Discovery of (S)-6-(3-cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-1-yl) propanamido)nicotinic acid as a hepatoselective glucokinase activator clinical candidate for treating type 2 diabetes mellitus

Article abstract of DOI:10.1021/jm2014887

Glucokinase is a key regulator of glucose homeostasis, and small molecule allosteric activators of this enzyme represent a promising opportunity for the treatment of type 2 diabetes. Systemically acting glucokinase activators (liver and pancreas) have bee

Full text of DOI:10.1021/jm2014887

Article
pubs.acs.org/jmc
Discovery of (S)-6-(3-Cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-
1-yl)propanamido)nicotinic Acid as a Hepatoselective Glucokinase
Activator Clinical Candidate for Treating Type 2 Diabetes Mellitus
Jeffrey A. Pfefferkorn,*,† Angel Guzman-Perez,‡ John Litchfield,‡ Robert Aiello,‡ Judith L. Treadway,‡
John Pettersen,‡ Martha L. Minich,‡ Kevin J. Filipski,† Christopher S. Jones,‡ Meihua Tu,† Gary Aspnes,‡
Hud Risley,‡ Jianwei Bian,‡ Benjamin D. Stevens,† Patricia Bourassa,‡ Theresa D’Aquila,‡ Levenia Baker,‡
Nicole Barucci,‡ Alan S. Robertson,‡ Francis Bourbonais,‡ David R. Derksen,‡ Margit MacDougall,‡
Over Cabrera,† Jing Chen,‡ Amanda Lee Lapworth,† James A. Landro,‡ William J. Zavadoski,‡
Karen Atkinson,‡ Nahor Haddish-Berhane,‡ Beijing Tan,‡ Lili Yao,‡ Rachel E. Kosa,‡
Manthena V. Varma,‡ Bo Feng,‡ David B. Duignan,‡ Ayman El-Kattan,‡ Sharad Murdande,‡
Shenping Liu,‡ Mark Ammirati,‡ John Knafels,‡ Paul DaSilva-Jardine,† Laurel Sweet,‡ Spiros Liras,†
and Timothy P. Rolph†
†Cambridge Laboratories, Pfizer Worldwide Research & Development, 620 Memorial Drive, Cambridge, Massachusetts 02139,
United States
‡Groton Laboratories, Pfizer Worldwide Research & Development, Eastern Point Road, Groton, Connecticut 06340, United States
ABSTRACT: Glucokinase is a key regulator of glucose
homeostasis, and small molecule allosteric activators of this
enzyme represent a promising opportunity for the treatment of
type 2 diabetes. Systemically acting glucokinase activators
(liver and pancreas) have been reported to be efficacious but
in many cases present hypoglycaemia risk due to activation of
the enzyme at low glucose levels in the pancreas, leading to
inappropriately excessive insulin secretion. It was therefore postulated that a liver selective activator may offer effective glycemic
control with reduced hypoglycemia risk. Herein, we report structure−activity studies on a carboxylic acid containing series of
glucokinase activators with preferential activity in hepatocytes versus pancreatic β-cells. These activators were designed to have
low passive permeability thereby minimizing distribution into extrahepatic tissues; concurrently, they were also optimized as
substrates for active liver uptake via members of the organic anion transporting polypeptide (OATP) family. These studies lead
to the identification of 19 as a potent glucokinase activator with a greater than 50-fold liver-to-pancreas ratio of tissue distribution
in rodent and non-rodent species. In preclinical diabetic animals, 19 was found to robustly lower fasting and postprandial glucose
with no hypoglycemia, leading to its selection as a clinical development candidate for treating type 2 diabetes.
complications and highlighting the need for novel therapeutic
options.
INTRODUCTION
■
Type 2 diabetes mellitus (T2DM) is a rapidly expanding public
epidemic affecting over 300 million people worldwide.1 This
disease is characterized by elevated fasting plasma glucose
(FPG), insulin resistance, abnormally elevated hepatic glucose
production (HGP), and reduced glucose-stimulated insulin
secretion (GSIS). Moreover, long-term lack of glycemic control
increases risk of complications from neuropathic, microvascular,
and macrovascular diseases. The standard of care for T2DM is
metformin followed by sulfonylureas, dipeptidyl peptidase-4
(DPP-IV) inhibitors, and thiazolidinediones (TZD) as second
line oral therapies.2 As disease progression continues, patients
typically require injectable agents such as glucagon-like peptide-
1 (GLP-1) analogues and, ultimately, insulin to help maintain
glycemic control.2 Despite these current therapies, many
patients still remain unable to safely achieve and maintain
tight glycemic control, placing them at risk of diabetic
Among the potential next generation therapies, small
molecule glucokinase activators offer a promising opportunity
for the treatment of T2DM patients.3 Glucokinase (GK)
converts glucose to glucose 6-phosphate (G-6-P). This enzyme
is unique among the members of the hexokinase family given its
low substrate affinity (Km ≈ 8 mM), positive substrate
cooperativity, and lack of product inhibition.4 Physiologically,
glucokinase functions as a key regulator of glucose homeostasis
in the pancreas, liver, and brain. Specifically, in the β-cells of the
pancreas, glucokinase acts as a glucostat, establishing the
threshold for glucose-stimulated insulin secretion (GSIS).5
Glucokinase is also expressed in the α-cells of the pancreas
Received: November 4, 2011
Published: December 23, 2011
© 2011 American Chemical Society
1318
dx.doi.org/10.1021/jm2014887 | J. Med. Chem. 2012, 55, 1318−1333

Journal of Medicinal Chemistry
Article
Figure 1. Representative structures of glucokinase activators.
where its role is less well characterized but may be involved in
regulating glucagon secretion.6 In hepatocyctes, glucokinase
represents the rate determining step for hepatic glucose uptake
and plays a central role in both glycogen synthesis and the
regulation of hepatic glucose production.7 In the brain,
glucokinase is expressed in glucose sensing neurons of the
ventromedial hypothalamus where it regulates the counter-
regulatory response (CRR) to hypoglycemia.8 Notably,
glucokinase is also found in the endocrine K and L cells of
the gut as well as certain pituitary cells where its functions are
less well characterized but may be involved in nutrient
sensing.9,10
Evidence for the importance of glucokinase in regulating
glucose metabolism can be found in prominent clinical
phenotypes associated with naturally occurring mutations of
the enzyme.11 As an example, heterozygous loss of function
mutations are associated with maturity-onset diabetes of the
young type 2 (MODY2) while homozygous loss of function
mutations are associated with permanent neonatal diabetes. By
contrast, human gain of function mutations that activate
glucokinase are associated with low blood glucose including
hyperinsulinemic hypoglycemia of infancy.
shown in Figure 1.15−21 These and other small molecule
activators of glucokinase have been recently reviewed.22
Consistent with genetic evidence, multiple glucokinase
activators have advanced to clinical studies and been shown
to lower both fasting and postprandial glucose in healthy
subjects and T2DM patients.23 However, during these same
clinical studies, hypoglycemia has been revealed as the key dose
limiting adverse effect of glucokinase activators.23 Several
clinical strategies have been employed to manage this
hypoglycemia risk including dose titration, dosing with meals,
and dosing multiple times a day (b.i.d., t.i.d., etc.) to minimize
Cmax-driven hypoglycemic events; however, more fundamental
changes to the design of activators are perhaps required to
resolve this risk and to maximize the therapeutic opportunity of
the mechanism.
To overcome the limitations imposed by hypoglycemia risk,
several design strategies have emerged to create glucokinase
activators with reduced potential for inducing hypoglycemia. As
previously reported, one such strategy has been the design of
partial activators that avoid reducing glucokinase’s glucose Km
to inappropriately low levels, thereby retaining increased
dependence of enzymatic activity on physiological glucose
concentrations.24 A second strategy to mitigate hypoglycemia
risk is to restrict enzyme activation to the liver, since the
hypoglycemia risk associated with glucokinase activation is
postulated to result from potentiation of pancreatic insulin
secretion at inappropriately low glucose levels.16,25 While
hypoglycemia risk is mitigated, liver selective activation is still
anticipated to offer efficacy given that >99% of the glucokinase
enzyme is found in the liver and obese diabetics reportedly lose
up to 50% of their hepatic glucokinase activity contributing to
reduced hepatic glycogen synthesis and improper regulation of
hepatic glucose production (HGP).26,27 Many diabetic animal
models similarly experience loss of hepatic glucokinase activity
Considering the central role of glucokinase in glucose
regulation, there has been significant interest in pharmaco-
logical activation of the enzyme as a potential treatment for
T2DM. In 2003, Grimsby and co-workers reported the binding
of small molecule activators to glucokinase, at an allosteric
pocket 20 Å remote from the active site, which influenced the
enzyme’s kinetic profile by modulating both Km for glucose
12
(also known as S0.5) and Vmax
.
These efforts resulted in the
identification of a phenylacetamide series of activators including
clinical candidates 1 and 2 (piragliatin).12−14 Since this early
work, a variety of other structurally diverse glucokinase
activators have been reported with representative examples
1319
dx.doi.org/10.1021/jm2014887 | J. Med. Chem. 2012, 55, 1318−1333

Journal of Medicinal Chemistry
Article
with increasing disease severity, and interestingly, normalization
of hepatic glucokinase activity via overexpression reduces
plasma glucose while also normalizing glycogen levels and
pancreatic tissue distribution. Activators were specifically
optimized along three parameters: (a) recognition by liver
specific OATP transporters for hepatic uptake, (b) low hepatic
oxidative metabolism to maximize residency time and thus
pharmacology, and (c) low passive permeability to minimize
distribution into peripheral tissues that lack liver specific OATP
transporters such as pancreas. Experimentally, evaluation of
molecules for OATP-mediated uptake was accomplished using
transporter transfected cell lines individually expressing either
human or rodent OATP transporters and comparing uptake of
analogues between transfected and wild type cells.38 Oxidative
metabolism was evaluated using human liver microsomes.
Passive permeability was experimentally evaluated using an
RRCK (low efflux MDCK) cell line.39 Additionally, functional
impairment of activators in pancreatic β-cells was evaluated
using a rat insulinoma (INS-1) cell line measuring activator
effects on glucose stimulated insulin secretion. The ratio of this
cellular to rat biochemical potency for a given activator was
taken as a surrogate measure of pancreatic impairment with
higher values suggesting increased impairment. Analogues that
successfully advanced through this screening cascade were then
progressed to in vivo pharmacokinetic, tissue distribution, and
pharmacology studies.
restoring regulation of HGP.28 Additionally, both Bodvarsdot
́
-
̈
tir25 and more recently Bebernitz16 (with compound 4, Figure
1) have reported glucose lowering efficacy of liver specific
glucokinase activators in preclinical diabetes models. Given the
therapeutic opportunity represented by hepatoselective gluco-
kinase activation, herein, we report the discovery of a liver
selective development candidate for the treament of T2DM.
HEPATOSELECTIVE GLUCOKINASE ACTIVATORS:
DESIGN STRATEGY AND SCREENING CASCADE
■
Since the liver and pancreatic forms of glucokinase are
kinetically and enzymatically indistinguishable (differing only
in sequence at the extreme amino terminus),7 we focused on a
hepatoselective tissue distribution strategy to achieve selective
liver activation. Liver selective drug discovery approaches have
been previously reported for various therapeutic classes
including HMG-CoA reductase inhibitors,29 stearoyl-CoA
desaturase inhibitors,30 glucocorticoid antagonists,31 thyroid
hormone receptor agonists,32 hepatitis B and C antivirals,33
caspase inhibitors,34 and nucleoside oncolytics.35 From these
examples, a variety of targeting strategies have been used to
achieve hepatoselectivity, including (a) optimizing molecules
for recognition and active uptake via liver specific transporters
such as the organic anion transporting polypeptide (OATP)
transporters and organic cation transporters (OCT), (b)
synthesizing conjugates with liver targeting motifs such as bile
acids or statins, and (c) designing prodrugs that undergo liver
specific metabolic activation.29−35 Among these options we
elected to pursue OATP-mediated hepatic uptake given the
precedent of preclinical-to-clinical translation with this strategy;
consequently, a screening cascade was built to enable parallel
optimization of molecules as both glucokinase activators and
OATP substrates.
SYNTHETIC CHEMISTRY
■
The synthesis of representative analogues 17 and 19 is
illustrated in Scheme 1. Initially, (R)-methyl oxirane-2-
carboxylate (10) was added to the in situ generated
organocuprate of cyclopentylmagnesium bromide to provide
11 in good yield.40 Hydroxy ester 11 was then alkylated with
inversion of stereochemistry to give 12 using a two-step
procedure of triflate formation followed by addition of 4-
trifluoromethylimidazole which had been premixed with
LiHMDS. The methyl ester of 12 was saponified to the
carboxylic acid 13 which was subsequently converted to the
corresponding acid chloride 14 under standard conditions.
Neutral amides were synthesized by coupling of 14 with
appropriate heteroarylamines as illustrated by coupling with 5-
methyl-2-aminopyridine to afford 17. Separately, the synthesis
of amides bearing acidic moieties was accomplished as
illustrated for the preparation of 19 starting from 6-amino-
nicotinic acid 15 which was first O-benzylated using benzyl
bromide and potassium carbonate to yield 16. Coupling of 16
with acid chloride 14 provided the penultimate benzyl-
protected 18 that underwent hydrogenolysis over catalytic
palladium on carbon to afford 19. By incorporation of
alternative heteroarylamines, this route enabled the synthesis
of all other analogues investigated in the current studies.
Our testing funnel began with an evaluation of biochemical
glucokinase activation where we sought potent activators (EC50
< 100 nM) that afforded substantial increases in glucokinase
Vmax (β > 1.5) and significant reductions in Km (α < 0.10). This
particular activation profile was targeted as a way to achieve
robust enzyme activation and to restore the levels of hepatic
glucokinase activity lost during progression of diabetes. The
glucokinase activation assay employed recombinant human
glucokinase and monitored the rate of glucose 6-phosphate
formation using G6PDH/NADP coupling. It was performed in
a matrix format wherein dose response determinations for an
activator (at 22 different activator concentrations from 0 to 100
μM) were conducted at 16 different glucose concentrations
(ranging from 0 to 100 mM).24,36 Analyzing this data using a
nonessential activator model enabled determination of an
activator’s maximum fold effect on reducing the glucokinase Km
for glucose defined as α and the maximum fold effect on
altering the enzyme’s Vmax defined as β.37 Values of α ranged
from 0 to 1 with lower values of α representing more
substantial reductions in the enzyme’s glucose Km. β > 1
STRUCTURE−ACTIVITY STUDIES
■
The starting point for our design efforts was compound 17
(Table 1), which originated from a previous investigation of
other N-heteroarylacetamides as systemically (i.e., liver and
pancreas) acting glucokinase activators.41 As a lead, activator 17
offered acceptable potency (EC50 = 114 nM) and a favorable
biochemical activation profile (α = 0.04, β = 1.53); additionally,
its relatively low molecular weight (MW = 366) made it
amenable to further structural modification. However, 17 was
metabolically unstable (HLM Clint > 120 mL min−1 kg−1), not a
substrate for OATP-mediated uptake (Table 1), and exhibited
high passive permeability measured in the RRCK assay (17.4 ×
represented activator induced increases in the enzyme’s Vmax
For example, β = 1.5 represented a 1.5-fold increase in Vmax
.
.
Activator potency or EC50 was formally defined as the
concentration affording a half-maximal reduction in Km.
Concurrent with optimization of biochemical potency we
also sought to maximize the therapeutic window against
hypoglycemia by achieving enhanced hepatic and impaired
1320
dx.doi.org/10.1021/jm2014887 | J. Med. Chem. 2012, 55, 1318−1333

Journal of Medicinal Chemistry
Article
minimizing passive permeability and oxidative metabolism. To
guide installation of the acid, structural biology studies were
undertaken to understand the binding of 17 to the allosteric
site of human glucokinase. As shown in Figure 2, 17 forms two
Scheme 1. Synthesis of Representative Glucokinase
Activators 17 and 19
a
Figure 2. Co-crystal structure of 17 bound to the allosteric site of
human glucokinase. Crystals of glucokinase in complex of glucose were
obtained following published protocols.17 Cocrystals were obtained by
overnight soaking these preformed protein crystals in crystallization
mother liquor containing 1 mM 17 before data collection at 17-ID
beamline at APS at 100 K, and the structure was determined using the
molecular replacement method. Coordinates for this structure have
been deposited into the PDB.
hydrogen bonds (donor−acceptor relationship) with the main
chain of Arg63. The cyclopentyl group of the activator is
located in a hydrophobic pocket formed by side chains of
Met210, Ile211, Tyr214, and Met235. Interestingly, the 5-
methyl substituent of the 2-aminopyridine is located at a
solvent-exposed channel lined with the side chain of Arg63,
suggesting that this vector might represent an opportunity to
incorporate polar substituents without compromising ligand−
protein binding interactions.42
To test this hypothesis, the analogue of 17 containing a
carboxylate at the 5-position of the 2-aminopyridine ring (19)
was synthesized as described previously in Scheme 1. As shown
in Table 1, carboxylic acid 19 offered comparable potency and
activation profile to its neutral counterpart 17; moreover, 19
exhibited low oxidative metabolism, improved aqueous
solubility, and reduced passive permeability. This reduced
passive permeability was also reflected by a significant reduction
in INS-1 cellular activity (77-fold cell/enzyme ratio versus 1.3-
a
Reagents and conditions: (a) cyclopentylmagnesium bromide,
Li2CuCl4, Et2O, THF, −78 → −50 °C, 30 min, 65−70%; (b) Tf2O,
2,6-lutidine, CH2Cl2, 0 °C, 45 min, 100%; (c) 4-trifluoromethylimi-
dazole, LiHMDS, THF, 23 °C, 2 h, 74%; (d) 6 N HCl, 95 °C, 16 h,
98%; (e) (COCl)2, cat. DMF, CH2Cl2, 23 °C, 90 min, 100%; (f) 5-
methyl-2-aminopyridine, pyridine, CH2Cl2, 23 °C, 16 h, 53%; (g)
benzyl bromide, K2CO3, DMF, 23 °C, 16 h, 65%; (h) pyridine,
CH2Cl2, 23 °C, 16 h, 74%; (i) H2, 10% Pd/C, ethyl acetate, ethanol,
23 °C, 50 psi, 90 min, 71%.
10−6 cm/s) and reflected in a 1.3-fold ratio of cellular activity
(INS-1) to biochemical potency.
Since acids are a common recognition element of OATP
transporters, we envisioned incorporation of a carboxylate into
17 as a strategy to elicit hepatic uptake while concurrently
Table 1. Comparison of Neutral (17) and Carboxylate (19) Containing Glucokinase Activators
a
c
d
human GK activation
INS-1 cellular activity
transporter substrate
b
permeability
kinetic
HLM Clint
RRCK Papp (10−6 solubility
cell/enzyme
ratio
compd EC50 (nM)
α
β
(mL min−1 kg−1)
cm/s)
(μM)
EC50 (nM)
153 0.05
OATP1B1 OATP1B3
17
19
114 41
90 45
0.04 0.01
0.03 0.01
1.53 0.08
1.65 0.14
>120
<8.0
17.4
1.0
174
524
1.3
77
no
no
6900 4090
yes
yes
a
b
Biochemical assay values reported as the arithmetic mean SD of n > 2 independent determinations. Passive permeability assessed in RRCK
c
canine kidney cell line. Determination of EC50 for stimulation of insulin secretion in INS-1 cells (rat insulinoma line) at 5 mM glucose. EC50 values
reported as the arithmetic mean SD of n > 2 independent determinations. Cell/enzyme ratio determined as INS-1 EC50 divided by the EC50 of
d
biochemical activation for rat glucokinase enzyme. Human OATP transporters overexpressed in HEK293 cells. Classified as substrate if fold
difference in uptake relative to the wild type control is >2.
1321
dx.doi.org/10.1021/jm2014887 | J. Med. Chem. 2012, 55, 1318−1333

Journal of Medicinal Chemistry
Article
Table 2. Structure−Activity Relationships for Glucokinase Activators 19−28
a
b
Biochemical assay values reported as the arithmetic mean SD of n > 2 independent determinations. Passive permeability assessed in RRCK
c
canine kidney cell line. Cell/enzyme ratio determined as INS-1 EC50 divided by the EC50 of biochemical activation for rat glucokinase enzyme.
d
Human OATP transporters overexpressed in HEK293 cells. Classified as substrate if fold difference in uptake relative to the wild type control is >2.
fold for parent compound 17). When screened in human
OATP1B1 and OATP1B3 transfected cells, 19 was found to be
a substrate for both transporters.
(26) was reasonably tolerated, EC50 = 284 nM and EC50 = 486
nM, respectively, and both remained OATP1B3 substrates;
however, these isosteres were susceptible to significant
oxidative metabolism and thus discontinued. Replacement of
the pyridine heterocycle of 19 was also explored examining
pyrazole (27) and pyrimidine (28), but these alternative
heterocycles were less active. On the basis of these and other
supporting structure−activity studies, activator 19 was selected
for additional pharmacokinetic and biological profiling given its
favorable balance of properties.
Structural variations of 19 were subsequently explored as
summarized in Table 2 to further characterize the structure−
activity trends in this series. Transposition of the carboxylate
from the 5-position of the aminopyridine (19) to either 6-
position (20) or 4-position (21) resulted in a significant loss of
activity (EC50 > 10 000 nM). Homologation of the carboxylate
motif (i.e., 19 → 22) resulted in 6-fold loss of potency, whereas
replacement with either a sulfonic acid (23) or phosphonic acid
(24) resulted in more substantial potency losses. Interestingly,
conversion of the acid to the tetrazole (25) or oxanilic acid
1322
dx.doi.org/10.1021/jm2014887 | J. Med. Chem. 2012, 55, 1318−1333

Journal of Medicinal Chemistry
Article
GLUCOKINASE ACTIVATOR 19:
PHARMACOKINETICS AND TISSUE DISTRIBUTION
■
The preclinical protein binding and pharmacokinetic properties
of activator 19 are summarized in Tables 3 and 4, respectively.
Table 3. Plasma and Tissue Protein Binding Properties of
Glucokinase Activator 19
fu
a
b
b
b
species
plasma
liver
pancreas
brain
rat
0.10
0.17
0.11
0.13
0.07
0.09
ND
ND
0.11
0.21
ND
ND
0.09
0.13
ND
ND
dog
monkey
human
Figure 3. pH dependent passive permeability profile of 19.
a
Plasma protein binding value determined at 50 μM using equilibrium
plasma, liver, pancreas, and brain drug concentrations of
animals at regular intervals (t = 0.25, 0.75, 1, 2, 4, 6, 8, and 24 h
postdose). As shown in Figure 4, 19 exhibited enhanced liver to
b
dialysis. Tissue protein binding values determined at 1 μM using
equilibrium dialysis. ND = not determined.
As shown in Table 3, 19 exhibited moderate plasma protein
binding (fu = 0.10−0.17) which was consistent across species;
additionally, its fraction unbound in tissues of pharmacological
interest (i.e., liver, pancreas, and brain) were comparable to
fraction unbound in plasma for both rat and dog.
Pharmacokinetic studies (Table 4) in rat, dog, and monkey
revealed 19 to have high systemic plasma clearance consistent
with active hepatic extraction. Evaluation of clearance
mechanisms in rat revealed biliary excretion of parent as the
primary pathway (62%) with a minor contribution from renal
clearance (11%). As suggested by the in vitro microsomal data,
in vivo oxidative metabolism of 19 was minimal. In all three
preclinical species activator 19 exhibited low to moderate
systemic bioavailability (18−35%, Table 4). Subsequent dosing
of 19 (5 mg/kg) to portal vein cannulated (PVC) rats revealed
a fraction absorbed of 53%. Given that 19 was designed for low
passive permeability as a strategy to minimize peripheral
distribution, its favorable oral absorption was attributed, in part,
to high thermodynamic solubility (4.2 mg/mL at pH 6.5) and a
beneficial pH-dependent passive permeability profile as shown
in Figure 3. Specifically, under physiological conditions (pH
7.4) 19 has low passive permeability but under more acidic
conditions (pH 5.5), which are representative of the upper
gastrointestinal tract, carboxylate protonation offers increased
passive permeability presumably contributing to oral absorp-
tion. Additionally, since 19 was determined to be an OATP
substrate (vida infra), active transport may also contribute to its
oral bioavailability.
Figure 4. Acute tissue distribution time course of 19 in rat after oral
administration. Tissue drug concentrations are expressed as the mean
SD (n = 3) for free drug concentrations using the fraction unbound
values reported in Table 3.
plasma and impaired pancreas to plasma distribution, affording
a substantial differential between liver and pancreas exposure.
In this same experiment, free brain concentrations of 19 were
determined to be exceedingly low and below the limit of
quantification at most time points. To further characterize this
tissue distribution, repeat dose studies were conducted in both
rat and dog, evaluating 28 and 4 days of dosing, respectively. At
4 h postdose on the last day of dosing free drug levels in
plasma, liver, pancreas, and brain were determined as
summarized in Table 5. Consistent with the observations in
the previous acute time course, enhanced liver and impaired
Having characterized the systemic pharmacokinetic proper-
ties of 19, we next sought to characterize its tissue distribution.
First, a single oral dose of 19 (50 mg/kg) was administered to
Wistar rats followed by sacrifice and determination of free
a
Table 4. Preclinical Pharmacokinetics of Glucokinase Activator 19
b
b
dose (mg/kg)
AUC(0−24) (ng·h/mL)
Cmax (ng/mL)
Cl (mL min−1 kg−1)
51
t1/2 (h)
VdSS (L/kg)
2.0
F (%)
rat iv
1
34.7
30.3
79.1
72.6
46.2
94.7
176
5.4
3.1
rat po
5
18
18
35
dog iv
1
479
23.3
219
13.6
35
20
2.2
1.6
0.7
dog po
monkey iv
monkey po
5
0.5
3
1.9
a
b
Pharmacokinetic parameters expressed as geometric mean of n = 2 animals per group unless otherwise noted. AUC(0−24) and Cmax reported as free
drug concentrations using the fraction of unbound values reported in Table 3.
1323
dx.doi.org/10.1021/jm2014887 | J. Med. Chem. 2012, 55, 1318−1333

Journal of Medicinal Chemistry
Article
a
Table 5. Tissue Distribution of Activator 19 in Rat and Dog after Repeat Oral Dosing
ratio
species
dose (mg/kg)
treatment duration (days)
liver/plasma
pancreas/plasma
brain/plasma
liver/pancreas
rat
100
50
28
4
10.5
14.5
0.14
0.25
BLQ
75
58
dog
0.009
a
Distribution ratios calculated based on free drug concentrations at 4 h postdose on the last day of treatment. BLQ = below limit of quantification.
Table 6. Evaluation of Uptake of Activator 19 into Suspended Rat and Human Hepatocytes
a
b
suspended rat hepatocycte uptake rate
rate, pmol min−1 (mg of protein)−1
suspended human hepatocycte uptake rate
rate, pmol min−1 (mg of protein)−1
c
c
compd
% inhibitable
% inhibitable
19
rosuvastatin
87 21
95
96
98
5
1
3
26
18
93
81
94
d
218 36
d
pravastatin
15
3
4.7
a
b
Experiment conducted in freshly isolated rat hepatocytes with data reported as the mean
SD (n = 3 or 4). Experiment conducted in
c
d
cyropreserved human hepatocytes with data reported from n = 1 determination. Inhibition with rifamycin as pan OATP inhibitor. Active control.
Table 7. Evaluation of 19 as a Substrate for Rat and Human Organic Anion Transporters
a
a
rat transporter
Oatp1a4
no
human transporter
OATP1B3
compd
Oatp1a1
substrate
Oatp1b2
substrate
OATP1B1
substrate
OATP2B1
substrate
19
substrate
a
Conducted in transfected CHO or HEK cells at 1 μM test article. Classified as substrate if fold difference in uptake relative to the wild type control
is >2. Active controls were pravastain (OATP1B1), rosuvastatin (OATP1B3), UK191005 (OATP2B1), pravastatin (Oatp1a1), fexofenedine
(Oatp1a4), and estradiol glucuriod (Oatp2b1). Midazolam was used as a negative control in all experiments.
pancreas drug levels (relative to plasma) were observed in both
species during these repeat dose experiments with a liver-to-
pancreas ratio of 75-fold for rat and 58-fold for dog.
we next undertook more detailed profiling of its biological
properties. First, selectivity screening revealed that 19 had no
effect on other members of the human hexokinase family (I, II,
or III) at concentrations >500-fold its EC50 for human
glucokinase (i.e., hexokinase IV).43 Additionally, no off-target
activity was observed for 19 when evaluated at 10 μM in a
broad CEREP screening panel.
In our studies of the functional and pharmacodynamic
activity of 19 (vida infra), we included previously reported
systemic (i.e., liver and pancreas acting) activators 30 and 31
(Figure 5) as reference compounds to illustrate similarities and
To characterize the active transport mechanism(s) respon-
sible for the selective liver distribution of 19 in rodent and
translational relevance to human, we evaluated its uptake into
both rat and human suspended hepatocyctes (Table 6). In
suspended rat hepatocyctes, the uptake rate of 19 was greater
than pravastatin but less than rosuvastatin; pravastatin and
rosuvastatin were used as active controls in the assay. Uptake of
19 was substantially inhibited by rifamycin SV, used as a pan
organic anion transporting polypeptide inhibitor. In human
hepatocyctes, the active uptake rate of 19 was greater than both
rosuvastatin and pravastatin and also was significantly inhibited
in the presence of rifamycin SV. This significant inhibition of
uptake by rifamycin SV in both rat and human hepatocyctes
indicated the importance of active hepatic uptake and
prompted subsequent studies to better understand transporter
substrate specificity of 19 in a panel of rat and human organic
anion transporting polypeptides as shown in Table 7.
Rosuvastatin/pravastatin and midazolam served as positive
and negative controls, respectively, in each assay. Compound
19 was found to be a substrate for rat Oatp1a1 and Oatp1b2
transporters but not Oatp1a4; furthermore, 19 was a substrate
for human OATP1B1, OATP1B3, and OATP2B1. Taken
together, the data from Tables 6 and 7 offered additional
confidence that the enhanced liver distribution of 19 observed
in rodent and dogs may be relevant to human.
Figure 5. Structures of reference systemic glucokinase activators 30
and 31.
differences between liver specific and systemic glucokinase
activation. These two reference compounds were potent (30,
EC50 = 188 nM; 31, EC50 = 212 nM) activators with high
passive permeability (30, RRCK Papp = 20.1 × 10−6 cm/s; 31,
RCK Papp = 22.3 × 10−6 cm/s) and moderate plasma protein
binding (30, rat fu = 0.24; 31, rat fu = 0.14).24 In contrast to the
hepatoselective activator, 30 and 31 were not substrates for the
OATP transporters and in rodent tissue distribution studies
these reference compounds were found to be equally
distributed between plasma, liver, and pancreas (ratio of
∼1:1:1) with 10-fold brain impairment relative to plasma.44
GLUCOKINASE ACTIVATOR 19:
■
PHARMACOLOGICAL EVALUATION AND
COMPARISON TO SYSTEMIC GLUCOKINASE
ACTIVATORS
Having identified 19 as a potent glucokinase activator with
preferential liver-to-pancreas distribution in preclinical species,
1324
dx.doi.org/10.1021/jm2014887 | J. Med. Chem. 2012, 55, 1318−1333

Journal of Medicinal Chemistry
Article
The functional activity of 19 versus the reference systemic
activator 30 was first compared using hepatocyctes and
pancreatic islets. In hepatocyctes glucokinase activity is
regulated, in part, though an interaction with glucokinase
regulatory protein (GKRP).7 Physiologically, under conditions
of low glucose GKRP binds the inactive conformation of
glucokinase and sequesters the enzyme to the nucleus. As
glucose concentrations increase, glucokinase is released from
GKRP and diffuses into the cytoplasm. Given that allosteric
glucokinase activators alter the protein conformation, they have
been shown to disrupt the GK−GKRP interaction analogous to
changes in glucose concentrations, and we therefore charac-
terized the effect of 19 on the subcellular localization of
glucokinase in cryopreserved rat hepatocytes. As shown in
Figure 6, dose response evaluations of 19 and 30 were
Figure 7. Effect of hepatoselective (19) and systemic (30) glucokinase
activators on insulin secretion in static cultured dispersed rat islets.
Shown is the glucose-stimulated insulin secretion in dispersed rat islets
in static culture at 11 mM glucose following administration of activator
19 or 30. Data are expressed as the mean
SD (n = 3). GKA
concentration is normalized by rat EC50 on the X-axis.
vivo efficacy evaluations were conducted using the Goto−
Kakizaki diabetic rat model. Goto−Kakizaki rats spontaneously
experience a condition similar to type 2 diabetes mellitus
(T2DM) starting at 4 weeks of life which includes (a) basal
hyperglycemia (nonfasting plasma glucose range of 180−250
mg/dL), (b) increased hepatic glucose production, and (c)
impaired postprandial insulin secretion. An initial study
evaluated the effect of 19 versus 31 on reducing fasting plasma
glucose after 28-day treatment of Goto−Kakizaki rats at 100
mg/kg. As shown in Figure 8A, the two activators offered
comparable efficacy in normalizing fasting plasma glucose at 14
days of treatment and this efficacy was durable through the end
of the study. A similar study was then conducted evaluating the
effects of both 19 and 31 on fasting plasma glucose levels in
normal, nondiabetic Sprague−Dawley rats, as shown in Figure
8B. In contrast to the results in the Goto−Kakizaki diabetic
rats, hepatoselective activator 19 had no effect on fasting
plasma glucose; however, systemic activator 31 reduced glucose
levels below euglycemia by the first week of treatment. These
results are consistent with the hepatoselective activator
correcting the dysregulated hepatic glucose metabolism in the
diabetic model while having little effect in normal healthy
animals that have intact regulation of hepatic glucose
metabolism. The glucose lowering effect of systemic activator
31 in normal, nondiabetic model (i.e., Figure 8B) may be
attributed to a leftward shift of the threshold for glucose-
stimulated insulin secretion. Notably, this same lack of glucose
lowering effect of 19 in normal, euglycemic animals was
reproduced in subsequent pharmacology and toxicology (rat,
dog, and monkey) studies, increasing confidence in the
minimized hypoglycemia risk of this profile relative to that
previously observed with systemically acting glucokinase
activators.46
Figure 6. Effect of hepatoselective (19) and systemic (30) glucokinase
activators on glucokinase translocation in cryopreserved rat
hepatocyctes. Shown is the dose dependent effect of activators 19
and 30 on the translocation of glucokinase from the nucleus to the
cytoplasm in cryopreserved rat hepatocytes cultured at 8.9 mM
glucose. Data represent the mean SD (n = 3). GKA concentration is
normalized by rat EC50 on the X-axis.
conducted in cryopreserved hepatocytes cultured in 8.9 mM
glucose and glucokinase translocation from the nucleus to the
cytoplasm was monitored via fluorescence based cellomics
analysis. Both activators dose-dependently increased glucoki-
nase translocation out of the nucleus with EC50 of 0.19 μM for
19 and EC50 of 0.90 μM for 30. As shown in Figure 6, the
potency normalized dose response of 19 was left-shifted relative
to 30, consistent with its active hepatic uptake.45
In the pancreatic β-cell, glucokinase serves as a glucostat
establishing the threshold for glucose-stimulated insulin
secretion. Systemically acting glucokinase activators are
known to enhance glucose stimulated insulin secretion, so we
sought to compare the effects of 19 and 30 on dispersed rat
islets in static culture at 11 mM glucose with data reported as
percentage of maximum secretion observed at high glucose (22
mM) for INS-1 cells. As shown in Figure 7, reference systemic
activator 30 dose-dependently increased glucose-stimulated
insulin secretion in these dispersed islets. By contrast
hepatoselective activator 19 demonstrated no significant effect
on insulin secretion even at concentrations up to 100-fold its
biochemical EC50, which is consistent with its poor passive
permeability.
The effect of hepatoselective activator 19 on reducing
postprandial glucose was also evaluated as illustrated in Figure
9. Activator 19 was again dosed once daily for 28 days (10 and
100 mg/kg) to Goto−Kakizaki rats. A vehicle treated group of
Wistar rats was included as a nondiabetic control. An oral
glucose tolerance test (OGTT) was performed on day 28 of
treatment with 19, demonstrating a dose-dependent decrease in
glucose excursion (Figure 9A) and a 36% decrease in glucose
AUC at 100 mg/kg (Figure 9B) with no occurrences of
Having demonstrated the differential functional activity of
hepatoselective activator 19 in isolated hepatocyctes versus
islets, we then evaluated its activity in whole animal models. In
1325
dx.doi.org/10.1021/jm2014887 | J. Med. Chem. 2012, 55, 1318−1333

Journal of Medicinal Chemistry
Article
a glucagon challenge experiment as illustrated in Figure 10. In
this model, normal (nondiabetic) jugular vein cannulated
Wistar rats were administered an iv-glucagon challenge (3 μg/
kg) to elicit a hyperglycemic glucose excursion via induction of
hepatic glycogenolysis.47 As shown in Figure 10, activator 19,
administered as a single oral dose 50 min prior to challenge,
dose-dependently reduced the glucagon-induced glucose
excursion. Interestingly, doses of 30 and 100 mg/kg 19 offered
almost complete suppression of the glucose excursion,
illustrating the strength of glucokinase activation in suppressing
glucagon-induced HGP. Since hyperglucagonemia is a key
driver of hyperglycemia in T2DM, these glucagon challenge
results combined with the efficacy previously observed in
Goto−Kakizaki rats (i.e., Figures 7 and 8) highlight the
potential for activator 19 to offer significant glycemic
reductions in diabetic patients.48
In conclusion we have described the design, synthesis, and
biological characterization of a hepatoselective glucokinase
activator for the treatment of type 2 diabetes. The favorable
tissue distribution profile of glucokinase activator 19 coupled
with its efficacy and low hypoglycemia risk differentiated it
from previous systemic activators, supporting its selection as a
development candidate that is currently under clinical
evaluation in T2DM patients.
EXPERIMENTAL SECTION
■
All reagents and solvents were used as received from commercial
sources. 1H NMR spectra were recorded on a Varian 400 MHz nuclear
1
magnetic resonance spectrometer. H NMR spectra were recorded in
CDCl3, CD3OD, or DMSO-d6, and chemical shifts are reported
relative to the residual solvent peak. The following abbreviations were
used to assign spectra: s = singlet, d = doublet, t = triplet, q = quartet,
quin = quintet, m = multiplet. Mass spectral analysis was conducted on
a Waters Micromass ZQ instrument.
Figure 8. Effect of once daily administration of glucokinase activators
19 and 31 on fasting plasma glucose in diabetic (A) and normal,
nondiabetic rats (B). (A) Glucokinase activators 19 and 31 were
administered orally, once daily for 28 days to Goto−Kakizaki diabetic
rats. Weekly fasting glucose samples were collected 1 h postdose, and
Purity of all final analogues for biological testing were confirmed to
be >95% as determined by HPLC analysis. HPLC analysis was
conducted according to one of the following methods with the
retention time (tR) expressed in min at UV detection of 254 or 210
nM. For HPLC method A, an Agilent 1100 series HPLC instrument
was used, with chromatography performed on an Xbridge 150 mm ×
4.6 mm, 5 μm C18 column with mobile phase gradient of 5−100%
acetonitrile in water containing 0.1% trifluoroacetic acid and with a
flow rate of 1.5 mL/min. For HPLC method B, UPLC was conducted
using solvent A consisting of water (0.05% trifluoroacetic acid),
solvent B consisting of acetonitrile (0.05% trifluoroacetic acid), and a
gradient from 5% to 95%.
Purification was done by either silica gel flash chromatography with
a Teledyne Isco CombiFlash Rf with RediSep Flash columns using a
gradient of ethyl acetate in heptanes or methanol in CH2Cl2, or similar
instrument or reverse phase preparative HPLC.
For experiments involving the use of animals, all procedures were
carried out in compliance with the NIH Guide for the Care and Use of
Laboratory Animals under a protocol approved by the Institutional
(Pfizer Worldwide Research and Development) Animal Care and Use
Committee.
(R)-Methyl 3-Cyclopentyl-2-hydroxypropanoate (11). A 0.2
M solution of Li2CuCl4 was prepared as follows: Anhydrous CuCl2
(26.9 g, 200 mol) and anhydrous LiCl (17.0 g, 400 mmol) were
dissolved in THF (1000 mL). A solution of Li2CuCl4 (0.2 M in THF,
125 mL, 25.0 mmol) was added slowly to a suspension of
cyclopentylmagnesium bromide (2 M in diethyl ether, 135 mL, 270
mmol) and THF (500 mL) at −50 °C over 2−3 min. The pale gray
suspension was then allowed to warm slowly to −10 °C over 30 min,
by which time the color had developed to a dark gray. The mixture was
recooled to −78 °C, and (R)-methyl oxirane-2-carboxylate (25.0 g,
245 mmol) was added neat via syringe over 90 s. The mixture was then
stirred at −78 °C for 20 min, before removing the ice bath and
data are expressed as the mean
standard error (n = 8 per dose
group). (B) Glucokinase activators 19 and 31 were administered orally
once daily for 21 and 14 days, respectively, to normal, nondiabetic
Sprague−Dawley rats. Weekly fasting glucose samples were collected 1
h postdose, and data are expressed as the mean standard error (n =
8 per dose group).
hypoglycemia. No changes in plasma insulin levels were
observed during the course of the OGTT study (Figure 9C).
Liver glycogen concentrations were also determined on day 28
for treated animals as shown in Figure 9D. Animals treated with
19 demonstrated a nonstatistically significant trend toward
increased liver glycogen, approaching the levels found in
normal, nondiabetic animals. This latter observation was in
agreement with previous glucokinase overexpression experi-
ments showing the restoration of glycogen synthesis upon
increases in hepatic glucokinase activity in diabetic animals.28
While not shown, a similar OGTT experiment was also
conducted in normal, nondiabetic rats undergoing treatment
with 19 once-daily for 21 days (10, 30, 100 mg/kg), and results
are consistent with the previous fasting glucose observations
(i.e., Figure 8B). 19 had no significant effects on reducing
postprandial glucose in nondiabetic animals with intact
regulation of hepatic glucose metabolism.
The above studies of hepatoselective activator 19 offering
glucose lowering efficacy in diabetic animals with dysregulated
hepatic glucose uptake and production but not in normal
animals illustrate the potential for this approach to normalize
hyperglycemia with minimal risk of inducing hypoglycemia. To
further characterize its effects on HGP, we also evaluated 19 in
1326
dx.doi.org/10.1021/jm2014887 | J. Med. Chem. 2012, 55, 1318−1333

Journal of Medicinal Chemistry
Article
Figure 9. Evaluation of in vivo efficacy of activator 19 during an oral glucose tolerance test in diabetic Goto−Kakizaki rats. Glucokinase activator 19
was administered orally once daily (10 and 100 mg/kg) for 28 days to Goto−Kakizaki diabetic rats. (A) Dose dependent effect of 19 on plasma
glucose following an oral glucose tolerance test conducted on day 28. Activator was administered at 60 min prior to the administration of a 2 g/kg
glucose bolus. Vehicle treated normal, nondiabetic Wistar rats were included for comparison. Data are expressed as the mean standard error (n = 5
per dose group). (B) Effect of 19 on glucose AUC following day 28 oral glucose tolerance test. Data are expressed as the mean standard error (n =
5 per dose group): (∗) P < 0.05; (∗∗∗) P < 0.001. (C) Effect of 19 on plasma insulin concentrations during an oral glucose tolerance test. Data are
expressed as the mean standard error (n = 5 per dose group). (D) Liver glycogen following 28 days of dosing with 19. Data are expressed as the
mean standard error (n = 4 per dose group).
Figure 10. Effect of glucokinase activator 19 on plasma glucose during a glucagon challenge in Wistar rats. Wistar rats were dosed orally with 19 (10,
30, 100 mg/kg) at t = 0 min. Glucagon challenge (3 μg/kg) was administered iv at t = 50 min. Data are expressed as the mean standard error (n =
6 per dose group).
allowing the mixture to warm to approximately −50 °C over 30 min.
Saturated NH4Cl (aq, 700 mL) was then added, and the mixture was
stirred for 30 min. The organic layer was collected and the aqueous
layer extracted with diethyl ether (2 × 250 mL). The combined
organics were washed with saturated NH4Cl (aq, 350 mL), dried
(MgSO4), and evaporated. Distillation of the crude residue (68−70 °C
at 0.8 mbar) afforded 11 as a pale yellow oil (65−70% over multiple
lutidine (9.5 g, 88 mmol) was added. A solution of triflic anhydride
(20.0 g, 71 mmol) in anhydrous CH2Cl2 (100 mL) was then added
dropwise, and the mixture was stirred for 45 min. The mixture was
concentrated, diluted with MTBE, and washed five times with 1 N
HCl and once with brine. The organic layer was dried over magnesium
sulfate, filtered, evaporated, and dried under high vacuum to afford the
crude (R)-methyl 3-cyclopentyl-2-(((trifluoromethyl)sulfonyl)oxy)-
propanoate (13.0 g, 100%), which was used without further
purification. 1H NMR (400 MHz, chloroform-d) δ 5.14 (dd, J =
3.62, 8.50 Hz, 1H), 3.85 (s, 3H), 2.04−2.16 (m, 1H), 1.91−2.02 (m,
2H), 1.79−1.90 (m, 2H), 1.50−1.73 (m, 4H), 1.08−1.23 (m, 2H).
4-Trifluoromethylimidazole (3.81 g, 28 mmol) was stirred in
anhydrous THF (150 mL) at room temperature under nitrogen. A
LiHMDS solution (1.0 M in THF, 25.0 mL, 25.0 mmol) was added
1
batches). H NMR (400 MHz, chloroform-d) δ 4.16−4.25 (m, 1H),
3.79 (s, 3H), 2.67 (d, J = 6.02 Hz, 1H), 1.95−2.11 (m, 1H), 1.47−1.91
(m, 8H), 1.03−1.21 (m, 2H).
(S)-Methyl 3-Cyclopentyl-2-(4-(trifluoromethyl)-1H-imida-
zol-1-yl)propanoate (12). To a 1 L flask was added 11 (7.29 g,
42 mmol) which was dissolved in anhydrous CH2Cl2 (300 mL) and
placed under nitrogen. The solution was stirred in an ice bath, and 2,6-
1327
dx.doi.org/10.1021/jm2014887 | J. Med. Chem. 2012, 55, 1318−1333

Journal of Medicinal Chemistry
Article
dropwise via addition funnel. After 50 min, a solution of crude (R)-
methyl 3-cyclopentyl-2-(((trifluoromethyl)sulfonyl)oxy)propanoate
(8.52 g, 28 mmol) in dry THF (20 mL) was added. The mixture
was stirred for 2 h, then quenched with saturated ammonium chloride.
The mixture was diluted with brine and ethyl acetate. The aqueous
layer was extracted with ethyl acetate, and the combined organics were
dried over sodium sulfate. The mixture was filtered and evaporated to
a brown oil. The oil was purified by silica gel chromatography (10%
ethyl acetate/heptane gradient to 80% ethyl acetate/heptane) to afford
Pd/C (350−380 mg) was added to each bottle, and the mixtures were
shaken under 50 psi hydrogen for 90 min. LCMS showed the reactions
to be complete. The mixtures were combined and filtered successively
through glass fiber filters until the filtrate was almost clear. The filtrate
was concentrated to a smaller volume and run through autovial filters,
then evaporated to a glass. Ether was added, and the mixture was
stirred for 18 h at room temperature. The resulting white precipitate
was filtered, washed with ether, and suction-dried for 20 min. It was
then dried under high vacuum at 50 °C for 3 h to afford 19 as a white
solid (3.22 g, 71%). 1H NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H),
8.86 (d, J = 1.95 Hz, 1H), 8.27 (dd, J = 2.24, 8.68 Hz, 1H), 8.13 (d, J =
8.78 Hz, 1H), 7.97 (d, J = 4.88 Hz, 2H), 5.27 (dd, J = 5.37, 9.66 Hz,
1H), 2.04−2.26 (m, 2H), 1.38−1.72 (m, 7H), 1.26−1.37 (m, 1H),
1.08 (td, J = 7.88, 11.75 Hz, 1H); LCMS m/z 397.5 (M + H)+. HPLC
purity (method A): tR = 7.690 min, 100%.
1
12 as a clear oil (5.98 g, 74%). H NMR (400 MHz, chloroform-d) δ
7.60 (s, 1H), 7.41 (s, 1H), 4.74 (dd, J = 6.24, 9.36 Hz, 1H), 3.79 (s,
3H), 2.02−2.19 (m, 2H), 1.45−1.84 (m, 7H), 1.04−1.24 (m, 2H);
LCMS m/z 290.9 (M + H)+.
(S)-3-Cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-1-yl)-
propanoic Acid (13). In a round-bottom flask, 12 (6.61 g, 22.8
mmol) was combined with 6 N HCl (140 mL) and heated to 95 °C
for 16 h before cooling. Solid potassium carbonate was added in
portions to bring the mixture to pH ∼4. Ethyl acetate was added to
dissolve the precipitate. The layers were separated, and the aqueous
layer was extracted with ethyl acetate. The combined organics were
washed with brine, dried over sodium sulfate, filtered, evaporated, and
(S)-3-Cyclopentyl-N-(5-methylpyridin-2-yl)-2-(4-(trifluoro-
methyl)-1H-imidazol-1-yl)propanamide (17). 1H NMR (400
MHz, chloroform-d) δ 8.44 (br s, 1H), 8.11 (br s, 1H), 8.05 (d, J =
8.39 Hz, 1H), 7.70 (s, 1H), 7.56 (dd, J = 1.85, 8.49 Hz, 1H), 7.53 (s,
1H), 4.71 (t, J = 7.51 Hz, 1H), 2.32 (s, 3H), 2.11−2.24 (m, 2H),
1.43−1.85 (m, 7H), 1.13 (td, J = 7.90, 11.71 Hz, 2H); LCMS m/z
366.9 (M + H)+. HPLC purity (method A): tR = 7.781 min, 99.49%*.
(S)-6-(3-Cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-1-
yl)propanamido)picolinic Acid (20). 20 was prepared according to
the method of 19, substituting commercially available 2-amino-
1
dried under high vacuum to afford 13 as a clear oil (6.15 g, 98%). H
NMR (400 MHz, chloroform-d) δ 7.84 (s, 1H), 7.41 (s, 1H), 4.76 (dd,
J = 5.37, 10.05 Hz, 1H), 2.17−2.27 (m, 1H), 2.04−2.15 (m, 1H),
1.45−1.86 (m, 7H), 1.05−1.24 (m, 2H); LCMS m/z 277.4 (M + H)+.
Benzyl 6-Aminonicotinate (16). To a stirred suspension of 6-
aminonicotinic acid (100 g, 0.72 mol) in DMF (700 mL) with brisk
mechanical stirring was added K2CO3 (150 g, 1.08 mol). The mixture
was stirred for 10 min before the portionwise addition of benzyl
bromide (95 mL, 0.80 mol). The mixture was stirred at room
temperature overnight. Then the solids were filtered off and washed
thoroughly with ethyl acetate. These organics were concentrated under
vacuum. The filter cake was dissolved in water and extracted with ethyl
acetate. The initial residue after concentration was combined with the
ethyl acetate extracts (total volume of 2 L of ethyl acetate). The
combined organic extracts were washed with brine (5 × 500 mL),
dried (MgSO4), and the solvent was removed under reduced pressure.
The crude product was refluxed with 1:1 Et2O/hexane for 30 min, and
then the solids were filtered off (warm), washed with Et2O/hexane
(1:1), and suction-dried. This solid was precipitated from hot toluene
1
pyridine-6-carboxylic acid for 6-aminonicotinic acid. H NMR (500
MHz, DMSO-d6) δ 11.42 (br s, 1H), 8.24 (d, J = 8.29 Hz, 1H), 7.92−
8.03 (m, 3H), 7.79 (d, J = 7.56 Hz, 1H), 5.28 (br s, 1H), 2.14−2.25
(m, 1H), 2.06−2.14 (m, 1H), 1.37−1.72 (m, 7H), 1.27−1.36 (m, 1H),
1.02−1.15 (m, 1H); LCMS m/z 397.1 (M + H)+. HPLC purity
(method A): tR = 7.662 min, 99.50%.
(S)-2-(3-Cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-1-
yl)propanamido)isonicotinic Acid (21). 21 was prepared according
to the method of 19, substituting commercially available 2-amino-
1
pyridine-4-carboxylic acid for 6-aminonicotinic acid. H NMR (500
MHz, DMSO-d6) δ 11.32 (s, 1H), 8.44−8.57 (m, 2H), 7.92−8.03 (m,
2H), 7.56 (d, J = 5.86 Hz, 1H), 5.25 (dd, J = 5.61, 8.78 Hz, 1H), 2.03−
2.25 (m, 2H), 1.37−1.73 (m, 7H), 1.31 (qd, J = 7.90, 11.92 Hz, 1H),
1.00−1.14 (m, 1H); LCMS m/z 397.1 (M + H)+. HPLC purity
(method A): tR = 7.371 min, 99.30%.
1
(S)-2-(6-(3-Cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-1-
yl)propanamido)pyridin-3-yl)acetic Acid (22). 22 was prepared
according to the method of 19, substituting 6-aminopyridine-3-acetic
and dried to afford 16 as an off-white solid (107.2 g, 65%). H NMR
(400 MHz, chloroform-d) δ 8.79 (d, J = 1.95 Hz, 1H), 8.05 (dd, J =
2.15, 8.59 Hz, 1H), 7.31−7.49 (m, 5H), 6.47 (d, J = 8.78 Hz, 1H),
5.34 (s, 2H), 4.91 (br s, 2H); LCMS m/z 229.2 (M + H)+.
acid49 for 6-aminonicotinic acid. H NMR (400 MHz, DMSO-d6) δ
1
12.45 (s, 1H), 11.08 (s, 1H), 8.23 (d, J = 1.95 Hz, 1H), 7.90−8.04 (m,
3H), 7.69 (dd, J = 2.15, 8.58 Hz, 1H), 5.13−5.32 (m, 1H), 3.60 (s,
2H), 2.02−2.24 (m, 2H), 1.37−1.76 (m, 7H), 1.21−1.35 (m, 1H),
0.99−1.15 (m, 1H); LCMS m/z 411.0 (M + H)+. HPLC purity
(method A): tR = 7.552 min, 99.33%.
(S)-6-(3-Cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-1-
yl)propanamido)pyridine-3-sulfonic Acid (23). To a solution of
14 (0.40 g, 1.35 mmol) in dichloromethane (10 mL) were added
commercially available 6-aminopyridinesulfonic acid (0.236 g, 1.35
mmol) and pyridine (0.32 mL, 4.1 mmol) at room temperature. The
mixture was refluxed for 12 h under a nitrogen atmosphere. The
reaction mixture was then concentrated in vacuo and the residue was
purified by chromatography on silica to afford 23 (47.2 mg, 8.1%) as a
white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.18 (s, 1H), 8.50 (s,
1H), 7.98 (m, 4H), 7.03 (s, 1H) 5.22 (m, 1H), 2.18 (m, 2H), 1.60 (m,
4H), 1.48 (m, 3H), 1.31 (m, 1H), 1.11 (m, 1H); LCMS m/z 431.4 (M
− H)−. HPLC purity (method A): tR = 6.819 min, 99.72%.
(S)-Benzyl 6-(3-Cyclopentyl-2-(4-(trifluoromethyl)-1H-imida-
zol-1-yl)propanamido)nicotinate (18). In a round-bottom flask, 13
(6.16 g, 22.3 mmol) was stirred in anhydrous CH2Cl2 (15 mL) at
room temperature under nitrogen. Oxalyl chloride (5.8 g, 46 mmol)
was added followed by one drop of DMF. After 90 min, the mixture
was evaporated and chased twice with 1,2-DCE to afford 14 which was
used without additional purification. Amine 16 (5.77 g, 25.3 mmol)
was combined with pyridine (3.8 g, 48 mmol) and anhydrous CH2Cl2
(200 mL). This was added to the acid chloride, using another 100 mL
of anhydrous CH2Cl2 to aid transfer. The mixture became cloudy. The
mixture was stirred for 16 h, then diluted with CH2Cl2 and water. A 1
M solution of potassium dihydrogen phosphate was then added. A
solid settled, which was suction-filtered off. The organic layer was
extracted with dilute potassium carbonate, washed with brine, dried
over sodium sulfate, filtered, and evaporated. The residue was purified
by silica gel chromatography (40% ethyl acetate/heptane) to afford 18
1
as a white solid (8.04 g, 74%). H NMR (400 MHz, DMSO-d6) δ
(S)-(6-(3-Cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-1-
yl)propanamido)pyridin-3-yl)phosphonic Acid (24). To a
solution of 14 (0.329 g, 1.12 mmol) in CH2Cl2 (8 mL) was added
intermediate 2-aminopyridin-5-ylphosphonic acid ethyl ester50 (0.257
g, 1.12 mmol) and pyridine (0.265 g, 3.36 mmol) at 25 °C for 12 h.
The reaction mixture was concentrated in vacuo and purified by silica
gel chromatography (10% petroleum ether/ethyl acetate) to provide
intermediate (S)-diethyl 6-(3-cyclopentyl-2-(4-(trifluoromethyl)-1H-
imidazol-1-yl)propanamido)pyridin-3-ylphosphonate (360 mg, 66.2%)
11.53 (s, 1H), 8.91 (d, J = 1.76 Hz, 1H), 8.34 (dd, J = 2.34, 8.78 Hz,
1H), 8.16 (d, J = 8.78 Hz, 1H), 7.93−8.01 (m, 2H), 7.45−7.53 (m,
2H), 7.31−7.44 (m, 3H), 5.36 (s, 2H), 5.27 (dd, J = 5.27, 10.15 Hz,
1H), 2.15−2.26 (m, 1H), 2.04−2.14 (m, 1H), 1.37−1.71 (m, 7H),
1.27−1.37 (m, 1H), 0.99−1.13 (m, 1H); LCMS m/z 487.5 (M + H)+.
(S)-6-(3-Cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-1-
yl)propanamido)nicotinic Acid (19). Intermediate 18 (5.58 g, 11.5
mmol) was divided into two equal portions. Each was dissolved in
ethyl acetate (35 mL) and ethanol (70 mL) in a Parr bottle. Then 10%
1328
dx.doi.org/10.1021/jm2014887 | J. Med. Chem. 2012, 55, 1318−1333

Journal of Medicinal Chemistry
Article
as a light yellow solid. To a solution of (S)-diethyl 6-(3-cyclopentyl-2-
(4-(trifluoromethyl)-1H-imidazol-1-yl)propanamido)pyridin-3-yl-
phosphonate (170 mg, 0.348 mmol) in CH2Cl2 (2 mL) was added
bromotrimethylsilane (2 mL) at room temperature, and the mixture
was stirred for 12 h at 25 °C under a nitrogen atmosphere. The
reaction mixture was quenched with methanol (2 mL), and the
mixture was concentrated in vacuo. The residue was purified by
preparative HPLC [Synergi 150 mm × 30 mm column, mobile phase
from 23% MeOH in water (0.225% formic acid) to 43% MeOH in
water (0.225% formic acid)] to afford 24 (47.1 mg, 30.0%) as a white
to 50% MeOH in water (0.225% formic acid)] to afford 26 (119.1 mg,
45.3%) as a white solid. H NMR (500 MHz, DMSO-d6) δ 11.10 (s,
1
1H), 10.88 (s, 1H), 8.74 (d, J = 1.71 Hz, 1H), 8.12 (dd, J = 1.95, 9.03
Hz, 1H), 8.01 (d, J = 8.78 Hz, 1H), 7.96 (d, J = 8.54 Hz, 2H), 5.14−
5.27 (m, 1H), 2.02−2.24 (m, 2H), 1.36−1.72 (m, 7H), 1.23−1.35 (m,
1H), 1.01−1.15 (m, 1H); LCMS m/z 440.2 (M + H)+. HPLC purity
(method A): tR = 7.522 min, 97.15%.
S)-2-(3-(3-Cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-1-
yl)propanamido)-1H-pyrazol-1-yl)acetic Acid (27). Carboxylic
acid 13 (145 mg, 0.525 mmol) was dissolved in dry CH2Cl2 (5 mL)
and stirred at room temperature under nitrogen. One drop of DMF
was added, followed by oxalyl chloride (0.095 mL, 1.1 mmol). After
bubbling had subsided, the mixture was left stirring for 90 min and
then evaporated. The residue was dissolved in two successive portions
of 1,2-dichloroethane and evaporated to remove excess oxalyl chloride,
and the residue was dissolved in dry CH2Cl2 (4 mL) to afford 14
which was used without additional purification. Commercially available
ethyl 2-(3-amino-1H-pyrazol-1-yl)acetate hydrochloride (130 mg,
0.630 mmol) and pyridine (0.130 mL, 1.61 mmol) were dissolved in
dry CH2Cl2 (5 mL) and added to the CH2Cl2 (5 mL) solution of acid
chloride 14. The mixture was stirred at room temperature under
nitrogen for 24 h. The mixture was then diluted with CH2Cl2 and
water, and the organic layer was separated, washed with brine, dried
over sodium sulfate, filtered, and evaporated. The residue was purified
by silica gel chromatography (50% ethyl acetate/heptane, linear
gradient to 75% ethyl acetate) to afford intermediate (S)-ethyl 2-(3-(3-
cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-1-yl)propanamido)-
1H-pyrazol-1-yl)acetate (153 mg, 68%) which was then combined
with lithium hydroxide (50.6 mg, 1.18 mmol) in THF/MeOH/water
(1:1:1, 3 mL) and stirred at 25 °C for 2 h. The reaction mixture was
then concentrated, and water and 1 N HCl were added to achieve
approximately pH 4. A thick precipitate settled. Ethyl acetate was
added, and the organic layer was washed with brine and dried over
sodium sulfate. The precipitate was filtered, washed, and dried under
high vacuum to afford 27 (31.5 mg, 22%). 1H NMR (400 MHz,
methanol-d4) δ 7.93 (s, 1H), 7.80 (s, 1H), 7.54 (br s, 1H), 6.58 (br s,
1H), 5.04 (t, J = 7.62 Hz, 1H), 4.83 (s, 2H), 2.17 (t, J = 7.33 Hz, 2H),
1.43−1.89 (m, 7H), 1.21−1.36 (m, 1H), 1.07−1.20 (m, 1H); LCMS
m/z 399.9 (M + H)+. HPLC purity (method B): tR = 0.61 min, 100%.
(S)-5-(3-Cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-1-
yl)propanamido)pyrazine-2-carboxylic Acid (28). 28 was pre-
pared according to the method of 19, substituting commercially
available 5-amino-2-pyrazinecarboxylic acid for 6-aminonicotinic acid.
1H NMR (400 MHz, DMSO-d6) δ 11.79 (s, 1H), 9.36 (s, 1H), 8.98 (s,
1
solid. H NMR (400 MHz, CD3OD) δ 8.64 (s, 1H), 8.15 (m, 2H),
7.93 (s, 1H), 7.81 (s, 1H), 5.12 (m, 1H), 2.18 (m, 2H), 1.80 (m, 1H),
1.70 (m, 4H), 1.55 (m, 2H), 1.30 (m, 1H), 1.25 (m, 1H); LCMS m/z
431.4 (M − H)−. HPLC purity (method A): tR = 6.763 min, 98.74%.
(S)-N-(5-(2H-Tetrazol-5-yl)pyridin-2-yl)-3-cyclopentyl-2-(4-
(trifluoromethyl)-1H-imidazol-1-yl)propanamide (25). To a
solution of 13 (140 mg, 0.31 mmol) in DMF (2 mL) was added
benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluoro-
phosphate (204.6 mg, 0.46 mmol), and the reaction mixture was
stirred for 1 h at 25 °C. Subsequently, 5-(1H-tetrazol-5-yl)pyridin-2-
amine51 (50 mg, 0.31 mmol) and NMM (47 mg, 0.46 mmol) were
added. The resulting mixture was stirred at 25 °C for 12 h. The
reaction mixture was quenched with saturated aqueous NH4Cl (1
mL), and the aqueous portion was extracted with EtOAc (20 mL × 3).
The combined organic layers were washed with brine, dried over
Na2SO4, and evaporated in vacuo. The residue was purified by
preparative HPLC [Xbridge 150 mm × 33 mm column, mobile phase
from 6% acetonitrile in water (ammonia added to pH 10) to 26%
acetonitrile in water (ammonia added to pH 10)] to afford 25 (10 mg,
10%) as a colorless solid. 1H NMR (500 MHz, DMSO-d6) δ 11.44 (s,
1H), 9.00 (d, J = 1.95 Hz, 1H), 8.39 (dd, J = 2.20, 8.78 Hz, 1H), 8.23
(d, J = 8.54 Hz, 1H), 7.99 (d, J = 6.34 Hz, 2H), 5.20−5.34 (m, 1H),
2.17−2.26 (m, 1H), 2.08−2.16 (m, 1H), 1.38−1.73 (m, 7H), 1.27−
1.37 (m, 1H), 1.02−1.15 (m, 1H); LCMS m/z 421.1 (M + H)+.
HPLC purity (method A): tR = 7.505 min, 99.78%.
(S)-2-(6-(3-Cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-1-
yl)propanamido)pyridin-3-ylamino)-2-oxoacetic Acid (26). To
a solution of 14 (2.13 g, 7.2 mmol) in CH2Cl2 (40 mL) were added 2-
amino-5-nitropyridine (1.0 g, 7.2 mmol) and Et3N (3.12 mL, 21.6
mmol), and the solution was stirred for 12 h at 25 °C under an
atmosphere of nitrogen. The reaction mixture was subsequently
concentrated under vacuum and filtered through a pad of silica gel to
afford intermediate (S)-3-cyclopentyl-N-(5-nitropyridin-2-yl)-2-(4-
(trifluoromethyl)-1H-imidazol-1-yl)propanamide (1.1 g, 38.7%) as a
light yellow solid. To a solution of (S)-3-cyclopentyl-N-(5-nitro-
pyridin-2-yl)-2-(4-(trifluoromethyl)-1H-imidazol-1-yl)propanamide
(0.62 mg, 1.56 mmol) in DMF (20 mL) were added zinc (1.02 g, 15.6
mmol) and a solution of FeCl3 (2.53 mg, 15.6 mmol) in water (20
mL) at 25 °C. The resulting mixture was heated to 100 °C with
stirring for 3 h. Subsequently, the reaction mixture was cooled, filtered
and the filtrate was concentrated under vacuum. Water (20 mL) was
added to the resulting residue, and the mixture was extracted with
EtOAc (20 mL × 3). The combined organic layers were washed with
brine, dried over Na2SO4, and concentrated to afford (S)-N-(5-
aminopyridin-2-yl)-3-cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-
1-yl)propanamide (0.58 g, ∼100%) as a light yellow solid that was
used without purification.
1H), 8.00 (s, 2H), 5.30 (dd, J = 4.59, 10.26 Hz, 1H), 2.18−2.31 (m,
1H), 2.04−2.16 (m, 1H), 1.27−1.75 (m, 8H), 0.97−1.14 (m, 1H);
LCMS m/z 398.3 (M + H)+. HPLC purity (method B): tR = 0.60 min,
99%.
Method for Determination of Activator Effects on GK
Translocation in Cryopreserved Rat Hepatocyctes. Rat primary
hepatocytes were obtained from Xenotech (Lenexa, KS) and isolated
using its isolation kit following their instructions. The cells were plated
into collagen coated 96-well plate with a cell density of 60 000 cells per
well in 100 μL per well of high glucose DMEM supplemented with
10% FBS, 100 nM insulin, 10 nM dexamathasone, and 2 mM sodium
pyruvate. The plates were incubated in a humidified incubator at 37
°C, 10% CO2. After 4 h, the medium was changed into fresh medium
with Matrigel. The plates were incubated overnight. On day 2, cells
were changed into DMEM (no glucose) with 10% FBS, 100 nM
insulin, 10 nM dexamethasone, 2 mM L-glutamine and incubated
overnight. On day 3, the medium was aspirated and an amount of 90
μL of testing glucose medium (8.9 mM glucose DMEM with 0.07%
BSA, 1 nM insulin, 10 nM dexamethasone) was added to the plate.
Test compounds were serial diluted in DMSO at 1000×. Compounds
at 10× of the final concentrations were diluted in the indicated glucose
medium, and an amount of 10 μL per well of these solutions was
added to the cell plate. Cells were incubated for 1 h at 37 °C, 5% CO2.
Cells in the plates were fixed by adding 100 μL per well of 8%
paraformaldehyde for 15 min and followed by 4% paraformaldehyde
for 30 min at 4 °C. Plates were then washed 6 times with 300 μL of
To a solution of intermediate (S)-N-(5-aminopyridin-2-yl)-3-
cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-1-yl)propanamide
(0.35 g, 0.95 mmol) in CH2Cl2 (20 mL) were added ethyl
chlorooxoacetate (130 mg, 0.95 mmol) and Et3N (0.41 mL, 2.85
mmol), and the reaction mixture was stirred for 12 h at 25 °C. The
reaction mixture was concentrated and the residue filtered through a
silica pad to afford (S)-ethyl 2-(6-(3-cyclopentyl-2-(4-(trifluorometh-
yl)-1H-imidazol-1-yl)propanamido)pyridin-3-ylamino)-2-oxoacetate
(130 mg) to which were subsequently added LiI (369 mg, 2.78 mmol)
and EtOAc (10 mL). This reaction mixture was heated to reflux for 24
h. The reaction mixture was concentrated under vacuum, and the
residue was purified by preparative HPLC [Synergi 150 mm × 33 mm
column, mobile phase from 30% MeOH in water (0.225% formic acid)
1329
dx.doi.org/10.1021/jm2014887 | J. Med. Chem. 2012, 55, 1318−1333

Journal of Medicinal Chemistry
Article
PBS per well per cycle using a Bio-Tek ELx 405 micoroplate washer
(Bio-Tek Instruments; Winooski, VT). After the last washing step, the
cells were permeablized using 0.1% TritonX-100 in PBS for 20 min at
room temperature. The plates were washed again 6 times with 100 μL
of PBS per well per cycle. The cells were then blocked by adding 50
μL per well of blocking solution (3% BSA, 1% goat serum in PBS) and
incubated at room temperature for 1 h. Primary antibody solution
(anti-glucokinase antibody diluted 1:250 in blocking buffer) was added
to the cells and incubated at 4 °C overnight. The plates were then
washed 6 times with 300 μL per well per cycle. Secondary antibody
solution (AF594-conjugated goat anti-rabbit antibody 1:500 and 2 μM
Hoechst in PBS) was added to the cells and incubated at room
temperature for 1 h. The plate was washed 6 times with 300 μL per
well per cycle before it was imaged in the ArrayScan (Cellomics).
Using GraphPad Prism, version 4.0, dose−response curves were fitted
to the following equation:
experiment was performed three times with three individual
preparations of rat dispersed islets. Insulin secretion data were
analyzed using GraphPad Prism. Data were normalized as % effect
relative to maximum glucose, which in these studies was 22 mM. Data
from the three individual experiments were averaged for graphing.
Evaluation of the Effect Glucokinase Activators on Fasting
and Postprandial Glucose in Goto−Kakizaki Rats. Rats arrived at
the facility at 7−8 weeks of age and were acclimated 1 week prior to
test article/vehicle administration. Following a 1 week acclimation, rats
were randomized by fed-state body weight into different treatment
groups and dosed daily in the with vehicle (0.5% methylcellulose) or
test article. Dosing was based on body weight at a concentration of 5
mL/kg and continued for 28 days. Blood samples (250 μL) were
collected in ethylenediaminetetraacetic acid (EDTA) treated micro-
tainers, mixed by inversion, and placed on ice. Plasma was isolated via
centrifugation at 14 000 rpm in an Eppendorf centrifuge 5417R at 4
°C for 2 min. The samples were then analyzed through the use of
Roche Diagnostics clinical analyzer. Insulin levels were also measured
using a diagnostics rat ultrasensitive insulin kit (Alpco). Rats
underwent an end-study oral glucose tolerance test (OGTT) on day
28. Sixty minutes after test article administration, a blood sample was
collected and the rats were immediately given a 2 g/kg oral bolus of
40% glucose in water. Subsequent blood samples were collected at 15,
30, 60, and 120 min after the oral glucose challenge.
f(x) = low + 1 + (high − low)/[10(log EC − x) × Hill]
50
where x is the concentration of the compound and Hill indicates the
Hill coefficient.
Method for Determination of Activator Effects on Insulin
Secretion in Dispersed Rat Islets. Male Sprague−Dawley rats
(200−275 g) were euthanized, and the pancreas was perfused via the
common bile duct with ice cold Hank’s balanced salt solution (HBSS,
Gibco, No. 14185) containing 1.2 mM CaCl2, 25 mM HEPES, 0.234
mg/mL liberase (Roche, No. 11815032001), and 0.07 mg/mL DNase
(Sigma, No. D4527-200KU). The pancreas was removed and digested
with stirring for 12 min at 37 °C on a submersible stir plate. Following
digestion ice cold quench (HBSS, 10% FBS, 10 mM HEPES) was
added and the pancreas was spun at 50g for 15 s. The supernatant was
removed, and the pellet was resuspended in ice-cold quench and
passed over a 250 μm screen. The digested pancreas was again spun at
50g for 15 s, and the supernatant was removed. The pellet was
resuspended in 8 mL of 27% Ficoll (Sigma, No. F9378) and
transferred to a 30 mL Corex tube. An amount of 4 mL of 23% Ficoll
was then layered in the tube followed by 4 mL of 20.5% Ficoll and 4
mL of 11% Ficoll. The sample was then spun at 500g for 10 min. The
islets were removed from the upper layers of Ficoll and washed twice
with HBSS without Ca2+. Following washing, 1 mM EDTA was added
and the islets were incubated for 8 min at room temperature. Next
0.00125% trypsin−EDTA and 2 μg/mL DNase I (final concen-
trations) were added, and the islets were incubated for 10 min at 30 °C
with shaking at 60 rpm. Following dispersion the islets were
dissociated via 50 passes with a 1 mL pipet. The islets were diluted
with culture medium (RPMI 1640, 10% FBS, L-glutamine, pen/strep,
and HEPES) and passed over a 63 μm nylon membrane. The
dispersed islets were pelleted, then washed again with culture medium.
After counting, the cells were suspended in culture medium and
seeded at 5000 cells per well (200 μL/well) in 96-well V bottom
plates. The plates were spun for 5 min at 200g and then placed in a 5%
CO2 cell culture incubator overnight.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 617-551-3169. Fax: 617-551-3084. E-mail: jeffrey.a.
pfefferkorn@pfizer.com.
ABBREVIATIONS USED
■
CRR, counter-regulatory response; FPG, fasting plasma
glucose; GK, glucokinase; GKA, glucokinase activator; GSIS,
glucose-stimulated insulin secretion; HGP, hepatic glucose
production; MODY, maturity onset diabetes of the young;
OATP, organic anion transporting polypeptide; OGTT, oral
glucose tolerance test; T2DM, type 2 diabetes mellitus
REFERENCES
■
(1) World Health Organization Diabetes Fact Sheet No. 312. http://
www.who.int/mediacentre/factsheets/fs312/en/ (accessed September
30, 2011).
(2) Lebovitz, H. E. Management of Hyperglycemia with Oral
Antihyperglycemic Agents in Type 2 Diabetes. In Joslin’s Diabetes
Mellitus, 14th ed.; Kahn, C. R., et al.et al., Eds.; Lippincott Williams &
Wilkins: Philadelphia, PA, 2005; pp 687−710.
(3) For reviews, see the following: (a) Fyfe, M. C. T.; Procter, M. J.
Glucokinase activators as potential antidiabetic drugs possessing
superior glucose-lowering efficacy. Drugs Future 2009, 34, 641−653.
(b) Matschinsky, F. Assessing the potential of glucokinase activators in
diabetes therapy. Nat. Rev. Drug Discovery 2009, 8, 399−416.
(4) Matschinsky, F., Magnuson, M. A., Eds. Glucokinase and Glycemic
Diseases: From Basics to Novel Therapeutics; Karger: Basel, Switzerland,
2004.
(5) Matschinsky, F. M.; Glaser, B.; Magnuson, M. A. Pancreatic β-cell
glucokinase: closing the gap between theoretical concepts and
experimental realities. Diabetes 1998, 47, 307−315.
(6) Heimberg, H.; De Vos, A.; Moens, K.; Quartier, E.; Bouwens, L.;
Pipeleers, D.; Van Schaftingen, E.; Madsen, O.; Schuit, F. The glucose
sensor protein glucokinase is expressed in glucagon-producing α-cells.
Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 7036−7041.
Test articles were solubilized in 100% DMSO at 10 mM. Serial
dilutions were made in 100% DMSO to 3, 1, 0.3, and 0.1 mM. The
compound was further diluted in incubation buffer to final
concentrations of 3, 1, 0.3, and 0.1 μM.
The medium was aspirated from each well and replaced with 100 μL
of 3 mM glucose in incubation buffer (115 mM NaCl, 5 mM KCl, 24
mM NaHCO3, 2.2 mM CaCl2, 1 mM MgCl2, 24 mM HEPES, 0.25%
BSA). The dispersed islet plates were spun at 200g to repellet the islets
and then incubated in a 37 °C water bath continuously gassed with
95% O2/5% CO2 for 45 min. The preincubation buffer was then
removed and replaced with 50 μL of incubation buffer containing 3, 1,
0.3, and 0.1 μM test article in 3, 5, 9, 11, 13, or 22 mM glucose, in
replicates of 8. All glucose concentrations containing 0.1% DMSO
were included on each plate as internal controls. The plates were spun
to pellet the islets and then incubated for 60 min in a 37 °C water bath
continuously gassed with 95% O2/5% CO2. Following the incubation
an amount of 35 μL of buffer was removed and transferred to a 96-well
polypropylene storage plate and frozen. Insulin content was analyzed
using a commercial EIA (Alpco, No. 80-INSRTH-E10). This
(7) (a) Agius, L. Glucokinase and molecular aspects of liver glycogen
metabolism. Biochem. J. 2008, 414, 1−18. (b) Iynedjian, P. B.
Molecular physiology of mammalian glucokinase. Cell. Mol. Life Sci.
2009, 66, 27−42.
(8) Dunn-Meynell, A. A.; Routh, V. H.; Kang, L.; Gaspers, L.; Levin,
B. E. Glucokinase is likely mediator of glucose sensing in both glucose-
1330
dx.doi.org/10.1021/jm2014887 | J. Med. Chem. 2012, 55, 1318−1333

Journal of Medicinal Chemistry
Article
excited and glucose-inhibited central neurons. Diabetes 2002, 51,
2056−2065.
(20) Yoshikawa, N.; Xu, F.; Arredondo, J. D.; Itoh, T. A large-scale
synthesis of potent glucokinase activator MK-0941 via selective O-
arylation and O-alkylation. Org. Process Res. Dev. 2011, 15, 824−830.
(21) Pike, K. G.; Allen, J. V.; Caulkett, P. W.; Clarke, D. S.; Donald,
C. S.; Fenwick, M. L.; Johnson, K. M.; Johnstone, C.; McKerrecher,
D.; Rayner, J. W.; Walker, R. P.; Wilson, I. Design of a potent, soluble
glucokinase activator with increased pharmacokinetic half-life. Bioorg.
Med. Chem. Lett. 2011, 21, 3467−3470.
(22) For recent reviews of glucokinase activators, see the following:
(a) Coghlan, M.; Leighton, B. Glucokinase activators in diabetes
management. Expert Opin. Invest. Drugs 2008, 17, 145−167.
(b) Sarabu, R.; Berthel, S. J.; Kester, R. F.; Tilley, J. W. Glucokinase
activators as new type 2 diabetes therapeutic agents. Expert Opin. Ther.
Pat. 2008, 18, 759−768. (c) Sarabu, R.; Berthel, S. J.; Kester, R. F.;
Tilley, J. W. Novel glucokinase activators: a patent review (2008−
2010). Expert Opin. Ther. Patents 2011, 21, 13−31.
(23) For reports of glucokinase activator clinical efficacy and safety,
see the following: (a) Zhi, J.; Zhai, S.; Mulligan, M.-E.; Grimsby, J.;
Arbet-Engels, C.; Boldrin, M.; Balena, R. A novel glucokinase activator
RO4389620 improved fasting and postprandial glucose in type 2
diabetic patients. Diabetologia 2008, 51 (Suppl. 1), S23, Abstract 42.
(b) Zhai, S.; Mulligan, M.-E.; Grimsby, J.; Arbet-Engels, C.; Boldrin,
M.; Balena, R.; Zhi, J. Phase 1 assessment of a novel glucokinase
activator RO4389620 in healthy male volunteers. Diabetologia 2008,
51 (Suppl. 1), S372, Abstract 928. (c) Bonadonna, R. C.; Heise, T.;
Arbet-Engels, C.; Kapitza, C.; Avogaro, A.; Grimsby, J.; Zhi, J.; Grippo,
J. F.; Balena, R. Piragliatin (RO4389620), a novel glucokinase
activator, lowers plasma glucose both in the postabsorptive state and
after a glucose challenge in patients with type 2 diabetes mellitus: a
mechanistic study. J. Clin. Endocrinol. Metab. 2010, 95, 5028−5036.
(d) Migoya, E.; Miller, J.; Moreau, M.; Reitman, C.; Maganti, L.;
Larson, P.; Gutierrez, M.; Morrow, L.; Denoia, E.; Gottesdiener, K.;
Wagner, J. A. Additional Glucose-Lowering Effects of Oral GK
Activator, MK-0941, in Patients with T2DM on Basal Insulin.
Presented at the 71st American Diabetes Association Meeting, San
Diego, CA, 2011. (e) Bue-Valleskey, J. M.; Schneck, K. B.; Sinha, V. P.;
Wonddmagegnehu, E. T.; Kapitza, C.; Miller, J. W. LY2599506, a
Novel Glucokinase Activator (GKA), Improves Fasting and Post-
prandial Glucose in Patients with Type 2 Diabetes Mellitus (T2DM).
Presented at the 71st American Diabetes Association Meeting, San
Diego, CA, 2011.
(9) (a) Jetton, T. L.; Liang, Y.; Pettepher, C. C.; Zimmerman, E. C.;
Cox, F. G.; Horvath, K.; Matschinsky, F. M.; Magnuson, M. A.
Analysis of upstream glucokinase promoter activity in transgenic mice
and identification of glucokinase in rare neuroendocrine cells in the
brain and gut. J. Biol. Chem. 1994, 269, 3641−3654. (b) Murphy, R.;
Tura, A.; Clark, P. M.; Holst, J. J.; Mari, A.; Hattersley, A. T.
Glucokinase, the pancreatic glucose sensor, is not the gut glucose
sensor. Diabetologia 2009, 52, 154−159.
(10) Zelent, D.; Golson, M. L.; Koeberlein, B.; Quintens, R.; van
Lommel, L.; Buettger, C.; Weik-Collins, H.; Taub, R.; Grimsby, J.;
Schuit, F.; Kaestner, K. H.; Matschinsky, F. M. A glucose sensor role
for glucokinase in anterior pituitary cells. Diabetes 2006, 55, 1923−
1929.
(11) For a review, seethe following: Gloyn, A. L. Glucokinase
(GCK) mutations in hyper- and hypoglycemia: maturity-onset
diabetes of the young, permanent neonatal diabetes, and hyper-
insulinemia of infancy. Hum. Mutat. 2003, 22, 353−362.
(12) Grimsby, J.; Sarabu, R.; Corbett, W. L.; Haynes, N. E.; Bizzarro,
F. T.; Coffey, J. W.; Guertin, K. R.; Hilliard, D. W.; Kester, R. F.;
Mahaney, P. E.; Marcus, L.; Qi, L.; Spence, C. L.; Tengi, J.; Magnuson,
M. A.; Chu, C. A.; Dvorozniak, M. T.; Matschinsky, F. M.; Grippo, J.
F. Allosteric activators of glucokinase: potential role in diabetes
therapy. Science 2003, 301, 370−373.
(13) Haynes, N. E.; Corbett, W. L.; Bizzarro, F. T.; Guertin, K. R.;
Hilliard, D. W.; Holland, G. W.; Kester, R. F.; Mahaney, P. E.; Qi, L.;
Spence, C. L.; Tengi, J.; Dvorozniak, M. T.; Railkar, A.; Matschinsky,
F. M.; Grippo, J. F.; Grimsby, J.; Sarabu, R. Discovery, structure−
activity relationships, pharmacokinetics, and efficacy of glucokinase
activator (2R)-3-cyclopentyl-2-(4-methanesulfonylphenyl)-N-thiazol-
2-yl-propionamide (RO0281675). J. Med. Chem. 2010, 53, 3618−
3625.
(14) (a) Kester, R. F.; Corbett, W. L.; Sarabu, R.; Mahaney, P. E.;
Haynes, N.-E.; Guertin, K. R.; Bizzarro, F. T.; Hilliard, D. W.; Qi, L.;
Tengi, J.; Grippo, J. F.; Grimsby, J.; Marcus, L.; Spence, C.;
Dvorozniak, M.; Racha, J.; Wang, K. Discovery of Piragliatin, a
Small Molecule Activator of GK. Presented at the 238th National
Meeting of the American Chemical Society, Washington, DC, August
16−20, 2009; MEDI-005. (b) Sarabu, R.; Tilley, J. W.; Grimsby, J. The
discovery of piragliatin, a glucokinase activator. RSC Drug Discovery
Ser. 2011, 4, 51−70.
(15) Bertram, L. S.; Black, D.; Briner, P. H.; Chatfield, R.; Cooke, A.;
Fyfe, M. C. T.; Murray, P. J.; Naud, F.; Nawano, M.; Procter, M. J.;
Rakipovski, G.; Rasamison, C. M.; Reynet, C.; Schofield, K. L.; Shah,
V. K.; Spindler, F.; Taylor, A.; Turton, R.; Williams, G. M.; Wong-Kai-
In, P.; Yasuda, K. SAR, pharmacokinetics, safety, and efficacy of
glucokinase activating 2-(4-sulfonylphenyl)-N-thiazol-2-ylacetamides:
discovery of PSN-GK1. J. Med. Chem. 2008, 51, 4340−4345.
(16) Bebernitz, G. R.; Beaulieu, V.; Dale, B. A.; Deacon, R.; Duttaroy,
A.; Gao, J.; Grondine, M. S.; Gupta, R. C.; Kakmak, M.; Kavana, M.;
Kirman, L. C.; Liang, J.; Maniara, W. M.; Munshi, S.; Nadkarni, S. S.;
Schuster, H. F.; Stams, T.; Denny, I. S.; Taslimi, P. M.; Vash, B.;
Caplan, S. L. Investigation of functionally liver selective glucokinase
activators for the treatment of type 2 diabetes. J. Med. Chem. 2009, 52,
6142−6152.
(17) Kamata, K.; Mitsuya, M.; Nishimura, T.; Eiki, J.; Nagata, Y.
Structural basis for allosteric regulation of the monomeric allosteric
enzyme human glucokinase. Structure 2004, 12, 429−438.
(18) Waring, M. J.; Johnstone, C.; McKerrecher, D.; Pike, K. G.;
Robb, G. Matrix-based multiparameter optimization of glucokinase
activators: the discovery of AZD1092. Med. Chem. Commun. 2011, 2,
775−779.
(19) Klapars, A.; Campos, C. R.; Waldman, J. H.; Zewge, D.;
Dormer, P. G.; Chen, C.-Y. Enantioselective Pd-catalyzed α-arylation
of N-Boc-pyrrolidine: the key to an efficient and practical synthesis of
a glucokinase activator. J. Org. Chem. 2008, 73, 4986−4993.
(24) Pfefferkorn, J. A.; Guzman-Perez, A.; Oates, P. J.; Litchfield, J.;
Aspnes, G.; Basak, A.; Benbow, J.; Berliner, M. A.; Bian, J.; Choi, C.;
Freeman-Cook, K.; Corbett, J. W.; Didiuk, M.; Dunetz, J. R.; Filipski,
K. J.; Hungerford, W. M.; Jones, C. S.; Karki, K.; Ling, A.; Li, J.-C.;
Patel, L.; Perreault, C.; Risley, H.; Saenz, J.; Song, W.; Tu, M.; Aiello,
R.; Atkinson, K.; Barucci, N.; Beebe, D.; Bourassa, P.; Bourbounais, F.;
Brodeur, A. M.; Burbey, R.; Chen, J.; D’Aquila, T.; Derksen, D. R.;
Haddish-Berhane, N.; Huang, C.; Landro, J.; Lee Lapworth, A.;
MacDougall, M.; Perregaux, D.; Pettersen, J.; Robertson, A.; Tan, B.;
Treadway, J. L.; Liu, S.; Qiu, X.; Knafels, J.; Ammirati, M.; Song, X.;
DaSilva-Jardine, P.; Liras, S.; Sweet, L.; Rolph, T. P. Designing
glucokinase activators with reduced hypoglycemia risk: discovery of
N,N-dimethyl-5-(2-methyl-6-((5-methylpyrazin-2-yl)-carbamoyl)-
benzofuran-4-yloxy)pyrimidine-2-carboxamide as a clinical candidate
for the treatment of type 2 diabetes mellitus. Med. Chem. Commun.
2011, 2, 828−839.
́
(25) Bodvarsdottir, T. B.; Wahl, P.; Santhosh, K. C.; Polisetti, D. R.;
̈
Guzel, M.; Fosgerau, Larsen, M. Ø.; Halberg, I. B.; Selmer, J.;
Andrews, R. C.; Mjalli, A. M. M.; Valcarce, V. TTP355, a Glucokinase
Activator. Presented at the 68th American Diabetes Association
Meeting, San Francisco, CA, 2008.
(26) Caro, J. F.; Triester, S.; Patel, V. K.; Tapscott, E. B.; Frazier, N.
L.; Dohm, G. L. Liver glucokinase: decreased activity in patients with
type II diabetes. Horm. Metab. Res. 1995, 27, 19−22.
(27) Basu, A.; Basu, R.; Shah, P.; Vella, A; Johnson, C. M.; Nair, K.
S.; Jensen, M. D.; Schwenk, W. F.; Rizza, R. A. Effects of type 2
diabetes on the ability of insulin and glucose to regulate splanchnic and
1331
dx.doi.org/10.1021/jm2014887 | J. Med. Chem. 2012, 55, 1318−1333

Journal of Medicinal Chemistry
Article
muscle glucose metabolism. Evidence for a defect in hepatic
glucokinase activity. Diabetes 2000, 49, 272−283.
(28) For an example, seethe following: Torres, T. P.; Catlin, R. L.;
Chan, R.; Fujimoto, Y.; Sasaki, N.; Printz, R. L.; Newgard, C. B.;
Shiota, M. Restoration of hepatic glucokinase expression corrects
hepatic glucose flux and normalizes plasma glucose in Zucker diabetic
fatty rats. Diabetes 2009, 58, 78−86.
receptor modulator-statin hybrids. Bioorg. Med. Chem. Lett. 2004, 14,
4173−4178.
(32) Boyer, S. H.; Jiang, H.; Jacintho, J. D.; Reddy, M. V.; Li, H.;
Godwin, J. L.; Schulz, W. G.; Cable, E. E.; Hou, J.; Wu, R.; Fujitaki, J.
M.; Hecker, S. J.; Erion, M. D. Synthesis and biological evaluation of a
series of liver-selective phosphonic acid thyroid hormone receptor
agonists and their prodrugs. J. Med. Chem. 2008, 51, 7075−7093.
(33) For a review, seethe following: Erion, M. D.; Bullough, D. A.;
Lin, C.-C.; Hong, Z. HepDirect prodrugs for targeting nucleotide-
based antiviral drugs to the liver. Curr. Opin. Invest. Drugs 2006, 7,
109−117.
(29) (a) Pfefferkorn, J. A.; Litchfield, J.; Hutchings, R.; Cheng, X.-M.;
Larsen, S. D.; Auerbach, B.; Bush, M. R.; Lee, C.; Erasga, N.; Bowles,
D. M.; Boyles, D. C.; Lu, G.; Sekerke, C.; Askew, V.; Hanselman, J. C.;
Dillon, L.; Lin, Z.; Robertson, A.; Olsen, K.; Boustany, C.; Atkinson,
K.; Goosen, T. C.; Sahasrabudhe, V.; Chupka, J.; Duignan, D. B.; Feng,
B.; Scialis, R.; Kimoto, E.; Bi, Y.-A.; Lai, Y.; El-Kattan, A.; Bakker-
Arkema, R.; Barclay, P.; Kindt, E.; Le, V.; Mandema, J. W.; Milad, M.;
Tait, B. D.; Kennedy, R.; Trivedi, B. K.; Kowala, M. Discovery of novel
hepatoselective HMG-CoA reductase inhibitors for treating hyper-
cholesterolemia: a bench-to-bedside case study on tissue selective drug
distribution. Bioorg. Med. Chem. Lett. 2011, 21, 2725−2731.
(b) Ahmad, S.; Madsen, C. S.; Stein, P. D.; Janovitz, E.; Huang, C.;
Ngu, K.; Bisaha, S.; Kennedy, L. J.; Chen, B. C.; Zhao, R.; Sitkoff, D.;
Monshizadegan, H.; Yin, X.; Ryan, C. S.; Zhang, R.; Giancarli, M.;
Bird, E.; Chang, M.; Chen, X.; Setters, R.; Search, D.; Zhuang, S.;
Nguyen-Tran, V.; Cuff, C. A.; Harrity, T.; Darienzo, C. J.; Li, T.;
Reeves, R. A.; Blanar, M. A.; Barrish, J. C.; Zahler, R.; Robl, J. A.
(3R,5S,E)-7-(4-(4-Fluorophenyl)-6-isopropyl-2-(methyl(1-methyl-1H-
1,2,4-triazol-5-yl)amino)pyrimidin-5-yl)-3,5-dihydroxyhept-6-enoic
acid (BMS-644950): a rationally designed orally efficacious 3-hydroxy-
3-methylglutaryl coenzyme-A reductase inhibitor with reduced
myotoxicity potential. J. Med. Chem. 2008, 51, 2722−2733.
(c) Pfefferkorn, J. A.; Choi, C.; Larsen, S. D.; Auerbach, B.;
Hutchings, R.; Park, W.; Askew, V.; Dillon, L.; Hanselman, J. C.;
Lin, Z.; Lu, G. H.; Robertson, A.; Sekerke, C.; Harris, M. S.; Pavlovsky,
A.; Bainbridge, G.; Caspers, N.; Kowala, M.; Tait, B. D. Substituted
pyrazoles as hepatoselective HMG-CoA reductase inhibitors: discovery
of (3R,5R)-7-[2-(4-fluoro-phenyl)-4-isopropyl-5-(4-methyl-benzylcar-
bamoyl)-2H-pyrazol-3-yl]-3,5-dihydroxyheptanoic acid (PF-3052334)
as a candidate for the treatment of hypercholesterolemia. J. Med. Chem.
2008, 51, 31−45.
(30) (a) Oballa, R. M.; Belair, L.; Black, W. C.; Bleasby, K.; Chan, C.
C.; Desroches, C.; Du, X.; Gordon, R.; Guay, J.; Guiral, S.; Hafey, M.
J.; Hamelin, E.; Huang, Z.; Kennedy, B.; Lachance, N.; Landry, F.; Li,
C. S.; Mancini, J.; Normandin, D.; Pocai, A.; Powell, D. A.; Ramtohul,
Y. K.; Skorey, K.; Sorensen, D.; Sturkenboom, W.; Styhler, A.;
Waddleton, D. M.; Wang, H.; Wong, S.; Xu, L.; Zhang, L.
Development of a liver-targeted stearoyl-CoA desaturase (SCD)
inhibitor (MK-8245) to establish a therapeutic window for the
treatment of diabetes and dyslipidemia. J. Med. Chem. 2011, 54, 5082−
5096. (b) Koltun, D. O.; Zilbershtein, T. M.; Migulin, V. A.;
Vasilevich, N. I.; Parkhill, E. Q.; Glushkov, A. I.; McGregor, M. J.;
Brunn, S. A.; Chu, N.; Hao, J.; Mollova, N.; Leung, K.; Chisholm, J.
W.; Zablocki, J. Potent, orally bioavailable, liver-selective stearoyl-CoA
desaturase (SCD) inhibitors. Bioorg. Med. Chem. Lett. 2009, 19, 4070−
4074. (c) Leclerc, J.-P.; Falgueyret, J.-P.; Girardin, M.; Guay, J.; Guiral,
S.; Huang, Z.; Li, C. S.; Oballa, R.; Ramtohul, Y. K.; Skorey, K.; Tawa,
P.; Wang, H.; Zhang, L. Conversion of systemically-distributed
triazole-based stearoyl-CoA desaturase (SCD) uHTS hits into liver-
targeted SCD inhibitors. Bioorg. Med. Chem. Lett. 2011, 21, 6505−
6509.
(34) Hoglen, N. C.; Chen, L.-S.; Fisher, C. D.; Hirakawa, B. P.;
Groessl, T.; Contreras, P. C. Characterization of IDN-6556 (3-{2-(2-
tert-butyl-phenylaminooxalyl)-amino]-propionylamino}-4-oxo-5-
(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid): a liver-targeted caspase
inhibitor. J. Pharmacol. Exp. Ther. 2004, 309, 634−640.
́
(35) Boyer, S. H.; Sun, Z.; Jiang, H.; Esterbrook, J.; Gomez-Galeno, J.
E.; Craigo, W.; Reddy, K. R.; Ugarkar, B. G.; MacKenna, D. A.; Erion,
M. D. Synthesis and characterization of a novel cyclic 1-aryl-1,3-
propanyl prodrug of cytarabine monophosphate (MB07133) for the
treatment of hepatocellular carcinoma. J. Med. Chem. 2006, 49, 7711−
7720.
(36) Brocklehurst, K. J.; Payne, V. A.; Davies, R. A.; Carroll, D.;
Vertigan, H. L.; Wightman, H. J.; Aiston, S.; Waddell, I. D.; Leighton,
B.; Coghlan, M. P.; Agius, L. Stimulation of hepatocyte glucose
metabolism by novel small molecule glucokinase activators. Diabetes
2004, 53, 535−541.
(37) Segel, I. H. Enzyme Kinetics: Behavior and Analysis of Rapid
Equilibrium and Steady State Enzyme Systems; Wiley: New York, 1993.
(38) Kalgutkar, A. S.; Feng, B.; Nguyen, H. T.; Frederick, K. S.;
Campbell, S. D.; Hatch, H. L.; Bi, Y. A.; Kazolias, D. C.; Davidson, R.
E.; Mireles, R. J.; Duignan, D. B.; Choo, E. F.; Zhao, S. X. Role of
transporters in the disposition of the selective phosphodiesterase-4
inhibitor (+)-2-[4-({[2-(benzo[1,3]dioxol-5-yloxy)-pyridine-3-carbon-
yl]-amino}-methyl)-3-fluoro-phenoxy]-propionic acid in rat and
human. Drug Metab. Dispos. 2007, 35, 2111−2118.
(39) Di, L.; Whitney-Pickett, C.; Umland, J. P.; Zhang, H.; Zhang, X.;
Gebhard, D. F.; Lai, Y.; Federico, J. J.; Davidson, R. E.; Smith, R.;
Reyner, E. L.; Lee, C.; Feng, B.; Rotter, C.; Varma, M. V.; Kempshall,
S.; Fenner, K.; El-Kattan, A. F.; Liston, T. E.; Troutman, M. D.
Development of a new permeability assay using low-efflux MDCKII
cells. J. Pharm. Sci. 2011, 100, 4974−4985.
(40) (a) Cryle, M. J.; Matovic, N. J.; De Voss, J. J. Products of
cytochrome P450BioI (CYP107H1)-catalyzed oxidation of fatty acids.
Org. Lett. 2003, 5, 3341−3344. (b) Larcheveque, M.; Petit, Y. A simple
preparation of R or S glycidic esters; application to the synthesis of
enantiomerically pure α-hydroxyesters. Tetrahedron Lett. 1987, 28,
1993−1996.
(41) Pfefferkorn, J. A.; Lou, J.; Minich, M. L.; Filipski, K. J.; He, M.;
Zhou, R.; Ahmed, S.; Benbow, J.; Guzman-Perez, A.; Tu, M.;
Litchfield, J.; Sharma, R.; Metzler, K.; Bourbonais, F.; Huang, C.;
Beebe, D.; Oates, P. J. Pyridones as glucokinase activators:
identification of a unique metabolic liability of the 4-sulfonyl-2-
pyridone heterocycle. Bioorg. Med. Chem. Lett. 2009, 19, 3247−3252.
(42) For other representative examples where structure-based
structure strategies have utilized solvent exposed pockets to modulate
molecular properties, see the following: (a) Sun, L.; Liang, C.;
Shirazian, S.; Zhou, Y.; Miller, T.; Cui, J.; Fukuda, J. Y.; Chu, J.-Y.;
Nematalla, A.; Wang, X.; Chen, H.; Sistla, A.; Luu, T. C.; Tang, F.;
Wei, J.; Tang, C. Discovery of 5-[5-fluoro-2-oxo-1,2- dihydroindol-
(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-
diethylaminoethyl)amide, a novel tyrosine kinase inhibitor targeting
vascular endothelial and platelet-derived growth factor receptor
tyrosine kinase. J. Med. Chem. 2003, 46, 1116−1119. (b) Plantan, I.;
Selic, L.; Mesar, T.; Stefanic Anderluh, P.; Oblak, M.; Prezelj, A.;
Hesse, L.; Andrejasic, M.; Vilar, M.; Turk, D.; Kocijan, A.; Prevec, T.;
Vilfan, G.; Kocjan, D.; Copar, A.; Urleb, U.; Solmajer, T. 4-Substituted
trinems as broad spectrum β-lactamase inhibitors: structure-based
(31) (a) von Geldern, T. W.; Tu, N.; Kym, P. R.; Link, J. T.; Jae, H.-
S.; Lai, C.; Apelqvist, T.; Rhonnstad, P.; Hagberg, L.; Koehler, K.;
̈
Grynfarb, M.; Goos-Nilsson, A.; Sandberg, J.; Osterlund, M.; Barkhem,
T.; Hoglund, M; Wang, J.; Fung, S.; Wilcox, D.; Nguyen, P.; Jakob, C.;
̈
̈
Hutchins, C.; Farnegardh, M.; Kauppi, B.; Ohman, L.; Jacobson, P. B.
̈
Liver-selective glucocorticoid antagonists: a novel treatment for type 2
diabetes. J. Med. Chem. 2004, 47, 4213−4230. (b) Link, J. T.;
Sorensen, B. K.; Lai, C.; Wang, J.; Fung, S.; Deng, D.; Emery, M.;
Carroll, S.; Grynfarb, M.; Goos-Nilson, A.; von Geldern, T. Synthesis,
activity, metabolic stability, and pharmacokinetics of glucocorticoid
1332
dx.doi.org/10.1021/jm2014887 | J. Med. Chem. 2012, 55, 1318−1333

Journal of Medicinal Chemistry
Article
design, synthesis, and biological activity. J. Med. Chem. 2007, 50,
4113−4121.
(43) For more detailed account of the steady state kinetic interaction
of 19 with the glucokinase enzyme, see the following: Bourbonais, F.
J.; Chen, J.; Huang, C.; Zhang, Y.; Pfefferkorn, J. A.; Landro, J. A.
Modulation of glucokinase by glucose, small molecule activator and
glucokinase regulatory protein: steady-state kinetic and cell-based
analysis. Biochem. J. [Online early access]. DOI: 10.1042/BJ20110721.
Published Online: Nov 1, 2011.
(44) Barucci, N.; Robertson, A. S.; Bourassa, P.; Baker, L.; Mather,
D.; Perregaux, D. G.; Treadway, J. L.; Walton, J.; Atkinson, K.;
Hellembaek, J.; Tan, B.; Pfefferkorn, J. A.; Boustany, C.; D’Aquila, T.;
Litchfield, J.; Aiello, R. Characterization of a Potent Glucokinase
Activator with Diminished Risk of Hypoglycemia. Presented at the
71st American Diabetes Association Meeting, San Diego, CA, 2011.
(45) Previous reports have found that cryopreservation and culture
conditions can significantly affect transporter expression in rat
hepatocyctes. For example, see the following: Jørgensen, L.; Van
Beek, J.; Lund, S.; Schousboe, A.; Badolo, L. Evidence of Oatp and
Mdr1 in cryopreserved rat hepatocytes. Eur. J. Pharm Sci. 2007, 30,
181−189. This should be considered during potency comparisons
between actively and nonactively transported compounds and when
interpreting absolute potencies in these systems.
(46) Pettersen, J.; Litchfield, J.; Neef, N.; Schmidt, S. P.; Shirai, N.;
Walters, K.; Enerson, B.; Pfefferkorn, J. The Relationship of
Glucokinase Activator Induced Hypoglycemia with Cardiac Arterio-
pathy, Neuronal Necrosis and Peripheral Neuropathy in Nonclinical
Toxicology Studies. Presented at the 50th Annual Meeting of the
Society of Toxicology, Washington, DC, March 6−10, 2011.
(47) Loxham, S. J. G.; Teague, J.; Poucher, S. M.; Schoolmeeser, J.
D.; Turnbull, A. V.; Carey, F. Glucagon challenge in the rat: a robust
method for the in vivo assessment of glycogen phosphorlyase inhibitor
efficacy. J. Phamcol. Toxicol. Methods 2007, 55, 71−77.
(48) (a) Unger, R. H.; Orci, L. Role of glucagon in diabetes. Arch.
Intern. Med. 1977, 137, 482−491. (b) Dunning, B. E.; Gerich, J. E. The
role of α-cell dysregulation in fasting and postprandial hyperglycemia
in type 2 diabetes and therapeutic implications. Endocr. Rev. 2007, 28,
253−283.
(49) Wachi, K.; Terada, A. Studies of 1,3-benzoxazines. I. Synthesis of
primary 2-aminopyridines via the reaction of imidoyl chlorides of 1,3-
benzoxazines with pyridine N-oxides. Chem. Pharm. Bull. 1980, 28,
465−472.
(50) Bessmertnykh, A.; Douaihy, C. M.; Muniappan, S.; Guilard, R.
Efficient palladium-catalyzed synthesis of aminopyridyl phosphonates
from bromopyridines and diethyl phosphite. Synthesis 2008, 1575−
1579.
(51) Holland, G. F.; Pereira, J. N. Heterocyclic tetrazoles, a new class
of lipolysis inhibitors. J. Med. Chem. 1967, 10, 149−154.
1333
dx.doi.org/10.1021/jm2014887 | J. Med. Chem. 2012, 55, 1318−1333