Discovery of novel 2-(pyridine-2-yl)-1H-benzimidazole derivatives as potent glucokinase activators

Article abstract of DOI:10.1016/j.bmcl.2009.05.038

The synthesis and structure-activity-relationships (SARs) of novel 2-(pyridine-2-yl)-1H-benzimidazole glucokinase activators are described. Systematic modification of benzimidazole lead 5a identified from a high-throughput screening led to the discovery o

Full text of DOI:10.1016/j.bmcl.2009.05.038

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4450–4454
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel 2-(pyridine-2-yl)-1H-benzimidazole derivatives as potent
glucokinase activators
*
Makoto Ishikawa , Katsumasa Nonoshita, Yoshio Ogino, Yoshikazu Nagae, Daisuke Tsukahara,
Hideka Hosaka, Hiroko Maruki, Sumika Ohyama, Riki Yoshimoto, Kaori Sasaki, Yasufumi Nagata,
Jun-ichi Eiki, Teruyuki Nishimura
Banyu Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd, Okubo 3, Tsukuba 300-2611, Ibaraki, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and structure–activity-relationships (SARs) of novel 2-(pyridine-2-yl)-1H-benzimidazole
glucokinase activators are described. Systematic modification of benzimidazole lead 5a identified from
a high-throughput screening led to the discovery of a potent and metabolically stable glucokinase acti-
vator 16p(R) with greater structural diversity from GKAs reported to date. The compound also demon-
strated acute oral glucose lowering efficacy in rat OGTT model.
Received 24 March 2009
Revised 11 May 2009
Accepted 12 May 2009
Available online 18 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Glucokinase
Glucokinase activator
Diabetes
Glucokinase (GK) is a hexokinase isoform (hexokinase IV) which
is predominantly expressed in liver and pancreatic beta-cells.1 It
functions as a key initial and rate-limiting step for glucose metab-
olism, that is, catalyzing the reaction of glucose to glucose-6-phos-
phate. In the liver, GK activation increases hepatic glucose uptake
and utilization, while GK activity is coupled to increase insulin
secretion in beta cells.2,3 Therefore, GK activators (GKAs) can be ex-
pected to work as hypoglycemic agents by both increasing glucose
uptake in liver and potentiating insulin secretion from pancreatic
beta-cells. Consistent with this rationale, several groups have de-
scribed the structures and SARs of various small-molecule
GKAs,4–9 exemplified by compounds 110 and 2.11 We also recently
reported the SARs on our own series of 2-aminobenzamide (3)12
and 3,6-disubstituted-2-pyridinecarboxiamide GKAs (4a, 4b),13
respectively, which function through binding to an allosteric site
of GK enzyme.14
tive molecule for further modification to improve GK potency and
develop structurally diverse GKAs. In this report, the synthesis and
SARs on a novel series of GKAs from 2-(pyridine-2-yl)-1H-benz-
imidazole are described.
The general synthetic route of benzimidazole derivatives re-
ported in this Letter is illustrated in Scheme 1. Commercially avail-
able 4-bromo-3-fluoroaniline 6 was coupled with 1-Boc-pyrrol-2-
boronic acid15 using Suzuki–Miyaura coupling to yield compound
7. Reduction of pyrrole 7 with Pt/C under hydrogen atmosphere
produced a racemic mixture of pyrrolidine derivative 8. Manipula-
tion of protecting group of Boc-pyrrolidine 8 and subsequent nitra-
tion by fuming nitric acid afforded nitrobenzene compound 9. The
trifluoroacetamide groups were then removed under basic condi-
tions and the resulting pyrrolidine amine was capped by t-butylc-
arbamate group (10), followed by displacement of the fluorine
atom on 5-position with 4-fluorophenol via nucleophilic aromatic
substitution, giving nitroaniline derivative 11. The nitro group was
reduced with Raney-nickel to provide o-phenylendiamine 12. Con-
densation of the diamine 12 with 2-pyridinecarboxaldehyde and
subsequent in situ thermal cyclization afforded the targeted 2-pyr-
idyl-1H-benzimidazole structure (13).16 Deprotection of the Boc
group on the pyrrolidine portion under acidic conditions gave
key intermediate 14a. Finally, reaction of the amine compound
14a with appropriate electrophiles gave the corresponding N-
substituted pyrrolidine derivatives 5a and 15a–g.
During our own effort to identify structurally diverse GKAs, a
benzimidazole compound 5a was identified as a lead with lM or-
der of GK activation from a high-throughput screen of our com-
pound collection. As shown in Figure 1, GKAs which have been
disclosed in the literature to date commonly possess the heteroar-
omatic amide structure as an important pharmacophore to bind to
the allosteric site of GK and show GK activation. Since the newly
identified benzimidazole template is entirely structurally distinct
from GKAs previously disclosed, compound 5a could be an attrac-
3-(pyridine-2-yl)-(5b) and 3-(phenyl-2-yl)-1H-benzimidazole
derivatives (5c) were prepared by the same reaction sequence as
those of compound 5a: coupling and cyclization reaction between
* Corresponding author. Tel.: +81 055 88 2430; fax: +81 055 89 0945.
E-mail address: makoto.ishikawa@mochida.co.jp (M. Ishikawa).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.038

M. Ishikawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4450–4454
4451
Figure 1. Structures of glucokinase activators (1–4a, b) reported in the literature and lead compound 5a.
Scheme 1. Reagents and conditions: (a) Pd(PPh)2Cl2, Na2CO3, 80 °C, DME, H2O, 63%; (b) Pt/C, H2, MeOH, 85%; (c) TFA; (d) (CF3CO)2O, pyridine; (e) fHNO3, 87% for three steps;
(f) K2CO3, MeOH–H2O; (g) Boc2O, Et3N, CHCl3, 67% for two steps; (h) Cs2CO3, 4-fluorophenol, NMP, 80 °C, 85%; (i) Raney-Ni, NH2NH2, MeOH, 90%; (j) 2-
pyridinecarboxaldehyde, EtOH, AcOH, 80 °C, 65%; (k) electrophile, CHCl3, TEA, room temperature.
the o-phenylendiamine intermediate 12 and 3-pyridinecarboxal-
dehyde for 5b or benzaldehyde for 5c, respectively. An N-methyl-
benzimidazole analogue 5d was prepared by methylation of 5a
with methyl iodide in the presence of NaH. The 5-phenoxy benz-
imidazole derivatives 16a–q shown in Table 3 were synthesized
in a similar manner to those of 4-F-phenyl compound 15e by reac-
tion of nitroaniline intermediate 10 with appropriate phenols in-
stead of 4-fluorophenol.
Prior to chemical modification of the benzimidazole lead, the
predicted binding mode of lead compound 5a to the allosteric site
of GK enzyme was examined by molecular modelling analysis. Pre-
viously we reported the results of co-crystal structure analysis and
the binding mode of GKAs 3 and 4b containing a 2-amino thiazole
amide scaffold as a common structural feature. According to the
co-crystal structure of GK complex with compound 4b (Fig. 2),
the amino-thiazole moiety makes two critical hydrogen-bonding
interactions with the enzyme to achieve potent GK activation: both
the NH and nitrogen atom of the compound with the C@O and NH
of the backbone Arg63. In contrast, a docking model of compound
5a into the allosteric binding site of amide GKAs led to the pro-
posed enzyme binding mode shown in Figure 3. This proposed
model suggests that the novel 2-(pyridine-2-yl)-1H-benzimidazole
core also can make key hydrogen-bonding interactions with back-
All synthesized compounds were tested for their GK activations
measured by glucose-6-phosphate dehydrogenase coupled contin-
uous spectrophotometric assay.17,18 Both EC50 values and maximal
effective responses (Emax)
19 are described for comparison of deriv-
atives in each assay. In addition, metabolic stability in microsomes
was measured to screen orally-available compounds before in vivo
efficacy study.20,21

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M. Ishikawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4450–4454
Table 1
SAR of Ar and R1 substituent
F
O
N
N
NR1
Ar
O
O
R1
EC50
(lM)
Emax (%)
a
b
Cpd.
Ar
5a
5b
5c
5d
2-Pyridyl
3-Pyridyl
Phenyl
H
H
H
Me
5.5
93
NAc
NAc
NAc
NAc
NAc
NAc
3-Pyridyl
a
Values are the mean of two or more independent assays.
See Ref. 19 for detailed description.
No activation at 30 lM.
b
c
Figure 2. X-ray co-crystal structure of 4b bound to glucokinase.
was examined (Table 2). The NH free pyrrolidine compound 14a
resulted in complete loss of potency. The analogues with N-alkyl
substituents such as methyl (15a) or ethyl (15b) also showed no
GK activation although they showed improved metabolic stability
in human microsomes compare with carbamate lead 5a. The po-
tency of N-methylsulfonylamide (15c) and N-ethylurea (15d)
derivatives decreased substantially compared to ethoxycarbonyl
compound 5a. During the course of the modification, acetyl group
(15e) was identified as an acceptable surrogate for the carbamate
group and resulted in improvement of both GK potency and meta-
bolic stability in human microsomes (25% for 15e vs 8% for 5a). The
increase in bulk of the substituent on the pyrrolidine part such as
propionyl (15f) and benzoyl group (15g) resulted in reduction of
potency.
Next we turned our attention to substitution on the phenoxy
moiety at the 5-position of the benzimidazole (15e) in order to im-
prove GK potency and identify a candidate for in vivo evaluation
(Table 3). First, we confirmed the effect of the regio-chemical
structure of a fluoro atom, a methoxy group and a cyano group
on GK potency. As a result, introduction of electron donating
(16e and f) or withdrawing (16h and i) groups onto the 3- or 4-po-
Figure 3. Predicted binding mode for 5a bound to glucokinase.
bone Arg63 as well as heteroaromatic amide GKAs, and two sub-
stituents on the benzimidazole seem to occupy each hydrophobic
interaction sites.
Table 2
SAR of N-substituted pyrrolidine derivatives
To clarify our hypothesis on the binding mode of a benzimid-
azole GKA (5a), we investigated the effect of the position of the
nitrogen atom in the aromatic ring (Ar) at the right hand side of
the molecule and a substituent (R1) on the NH in the benzimid-
azole core to their GK potency (Table 1). The replacement of 2-pyr-
idyl (5a) moiety with 3-pyridyl (5b) or phenyl (5c) resulted in a
complete loss of GK potency. In addition, 1-methybenzimidazole
derivative 5d also completely lost its potency. Because these de-
creases in the GK potency were presumably due to the loss of
hydrogen bonding interaction of the nitrogen atom at 2-pyridine
moiety or NH in the benzimidazole core with Arg 63 on GK en-
zyme, the 2-(pyridine-2-yl)-1H-benzimidazole template in this
lead structure (5a) would be an important pharmacophore for GK
activation.
To explore the detailed SARs of the substitutions at the 5- or 6-
position on the benzimidazole portion, 2-(pyridine-2-yl)-1H-benz-
imidazole core structure was kept based on the predicted binding
mode and primary SARs on the lead compound 5a. Initially, the im-
pact of the substitution (R2) on the pyrrolidine moiety at the 6-po-
sition of the benzimidazole on GK potency and metabolic stability
F
O
N
N
N
NR2
H
a
b
Cpd.
R2
EC50 (lM)
Emax (%)
HM stabilityc % remaining
5a
CO2Et
H
Me
5.47
NAd
NAd
NAd
19.1
>30
1.1
93
8
69
77
66
26
37
26
6
14a
15a
15b
15c
15d
15e
15f
15g
NAd
NAd
NAd
132
64
Et
SO2Me
CONHEt
COMe
COEt
COPh
93
76
44
6.1
>30
44
a
Values are the mean of two or more independent assays.
See Ref. 19 for detailed description.
See Ref. 20 for detailed description.
b
c
d
No activation at 30 lM.

M. Ishikawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4450–4454
4453
Table 3
16p(R) and 16p(S), respectively. The absolute configuration of
16p(R) and 16p(S) were determined based on authentic samples
prepared by reported asymmetric synthesis.23,24 Between these
two enantiomers, one isomer 16p(R) was identified as an active
Effects of substitution on the benzene ring
R3
isomer with 0.36 lM EC50 value (Table 3).
O
N
We describe here the glucose lowering efficacy of compound
16p(R) in male Wistar rat. In rat Oral Glucose Tolerance Test
(OGTT),25 oral administration of compound 16p(R) demonstrated
glucose lowering efficacy in a dose-dependent manner from
3 mpk to 30 mpk (Fig. 4a), and showed significant reduction of
plasma glucose at 3 mg/kg (Fig. 4b).
In summary, we have identified novel 2-(pyridine-2-yl)-1H-
benzimidazole GKAs which are entirely structurally distinct from
GKAs reported so far with heteroaromatic amine templates. Sys-
tematic modifications on lead compound 5a led to an improve-
ment in GK potency and metabolic stability. Further we
identified an N-acetyl-pyrrolidinylbenzimidazole derivative
N
H
N
NAc
Cpd.
R3
EC50
(lM)
Emax (%)
HM stabilityc % remaining
a
b
16a
16b
16c
15e
16d
16e
16f
16g
16h
16i
16j
16k
16l
16m
16n
16o
16p
16p(R)
16p(S)
16q
H
2-F
3-F
4-F
2-OMe
3-OMe
4-OMe
2-CN
3-CN
4-CN
1.1
3.0
0.93
1.1
9.6
2.4
1.7
>30
1.9
1.9
7.4
1.5
1.6
2.7
1.9
1.1
0.63
0.36
2.3
1.5
110
87
138
93
103
111
109
28
18
19
17
26
24
28
47
17
46
48
62
53
82
46
60
76
61
41
—
83
110
88
3-Ph
4-Ph
89
4-CONH2
4-CO2Et
4-NO2
4-SO2Me
4-SO2Et
100
100
100
113
98
113
67
4-SO2iPr
100
30
a
Values are the mean of two or more independent assays.
See Ref. 19 for detailed description.
See Ref. 20 for detailed description.
b
c
sition of the benzene ring at the phenoxy portion were tolerable. In
contrast, installation of these substituents at 2-position of the phe-
nyl ring reduced GK potency (16b, d and g). Incorporation of a
bulky substituent like phenyl into the 3-position (16j) resulted in
substantial loss of GK activity compared to the 4-substituted deriv-
ative (16k). In addition, para-substituted analogues tended to show
significantly improved metabolic stability in human microsomes.
From these results, our modification effort focused on the 4-posi-
tion of the phenoxy moiety and we examined hydrophilic substitu-
tions (16l–q) to improve metabolic stability in human microsomes.
Among these analogues, 4-ethylsulfonylphenoxy derivative 16p
exhibited
(EC50 = 0.63
a
l
satisfactory balance between GK potency
M) and metabolic stability in human microsomes
(61% remaining) to address the lead potential of this structure class
in animal model.
The next logical step was to synthesize and compare two indi-
vidual enantiomers of analogue 16p. Key compound 14b (Scheme
2) was prepared from intermediate 10 in a similar manner as 4-flu-
oro-phenoxy compound 14a using 4-ethylsulfonylphenol in place
of 4-fluorophenol. Racemic pyrrolidine compound 14b was sepa-
rated by chiral HPLC to identify each enantiomer.22 Subsequently,
both isomers were converted to corresponding acetyl derivatives
Figure 4. In vivo efficacy data of compound 16p(R) in male Wister rat OGTT.25 (a)
Plasma glucose lowering and (b) AUC reduction. *Values of p < 0.05 were considered
statistically significant for vehicle group.
Scheme 2. Reagents and conditions: (a) chiral separation by HPLC eluted with n-hexane/iPrOH (9/1)/0.1% Et2NH; (b) Ac2O, pyridine.

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M. Ishikawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4450–4454
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maximal increased enzymatic activities of glucokinase by the compound at
2.5 mM glucose. Compound 3 was used an internal control across all assay
16p(R) which showed significant in vivo glucose lowering efficacy
in rat OGTT model. The predicted binding mode by molecular mod-
elling and our modification results on lead compound 5a suggested
that the 2-pyridyl-benzimidazole pharmacophore would be an
alternative allosteric GKA instead of the heteroaromatic amide
template. Co-crystal structure analysis of benzimidazole deriva-
tives with GK protein are underway to confirm the binding mode
of this novel structure class of GKAs, and further modification re-
sults on this series to improve GK potency will be reported in
due course.
Acknowledgement
plates for data validation. The EC50 value of compound 3 is 0.42 0.09 lM.
Detailed assay methods and conditions are described in Ref. 18.
We would like to acknowledge the contributions of colleagues
in biochemistry, pharmacology and pharmacokinetics in generat-
ing and interpreting the data reported in this communication.
We thank Yukari Tachibana for the binding mode prediction of
GKAs.
18. Futamura, M.; Hosaka, H.; Kadotani, A.; Shimazaki, H.; Sasaki, K.; Ohyama, S.;
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compound 3 and the maximal response of test compounds was compared to
that of compound 3.
20. Human microsomal stability was determined by % parent compound (1 lM)
remaining after 30 min (37 °C) incubation with human liver microsomes
(0.25 mg protein/ml).
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