The design and synthesis of indazole and pyrazolopyridine based glucokinase activators for the treatment of Type 2 diabetes mellitus

Article abstract of DOI:10.1016/j.bmcl.2012.09.082

Glucokinase activators represent a promising potential treatment for patients with Type 2 diabetes. Herein, we report the identification and optimization of a series of novel indazole and pyrazolopyridine based activators leading to the identification of 4-(6-(azetidine-1-carbonyl)-5-fluoropyridin-3- yloxy)-2-ethyl-N-(5-methylpyrazin-2-yl)-2H-indazole-6-carboxamide (42) as a potent activator with favorable preclinical pharmacokinetic properties and in vivo efficacy.

Full text of DOI:10.1016/j.bmcl.2012.09.082

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7100–7105
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The design and synthesis of indazole and pyrazolopyridine based
glucokinase activators for the treatment of Type 2 diabetes mellitus
Jeffrey A. Pfefferkorn a, , Meihua Tu a, Kevin J. Filipski a, Angel Guzman-Perez b, Jianwei Bian b,
⇑
Gary E. Aspnes b, Matthew F. Sammons a, Wei Song b, Jian-Cheng Li b, Christopher S. Jones b, Leena Patel b,
Tim Rasmusson b, Dongxiang Zeng b, Kapil Karki b, Michael Hamilton b, Richard Hank b, Karen Atkinson b,
John Litchfield b, Robert Aiello b, Levenia Baker b, Nicole Barucci b, Patricia Bourassa b,
Francis Bourbounais b, Theresa D’Aquila b, David R. Derksen b, Margit MacDougall b, Alan Robertson b
a Pfizer Cardiovascular, Metabolic and Endocrine Disease Research Unit, Pfizer Worldwide Research and Development, 620 Memorial Drive, Cambridge, MA 02139, USA
b Pfizer Worldwide Research and Development, Eastern Point Road, Groton, CT 06442, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Glucokinase activators represent a promising potential treatment for patients with Type 2 diabetes.
Herein, we report the identification and optimization of a series of novel indazole and pyrazolopyridine
based activators leading to the identification of 4-(6-(azetidine-1-carbonyl)-5-fluoropyridin-3-yloxy)-2-
ethyl-N-(5-methylpyrazin-2-yl)-2H-indazole-6-carboxamide (42) as a potent activator with favorable
preclinical pharmacokinetic properties and in vivo efficacy.
Received 20 August 2012
Revised 18 September 2012
Accepted 21 September 2012
Available online 29 September 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Glucokinase activator
Type 2 diabetes mellitus
Type 2 diabetes mellitus (T2DM) is a rapidly growing public
health problem affecting more than 300 million people world-
wide.1 Despite the availability of various anti-diabetic therapies,
many patients fail to achieve and maintain treatment targets
increasing their long term risk of microvascular and macrovascular
complications. Hence, there remains a significant medical need for
novel treatments.2 Among the new therapies currently under
investigation, small molecule activators of glucokinase (GK) en-
zyme represent a promising opportunity to help patients achieve
improved glucose control.3
Glucokinase is responsible for the conversion of glucose to
glucose-6-phosphate (G-6-P) and is an important regulator of
glucose homeostasis.4 In the liver, glucokinase regulates hepatic
glucose uptake and output, whereas in the pancreas it functions
as a glucostat establishing the threshold for b-cell glucose-stimu-
lated insulin secretion. Glucokinase is also found in glucose sensing
neurons of the ventromedial hypothalamus where it regulates the
counter regulatory response (CRR) to hypoglycemia.5 Therapeuti-
cally, it is envisioned that activation of glucokinase in the liver
and pancreas would be an effective strategy for lowering blood
glucose by up-regulating hepatic glucose utilization, down-regu-
lating hepatic glucose output and enhancing glucose-stimulated
insulin secretion.
In 2003, Grimsby and coworkers reported the first small mole-
cule glucokinase activator which allosterically bound to the en-
zyme, increasing activity through modulation of both Km (i.e.
S
0.5) and Vmax.6 Subsequently, many structurally diverse small mol-
ecule activators of glucokinase have been reported.7 These glucoki-
nase activators have been shown to effectively lower blood glucose
in a variety of diabetic animal models and multiple candidates
have advanced into Phase 1 and 2 clinical studies where they have
been found to effectively lower both fasting and postprandial glu-
cose in T2DM patients.8 However, despite the promising efficacy of
this mechanism, there has been significant attrition in the clinical
development of glucokinase activators driven by narrow therapeu-
tic windows against hypoglycemia as well as concerns around
durability and chemotype-specific safety issues.8 These issues with
early activators have fostered investigations into structurally
diverse second generation activators designed to reduce hypogly-
cemia risk. We recently reported structure–activity studies of a
2-methyl benzofuran activator series (1, Fig. 1) culminating in
the selection of 2 as a clinical development candidate.9 Benzofuran
2 was specifically selected as a ‘partial activator’ of glucokinase de-
signed to cause a less significant reduction in the glucokinase Km
relative to earlier candidates; this profile maintained increased
glucose dependency of activation thereby reducing the risk of
hypoglycemia. To expand upon this work, we sought to explore
the use of various nitrogen-containing 5,6-heterocyclic scaffolds
to identify novel activators with potentially increased lipophilic
⇑
Corresponding author.
E-mail address: jeffrey.a.pfefferkorn@pfizer.com (J.A. Pfefferkorn).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.082

J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7100–7105
7101
Br
F
Br
F
Br
F
N
N
O
a
b
O
O
O
N
H
F
5
F
6
OMe
7
O
X
N
c
Y
N
H
H
N
1: X = N; Y = CH
2: X = CH; Y = N
CO2Me
CO2Me
Br
N
N
N
N
e
d
O
N
N
N
N
N
OMe
OMe
OMe
O
O
10
9
8
N
N
N
R1
N
R1
f
CO2Me
N
N
Cl
12: X = OH
13: X = Cl
g
h
3
4
+
X
14: X = NMe2
Figure 1. Structure benzofuran (1,2), indazole (3) and pyrazolopyridine (4) based
OH
O
glucokinase activators.
11
i
efficiency (LipE).10 To help guide these efforts, we utilized the
recently reported modeling tool NEAT (Novel and Electronically-
equivalent Aromatic Template) to evaluate the electronic
properties of a diverse array of substituted 5,6-heterocyclic ring
systems.11 This in silico analysis suggested that, among the
nitrogen-containing heterocyclic systems examined, indazole (3)
and pyrazolopyridine (4) were the most electronically comparable
to the parent benzofuran (2). Herein, we report the synthesis and
evaluation of activators based upon these two heterocyclic
templates.
Schemes 1 and 2 illustrate the synthesis of prototype indazole
and pyrazolopyridine activators 16 and 26, respectively. As shown
in Scheme 1 1-bromo-3,5-difluorobenzene (5) was selectively lith-
iated and then quenched with DMF to give benzaldehyde 6.12 Sub-
sequent mono-displacement of an ortho-fluorine with sodium
methoxide afforded 7. Treatment of 7 with hydrazine resulted in
a one pot condensation and cyclization to form indazole 8 in good
yield. Palladium-catalyzed carbonylation provided ester 9 which
was then selectively alkylated at the N-2 position using tri-meth-
yloxonium tetrafluoroborate.13 Hydroxyindazole 11 was formed
by global demethylation with boron tribromide followed by re-
esterification of the carboxylate with methyl alcohol and catalytic
acid. Separately, chloropyrazine 14 was formed by amidation of the
acid chloride formed from 5-chloropyrazine-2-carboxylic acid (12).
In the penultimate step, biaryl ether bond formation was accom-
plished via base-mediated nucleophilic displacement of 2-chloro-
pyrazine 14 with the indazole-phenol 11 to generate 15. Finally,
transamidation of 14 with 2-amino-5-methylpyrazine in the pres-
ence of Me2AlCl afforded indazole 16.
As shown in Scheme 2, pyrazolopyridine activator 26 was syn-
thesized by initial chlorination of methyl-5-hydroxynicotinate (17)
with bleach14 to chloro-pyridine 18. After benzyl protection of the
phenol under standard conditions, a palladium-catalyzed coupling
of 19 with the tributyltin enolate of isopropenyl acetate afforded
ketone 20.15 Conversion of 20 to indazole 23 was accomplished
by treatment with hydroxylamine hydrochloride to form interme-
diate oxime 21 which, upon treatment with trifluoroacetic anhy-
dride and triethylamine, was converted to the azirine 22
followed by FeCl2 catalyzed rearrangement.16 Hydrogenolysis of
the benzyl protecting group with catalytic palladium on carbon
afforded 24 which underwent subsequent base-mediated coupling
with 14 (from Scheme 1) to afford heteroaryl ether 25. Aluminum-
mediated transamidation of 25 with 2-amino-5-methylpyrazine
then provided pyazolopyridine 26. Through incorporation of alter-
native starting materials, the methods outlined in Schemes 1 and 2
N
O
CO2Me
N
N
N
H
N
j
O
N
N
O
N
N
N
N
O
O
16
15
Scheme 1. Synthesis of representative indazole 16. Reagents and conditions: (a)
diisopropylamine, n-BuLi, THF, DMF, À70 °C, 2 h, 63%; (b) NaOMe, MeOH, 0 °C –
reflux, 3.5 h, 71%; (c) N2H4–H2O, ethylene glycol, 95 °C, 24 h, 83%; (d) CO, Pd(OAc)2,
BINAP, Et3N, MeOH, 50 psi, 80 °C, 24 h, 87%; (e) trimethyloxonium tetrafluorobo-
rate, ethyl acetate, 23 °C, 16 h, 86%; (f) i) BBr3, DCM, 0 °C, 48 h; ii) MeOH, cat. H2SO4,
reflux, 2 h, 78%; (g) (COCl)2, cat. DMF, DCM, 23 °C, 18 h, quant.; (h) Me2N–HCl, Et3N,
DCM, 0 – 23 °C, 4 h, 85%; (i) Cs2CO3, CuI, DMF, 110 °C, 3 h, 63%; (j) 2-amino-5-
methylpyrazine, Me2AlCl (1.0 M in hexanes), DME, 90 °C, 16 h, 46%.
subsequently enabled the synthesis of all additional analogs inves-
tigated in these studies.
Biochemical evaluation of these prototype activators and subse-
quent analogs utilized recombinant human glucokinase, monitor-
ing the rate of glucose 6-phosphate formation using G6PDH/
NADP coupling. As previously described, this assay was performed
in a matrix format wherein dose response determinations for an
activator (at 22 different activator concentrations from 0 to
100 lM) were conducted at 16 different glucose concentrations
(ranging from 0 to 100 mM).9 Analyzing this data using a nones-
sential activator model enabled determination of an activator’s
maximum fold effect on reducing the glucokinase Km for glucose
defined as
Vmax defined as b.17 As previously described,
zyme’s Km at maximum activator concentration divided by the en-
zyme’s Km value in the absence of activator.9 Values of
range
from 0 to 1 with lower values of representing more substantial
a
and the maximum fold effect on altering the enzyme’s
a
is the ratio of the en-
a
a
reductions in the enzyme’s glucose Km. Activator potency or EC50
was formally defined as the concentration of activator affording a
half-maximal reduction in Km. The b value of an activator is defined
as the ratio of the enzyme’s velocity at maximum activator concen-
tration divided by the enzyme’s velocity in the absence of activa-
tor. Values of b > 1 represent activator induced increases in the
enzyme’s Vmax whereas values of b < 1 represented decreases in
the enzyme’s Vmax. Activators with b = 1 have no effect on Vmax
Human microsomal (HLM) stability, passive permeability and
kinetic solubility of the activators were also evaluated.
.

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J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7100–7105
CO2Me
CO2Me
CO2Me
indazole 16, it had a non-optimal activation profile given its more
significant suppression of Km as indicated by = 0.05. Previous
studies had suggested that glucokinase activators with P 0.1
may offer reduced hypoglycemia risk relative to those with lower
values.9 Additionally, pyrazolopyridine 26 did not offer an im-
proved LipE relative to the parent benzofuran (e.g. 1). Based on
these observations, our subsequent structure–activity efforts fo-
cused on the indazole template with the goal of increasing potency
while retaining a favorable activation profile.
As summarized in Table 2, initial optimization efforts explored
the structure–activity relationships of the A-Ring examining a vari-
ety of substituted aryl and heteroaryl rings. As a base case, indazole
27 bearing a 4-dimethyl amide substituted phenyl ring was found
N
N
O
N
a
c
a
Cl
a
OH
N
OR
OBn
20
18: X = H
17
b
19: X = Bn
a
d
CO2Me
CO2Me
HO
CO2Me
N
N
N
N
f
e
N
OBn
OBn
OBn
23
22
21
g
to have moderate potency (EC50 = 0.71 lM), but also a low b of
CO2Me
N
N
Cl
N
N
0.65 suggesting the potential of the molecule to inhibit glucokinase
at high glucose concentrations. Subsequent modification of 27,
sought to improve potency, increase b and reduce metabolic insta-
bility by examining alternative substituents and heterocyclic ring
systems. Initially, the amide of 27 was replaced by a series of sulf-
ones and sulfonamides as illustrated by 28–33. Relative to 27, both
the sulfones and sulfonamides offered increases in Vmax (as re-
flected by b > 1), improved metabolic stability and comparable per-
meability; however, these sulfur containing activators also had
reduced aqueous solubility with the exception of methyl sulfone
28. Only cyclopropyl sulfone 30 and dimethyl sufonamide 31 of-
fered potency improvements relative to 27. Unable to overcome
the limitations of these sulfone substituents, we next explored het-
erocyclic amides 34–36 (and prototype 16) bearing pyridine, pyra-
zine and pryrimidine A-rings. Pyridines 34 and 35 offered
moderate improvements in potency and metabolic turnover rela-
tive to 27. Both pyrazine 16 and pyrimidine 36 also offered favor-
able properties but only moderate potency.
+
N
OH
O
24
14
h
N
O
CO2Me
N
N
N
N
N
N
H
N
i
O
O
N
N
N
N
N
O
O
26
25
Scheme 2. Synthetic method for the synthesis of 26. Reagents and conditions: (a)
NaOCl, water, 0 °C, 1 h; (b) benzyl bromide, NaH, DMF, 23 °C–reflux, 40 min, 30%;
(c) isopropenyl acetate, Bu3SnOMe, Pd(dba)2, S–Phos, toluene, 100 °C, 16 h, 36%; (d)
NH4OH-HCl, MeOH, aq. NaOH, 70 °C, 1 h; (e) trifluoroacetic anhydride, Et3N, DME,
23 °C, 16 h; (f) FeCl2, 75 °C, 2 h, 36% over 3 steps; (g) H2, 10% Pd/C, EtOH, ethyl
acetate, 30 psi, 23 °C, 16 h; (h) K2CO3, DMF, 60 °C, 16 h; (i) 2-amino-5-methylpyr-
azine, Me2AlCl (1.0 M in hexanes), DME, reflux, 16 h, 43%.
To explore additional opportunities for potency improvements,
we next examined the effects of substitution at the N-2 position of
the indazole as illustrated in Table 3.
For the purposes of synthetic feasibility, the A-ring was
held constant as a 4-methylsulfone phenyl and the heterocyclic
amide as 5-methyl-aminopyrazine. Increasing steric bulk at N-2
offered improvements in potency as illustrated by 38 (R1 = nPr,
As shown in Table 1, indazole 16 and pyrazolopyridine 26, were
found to be 5- and 3-fold less potent, respectively, than benzofuran
1. Encouragingly, despite its reduced potency, indazole 16 had a
EC50 = 0.31
1.93 M); however, these potency improvements were accompa-
l l =
M) >37 (R1 = Et, EC50 = 0.77 M) >28 (R1 = Me, EC50
favorable activation profile (a = 0.11, b = 0.87) and its reduced lipo-
l
philicity (LogD = 0.83) afforded an increase in lipophilic efficiency
(LipE) relative to benzofuran 1, suggesting that it may offer a favor-
able starting point for further optimization. While pyrazolopyri-
nied by a significant loss in aqueous solubility. Attempts to
incorporate a hydroxyl ethyl group at R1 (e.g. 39) did not offer
improvements in solubility or potency.
dine 26 offered
a slight potency improvement relative to
Table 1
Properties of prototype indazole (16) and pyrazolopyridine (26) glucokinase activators relative to benchmark benzofuran (1)
N
N
N
N
O
O
O
O
N
N
N
H
N
N
H
N
N
N
H
N
O
N
N
O
N
N
O
N
N
N
N
N
1
16
26
O
O
O
Biochemical activationa
LogDb
LipEc
HLM Clint(ml/min/kg)
Permeabilityd (10À6cm/s)
Solubility(lM)
EC50
(l
M)
a
b
1
16
26
0.22
0.99
0.62
0.09
0.11
0.05
0.97
0.87
0.91
2.16
0.83
1.87
4.47
5.18
4.33
<8.0
<8.5
13.7
22.3
20.8
17.0
176
192
123
a
b
c
Biochemical data reported as the geometric mean of >2 determinations.
Experimental LogD determined by shake flask method.
LipE = Àlog (EC50)ÀLogD.
d
Passive permeability assessed in RRCK cell line.

J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7100–7105
7103
Table 2
Biochemical properties of glucokinase activators 16 and 27–36
N
N
O
N
N
H
N
W
O
X
A
Z
R1
Y
A-Ring
Compound
R1
Biochemical activationa
HLM Clint(ml/min/kg)
Permeabilityb (10À6cm/s)
Kinetic solubility(lM)
EC50
(l
M)
a
b
O
27
C(O)NMe2
0.71
0.16
0.65
12.3
14.6
351
28
29
30
31
32
33
SO2Me
SO2Et
SO2cPr
SO2NMe2
SO2NHMe
SO2NH2
1.93
0.98
0.51
0.44
0.88
2.32
0.20
0.11
0.09
0.10
0.07
0.50
1.05
1.07
1.01
1.14
1.18
1.14
<8.0
<8.0
<8.0
<8.0
<8.6
NT
20.4
10.6
17.8
13.6
NT
454
69
73
134
27
NT
NT
F
O
34
35
16
C(O)NMe2
0.40
0.34
0.99
0.11
0.14
0.11
0.80
0.71
0.86
<8.0
<8.0
<8.0
20.2
11.9
20.8
328
70
N
N
O
N
N
O
O
C(O)NMe2
C(O)NMe2
192
N
36
0.91
0.13
0.82
<8.0
11.5
446
N
a
Biochemical data reported as the geometric mean of >2 determinations.
Passive permeability assessed in RRCK cell line; NT = not tested.
b
Table 3
Properties of glucokinase activators 28 and 37–40
N
O
N
N
H
N
R1
S
N
O
O
O
R1
Biochemical Activationa
HLM Clint(ml/min/kg)
Permeabilityb (10À6cm/s)
Kinetic solubility(
l
M)
EC50
(l
M)
a
b
28
37
38
Me
Et
nPr
1.93
0.77
0.31
0.20
0.15
0.06
1.05
1.19
1.20
<8.0
<8.0
<8.0
20.4
13.4
14.2
454
2.1
2.3
OH
39
40
1.54
2.03
0.10
0.13
1.07
1.16
<8.9
<8.0
6.3
15.5
24.4
0.5
cPr
a
Biochemical data reported as the geometric mean of >2 determinations.
Passive permeability assessed in RRCK cell line.
b
In order to maintain the potency advantage offered by increas-
N-dimethylpicolinamide (41) or the corresponding azetidine analog
(42) offered an encouraging potency improvement. Comparing 41
and 42 to the original indazole prototype 16 revealed that the po-
tency improvements of 41 and 42 came at the expense of increased
ing steric bulk at N-2 but improve solubility, the N-2 ethyl core was
then combined with several heterocyclic A-ring systems as sum-
marized in Table 4. In particular, combination with 3-fluoro-N,

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J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7100–7105
Table 4
Properties of glucokinase activators 16, 41 and 42
N
O
N
N
H
N
R1
N
X
O
Y
A
R2
Z
R1
Et
A-Ring Heterocycle
Biochemical activationa
LogDb
LipEc
HLM Clint (ml/min/kg)
Permeabilityd (10À6cm/s)
Kinetic solubility(lM)
EC50
(l
M)
a
b
F
O
41
42
16
0.23
0.06
0.80
1.80
4.83
4.91
5.18
10.4
13.8
367
N
N
O
F
O
Et
0.12
0.99
0.12
0.11
0.82
0.87
2.01
0.83
<8.0
<8.0
16.3
20.8
84.8
192
N
N
O
N
N
O
Me
N
O
a
b
c
Biochemical data reported as the geometric mean of >2 determinations.
Experimental LogD determined by shake flask method.
LipE = Àlog (EC50)ÀLogD.
d
Passive permeability assessed in RRCK cell line.
lipophilicity such that the LipE of these analogs (41: LipE = 4.83;
42: LipE = 4.91) was slightly less than the indazole prototype (16:
LipE = 5.18) yet still better than parent benzofuran (1: LipE = 4.47).
Based on its overall balance of potency, activation profile, clearance
(low), permeability (high) and solubility (moderate), activator 42
was selected for further pharmacological and pharmacokinetic
characterization.
was first determined using the method described above for human
glucokinase. Activator 42 was found to have an EC50 = 0.35 M for
l
rat glucokinase with an activation profile similar to that observed
for the human glucokinase. Since within the pancreatic b-cell glu-
cokinase establishes the threshold for glucose-stimulated insulin
secretion, the effect of 42 on glucose-stimulated insulin secretion
was next evaluated in a rat insulinoma (INS-1) cell line as shown
in Figure 2. In this experiment, INS-1 cells were incubated in the
presence of 5 mM glucose and then treated with increasing con-
centrations of 42, demonstrating a dose dependent enhancement
To support the further characterization of compound 42 in rat
models, its potency for the recombinant rat glucokinase enzyme
of glucose-stimulated insulin secretion (EC50 = 0.17 lM).
250
200
150
The pharmacological selectivity and early safety of 42 was eval-
uated using a series of in vitro assessments. First, compound 42
was evaluated against a CEREP panel of 69 receptors, ion channels
and enzymes; testing at 10 lM using a 30% threshold revealed no
off target pharmacology. Activator 42 was negative for genetic tox-
icity in both Ames and in vitro micronucleus (IVMN) assessments.
Finally, in a hERG patch clamp assay, 42 had an IC50 >300 lM.
100
EC50 = 0.17 µM
The pharmacokinetic properties of 42 were next characterized
in rat as summarized in Table 5. Compound 42 was found to have
low clearance (10.5 ml/min/kg) and good oral bioavailability (69%).
After the i.v. dose of 42, less than 1% of the dose was excreted in
urine suggesting negligible renal clearance. Activator 42 was also
found to have moderate protein binding in rat (fu = 0.18) and hu-
man (fu = 0.20) plasma.
50
0
.01
.1
1
10
Compound 42 (µM)
Figure 2. Effect of 42 on glucose-stimulated insulin secretion in rat INS-1 cells at
5 mM glucose.
Table 5
Compound 42 pharmacokinetic propertiesa
Route
Dose (mg/kg)
Cmax(ng/mL)
AUC (ng hr/mL)
t1/2 (h)
CL (mL/min/kg)
Vdss (L/kg)
F (%)
i.v.
p.o.
1
5
1820
1490
1580
5440
1.0
2.6
10.5
—
0.61
—
—
69%
a
Wistar-Han rats with n = 3 animals/group.

J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7100–7105
7105
benchmark clinical candidate 2; hence, further clinical develop-
ment of 42 was not pursued.
Vehicle
10 mg/kg
25 mg/kg
100 mg/kg
250 mg/kg
200
150
100
50
References and notes
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(b) Coghlan, M.; Leighton, B. Expert Opin. Investig Drugs 2008, 17, 145.
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048335.
-60
0
15 30
60
120
Time (Minutes)
Figure 3. Oral glucose tolerance test (OGTT) for compound 42. Dose dependent
effect of 42 on plasma glucose following a single oral dose of 10, 25, 100 or 250 mg/
kg during an oral glucose tolerance test. Glucose excursion data expressed as
Means Standard Error (n = 7 animals/group).
To evaluate the in vivo efficacy and hypoglycemia safety, 42
was evaluated using an oral glucose tolerance test (OGTT) con-
ducted in Sprague-Dawley rats as shown in Figure 3. Animals were
fasted overnight and orally dosed at t = À60 min with 42 (10, 25,
100 and 250 mg/kg) as a suspension in 30% sulfobutylether/water.
To evaluate efficacy, an oral glucose challenge (2 g/kg) was admin-
istered at t = 0 min and the effect of the activator at reducing the
glucose AUC excursion was determined. As shown in Figure 3, 42
offered relatively dose-dependent glucose lowering with a 22%
reduction in glucose AUC relative to vehicle control observed at
the highest dose (250 mg/kg) evaluated. There were no occur-
rences of hypoglycemia (defined as blood glucose <60 mg/dL) in
this study. For comparison, previously reported clinical candidate
benzofuran 2 evaluated at doses of 10, 30 and 100 mg/kg in the
same in vivo model offered glucose AUC reductions of 25, 38 and
43%, respectively.
13. Cheung, M.; Boloor, A.; Stafford, J. A. J. Org. Chem. 2003, 68, 4093.
14. Zhu, Y.; Loso, M. R.; Nugent, B. M.; Huang, J. X.; Rogers, R. B.; US2008/108667,
2008.
15. Kosugi, M.; Hagiwara, I.; Sumiya, T.; Migita, T. Bull. Chem. Soc. Jpn. 1984, 57,
242–246.
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C. J.; Boyd, F. L., Jr.; Peel, M. R. Tetrahedron 2003, 59, 9001.
17. Segel, I. H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and
Steady State Enzyme Systems; Wiley: New York, 1993.
In conclusion, herein we have reported the identification of a
series of indazole-based glucokinase activators. Optimization ef-
forts led to the identification of 42 as a potent activator with good
preclinical pharmacokinetic properties. While 42 offered in vivo
efficacy, the magnitude of this efficacy was inferior to that of