The design and optimization of a series of 2-(pyridin-2-yl)-1H-benzimidazole compounds as allosteric glucokinase activators

Article abstract of DOI:10.1016/j.bmc.2009.05.037

The optimization of a series of benzimidazole glucokinase activators is described. We identified a novel and potent achiral benzimidazole derivative as an allosteric GK activator. This activator was designed and synthesized via removal of the chiral center of the lead compound, 6-(N-acylpyrrolidin-2-yl)benzimidazole. The activator exhibited good PK profiles in rats and dogs, and significant hypoglycemic efficacy at 1 mg/kg po dosing in a rat OGTT model. The binding site and binding mode of the benzimidazole class of GKA with GK protein was confirmed by X-ray crystallographic analysis.

Full text of DOI:10.1016/j.bmc.2009.05.037

Bioorganic & Medicinal Chemistry 17 (2009) 7042–7051
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The design and optimization of a series of 2-(pyridin-2-yl)-1H-benzimidazole
compounds as allosteric glucokinase activators
*
Keiji Takahashi , Noriaki Hashimoto, Chisato Nakama, Kenji Kamata, Kaori Sasaki, Riki Yoshimoto,
Sumika Ohyama, Hideka Hosaka, Hiroko Maruki, Yasufumi Nagata, Jun-ichi Eiki, Teruyuki Nishimura
Banyu Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd, 3 Okubo, Tsukuba, Ibaraki 300-2611, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The optimization of a series of benzimidazole glucokinase activators is described. We identified a novel
and potent achiral benzimidazole derivative as an allosteric GK activator. This activator was designed and
synthesized via removal of the chiral center of the lead compound, 6-(N-acylpyrrolidin-2-yl)benzimid-
azole. The activator exhibited good PK profiles in rats and dogs, and significant hypoglycemic efficacy
at 1 mg/kg po dosing in a rat OGTT model. The binding site and binding mode of the benzimidazole class
of GKA with GK protein was confirmed by X-ray crystallographic analysis.
Received 8 April 2009
Revised 14 May 2009
Accepted 15 May 2009
Available online 21 May 2009
Keywords:
Ó 2009 Published by Elsevier Ltd.
Glucokinase activator
Benzimidazole
Hypoglycemic
Type II diabetes
1. Introduction
Our group has also reported SAR analyses of several structural
classes of GKAs.11 In our previous article, we described the discovery
Glucokinase (GK), a member of the hexokinase family,1 is ex-
pressed in the liver and in pancreatic b-cells, and catalyzes the first
step in glycolysis. This step involves the phosphorylation of glu-
cose to glucose 6-phosphate. GK plays an important role as a glu-
cose sensor in maintaining plasma glucose homeostasis by
enhancing both glucose uptake in the liver and insulin secretion
from pancreatic b-cells.2 Therefore, a GK activator (GKA) can be ex-
pected to function as a hypoglycemic drug in the treatment of type
II (insulin-independent) diabetes mellitus.
It was recently discovered that GKAs bind to an allosteric site on
GK, 20 Å away from the glucose binding site.3–5 These GKAs cause
potent hypoglycemic reactions in rodents, both by increasing pan-
creatic insulin secretion and by augmenting hepatic glucose
metabolism.6
of 6-[(2R)-1-acetylpyrrolidin-2-yl]-5-aryloxy-2-pyridine-yl-1H-
benzimidazole GKAs, represented by compound 1. In this earlier
study, detailed SARs for the pyridine ring at the 2-position and the
phenoxy portion at the 5-position of the benzimidazole pharmaco-
phore were examined. However, modification of the N-acylpyrroli-
dine portion of lead compound 1 was not performed due to
synthetic complexities caused by the chiral center.12 From the point
of view of drug discovery, removal of the chiral center should ex-
pand the scope of modification strategies and further development
of potent benzimidazole GKAs. In this paper, we describe detailed
SAR analysis of novel benzimidazole GKAs generated by replacing
the chiral N-acetylpyrrolidin-2-yl group at the 6-position of lead 1
with appropriate achiral substituents. This approach led to the iden-
tification of the potent benzimidazole derivative, 3g. In addition, the
binding mode of benzimidazole GKA with GK protein, determined
by X-ray crystallographic analysis, is reported.
To date, several amide classes of GKAs, including RO0281675
(Roche),2,5 GKA50 (AstraZeneca),7 LY2121260 (Lilly)8 and PSN-
GK1 (Prosidion/OSI)9 have been disclosed, and some of these com-
pounds have entered human clinical trials (Scheme 1).10
2. Chemistry
Preparation of compounds 2a–c and 3a–k are described in
Scheme 2. First, an o-nitroanilide 9 was obtained from commercially
available 2-fluoro-4-nitrobenzoic acid (4) in 60% yield in five steps
via esterification, reduction of the nitro group with Raney Ni, amida-
tion with 2-picolinic acid in the presence of WSCI, nitration with
fuming HNO3 and substitution by 4-(ethylsulfonyl)phenol (27).
Then, reduction of the nitro group in anilide 9 with SnCl2 directly
afforded a cyclized benzimidazole product, 10. Next, benzimidazole
Abbreviations: rt, room temperature; CDI, 1,10-carbonylbis-1H-imidazole; DEAD,
diethyl azodicarboxylate; HOBt, 1-hydroxybenzotriazole; LDA, lithium diisopropyl
amide; PdCl2(dppf)-CH2Cl2, 1,10-bis(diphenylphosphino)ferrocenedichloro palla-
dium(II) dichloromethane complex; SEM-Cl, 2-(trimethylsilyl)ethoxymethyl chlo-
ride; TFA, trifluoroacetic acid; WSCI, 1-ethyl3-(3-dimethylaminopropyl)
carbodiimide hydrochloride.
* Corresponding author. Tel.: +81 29 877 2000; fax: +81 29 877 2029.
E-mail addresses: g94e079@ruri.waseda.jp, keiji_takahashi@merck.com (K. Ta-
kahashi).
0968-0896/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2009.05.037

K. Takahashi et al. / Bioorg. Med. Chem. 17 (2009) 7042–7051
7043
Scheme 1. Structures of GK activators.
Scheme 2. Reagents and conditions: (a) catH2SO4, MeOH, reflux, 94%; (b) H2 (balloon), Raney Ni (cat), MeOH, THF, rt, 99%; (c) 2-picolinic acid, WSCI, pyridine, rt, 92%; (d)
fuming HNO3, 0 °C to rt, 85%; (e) phenol 27, K2CO3, DMF, 80 °C, 82%; (f) SnCl2–2H2O, HCl, MeOH, DMF, 80 °C; (g) SEMCl, NaH, DMF, rt, 73% in two steps; (h) LiAlH4, THF, 0 °C,
80%; (i) MsCl, NEt3, THF, 0 °C; (j) NaN3, DMF; (k) CuSO4/5H2O, NaBH4, MeOH, rt, 51% in two steps; (l) acetyl chloride, NEt3, CHCl3, rt, 97%; (m) MeI, NaH, DMF, rt, 47%; (n) EtI,
NaH, DMF, rt, 28%; (o) TFA, H2O, rt; (p) Nucleophiles, NaH, DMF, rt. (q) imides, 40 wt % DEAD in toluene, PPh3, THF, rt; (r) NaCN, DMF, rt, 66%; (s) NaOH, EtOH, reflux, 58%; (t)
Me2NH, WSCI, HOBt, CHCl3, rt, 56%; (u) TMSN3, Bu2SnO, Toluene; (v) MeI, NaH, DMF, 0 °C.

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K. Takahashi et al. / Bioorg. Med. Chem. 17 (2009) 7042–7051
SO2Et
O
SO2Et
SO2Et
Conformationally
rigidification
Removal of
chiral center
N
O
5
N
O
N
N
H
N
*
N
H
N
N
H
6
N
N
O
N
O
N
O
O
3g
2a
1
Figure 1. Modification strategy from chiral (N-acetylpyrrolidin-2-yl)benzimidazole lead 1 to achiral succinimide derivative 3g.
10 was protected with a 2-(trimethylsilyl)ethoxymethyl (SEM)
group before the carboxyester group was reduced with LiAlH4 to
give hydroxymethyl analog 12 as a key intermediate in 58% yield.
The mesylate of 12 was substituted by NaN3 and reduced with
CuSO4–5H2O to give aminomethyl derivative 15 in 51% yield. Acet-
ylation of 15 followed by alkylation (if necessary) and deprotection
of the SEM group afforded acetylamino derivatives 2a–c. On the
other hand, the mesylate of 12 was substituted by appropriate
nucleophiles followed by deprotection to afford the corresponding
targeted compounds 3a–f, 3j and 3k. Preparation of compounds 2d
and 3l were also achieved via cyanation of the mesylate of 12 fol-
lowed by hydrolysis of the CN group or reaction with TMSN3. Each
reaction is described in Section 5.
first replaced the chiral N-acetylpyrrolidin-2-yl group with the
achiral (N-acetyl-N-ethylamino)methyl group, as shown in Figure
1. N-Acetyl-N-ethylamino derivative 2a showed GK activating po-
tency comparable to compound 1. This result suggested the utility
of screening acylamino groups at the 6-position. Noteworthy data
are summarized in Table 1. As for substituents on the nitrogen
atom, a methyl group (2b) resulted in higher potency than an ethyl
group (2a), whereas removal of the substituent (2c) reduced po-
tency. Replacement of the acylamino moiety with the aminocar-
bonyl group maintained potency (2d).
Initial modification of lead 1, the transformation of the chiral N-
acetylpyrrolidin-2-yl group to the achiral acetylamino group at the
6-position, led to the identification of two promising compounds,
2a and 2b. We then examined rigid cyclic structures, as shown in
Figure 1, in order to increase potency. The GK potencies of com-
pounds 3a–l are summarized in Table 2. The pyrrolidin-2-one
(3a) and 1,3-oxazolidine-2-one (3b) derivatives showed GK po-
tency comparable to the acetyl(methyl)amino derivative (2b).
Although the 3-methyl-1,3-imidazolidin-2-one (3c), 1,1-dioxido-
isothiazolidin (3e) and morpholin-2-one (3f) derivatives also
showed acceptable EC50 values, their Emax values were lower than
those of compounds 3a and 3b. 1,3-Imidazolidin-2-one derivative
(3d), which has a proton donor on the nitrogen atom, showed no
GK potency.
Mitsunobu reaction of the hydroxymethyl intermediate 12 with
succinimide followed by deprotection provided succinimide deriv-
ative 3g. Other imide derivatives (3h and 3i) were also prepared by
Mitsunobu reaction with appropriate imides.
3. Results and discussion
In vitro GK assays were conducted at two glucose concentra-
tions, 2.5 mM and 10 mM, which simulate low and high postpran-
dial blood glucose condition, respectively. To remove the chiral
center of (N-acetylpyrrolidin-2-yl)benzimidazole derivative 1, we
Further SAR development showed that the incorporation of an-
other carbonyl group into the cyclic amide moiety of compounds
3a–c enhanced potency. Imide derivatives 3g-i exhibited two to
three times higher GK potency than the corresponding amide
derivatives 3a–c. In contrast, removal of the carbonyl group from
the pyrrolidine-2-one ring resulted in complete loss of potency.
These results suggest that at least one carbonyl group on the sub-
stituent is important for potency.
Table 1
EC50 of the benzimidazole derivatives with an aliphatic amide moiety at the 6-
position6
O
R
N
N
N
EtO2S
H
Modification of the substitution at the 6-position of the benz-
imidazole led to the identification of potent benzimidazole GKAs
with an achiral substituent: pyrrolidin-2-one derivative 3a, 1,3-
oxazolidin-2-one derivative 3b and the corresponding imide ana-
logs, 3g and 3h. To select candidates for further evaluation, the
in vivo acute glucose lowering efficacies of these compounds were
examined in high-fat diet (HFD) mice (Fig. 2). Both the pyrolidin-2-
one (3a) and succinimide (3g) derivatives showed more potent glu-
cose lowering effect than the aliphatic acetylamino derivative 2b at
10 mg/kg po. On the other hand, the glucose lowering effects of
compounds 3b and 3h, derivatized with substituents containing
the oxazolidine component, were significantly weaker than those
of other derivatives (2b, 3a and 3g).
These results led to the choice of succinimide derivative 3g for
pharmacokinetic studies and oral glucose tolerance tests (OGTTs).
The PK profiles determined in male SD rats and beagle dogs are
shown in Table 3. Compound 3g exhibited moderate and accept-
able bioavailability in both species. In Wister rat OGTTs, oral
administration of compound 3g demonstrated hypoglycemic effi-
cacy at 1 and 3 mg/kg, resulting in 19% and 27% reduction in the
area under the glucose curve, respectively (Fig. 3).
Compounds
R
2.5 mM Glc EC50 (Emax
M (fold)a
)
10 mM Glc EC50 (Emax
l
M (fold)a
)
l
Me
N
2a
0.50 (1.04)
0.30 (1.02)
1.41 (0.98)
0.62 (1.00)
0.11 (0.95)
0.07 (1.13)
0.22 (0.94)
0.12 (0.98)
Et
O
O
Me
N
2b
2c
Me
Me NH
O
O
2d
NMe2
a
Values are the means of those obtained from two or more independent assays.
Compound A (see Section 5) was used as the internal control across all assay plates
for data validation. The EC50 values of A were 0.42 0.09 lM and 0.14 0.04 lM at
2.5 mM and 10 mM glucose conditions, respectively. Emax values of each compound
were calculated as a ratio of the maximal response evoked by the compound
compared to compound A at each concentration.

K. Takahashi et al. / Bioorg. Med. Chem. 17 (2009) 7042–7051
7045
Table 2
260
EC50 of the benzimidazole derivatives with
a
pyrrolidin-2-one, azolidin-2-ones,
morpholin-2-one, imides, pyrrolidine or aromatic moiety at the 6-position6
240
220
200
180
160
140
120
100
80
O
N
N
N
EtO2S
H
R
Compounds
R
2.5 mM Glc EC50 (Emax
l
)
10 mM Glc EC50 (Emax
l
M (fold)a
)
M (fold)a
2b
3a
3b
3g
3h
N
N
3a
0.35 (1.07)
0.25 (1.00)
0.32 (0.96)
0.06 (1.00)
0.06 (1.03)
0.08 (0.69)
O
O
O
3b
3c
3d
3e
3f
60
O
pre
1 hr
2 hr
3 hr
4 hr
Time (h)
N
N
Figure 2. Acute glucose lowering effect in HFD mice of compounds 2b, 3a, 3b, 3g
and 3h.
Me
N
N
O
>5
>5
Table 3
NH
PK data for 3g
O
O
Species
AUC (
l
M h)
CLp (mL/min/kg)
Cmax
(
lM)
T1/2 (h)
F (%)
0.46 (0.80)
0.28 (0.88)
0.16 (1.00)
0.09 (1.08)
0.18 (0.90)
0.09 (0.63)
0.10 (0.85)
0.04 (1.09)
0.02 (1.02)
0.06 (0.74)
S
Rata
0.5
4.0
32
0.4
0.4
0.6
6.4
16
41
Dogb
3.6
N
O
O
a
Doses: 1 mg/kg iv, 3 mg/kg po.
Doses: 0.3 mg/kg iv, 1 mg/kg po.
b
N
3g
3h
3i
O
O
O
O
O
O
N
O
N
N
Me
N
3j
>5
>5
N
N
3k
0.43 (1.03)
0.41 (1.10)
0.12 (0.99)
0.07 (1.07)
Figure 3. Wister Rat OGTT using compound 3g.
Me
N
3l
N
N N
a
Values are the means of those obtained from two or more independent assays.
Compound A (see Section 5) was used as the internal control across all assay plates
for data validation. The EC50 values of A were 0.42 0.09 lM and 0.14 0.04 lM at
2.5 mM and 10 mM glucose conditions, respectively. Emax values of each compound
were calculated as a ratio of the maximal response evoked by the compound
compared to compound A at each concentration.
The co-crystal structure of GK protein with GKA 3g was deter-
mined, revealing the binding mode of benzimidazole GK activators
to GK protein, as well as the structural requirements for efficacy
(Fig. 4). 2-(Pyridine-2-yl)-1H-benzimidazole 3g binds to the same
allosteric site of GK protein as heteroaromatic amide GKAs bind,
as described in our earlier report.11a The NH of the imidazole and
the nitrogen atom in the pyridine ring make key hydrogen bonds
with the backbone C@O and N–H of Arg63, respectively. In addi-
tion, the plenary succinimide moiety and the aromatic ring of
Tyr214 are parallel to each other, which probably increase potency
Figure 4. GK activator 3g bound to Glucokinase. Final 2Fo–Fc electron density (1.2
level) for compound 3g is shown as orange mesh. This figure was prepared using
the CCP4 package13 and PyMOL.14
r

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K. Takahashi et al. / Bioorg. Med. Chem. 17 (2009) 7042–7051
due to van der Waals interactions. In fact, the imide derivatives 3g–
i, which have an additional carbonyl group in the ring structure,
showed higher potency than the corresponding amide derivatives,
3a–c. The carbonyl group in the ring system at the 6-position of our
GKAs is important functional group to support the planarity of the
ring structure and interact with Tyr214 on GK enzyme. The intro-
duction of plenary heteroaromatic rings such as imidazole (3k) and
tetrazole (3l) in place of the imide moiety was also compatible
with potency (Table 2).
filtration through Celite, the mixture was concentrated in vacuo
to give 6 (119 g, 704 mmol, 99%).
5.1.1.3. Methyl 2-fluoro-4-[(pyridin-2-ylcarbonyl)amino]ben-
zoate (7). To a stirred solution of 6 (18.9 g, 112 mmol) and 2-
picolinic acid (16.5 g, 134 mmol) in pyridine (500 mL) was added
WSCI (32.0 g, 167 mmol), and the mixture was stirred at rt for
2 h. After concentration in vacuo, the residue was dissolved into
EtOAc (600 mL), and washed with 0.25 M aq HCl, 0.25 M aq NaOH
and brine, dried over Na2SO4 and concentrated in vacuo. The resi-
due was triturated with EtOAc/n-hexane and the resulting partici-
pate was collected, dried in vacuo to give 7 (28.3 g, 103 mmol, 92%)
as a white solid.
4. Conclusion
In conclusion, we have identified several potent GK activators
with a cyclic amide or imide moiety on the benzimidazole struc-
ture. These compounds, exemplified by 3g, were designed and dis-
covered by removing the chiral center of (N-acetylpyrrolidin-2-
yl)benzimidazole lead 1, followed by conformational rigidification
of the resulting acetylamino group in compound 2a. Succinimide
derivative 3g demonstrated significant glucose lowering effect in
rat OGTT at 1 mg/kg po, and showed acceptable pharmacokinetic
profiles in beagle dogs. Further in-depth evaluation of compound
3g is ongoing. Furthermore, X-ray crystallographic analysis of the
GKA 3g/GK protein complex revealed the binding mode of this no-
vel benzimidazole GK activator to the allosteric binding site of GK.
5.1.1.4. Methyl 2-fluoro-5-nitro-4-[(pyridin-2-ylcarbonyl)amino]
benzoate (8). Fuming HNO3 (110 mL) was slowly added to 7
(27.7 g, 101 mmol) at 0 °C, and the mixture was stirred at rt for
1.5 h. Then the mixture was poured into a cooled solution of Na2CO3
(138 g) in water (2.00 L), and the resulting precipitate was collected,
dried in vacuo to give 8 (27.5 g, 86.1 mmol, 85%) as a yellow solid.
5.1.1.5. Methyl 2-[4-(ethylsulfonyl)phenoxy]-5-nitro-4-[(pyri-
din-2-ylcarbonyl)amino]benzoate (9). To a stirred solution of 8
(6.00 g, 18.8 mmol) and 4-(ethylsulfonyl)phenol (27, 3.48 g,
18.7 mmol) in DMF (110 mL) was added K2CO3 (3.50 g, 25.3 mmol),
and the mixture was stirred at 80 °C for 30 min. The mixture was
cooled and poured into water (300 mL). The resulting precipitate
was collected, dried in vacuo to give 9 (7.46 g, 15.4 mmol, 82%) as
a yellow solid.
5. Experimental
5.1. Chemistry
All solvents, chemicals and reagents were commercially avail-
able unless otherwise noted. 1H NMR spectra were recorded on a
Varian MERCURY-400 (400 MHz) or a JEOL JMN-AL400 (400 MHz)
spectrometer. Chemical shifts are expressed in ppm units relative
to tetramethylsilane, methanol-d4 or dimethylsulfoxide-d6 as the
internal standard. Coupling constants are expressed in units of hertz
(Hz). Splitting patterns are designated as: s, singlet; d, doublet; t.
triplet; q, quartet; m, multiplet; br, broad. Mass spectra were re-
corded on a Waters Micromass ZQ2000 spectrometer under elec-
tronspray ionization (ESI) or atmospheric pressure chemical
ionization (APCI) conditions. High resolution mass spectra (HRMS)
were recorded on a Micromass Q-Tof-2 spectrometer under ESI con-
ditions. Column chromatography was performed with pre-packed
silica gel columns (KP-Sil silica) purchased from Biotage Japan, Ltd.
Preparative thin-layer chromatography (TLC) was performed on a
TLC Silica gel 60 F (Merck5744) purchased from Merck KGaA. Pre-
parative HPLC purification was performed on a YMC-CombiPrep
5.1.1.6. Methyl 5-[4-(ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-
1H-benzimidazole-6-carboxylate (10). To a stirred suspension
of 9 (7.46 g, 15.4 mmol) in DMF (37.0 mL) and MeOH (37.0 mL)
were added SnCl2–2H2O (17.3 g, 76.7 mmol) and HCl (15.0 mL),
and the mixture was stirred at 80 °C for 40 min. After cooling,
the mixture was neutralized with aq Na2CO3, diluted with EtOAc
and stirred at rt for 30 min. The resulting insoluble solid was fil-
tered off. The organic layer was separated, washed with water
and brine, dried over Na2SO4, and concentrated in vacuo to give
a crude product containing 10 (6.90 g) as a yellow solid. This
was used in the next step without further purification.
5.1.1.7. A mixture of methyl 5-[4-(ethylsulfonyl)phenoxy]-2-
(pyridin-2-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-benz-
imidazole-6-carboxylate and methyl 6-[4-(ethylsulfonyl)phen-
oxy]-2-(pyridin-2-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-
benzimidazole-5-carboxylate (11). To a solution of 10 (6.90 g) in
DMF (70.0 mL) were added SEM-Cl (4.00 mL, 22.6 mmol) and NaH
(70% dispersion in paraffin liquid, 920 mg, 26.8 mmol), and the mix-
ture was stirred at rt for 30 min. After cooling, the mixture was par-
titioned between saturated aq NH4Cl and EtOAc. The organic layer
was separated, washed with water and brine, dried over Na2SO4,
andconcentratedinvacuo. Theresiduewaspurifiedbycolumnchro-
matography on silica gel eluted with EtOAc/n-hexane to give 11
(6.43 g, 11.3 mmol, 73% in two steps) as a yellow solid.
Pro C18 (50 mm ꢀ 30 mm id, S-5
lm, 12 nm), eluting with a gradi-
ent of 0.1% aqueous TFA/CH3CN at a flow rate of 40 mL/min under
appropriate conditions.
5.1.1. A mixture of (5-[4-(ethylsulfonyl)phenoxy]-2-(pyridin-2-
yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol-6-yl)
methanol and (6-[4-(ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1-
{[2-(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol-5-yl)
methanol (12): Preparation of the key intermediate
5.1.1.1. Methyl 2-fluoro-4-nitrobenzoate (5). To a stirred solu-
tion of 2-fluoro-4-nitrobenzoic acid (4, 140 g, 756 mmol) in MeOH
(1.30 L) was added H2SO4 (5.00 mL), and the mixture was refluxed
for 48 h. After concentration in vacuo, the residue was triturated
with water. The resulting precipitate was collected, dried in vacuo
to give 5 (141 g, 708 mmol, 94%) as a yellow solid.
5.1.1.8. A mixture of (5-[4-(ethylsulfonyl)phenoxy]-2-(pyridin-
2-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol-
6-yl)methanol and (6-[4-(ethylsulfonyl)phenoxy]-2-(pyridin-2-
yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol-5-
yl)methanol (12a and 12b). To a stirred cooled solution of LiAlH4
(990 mg, 26.1 mmol) in THF (60.0 mL) was slowly added a solution
of 11 (5.90 g, 10.4 mmol) in THF (50.0 mL), and the mixture was
stirred at rt for 15 min. The mixture was cooled to 0 °C, and sodium
sulfate decahydrate was slowly added until the foaming quieted
down. After diluting with EtOAc, the mixture was stirred at rt for
5.1.1.2. Methyl 4-amino-2-fluorobenzoate (6). A solution of 5
(141 g, 708 mmol) in MeOH (1.00 L) and THF (400 mL) was hydro-
genated at rt overnight in the presence of Raney Ni (20.0 g). After

K. Takahashi et al. / Bioorg. Med. Chem. 17 (2009) 7042–7051
7047
1 h. The resulting insoluble solid was filtered off. The filtrate was
concentrated in vacuo. The residue was purified by column chro-
matography on silica gel eluted with EtOAc/n-hexane to give 12
(4.50 g, 8.34 mmol, 80%) as a yellow amorphous mass. In some
cases, regiomixture 12 was separated by column chromatography
on silica gel to give 12a and 12b as a pale yellow solid respectively.
5.1.2.6. N-({5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1H-
benzimidazol-6-yl}methyl)-N-methylacetamide (2b). TFA
(0.500 mL) was added to the compound 17 (18.1 mg,
30.0 lmol), and the mixture was stirred at rt for 2 h. The sol-
vent was evaporated under reduced pressure. The residue was
dissolved into CHCl3, and then Et3N was added. The solvent
was evaporated under reduced pressure. The residue was
purified by preparative TLC eluted with MeOH/CHCl3 to give
5.1.2. N-({5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-
1H-benzimidazol-6-yl}methyl)-N-methylacetamide (2b)
5.1.2.1. (5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1-{[2-(tri-
methylsilyl)ethoxy]methyl}-1H-benzimidazol-6-yl)methyl meth-
2b (13.2 mg, 28.0 lmol, 93%) as a colorless amorphous mass.
1H NMR (CDCl3) d: 10.8 (1H, br), 8.70–8.65 (1H, m), 8.42–
8.37 (1H, m), 7.90–7.82 (3H, m), 7.73–7.17 (total 3H, m),
7.10–7.06 (2H, m), 4.67–4.56 (total 2H, m), 3.16–3.09 (2H,
m), 2.96 and 2.99 (total 3H, s), 2.05 and 2.08 (total 3H, s),
1.33–1.24 (3H, m). HRMS m/z: Calcd for C24H25N4O4S
([M+H]+) 465.1597. Found: 465.1593.
anesulfonate (13a). To
0.186 mmol) and Et3N (52.0
added MsCl (29.0
a
stirred solution of 12a (100 mg,
L, 0.370 mmol) in THF (1.00 mL) was
l
l
L, 0.370 mmol) at 0 °C, and then the mixture
was stirred at the same temperature for 30 min. The mixture was
partitioned between water and EtOAc. The organic layer was sepa-
rated, washed with brine, dried over MgSO4, and concentrated in va-
cuo to give a crude product containing 13a. This was used in the
next step without further purification.
5.1.3. N-({5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1H-
benzimidazol-6-yl}methyl)acetamide (2c)
Compound 2c was prepared in a similar manner to the step of
Section 5.1.2.6 using 16 instead of 17. 1H NMR (CDCl3) d: 10.8
(1H ꢀ 1/2, br), 10.7 (1H ꢀ 1/2, br), 8.70–8.65 (1H, m), 8.39–8.36
(1H, m), 7.90–7.83 (3H, m), 7.90–7.83 (1H ꢀ 1/2, m), 7.65
(1H ꢀ 1/2, s), 7.46 (1H ꢀ 1/2, s), 7.41–7.38 (1H, m), 7.15 (1H ꢀ 1/
2, s), 5.97 (1H ꢀ 1/2, br), 5.83 (1H ꢀ 1/2, br), 4.50–4.48 (2H, m),
3.11 (2H, q, J = 7.4 Hz), 1.95 (3H, s), 1.30 (3H, t, J = 7.4 Hz). HRMS
m/z: Calcd for C23H23N4O4S ([M+H]+) 451.1440. Found: 451.1437.
5.1.2.2. 6-(Azidomethyl)-5-[4-(ethylsulfonyl)phenoxy]-2-(pyri-
din-2-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-benzimid-
azole (14). To a stirred cooled solution of 13a in DMF (1.00 mL)
was added NaN3 (60.0 mg, 0.930 mmol), and then the mixture
was stirred at rt. The mixture was partitioned between water
and EtOAc, washed with brine, dried over MgSO4, and concentrated
in vacuo to give a crude product containing 14 (136 mg). This was
used in the next step without further purification.
5.1.4. N-Ethyl-N-({5-[4-(ethylsulfonyl)phenoxy]-2-(pyridin-2-
yl)-1H-benzimidazol-6-yl}methyl)acetamide (2a)
To a stirred cooled solution of 16 (90.0 mg, 0.155 mmol) in DMF
(1.00 mL) were added NaH (55% dispersion in paraffin liquid,
12.0 mg, 0.310 mmol) and EtI (37.0 mg, 0.310 mmol), and the mix-
ture was stirred at rt for 2 h. The mixture was partitioned between
aq NH4Cl and EtOAc. The organic layer was separated, washed with
brine, dried over MgSO4, and concentrated in vacuo. The residue
was purified by preparative HPLC to give N-ethyl-N-[(5-[4-(ethyl-
sulfonyl)phenoxy]-2-(pyridin-2-yl)-1-{[2-(trimethylsilyl)ethoxy]-
methyl}-1H-benzimidazol-6-yl)methyl]acetamide (18). TFA (0.500 mL)
was added to 18, and the mixture was stirred at rt for 2 h. After con-
centration, the residue was dissolved into CHCl3 and neutralized with
aq NaHCO3. The organic layer was separated, washed with brine, dried
over MgSO4, and concentrated in vacuo. The residue was purified by
preparative TLC eluted with MeOH/CHCl3 to give 2a (12.5 mg,
5.1.2.3. 1-(5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1-{[2-
(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol-6-yl)methan-
amine (15). To a stirred solution of 14 (136 mg) in MeOH
(2.00 mL) were added CuSO4/10H2O (2.00 mg, 8.00 lmol) and
NaBH4 (35.0 mg, 0.930 mmol), and the mixture was stirred at rt
for 30 min. The mixture was partitioned between aq NH4Cl, aq
NH3 and EtOAc. The organic layer was separated, washed with
brine, dried over MgSO4, and concentrated in vacuo. The residue
was purified by column chromatography on silica gel eluted with
MeOH/CHCl3 to give 15 (81.5 mg, 0.151 mmol, 51% in two steps)
as yellow oil.
5.1.2.4. N-[(5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1-{[2-
(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol-6-yl)methyl]
acetamide (16). To a stirred solution of 15 (400 mg, 0.744 mmol)
in CHCl3 (4.00 mL) were added Et3N (0.208 mL, 1.49 mmol) and
AcCl (0.104 mL, 1.49 mmol), and the mixture was stirred at rt for
30 min. The mixture was partitioned between water and EtOAc,
and diluted with aq NaHCO3. The organic layer was separated,
washed with brine, dried over MgSO4 and, and concentrated in va-
cuo. The residue was purified by column chromatography on silica
gel to give 16 (387 mg, 0.718 mmol, 97%) as a yellowish brown
amorphous mass.
26.0 lmol, 17% in two steps) as a white amorphous mass.
1H NMR (CDCl3) d: 10.8 (1H, br), 8.70–8.64 (1H, m), 8.41–8.39
(1H, m), 7.90–7.00 (1H ꢀ 3/5, br m), 7.88–7.82 (3H, m), 7.64
(1H ꢀ 3/5, s), 7.42–7.38 (1H, m), 7.42–7.38 (1H ꢀ 2/5, m), 7.11–
7.00 (2H, m), 7.11–7.00 (1H ꢀ 2/5, m), 4.66 (2H ꢀ 3/5, s), 4.56
(2H ꢀ 2/5, s), 3.48 (2H ꢀ 2/5, q, J = 7.2 Hz), 3.30 (2H ꢀ 3/5, q,
J = 7.2 Hz), 3.16–3.09 (2H, m), 2.11 (3H ꢀ 3/5, s), 2.07 (3H ꢀ 2/5,
s), 1.32–1.27 (3H, m), 1.15–1.07 (3H, m). HRMS m/z: Calcd for
C25H27N4O4S ([M+H]+) 479.1753. Found: 479.1751.
5.1.5. 1-({5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1H-
benzimidazol-6-yl}methyl)pyrrolidin-2-one (3a): General
Procedure A
5.1.5.1. (6-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1-{[2-
(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol-5-yl)methyl
methanesulfonate (13b). To a stirred solution of 12b (100 mg,
5.1.2.5. N-[(5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1-{[2-
(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol-6-yl)methyl]-
N-methylacetamide (17). To a stirred cooled solution of 16
(38.0 mg, 0.0650 mmol) in DMF (0.300 mL) were added NaH (55%
dispersion in paraffin liquid, 5.20 mg, 0.130 mmol) and MeI
(18.4 mg, 0.130 mmol), and the mixture was stirred at rt for 2 h.
The mixture was partitioned between aq NH4Cl and EtOAc. The or-
ganic layer was separated, washed with brine, dried over MgSO4,
and concentrated in vacuo. The residue was purified by preparative
HPLC followed by usual work-up to give 17 (18.1 mg, 0.030 mmol,
47%) as colorless oil.
0.186 mmol) and Et3N (50.0
lL, 0.360 mmol) in THF (1 mL) was
added MsCl (28.0 L, 0.360 mmol) at 0 °C, and the mixture was stir-
l
redatthesametemperaturefor30 min. Themixturewaspartitioned
between water and EtOAc. The organic layer was separated, washed
with brine, dried over MgSO4, and concentrated in vacuo to give a
crude product containing 13b (110 mg) as pale orange oil. This was
used in the next step without further purification.

7048
K. Takahashi et al. / Bioorg. Med. Chem. 17 (2009) 7042–7051
5.1.5.2. 1-[(6-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1-{[2-
(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol-5-yl)methyl]pyrr-
olidin-2-one (19). To a stirred solution of 13b (110 mg) and
5.1.9. 6-[(1,1-Dioxidoisothiazolidin-2-yl)methyl]-5-[4-(ethylsulfo-
nyl)phenoxy]-2-(pyridin-2-yl)-1H-benzimidazole (3e)
Isothiazolidine 1,1-dioxide15 instead of 2-pyrrolidone was used
at the step of Section 5.1.5.2 followed by Section 5.1.5.3. 1H NMR
(CDCl3) d: 10.7 (1H, br), 8.70–8.65 (1H, m), 8.41–8.39 (1H, m),
7.96 (1H ꢀ 1/2, br s), 7.91–7.86 (1H, m), 7.84 (2H, d, J = 8.9 Hz),
7.71 (1H ꢀ 1/2, br s), 7.50 (1H ꢀ 1/2, br s), 7.43–7.39 (1H, m),
7.19 (1H ꢀ 1/2, br s), 7.08 (2H, d, J = 8.9 Hz), 4.28 (2H, br s), 3.20–
3.05 (4H, m), 3.12 (2H, q, J = 7.4 Hz), 2.30–2.20 (2H, m), 1.30 (3H,
t, J = 7.4 Hz). HRMS m/z: Calcd for C24H25N4O5S2 ([M+H]+)
513.1266. Found: 513.1265.
2-pyrrolidone (68.0 lL, 0.900 mmol) in DMF (2.00 mL) was added
NaH (55% dispersion in paraffin liquid, 21.6 mg, 0.495 mmol) at
0 °C, and the mixture was stirred at rt for 30 min. The mixture
was partitioned between aq NH4Cl and EtOAc. The organic layer
was separated, washed with brine, dried over MgSO4, and con-
centrated in vacuo to give a crude product containing 19 as
yellow oil. This was used in the next step without further
purification.
5.1.10. 4-({5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1H-
benzimidazol-6-yl}methyl)morpholin-3-one (3f)
5.1.5.3. 1-({5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1H-
benzimidazol-6-yl}methyl)pyrrolidin-2-one (3a). TFA (1.00 mL)
was added to the compound 19, and the mixture was stirred at rt
for 1 h. The solvent was evaporated under reduced pressure. The
residue was purified by preparative HPLC (YMC-CombiPrep Pro
C18, eluting CH3CN/water + 0.1%TFA) followed by usual work-up
to give 3a (26.5 mg, 0.0556 mmol, 31%) as a white solid. 1H NMR
(CDCl3) d: 10.86 (1H, br s), 8.64 (1H, d, J = 4.3 Hz), 8.39 (1H, d,
J = 7.8 Hz), 7.91–7.78 (2H, m), 7.84 (2H, d, J = 8.4 Hz), 7.61–7.45
(1H, m), 7.42–7.37 (1H, m), 7.06 (2H, d, J = 8.4 Hz), 4.54 (2H, s),
3.30 (2H, t, J = 7.1 Hz), 3.11 (2H, q, J = 7.4 Hz), 2.34 (2H, t,
J = 8.1 Hz), 1.93 (2H, tt, J = 8.1, 7.1 Hz), 1.29 (3H, t, J = 7.4 Hz). HRMS
m/z: Calcd for C25H25N4O4S ([M+H]+) 477.1597. Found: 477.1591.
The compounds 3b–3f, 3j and 3k were prepared in a similar
manner to that described for the preparation of 3a.
Morpholin-3-one16 instead of 2-pyrrolidone was used at the
step of Section 5.1.5.2 followed by Section 5.1.5.3. 1H NMR (CDCl3)
d
:
10.8 (1H ꢀ 1/2, br), 10.7 (1H ꢀ 1/2, br), 8.70–8.66 (1H, m), 8.42–
8.37 (1H, m), 7.84 (2H, d, J = 8.8 Hz), 7.90–7.80 (1H, m), 7.90–7.80
(1H ꢀ 1/2, m), 7.68 (1H ꢀ 1/2, s), 7.49 (1H ꢀ 1/2, s), 7.42–7.39 (1H,
m), 7.18 (1H ꢀ 1/2, s), 7.09–7.05 (2H, m), 4.73–4.70 (2H, m), 4.18–
4.13 (2H, m), 3.85–3.81 (2H, m), 3.36–3.34 (2H, m), 3.11 (2H, q,
J = 7.4 Hz), 1.30 (3H, t, J = 7.4 Hz). HRMS m/z: Calcd for
C25H25N4O5S ([M+H]+) 493.1546. Found: 493.1546.
5.1.11. 5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-6-(pyrroli-
din-1-ylmethyl)-1H-benzimidazole (3j)
Pyrrolidine instead of 2-pyrrolidone was used at the step of Sec-
tion 5.1.5.2 followed by Section 5.1.5.3. 1H NMR (CDCl3) d: 10.73
(1H, br s), 8.65 (1H, d, J = 3.9 Hz), 8.40 (1H, d, J = 7.3 Hz), 8.06–
7.11 (1H, m), 7.91–7.78 (2H, m), 7.82 (2H, d, J = 8.8 Hz), 7.42–
7.36 (1H, m), 7.05 (2H, d, J = 8.8 Hz), 3.77 (2H, br s), 3.12 (2H, q,
J = 7.5 Hz), 2.65 (4H, br s), 1.79 (4H, s), 1.29 (3H, t, J = 7.5 Hz). MS
m/z: 463 ([M+H]+).
5.1.6. 3-({5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1H-
benzimidazol-6-yl}methyl)-1,3-oxazolidin-2-one (3b)
1,3-Oxazolidin-2-one instead of 2-pyrrolidone was used at the
step of Section 5.1.5.2 followed by Section 5.1.5.3. 1H NMR (CDCl3)
d: 10.68–10.59 (1H, br m), 8.70–8.61 (1H, m), 8.43–8.35 (1H, m),
7.93–7.81 (1H, m), 7.85 (2H, d, J = 8.8 Hz), 7.65 (1H ꢀ 1/2, s), 7.51
(1H ꢀ 1/2, s), 7.44–7.38 (1H ꢀ 1/2, m), 7.44–7.38 (1H, m), 7.12–
7.04 (1H ꢀ 1/2, m), 7.09 (2H, d, J = 9.8 Hz), 4.52 (2H, s), 4.24 (2H,
t, J = 8.3 Hz), 3.54–3.40 (2H, m), 3.12 (2H, q, J = 7.4 Hz), 1.30 (3H,
t, J = 7.4 Hz). HRMS m/z: Calcd for C24H23N4O5S ([M+H]+)
479.1389. Found: 479.1386.
5.1.12. 5-[4-(Ethylsulfonyl)phenoxy]-6-(1H-imidazol-1-
ylmethyl)-2-(pyridin-2-yl)-1H-benzimidazole (3k)
Imidazole instead of 2-pyrrolidone was used at the step of Sec-
tion 5.1.5.2 followed by Section 5.1.5.3. 1H NMR (CDCl3) d: 10.8
(1H ꢀ 1/2, br), 10.7 (1H ꢀ 1/2, br), 8.70–8.60 (1H, m), 8.44–8.35
(1H, m), 7.94–7.80 (3H, m), 7.80–7.70 (1H, m), 7.60–7.45 (2H, m),
7.45–7.40 (1H, m), 7.20–6.88 (4H, m), 5.20 (2H, s), 3.13 (2H, q,
J = 7.4 Hz), 1.34 (3H, t, J = 7.4 Hz). HRMS m/z: Calcd for
C24H22N5O3S ([M+H]+) 460.1443. Found: 460.1447.
5.1.7. 1-({5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1H-
benzimidazol-6-yl}methyl)-3-methylimidazolidin-2-one (3c)
1-Methylimidazolidin-2-one instead of 2-pyrrolidone was used
at the step of Section 5.1.5.2 followed by Section 5.1.5.3. 1H NMR
(CDCl3) d: 10.84 (1H ꢀ 1/2, br s), 10.81 (1H ꢀ 1/2, br s), 8.64
(1H ꢀ 1/2, d, J = 4.7 Hz), 8.62 (1H ꢀ 1/2, d, J = 4.7 Hz), 8.40
(1H ꢀ 1/2, d, J = 7.8 Hz), 8.37 (1H ꢀ 1/2, d, J = 7.8 Hz), 7.89–7.84
(1H, m), 7.80 (2H, d, J = 9.0 Hz), 7.61 (1H ꢀ 1/2, s), 7.47 (1H ꢀ 1/2,
s), 7.40–7.37 (1H, m), 7.16 (1H ꢀ 1/2, s), 7.06 (1H ꢀ 1/2, s), 7.03
(2H, d, J = 9.0 Hz), 4.43 (2H ꢀ 1/2, s), 4.43 (2H ꢀ 1/2, s), 3.20–3.16
(4H, m), 3.10 (2H, q, J = 7.4 Hz), 2.75 (3H ꢀ 1/2, s), 2.73 (3H ꢀ 1/2,
s),1.28 (3H, t, J = 7.4 Hz). HRMS m/z: Calcd for C25H26N5O4S
([M+H]+) 492.1706. Found: 492.1704.
5.1.13. 1-({5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1H-
benzimidazol-6-yl}methyl)pyrrolidine-2,5-dione (3g): General
Procedure B
5.1.13.1. A mixture of 1-[(5-[4-(ethylsulfonyl)phenoxy]-2-(pyri-
din-2-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol-
6-yl) methyl]pyrrolidine-2,5-dione and 1-[(6-[4-(ethylsulfonyl)-
phenoxy]-2-(pyridin-2-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-
1H-benzimidazol-5-yl)methyl]pyrrolidine-2,5-dione (20). To a
stirred solution of 12 (2.40 g, 4.45 mmol), succinimide (1.30 g,
13.1 mmol) and PPh3 (13.3 mmol) in THF (24.0 mL) was added a
40 wt % solution of DEAD in toluene (5.80 mL, 12.7 mmol) at 0 °C,
and the mixture was stirred at rt for 1 h. After concentration in va-
cuo, the residue was purified by column chromatography on silica
gel eluted with EtOAc/n-hexane to give 20 (2.30 g, 3.70 mmol, 83%)
as a yellow amorphous mass.
5.1.8. 1-({5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1H-
benzimidazol-6-yl}methyl)imidazolidin-2-one (3d)
Imidazolidin-2-one instead of 2-pyrrolidone was used at the
step of Section 5.1.5.2 followed by Section 5.1.5.3. 1H NMR (CDCl3,
one drop of CD3OD) d: 8.65–8.61 (1H, m), 8.37 (1H, d, J = 7.4 Hz),
7.90–7.85 (1H, m), 7.82 (2H, d, J = 8.2 Hz), 7.77 (1H ꢀ 1/2, s), 7.59
(1H ꢀ 1/2, s), 7.45 (1H ꢀ 1/2, s), 7.41–7.37 (1H, m), 7.20 (1H ꢀ 1/
2, s), 7.05 (2H, d, J = 8.2 Hz), 4.65 (1H ꢀ 1/2, s), 4.63 (1H ꢀ 1/2, s),
4.44 (2H ꢀ 1/2, s), 4.41 (2H ꢀ 1/2, s), 3.28–3.25 (4H, m), 3.11 (2H,
q, J = 7.2 Hz), 1.28 (3H, t, J = 7.2 Hz). HRMS m/z: Calcd for
C24H24N5O4S ([M+H]+) 478.1549. Found: 478.1559.
5.1.13.2. 1-({5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1H-
benzimidazol-6-yl}methyl)pyrrolidine-2,5-dione (3g). TFA
(15.0 mL) was added to the compound 20 (2.30 g, 3.70 mmol),
and the mixture was stirred at rt for 2 h. After concentration,
the residue was purified by column chromatography on silica

K. Takahashi et al. / Bioorg. Med. Chem. 17 (2009) 7042–7051
7049
gel eluted with MeOH/CHCl3 followed by crystallization from
EtOAc to give 3g (1.02 g, 2.08 mmol, 68%) as a white crystal.
1H NMR (CDCl3) d: 10.58 (1H ꢀ 1/2, s), 10.52 (1H ꢀ 1/2, s),
8.65 (1H ꢀ 1/2, d, J = 4.9 Hz), 8.63 (1H ꢀ 1/2, d, J = 4.1 Hz), 8.38
(1H ꢀ 1/2, d, J = 7.8 Hz), 8.36 (1H ꢀ 1/2, d, J = 8.0 Hz), 7.90–
7.81 (1H, m), 7.86 (2H ꢀ 1/2, d, J = 8.8 Hz), 7.83 (2H ꢀ 1/2, d,
J = 9.0 Hz), 7.76 (1H ꢀ 1/2, s), 7.64 (1H ꢀ 1/2, s), 7.45 (1H ꢀ 1/
2, s), 7.42–7.37 (1H, m), 7.12 (1H ꢀ 1/2, s), 7.11 (2H ꢀ 1/2, d,
J = 8.8 Hz), 7.08 (2H ꢀ 1/2, d, J = 9.0 Hz), 4.80 (2H ꢀ 1/2, s),
4.79 (2H ꢀ 1/2, s), 3.12 (2H ꢀ 1/2, q, J = 7.4 Hz), 3.11 (2H ꢀ 1/2,
q, J = 7.4 Hz), 2.65 (2H, s), 2.54 (2H, s), 1.30 (3H ꢀ 1/2, t,
J = 7.4 Hz), 1.29 (3H ꢀ 1/2, t, J = 7.4 Hz). HRMS m/z: Calcd for
C25H23N4O5S ([M+H]+) 491.1389. Found: 491.1387.
off. The organic layer was separated, washed with brine, dried over
Na2SO4 and concentrated in vacuo. The residue was purified by
preparative HPLC to give 22 (17.7 mg, 32.0 lmol, 58%).
5.1.16.3. A mixture of 2-(5-[4-(ethylsulfonyl)phenoxy]-2-
(pyridin-2-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-ben-
zimidazol-6-yl)-N,N-dimethylacetamide and 2-(6-[4-(ethyl-
sulfonyl)phenoxy]-2-(pyridin-2-yl)-1-{[2-(trimethylsilyl)eth-
oxy]methyl}-1H-benzimidazol-5-yl)-N,N-dimethylacetamide
(23)
.
To
a
suspension of 22 (17.7 mg, 32.0
l
mol) in CHCl3
(1.00 mL) were added HOBt (8.70 mg, 64.0
lmol) and WSCI
(12.3 mg, 0.064 mmol), and the mixture was stirred at rt for
10 min. Then to the mixture was added 2.0 M solution of (CH3)2NH
in THF (48.0 lL, 96.0 lmol), and the resulting mixture was stirred
at rt for 1.5 h. The mixture was cooled to 0 °C, and partitioned be-
tween CHCl3 and water. The organic layer was separated, washed
with aq NaHCO3 and brine, dried over Na2SO4 and concentrated
in vacuo. The residue was purified by preparative TLC eluted with
The compounds 3h and 3i were prepared in a similar manner to
that described for the preparation of 3g.
5.1.14. 3-({5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1H-
benzimidazol-6-yl}methyl)-1,3-oxazolidine-2,4-dione (3h)
1,3-Oxazolidine-2,4-dione17 instead of succinimide was used at
the step of Section 5.1.13.1 followed by Section 5.1.13.2. 1H NMR
(CDCl3) d: 10.90 (1H ꢀ 1/2, br s), 10.85 (1H ꢀ 1/2, br s), 8.67–8.62
(1H, m), 8.42–8.37 (1H, m), 7.92–7.83 (1H ꢀ 1/2, m), 7.92–7.83
(3H, m), 7.70 (1H ꢀ 1/2, s), 7.47 (1H ꢀ 1/2, s), 7.44–7.38 (1H, m),
7.11 (1H ꢀ 1/2, s), 7.10 (2H, d, J = 8.2 Hz), 4.83 (2H ꢀ 1/2, s), 4.81
(2H ꢀ 1/2, s), 4.59 (2H ꢀ 1/2, s), 4.52 (2H ꢀ 1/2, s), 3.12 (2H, q,
J = 7.4 Hz), 1.30 (3H, t, J = 7.4 Hz). HRMS m/z: Calcd for
C24H21N4O6S ([M+H]+) 493.1182. Found: 493.1176.
MeOH/CHCl3 to give 23 (10.8 mg, 18.0 lmol, 56%).
5.1.16.4. 2-{5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1H-
benzimidazol-6-yl}-N,N-dimethylacetamide (2d). TFA (1.00 mL)
was added to the compound 23 (10.8 mg, 18.0 lmol), and the mix-
ture was stirred at rt for 1 h. After concentration, the residue was
dissolved into CHCl3, neutralized with aq NaHCO3 and extracted
with CHCl3. The combined organic layer was dried over Na2SO4
and concentrated in vacuo. The residue was purified by preparative
TLC eluted with MeOH/CHCl3 to give 2d (7.00 mg, 15.1 lmol, 84%)
5.1.15. 3-({5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1H-
benzimidazol-6-yl}methyl)-1-methylimidazolidine-2,4-dione
(3i)
as an amorphous mass. 1H NMR (CDCl3) d: 10.94 (1H ꢀ 1/2, br s),
10.88 (1H ꢀ 1/2, br s), 8.65–8.61 (1H, m), 8.42–8.36 (1H, m), 7.82
(2H, d, J = 9.0 Hz), 7.89–7.80 (1H, m), 7.77 (1H ꢀ 1/2, s), 7.55
(1H ꢀ 1/2, s), 7.44 (1H ꢀ 1/2, s), 7.40–7.37 (1H, m), 7.12 (1H ꢀ 1/
2, s), 7.08 (2H, d, J = 9.0 Hz), 3.74 (2H ꢀ 1/2, s), 3.73 (2H ꢀ 1/2, s),
3.11 (2H, q, J = 7.4 Hz), 2.98 (3H, s), 2.91 (3H ꢀ 1/2, s), 2.90
(3H ꢀ 1/2, s), 1.29 (3H, t, J = 7.4 Hz). HRMS m/z: Calcd for
C24H25N4O4S ([M+H]+) 465.1597. Found: 465.1600.
1-Methylhydantoin instead of succinimide was used at the step
of Section 5.1.13.1 followed by Section 5.1.13.2. 1H NMR (CDCl3) d:
10.67 (1H ꢀ 1/2, br s), 10.63 (1H ꢀ 1/2, br s), 8.65–8.60 (1H, m),
8.37 (1H, t, J = 8.2 Hz), 7.89–7.79 (1H ꢀ 1/2, m), 7.89–7.79 (3H,
m), 7.66 (1H ꢀ 1/2, s), 7.45 (1H ꢀ 1/2, s), 7.40–7.36 (1H, m), 7.10
(1H ꢀ 1/2, s), 7.06 (2H, d, J = 9.0 Hz), 4.78 (2H ꢀ 1/2, s), 4.77
(2H ꢀ 1/2, s), 3.74 (2H ꢀ 1/2, s), 3.59 (2H ꢀ 1/2, s), 3.11 (2H, q,
J = 7.0 Hz), 2.92 (3H ꢀ 1/2, s), 2.85 (3H ꢀ 1/2, s), 1.29 (3H, t,
J = 7.0 Hz). HRMS m/z: Calcd for C25H24N5O5S ([M+H]+) 506.1498.
Found: 506.1502.
5.1.17. 5-[4-(Ethylsulfonyl)phenoxy]-6-[(1-methyl-1H-tetrazol-
5-yl)methyl]-2-(pyridin-2-yl)-1H-benzimidazole (3l)
5.1.17.1. 5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-6-(2H-
tetrazol-5-ylmethyl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-
benzimidazole (24a) and 6-[4-(ethylsulfonyl)phenoxy]-2-(pyri-
din-2-yl)-5-(2H-tetrazol-5-ylmethyl)-1-{[2-(trimethylsilyl)eth-
oxy]methyl}-1H-benzimidazole (24b). To a stirred solution of 21
(100 mg, 0.183 mmol) in toluene (1.00 mL) were added TMSN3
(0.0485 mL, 0.365 mmol) and dibutyltin(IV) oxide (9.20 mg,
0.037 mmol), and the mixture was stirred at 115 °C for 24 h. The
mixture was cooled to 0 °C, diluted with aq NaHCO3 and extracted
with CHCl3 repeatedly. The combined organic layers were dried
over Na2SO4 and concentrated in vacuo. The residue was purified
by column chromatography on silica gel eluted with MeOH/CHCl3
to give 24a (14.0 mg, 0.024 mmol, 13%) and 24b (13.3 mg,
0.022 mmol, 12%).
5.1.16. 2-{5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-2-yl)-1H-
benzimidazol-6-yl}-N,N-dimethylacetamide (2d)
5.1.16.1. A mixture of (5-[4-(ethylsulfonyl)phenoxy]-2-(pyridin-
2-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol-6-
yl)acetonitrile and (6-[4-(ethylsulfonyl)phenoxy]-2-(pyridin-2-
yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol-5-yl)-
acetonitrile (21). Compound 13 was prepared in a similar manner
to the step of Section 5.1.2.1 using 12 instead of 12a. Then the com-
pound 13 was dissolved into DMF (20.0 mL) at 0 °C and NaCN
(254 mg, 5.19 mmol) was added. The resulting mixture was stirred
at rt for 3 h. The mixture was partitioned between aq NaHCO3 and
EtOAc. The organic layer was separated, washed with water and
brine, dried over Na2SO4 and concentrated in vacuo. The residue
was purified by column chromatography on silica gel eluted with
EtOAc/n-hexane to give 21 (623 mg, 1.14 mmol, 66%).
5.1.17.2. 5-[4-(Ethylsulfonyl)phenoxy]-6-[(1-methyl-2H-tetra-
zol-5-yl)methyl]-2-(pyridin-2-yl)-1-{[2-(trimethylsilyl)ethoxy]
methyl}-1H-benzimidazole (25). To a stirred solution of 24a
(14.0 mg, 0.0240 mmol) in DMF (0.500 mL) were added NaH (55%
dispersion in paraffin liquid, 1.60 mg, 0.0360 mmol) and MeI
5.1.16.2. A mixture of (5-[4-(Ethylsulfonyl)phenoxy]-2-(pyridin-
2-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol- 6-
yl)acetic acid and (6-[4-(ethylsulfonyl)phenoxy]-2-(pyridin-2-
yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-benzimidazol-5-yl)-
(4.60 mg, 32.0 lmol), and the mixture was stirred at rt for 1 h.
The mixture was partitioned between aq NH4Cl and EtOAc. The or-
ganic layer was separated, washed with water and brine, dried
over Na2SO4 and concentrated in vacuo. The residue was purified
by preparative TLC eluted with MeOH/CHCl3 to give 25 (3.50 mg,
acetic acid (22). To a solution of 21 (30.0 mg, 55.0 lmol) in EtOH
(1.00 mL) was added 2.5 M aq NaOH (1.00 mL), and the mixture
was refluxed overnight. The mixture was cooled to 0 °C and acidi-
fied with 10% citric acid. The resulting insoluble solid was filtered
5.80 lmol, 24%) and its regioisomer 26 (4.10 mg, 6.80 lmol, 28%).

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K. Takahashi et al. / Bioorg. Med. Chem. 17 (2009) 7042–7051
5.1.17.3. 5-[4-(Ethylsulfonyl)phenoxy]-6-[(1-methyl-1H-tetra-
zol-5-yl)methyl]-2-(pyridin-2-yl)-1H-benzimidazole (3l). Com-
pound 3l was prepared in a similar manner to the step of Section
5.1.2.6 using 25 instead of 17. 1H NMR (CDCl3) d: 10.65 (1H, br
s), 8.67–8.63 (1H, m), 8.40–8.35 (1H, m), 7.91–7.85 (1H, m), 7.84
(2H, d, J = 8.6 Hz), 7.70 (1H ꢀ 1/2, s), 7.48 (1H ꢀ 1/2, s), 7.46
(1H ꢀ 1/2, s), 7.43–7.39 (1H, m), 7.17 (1H ꢀ 1/2, s), 7.05 (2H ꢀ 1/
2, d, J = 8.6 Hz), 7.02 (2H ꢀ 1/2, d, J = 8.6 Hz), 4.34 (2H, s), 3.91
(3H ꢀ 1/2, s), 3.90 (3H ꢀ 1/2, s), 3.13 (2H, q, J = 7.4 Hz),1.31 (3H,
t, J = 7.4 Hz). HRMS m/z: Calcd for C23H22N7O3S ([M+H]+)
476.1505. Found: 476.1507.
(2 g/kg) and either compound 3g or vehicle alone (0.5% methylcel-
lulose solution) at the same time. Blood glucose concentrations
were measured by OneTouch Ultra (LifeScan, Inc., Milpitas, CA) just
prior to and following the glucose challenge (30, 60, 90, and
120 min). The area under the curve (AUC) values were calculated
from the data (from 0 min to 120 min). Statistical analysis was per-
formed using Prism Software (version 4.00; GraphPad Software
Inc.). One-way analysis of variance was used followed by Dunnett’s
test for multiple comparisons. Values of p < 0.05 were considered
statistically significant.
5.3. X-ray crystallography
5.1.18. 4-(Ethylsulfonyl)phenol (27)
To a stirred cooled solution of 4-mercaptophenol (5.00 g,
39.6 mmol) in acetone (50.0 mL) was added K2CO3 (5.74 g,
41.6 mmol) and EtI (4.75 mL, 59.4 mmol), and the mixture was
stirred at rt for 3 h. The insoluble solid was filtered off and the fil-
trate was concentrated in vacuo. The residue was dissolved into
Et2O and extracted with 2 M aq NaOH. The aqueous layer was sep-
arated, acidified with 6 M aq HCl, and then extracted with Et2O.
The organic layer was separated, washed with brine, dried over
MgSO4, concentrated in vacuo to give a crude product containing
4-(ethylsulfanyl)phenol (7.21 g) as brown oil. This was dissolved
into AcOH (50.0 mL), and 30% aq H2O2 was carefully dropped.
And the reaction mixture was stirred at 100 °C (bath temperature)
for 1 h. Then the mixture was cooled to rt, poured into saturated aq
NaHCO3 on crushed ice and repeatedly extracted with EtOAc. The
combined organic layers were washed with brine, dried over
MgSO4 and concentrated in vacuo. The residue was purified by col-
umn chromatography on silica gel eluted with EtOAc/n-hexane to
give 27 (8.29 g, 44.5 mmol, 112%) as pale yellow oil.
Protein and protein crystals were obtained according to estab-
lished procedures.3 Crystals were soaked in 0.5 mM compound
3g overnight in mother liquor containing 5% DMSO. Diflayer data
were collected on beamline BL32B2 at the SPring-8, at 100 K (a Rig-
aku R-axis V imaging plate). Data processing and data reduction
were carried out using programs from the HKL2000 (HKL Research,
Inc.) and the CCP4 package. Compound 3g was modeled into the
electron density using Afitt (OpenEye Scientific Software). The pro-
tein-compound complex model was refined using REFMAC5 of the
CCP4 package. The final structure has been deposited in the Protein
Data Bank under deposition code 3H1 V, together with structure
factors and detailed experimental conditions.
Crystallographic statistics for the GK-compound 3g complex are
as follows: space group P6522, unit cell 79.8, 79.8, 326.1 Å, resolu-
tion 2.11 Å, 33,083 reflections from 185,069 observations give
94.7% completeness with Rsym of 4.8% and mean I/r (I) of 31.9.
The final model containing 3493 protein, 120 water, 1 salt, 12 glu-
cose and 34 compound atoms has an R-factor of 22.1% (Rfree using
5% of the data 27.5%). Mean temperature factors for the protein and
the ligand are 40.2 and 31.5 Å2, respectively.
5.2. Biology and pharmacology
5.2.1. GK assay
Acknowledgments
The recombinant human liver GK used in this assay was ex-
pressed in Escherichia coli as a FLAG fusion protein. GK activity
was measured by the glucose-6-phosphate dehydrogenase coupled
continuous spectrophotometric assay. GK was incubated with
DMSO in assay buffer containing 25 mM Hepes, pH 7.2, 1 mM
dithiothreitol, 0.5 mM thionicotinamide adenine dinucleotide,
2 mM MgCl2, 1 mM ATP, 2 U/mL glucose-6-phosphate dehydroge-
nase and 2.5 or 10 mM glucose at 30 °C. Reaction velocities were
obtained from the rate of increase in absorbance at 405 nm after
5 min of reaction. The OD values were measured at each concentra-
tion of the evaluated compound, using the OD value of the DMSO
The authors thank Hirokazu Ohsawa and Shigeru Nakajima for
the analytical and spectral studies, and Hideyuki Mukai and Takuro
Hasegawa for the PK study.
References and notes
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)
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mice fed a high-fat diet (HFD) were freely fed before performing
the test. To the mice were orally administrated each compound
2b, 3a, 3b, 3g, 3h or vehicle alone (0.5% methylcellulose solution).
Blood glucose concentrations were measured just prior to and fol-
lowing oral dosing (1, 2, 3 and 4 h).
5.2.3. Rat OGTT study
Nine-week-old male Wistar rats were fasted overnight before
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