (3H, s),
3.61–3.75 (2H, m), 3.36–3.45 (1H, m), 3.27–3.30 (2H, m), 1.38–
1.66 (4H, m), 1.44 (9H, s).
(R)-Cyclopropyl-{3-[4-(5-methoxy-2-naphthalen-2-yl-ben-
zoimidazol-1-yl)-pyrimidin-2-ylamino]-piperidin-1-yl}-metha-
none (15b) was synthesized by identical method with compound
7a except for use of compound 13f (19 mg, 0.035 mmol). Purifica-
tion by flash column chromatography on silica gel using EA/
Hex = 2:1 as the eluent produced compound 15b as white solid
(74%); 1H NMR (400 MHz, CDCl3) d 8.18–8.21 (2H, m), 7.84–7.86
(2H, m), 7.69–7.79 (1H, m),7.49–7.56 (3H, m), 7.37 (1H, d,
J = 2.4 Hz), 6.98–7.02 (1H, m), 6.18–6.42 (1H, m), 5.44 (1H, br,
s),3.90 (3H, s), 3.55–3.78 (2H, m), 3.19–3.46 (3H, m), 1.39–1.72
(5H, m), 0.69–0.94 (4H, m); HRMS (ESI) calcd for C31H31N6O2
[M+H]+: 519.2503, found 519.2500.
3.66–3.69 (2H, m), 3.42–3.47 (1H, m), 3.26–3.36 (2H, m), 1.88
(2H, br, s), 1.62 (2H, br, s), 1.41 (9H, s).
(R)-Cyclopropyl-(3-{4-[2-(3,4-dichloro-phenyl)-benzoimidazol-
1-yl]-pyrimidin-2-ylamino}-piperidin-1-yl)-methanone (15d) was
synthesized by identical method with compound 7a except for
use of compound 13k (16 mg, 0.031 mmol). Purification by flash
column chromatography with EA/Hex = 2:1 ? 1:1 as the eluent
produced compound 15d as white solid (60%); 1H NMR
(400 MHz, CDCl3) d 8.36 (1H, br, s), 7.87 (1H, d, J = 8.0 Hz), 7.70–
7.76 (2H, m), 7.48 (1H, d, J = 8.0 Hz), 7.39–7.43 (3H, m), 6.48 (1H,
br, s), 5.35–5.80 (1H, m), 4.04–4.14 (1H, m), 3.33–3.80 (4H, m),
2.01–2.14 (1H, m), 1.76 (2H, br, s), 1.57 (2H, br, s), 0.80–0.99
(4H, m); HRMS(ESI) calcd for C26H25Cl2N6O [M+H]+: 507.1461,
found 507.1473.
6.23. Synthesis of 1-(2-cyclohexylamino-pyrimidin-4-yl)-2-
naphthalen-2-yl-1H-benzoimidazol-5-ol (16a)
Compound 16a was synthesized by identical method with com-
pound 8a except for using compound 13d (12 mg, 0.027 mmol) in-
stead of compound 6e as substrate. Purification by flash column
chromatography with CH2Cl2/MeOH (40:1)as eluent produced
compound 16a as white solid (52%); 1H NMR (400 MHz, DMSO-
d6) d 9.35 (1H, s), 8.40 (1H, s), 8.21 (1H, s), 7.95–8.00 (2H, m),
7.52–7.80 (4H, m), 7.29 (1H, s), 7.14 (1H, s), 6.87 (1H, dd,
J = 8.8 Hz, J = 2.4 Hz), 6.71 (1H, s), 5.22 (1H, br, s), 2.90 (1H, br, s),
1.15–1.25 (6H, m), 0.67–0.91 (4H, m);HRMS(ESI) calcd for
6.21. Synthesis of (R)-cyclopropyl-{3-[4-(6-methoxy-2-
naphthalen-2-yl-benzoimidazol-1-yl)-pyrimidin-2-ylamino]-
piperidin-1-yl}-methanone (15c)
C
27H26N5O [M+H]+: 436.2132, found 436.2127.
(R)-3-[4-(6-Methoxy-2-naphthalen-2-yl-benzoimidazol-1-yl)-
pyridin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester
(13i) as white solid (35%) was synthesized by identical method
with compound 13f except for use of compound 12c; 1H NMR
(400 MHz, CDCl3) d 8.19 (1H, d, J = 5.2 Hz), 8.18 (1H, s), 7.81–7.85
(2H, m), 7.79 (1H, d, J = 8.8 Hz), 7.75 (1H, d, J = 8.8 Hz), 7.49–7.54
(3H, m), 7.34 (1H, d, J = 2.4 Hz), 6.99 (1H, dd, J = 8.8 Hz,
J = 2.4 Hz), 6.26 (1H, s), 5.44 (1H, br, s), 3.91 (3H, s), 3.62–3.75
(2H, m), 3.25–3.50 (3H, m), 1.44 (9H, s), 1.22–1.61 (4H, m).
(R)-Cyclopropyl-{3-[4-(6-methoxy-2-naphthalen-2-yl-ben-
6.24. Synthesis of 1-(2-cyclohexylamino-pyrimidin-4-yl)-2-
naphthalen-2-yl-1H-benzoimidazol-6-ol (16b)
Compound 16b was synthesized by identical method with com-
pound 8a except for using compound 13g (14 mg, 0.027 mmol) in-
stead of compound 6e as substrate and 10 equiv of
borontribromde. Purification by flash column chromatography
with CH2Cl2/MeOH (40:1) as eluent produced compound 16b as
white solid (13%) and recovered substrate 13g (20%); 1H NMR
(400 MHz, DMSO-d6) d 8.42 (1H, s), 8.15 (1H, s), 7.93–7.95 (4H,
m), 7.28–7.62 (5H, m), 7.07 (1H, s), 6.85 (1H, dd, J = 8.8 Hz,
J = 2.4 Hz), 6.72 (1H, –NH, s), 2.89 (1H, br, s), 1.23 (6H, m), 0.66–
0.85 (4H, m);HRMS(ESI) calcd for C27H26N5O [M+H]+: 436.2132,
found 436.2128.
zoimidazol-1-yl)-pyrimidin-2-ylamino]-piperidin-1-yl}-metha-
none (15c) was synthesized by identical method with compound
7a except for use of compound 13i (22 mg, 0.04 mmol). Purifica-
tion by flash column chromatography on silica gel using EA/
Hex = 2:1 as the eluent produced compound 15c as white solid
(59%); 1H NMR (400 MHz, CDCl3) d 8.20 (1H, s), 8.19 (1H, s),7.80–
7.85 (3H, m), 7.78 (1H, d, J = 8.8 Hz), 7.51–7.54 (3H, m), 7.30 (1H,
s), 7.02 (1H, dd, J = 8.8 Hz, J = 2.4 Hz), 6.20–6.38 (1H, m), 5.47
(1H, br, s), 3.87 (3H, s), 3.69–3.89 (2H, m), 3.40–3.62 (2H, m),
3.12–3.31 (1H, m), 1.54–1.78 (5H, m), 0.93–0.97 (2H, m), 0.72–
6.25. Synthesis of 2-naphthalen-2-yl-1-[2-(tetrahydro-pyran-4-
ylamino)-pyrimidin-4-yl]-1H-benzoimidazol-5-ol (16c)
[4-(5-Methoxy-2-naphthalen-2-yl-benzoimidazol-1-yl)-pyrim-
idin-2-yl]-(tetrahydro-pyran-4-yl)-amine (13e) was synthesized
by identical method with compound 13b except for use of com-
pound 12b (28 mg, 0.062 mmol). The reaction terminated after
4 h. Purification by flash column chromatography with Hex/ace-
tone (2:1) as eluent produced compound 13e as white solid
(86%); 1H NMR (400 MHz, CDCl3) d 8.26 (1H, s), 8.20 (1H, d,
J = 1.6 Hz), 7.76–7.85 (3H, m), 7.63 (1H, s), 7.49–7.54 (3H, m),
7.36 (1H, d, J = 2.4 Hz), 6.98 (1H, dd, J = 8.8 Hz, J = 2.4 Hz), 6.34
(1H, br, s), 5.34 (1H, d, J = 7.6 Hz), 3.89 (3H, s), 3.49–3.62 (2H, m),
3.24–3.42 (1H, m), 2.80–3.03 (2H, m), 1.07–1.46 (4H, m, mixed
with EtOAc peak).
2-Naphthalen-2-yl-1-[2-(tetrahydro-pyran-4-ylamino)-pyrimi-
din-4-yl]-1H-benzoimidazol-5-ol (16c) was synthesized by identi-
cal method with compound 8a except for using compound
13e(24 mg, 0.053 mmol) instead of compound 6e as substrate.
Purification by flash column chromatography with CH2Cl2/MeOH
(40:1 ? 5:1 gradient) as eluent produced compound 16c as white
solid (23%); 1H NMR (400 MHz, DMSO-d6) d 9.34 (1H, s), 8.45 (1H,
0.75 (2H, m); HRMS (ESI) calcd for
519.2503, found 519.2500.
C
31H31N6O2 [M+H]+:
6.22. Synthesis of (R)-cyclopropyl-(3-{4-[2-(3,4-dichloro-
phenyl)-benzoimidazol-1-yl]-pyrimidin-2-ylamino}-piperidin-
1-yl)-methanone (15d)
(R)-3-{4-[2-(3,4-Dichloro-phenyl)-benzoimidazol-1-yl]-pyrimi-
din-2-ylamino}-piperidine-1-carboxylic acid tert-butyl ester
(13k)was synthesized by identical method with compound 13c ex-
cept for use of compound 12d (33 mg, 0.08 mmol). The reaction
time up to limit of substrate consumption was 10 hr. Purification
by flash column chromatography with Hex/acetone (2:1) as eluent
produced compound 13k as white solid (40%) and recovered sub-
strate 12d (50%); 1H NMR (400 MHz, CDCl3) d 8.36 (1H, d,
J = 4.8 Hz), 7.84–7.87 (2H, m), 7.74–7.76 (1H, m), 7.46 (1H, d,
J = 8.0 Hz), 7.33–7.41 (3H, m), 6.42 (1H, br, s), 5.62 (1H, br, s),

2284
M.-h. Kim et al. / Bioorg. Med. Chem. 21 (2013) 2271–2285
s), 8.21 (1H, s), 7.92–8.00 (3H, m), 7.37–7.61 (5H, m), 7.13 (1H, d,
J = 2.0 Hz), 6.86 (1H, dd, J = 8.8 Hz, J = 2.0 Hz), 4.78 (1H, br, s),
3.00–3.03 (1H, m), 2.60–2.66 (1H, m), 1.96 (1H, d, J = 13.6 Hz),
1.75–1.78 (1H, m), 1.45 (1H, s), 1.23–1.34 (2H, m), 1.14 (2H, m);
HRMS(ESI) calcd for
438.1918.
105.4, 97.5, 49.1, 48.0, 45.1, 29.6, 22.9, 10.5, 6.9 ppm; HRMS (ESI)
calcd for C30H29N6O2 [M+H]+: 505.2347, found 505.2340
7. Preparation of JNK3 and Kd measurement with JNK 3
C
26H24N5O2 [M+H]+: 438.1925, found
JNK3 was cloned, expressed and purified as described in Xie
et al.13 Briefly, a DNA fragment encoding the catalytic domain of
human JNK3 spanning from S40 to E402 was amplified by PCR
and sub cloned into the pET15b (Merch, Germany) vector, which
has Nco I (NEB, MA, USA) and BamH I (NEB, MA, USA) restriction
sites. A recombinant protein containing an N-terminal 6 histidine
tag and the tobacco etch viral protease (TEV) cleavage site was ex-
pressed in Escherichia coli BL21(DE3). Ni-NTA affinity chromatog-
raphy and ion-exchange chromatography using Mono-Q resin were
conducted for purification of the His-tagged fusion protein. After
TEV cleavage, the catalytic domain of JNK3 was purified by size-
exclusion chromatography. The final yield of the protein was more
than 10 mg from 1 l of culture. As a direct binding assay for JNK3,
surface Plasmon resonance (SPR) measurements were performed
on a ProteOn™ XPR36 instrument (Bio-Rad Laboratories Inc.). All
reagents and buffers used were purchased from Bio-Rad Laborato-
ries Inc. Kinase was synthesized by a method described in the lit-
erature.6 JNK3 was immobilized (average 25,000RU) by standard
amine coupling chemistry on a GLH chip. Binding experiments
were performed using PBST buffer (phosphate buffered saline, pH
7.4, 0.005% Tween 20) as the running buffer. Compounds were dis-
solved in 10 mM DMSO and subsequently diluted with PBST buffer
to produce a 1% DMSO solution. In order to calculate Kd value, each
6.26. Synthesis of 2-naphthalen-2-yl-1-[2-(tetrahydro-pyran-4-
ylamino)-pyrimidin-4-yl]-1H-benzoimidazol-6-ol (16d)
[4-(6-Methoxy-2-naphthalen-2-yl-benzoimidazol-1-yl)-pyrim-
idin-2-yl]-(tetrahydro-pyran-4-yl)-amine (13h) as white solid
(71%) was synthesized by identical method with compound 13e
except for use of compound 12c (30 mg, 0.066 mmol); 1H NMR
(400 MHz, CDCl3) d 8.26 (1H, d, J = 4.8 Hz), 8.19 (1H, s), 7.73–7.86
(4H, m), 7.47–7.54 (3H, m), 7.24–7.26 (1H, mixed with CDCl3),
7.01 (1H, dd, J = 8.8 Hz, J = 2.4 Hz), 6.48 (1H, s), 5.57 (1H, s), 3.87
(3H, s), 3.68 (2H, d, J = 10.8 Hz), 3.47–3.66 (1H, m), 2.94–3.14
(2H, m), 1.37–1.45 (2H, m), 1.22–1.32 (2H, m, mixed with EtOAc).
2-Naphthalen-2-yl-1-[2-(tetrahydro-pyran-4-ylamino)-pyrimi-
din-4-yl]-1H-benzoimidazol-6-ol (16d) was synthesized by identi-
cal method with compound 8a except for using compound 13h
(21 mg, 0.047 mmol) instead of compound 6e as substrate and
10 equiv of borontribromde. Purification by flash column chroma-
tography with CH2Cl2/MeOH (40:1 ? 5:1 gradient)as eluent pro-
duced compound 16d as white solid (33%); 1H NMR (400 MHz,
DMSO-d6) d 9.54 (1H, s), 8.47 (1H, s), 8.16 (1H, s), 7.91–7.97 (3H,
m), 7.39–7.62 (5H, m), 7.06 (1H, s), 6.84–6.87 (1H and –NH, m),
3.03–3.07 (1H, m), 2.63–2.67(1H, m), 1.98 (1H, d, J = 14.0 Hz),
1.72–1.75 (1H, m), 1.46 (1H, s), 1.14–1.24 (2H, m), 1.02–1.10
(2H, m); HRMS(ESI) calcd for C26H24N5O2 [M+H]+: 438.1925, found
438.1919.
100
lM test compound in 1% DMSO PBST solution was serially di-
luted threefold from 100 to 0.01
lM using 1% DMSO in PBST solu-
tion to adjust the final DMSO content and the concentration of the
respective compound. Five concentrations were selected from
among the nine serial concentrations and blank PBST with 1%
DMSO was injected into six analytes in the SPR instrument. The
optimal concentration range was finally determined from at least
three measurements. To obtain a reliable deviation value of the re-
sponse unit between apo protein and binding protein with com-
pound, the baseline was standardized using inter spot references
and a blank solution containing 1% DMSO. A curve was fit to the
raw data using the Langmuir equation in ProteOn Manager™.
6.27. Synthesis of (R)-cyclopropyl-{3-[4-(5-hydroxy-2-
naphthalen-2-yl-benzoimidazol-1-yl)-pyrimidin-2-ylamino]-
piperidin-1-yl}-methanone(16e)
Compound 16e was synthesized by identical method with com-
pound 8a except for using compound 15b (12 mg, 0.023 mmol) in-
stead of compound 6e as substrate and 5 equiv of borontribromde.
Purification by flash column chromatography with CH2Cl2/MeOH
(40:1 ? 10:1 gradient)as eluent produced compound 16e as white
solid (55%); 1H NMR (400 MHz, DMSO-d6) d 9.36 (1H, s), 8.18–8.42
(2H, m), 7.94–7.96 (3H, m), 7.54–7.61 (5H, m),7.11 (1H, d,
J = 2.4 Hz), 6.84 (1H, d, J = 7.6 Hz), 6.25–6.67 (1H, m), 4.78 (1H,
br, s), 3.84–4.14 (2H, m), 2.85–3.17 (2H, m),1.91–1.97 (2H, m),
1.75 (1H, br, s), 1.14–1.45 (4H, m), 0.69–0.85 (2H, m); HRMS(ESI)
calcd for C30H29N6O2 [M+H]+: 505.2347, found 505.2343.
8. Cell-based assay
8.1. Cell culture
Human neuroblastoma SH-SY5Y cells (Korean Cell Line Bank,
Korea) were cultured at 37 °C in Dulbecco’s modified Eagle’s med-
ium (DMEM; WelGene, Korea) supplemented with 10% heat-inac-
tivated fetal bovine serum (WelGene) in a humidified 95% air, 5%
CO2 incubator. After cells reached 70–80% confluence, they were
6.28. Synthesis of (R)-cyclopropyl-{3-[4-(6-hydroxy-2-
naphthalen-2-yl-benzoimidazol-1-yl)-pyrimidin-2-ylamino]-
piperidin-1-yl}-methanone (16f)
treated with 10
treatment of 10
l
M of various JNK inhibitors for 1 h prior to the
l
g/ml anisomycin (Sigma, USA) under serum-free
conditions for 30 min. The JNK inhibitor SP600125 (Sigma) was
used as a positive control. All treatment chemicals were dissolved
in dimethyl sulfoxide (DMSO) and the final concentration of DMSO
was 0.2%.
Compound 16f was synthesized by identical method with com-
pound 8a except for using compound 15c (11 mg, 0.022 mmol)
instead of compound 6e as substrate and 10 equiv of boro-
ntribromde. Purification by flash column chromatography with
CH2Cl2/MeOH (40:1 ? 10:1 gradient) as eluent produced com-
pound 16f as white solid (62%); 1H NMR (400 MHz, DMSO-d6) d
9.52 (1H, s), 8.15–8.45 (2H, m), 7.92–7.95 (3H, m), 7.55–7.61
(5H, m), 6.84 (1H, dd, J = 8.8 Hz, J = 2.0 Hz), 6.34–6.67 (1H, m),
3.87–4.39 (3H, m), 3.30 (1H, shielded by H2O), 2.80–3.05 (1H, m),
1.98 (1H, m), 1.34–1.51 (4H, m), 0.70–0.85 (4H, m); 13C NMR
(100 MHz, DMSO-d6) d 162.2, 160.4, 157.3, 155.0, 136.1, 132.9,
132.5, 128.4 127.8, 127.6, 127.2, 126.8, 125.7, 120.1, 113.2, 113.0,
8.2. Cell-based JNK activity measurement by immunoblotting
After treatment, the cells were scraped from the plates and
lysed in ice-cold lysis buffer. The protein contents of the lysates
were determined using the Bio-Rad Protein Assay Reagent, and
equal amounts of protein were loaded into the lanes of a SDS–PAGE
gel, separated, and blotted onto a PVDF membrane (Millipore,
USA). After the membranes had been blocked with either 5% nonfat

M. Kim et al. / Bioorg. Med. Chem. 21 (2013) 2271–2285
2285
milk or bovine serum albumin, they were probed with anti-phos-
pho-c-Jun (p-Ser73; Cell Signaling, USA), anti-c-Jun (Santa Cruz
Biotechnology Inc., USA), or anti-b-actin (Sigma) as a primary anti-
body. Membranes were then incubated with a horseradish-perox-
idase-linked secondary antibody for chemiluminescent detection.
The band intensities were quantified with Quantity One software
(Bio-Rad).
All water molecules were removed from the structure and it was
selected as a template. The structures of JNK inhibitors were drawn
using Chemdraw, and their 3D conformations were generated
using the Schrödinger LigPrep program with the OPLS 2005 force
field. Molecular docking of compound 1 and 16f into the structure
ofJNK3 (PDB code: 3oy1) was carried out using Schrodinger Glide
(whole software package; Version 9.2).When making grid file, de-
fault condition was selected except for hinge hydrogen bonding
constraint. In order to acquire high docking score and more plausi-
ble docking pose, the docking result of compounds iteratively con-
ducted in the condition of SP (standard precision) and XP (extra
precision) also was followed by revision of docking pose through
substructure energy minimization using Schrodinger Macro Model.
8.3. Cell-based IC50 determination by quantitative real-time PCR
IC50 values were determined by measurement of tumor necrosis
factor-a (TNF-
analysis. SH-SY5Y cells were pre-treated for 1 h with a compound
16f (0, 0.1, 0.5, 1, 5, and 10 M) prior to stimulating with anisomy-
cin (10 g/ml) for 30 min. Total RNA was isolated using RNAiso
a) mRNA levels using SYBR Green real-time PCR
l
l
Acknowledgment
(Takara, Japan) and cDNA was synthesized using 2 g of total RNA
with the SuperScript Fist-strand Synthesis System for RT-PCR
(Invitrogen, USA) according to the manufacturers’ instructions.
For amplification of cDNA, all reactions were performed using a
StepOne™ Real Time PCR System (Applied Biosystems, USA). To
determine gene expression, the amplification reactions were per-
formed using SYBRÒ Green PCR Master Mix (Applied Biosystems)
according to the manufacturers’ protocols. PCR products were ver-
ified by melting curve analysis. Human GAPDH (glycealdehyde 3-
phosphate dehydrogenase) was a used as reference gene to nor-
malize for differences in the amount of total RNA in each sample.
This work was supported by the Hanyang University research
fund (HY-2012-N).
References and notes
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Rakic, P.; Flavell, R. A. Nature 1997, 389, 865.
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Jan 2004.
10. Kumar, R.; Joshi, Y. C. Eur. J. Chem. 2007, 4, 606.
The following primers were used: tumor necrosis factor
a (TNF-
a
: upstream primer 50-TTTGATCCCTGACATCTGGA-30, downstream
primer 50-GGCCTAAGGTCCACTTGTGT-30), GAPDH (upstream pri-
mer 50-GAGTCCACTGGCGTCTTCAC-30, downstream primer 50-
GTTCACACCCATGACGAACA-30). Relative gene expression levels
DD
were analyzed using the 2- Ct method.
11. In the case of Scheme 2, oxidative cyclization of primary diamine proceeded
without CAN. Compound 10b existed as tautomer in the ratio of 9.5:1.
12. The coupling products of compound 10b were compound 11b and compound
11c in the ratio of 1:1.
9. Docking simulations
13. Xie, X.; Gu, Y.; Fox, T.; Coll, J. T.; Fleming, M. A.; Markland, W.; Caron, P. R.;
Wilson, K. P.; Su, M. S. Structure 1998, 6, 983.
14. Bennett, B. L.; Sasaki, D. T.; Murray, B. W.; O’Leary, E. C.; Sakata, S. T.; Xu, W.;
Leisten, J. C.; Motiwala, A.; Pierce, S.; Satoh, Y.; Bhagwat, S. S.; Manning, A. M.;
Anderson, D. W. Proc. Natl. Acad. Sci. U.S.A. 1998, 13681.
The 3D X-ray protein structure of JNK3 as a complex with a li-
gand was obtained from the PDB (code: 3oy1) and prepared using
Protein Preparation Wizard of the Schrödinger Maestro program.