Discovery, structure-activity relationships, pharmacokinetics, and efficacy of glucokinase activator (2 R)-3-cyclopentyl-2-(4-methanesulfonylphenyl)-N- thiazol-2-yl-propionamide (RO0281675)

Article abstract of DOI:10.1021/jm100039a

Glucokinase (GK) is a glucose sensor that couples glucose metabolism to insulin release. The important role of GK in maintaining glucose homeostasis is illustrated in patients with GK mutations. In this publication, identification of the hit molecule 1 and its SAR development, which led to the discovery of potent allosteric GK activators 9a and 21a, is described. Compound 21a (RO0281675) was used to validate the clinical relevance of targeting GK to treat type 2 diabetes.

Full text of DOI:10.1021/jm100039a

3618 J. Med. Chem. 2010, 53, 3618–3625
DOI: 10.1021/jm100039a
Discovery, Structure-Activity Relationships, Pharmacokinetics, and Efficacy of Glucokinase
Activator (2R)-3-Cyclopentyl-2-(4-methanesulfonylphenyl)-N-thiazol-2-yl-propionamide
(RO0281675)
Nancy-Ellen Haynes,† Wendy L. Corbett,† Fred T. Bizzarro,† Kevin R. Guertin,† Darryl W. Hilliard,† George W. Holland,†
Robert F. Kester,† Paige E. Mahaney,† Lida Qi,† Cheryl L. Spence,§ John Tengi,† Mark T. Dvorozniak,§ Aruna Railkar,
Franz M. Matschinsky,‡ Joseph F. Grippo,§ Joseph Grimsby,§ and Ramakanth Sarabu*,†
†Departments of Discovery Chemistry, §Metabolic and Vascular Diseases, Non-Clinical Drug Safety, and Discovery Technologies,
Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, New Jersey 07110, and ‡Department of Biochemistry and Diabetes Center,
University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Received January 12, 2010
Glucokinase (GK) is a glucose sensor that couples glucose metabolism to insulin release. The important
role of GK in maintaining glucose homeostasis is illustrated in patients with GK mutations. In this
publication, identification of the hit molecule 1 and its SAR development, which led to the discovery of
potent allosteric GK activators 9a and 21a, is described. Compound 21a (RO0281675) was used to
validate the clinical relevance of targeting GK to treat type 2 diabetes.
Introduction
leading to insulin release. In the liver, GK catalyzes the first
step in glucose metabolism and plays an important role in
determining net glucose utilization and production in this
organ. GK is also expressed in pancreatic R-cells, enteroendo-
crine K and L cells, glucose excited/glucose inhibited neurons of
the hypothalamus and brainstem, and anterior pituitary cells,
but its physiological role in these tissues is less well understood.
The enzymatic parameters and activity of GK (kcat, glucose
Maintenance of tight glycemic control requires a complex
interplay between an individual’s lifestyle, genes, environ-
ment, and neuroendocrine and endocrine systems. Defects
in the ability of pancreatic β-cells to secrete insulin in response
to meals and the diminished capacity of the liver and skeletal
muscle to take up glucose in response to insulin represent core
pathophysiological defects associated with type 2 diabetes
(T2Da). Other tissues such as fat cells, gastrointestinal tract,
kidney, and brain also contribute to dysglycemia in patients
with T2D.1 As T2D progresses, patients rely on higher doses
of antidiabetic agents and multiple oral antidiabetic drugs and
progress to exogenous insulin therapy to gain adequate
glycemic control. Therefore, more effective therapeutic agents
that target multiple pathogenic abnormalities with sustained
efficacy represent a major unmet medical need.
Glucokinase (GK), alsocalledhexokinase IV, is a glycolytic
enzyme that catalyzes the oxidative phosphorylation of
hexoses by ATP. Unlike its isozymes (hexokinases I-III) that
are fully saturated at physiologically glucose levels, GK
respondsfunctionally toplasmaglucosefluctuationsobserved
during preprandial and postprandial periods. This enables
pancreatic β-cell GK to function as a molecular sensor that
couples changes in blood glucose levels with insulin release to
maintain glucose homeostasis. As a sensor, it determines the
rate and threshold concentration of glucose (∼5 mM in
healthy individuals) required to initiate the signaling cascade
S0.5, and positive kinetic cooperativity) are fundamental to its
role as a glucose sensor, and this is best illustrated in humans
with diseases related tomutations of GK. For instance, loss-of-
function mutations in one or both alleles of the GK gene cause
maturity onset diabetes of the young type 2 (MODY-2) and
permanent neonatal diabetes mellitus, respectively. In con-
trast, a gain-of-function mutation in a single GK allele causes
persistent hyperinsulinemic hypoglycemia of infancy (PHHI)
due to excessive insulin release. On the basis of the pivotal role
of GK as a glucose sensor in pancreatic β-cells and its key role
in regulating glucose production in the liver, allosteric activa-
tors of this enzyme are being actively pursued as antidiabetic
agents based on the dual effects on augmenting insulin release
and suppressing excessive hepatic glucose production.2-4
Screening of Roche’s compound library led to the identifi-
cation of 1, which was shown to increase GK enzymatic
activity by 1.5-fold at 24 μM (SC1.5). This molecule had
desired “lead-like” properties (i.e, low molecular weight and
a combination of polar and nonpolar moieties) to warrant
further optimization. RO0281675, 21a, a prototypical GK
activator (GKA) that emerged from this effort, causes robust
glucose lowering effects in preclinical models of T2D.5 These
findings have stimulated intense interest within the pharma-
ceutical industry leading to several structural mimics of 21a.6,7
Here we report on the optimization of the original screening
hit 1, which led to the identification of two lead molecules, the
N-acylurea (RO0274375) 9a and 21a (Figure 1).
*To whom correspondence should be addressed. Phone: 973-235-
3984. Fax: 973-235-2448. E-mail: ramakanth.sarabu@roche.com.
a Abbreviations: ATP, adenosine triphosphate; GK, glucokinase;
GKA, glucokinase activator; GSIR, glucose-stimulated insulin release;
MODY-2, maturity onset diabetes of the young type 2; OGTT, oral
glucose tolerance test; PHHI, persistent hyperinsulinemic hypoglycemia
of infancy; PK, phramacokinetics; SC1.5, concentration of a compound
to increase GK activity by 1.5 times; T2D, type 2 diabetes.
r
pubs.acs.org/jmc
Published on Web 04/21/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3619
Chemistry
The target compounds, N-acylurea hit 1, analogues, 5a-zf,
and the corresponding 2-aminothiazole amides 8a-z de-
scribed in this report were prepared following a two to four
step sequence outlined in Scheme 1. Starting from commer-
cially available arylacetic acids 2, direct alkylation or alkyla-
tion of the corresponding alkyl ester 6 with an alkyl iodide
yielded alkylphenylacetic acids 3 or the esters 7, respectively.
A few ester analogues of 7 were also prepared via the
esterification of the corresponding acids 3. The aryl acetic
acid esters 7 were then reacted with urea or alkylureas in the
presence of magnesium alkoxide to prepare the unsubstituted,
methyl, or ethyl-N-acylurea analogues 5.8 Additional analo-
gues of 5 containing larger alkyl groups and polar moieties in
the R3 region of the molecule were accessible via the reaction
of the phenylacetamide derivative 4 with the appropriate
isocyanates.9 The enantiomers 9a and 9b of the racemic 5h
were prepared from stereochemically pure carboxylic acids,
Figure 1. Glucokinase activator hit molecule 1 and optimized
molecules 9a and 21a.
Scheme 1. Synthesis of N-Acylurea Analoguesa
a (i) LDA/HMPA or DMPU, THF, R2-CH2-I, -78 °C to room temp; (ii) cat. DMF, (COCl)2, CH2Cl2, HMDS, 0 °C to room temp, aq HCl;
(iii) R3-N=C=O, toluene, reflux; (iv) cat. H2SO4, R0OH, reflux; (v) LDA/HMPA, R2-CH2-I; (vi) Mg(OCH3)2, methyl or ethyl urea, CH3OH,
reflux.

3620 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Scheme 2. Synthesis of 9a and 21aa
Haynes et al.
a (i) (CH3)3COCl, Et3N, then n-BuLi, (S)-4-isopropyl-2-oxazolidinone, THF 28% (for 3a to 10a); (ii) 30% H2O2, LiOH, 72% for 11a and 89% for
14a; (iii) oxalyl chloride, HMDS, 0 °C, 58%; (iv) MeNCO, toluene, reflux, 63%; (v) (CH3)3COCl, Et3N, then n-BuLi, (R)-4-benzyl-2-oxazolidinone,
49%, (for 3g to 13a); (vi) N-bromosuccinimide, triphenylphosphine.
Scheme 3. Synthesis of 2-Aminothiazole Amidesa
a (i) 2-Aminothiazole, NBS, PPh3, CH2Cl2, room temp; (ii) LiOH, THF/H2O, room temp; (iii) 2-aminothiazole, Mg(OCH3)2, CH3OH, reflux.
11a and 11b, which were prepared by resolution of the
corresponding chiral diastereomeric oxazolidinoneimides 10a
and 10b (Scheme 2). A single crystal X-ray structure of 9a
was obtained, and the absolute stereochemistry was shown
to be R (see Figure 1 in Supporting Information). The
2-aminothiazoleamides (8a-z) were prepared from the
arylacetic acids 3 using a N-bromosuccinimide/triphenyl-
phosphine coupling method (Scheme 3).10 Enantiomerically
pure 21a was prepared starting from the corresponding racemic
carboxylic acid 3g via chromatographic separation of diastere-
omeric benzyloxazolidine derivatives 13a and 13b, as shown in
Scheme 2. Several structural variants of 5h were prepared to
explore its active conformation.
centered on the identification of the optimal R2 alkyl group
of the hit molecule 1. A systematic evaluation of several R2
analogues that varied in size and polarity were explored
(Table 1). The original isopropyl group present in 1 remained
the preferred group among the aliphatic groups investigated,
while incorporation of cyclohexyl (5d) or cyclopentyl (5c)
moieties were optimal among the alicyclic groups. The latter
improved potency by nearly 6-fold over 1. Larger alkyl,
cycloalkyl, aryl groups and groups with polar substitutents
significantly reduced activity or were inactive. For example,
neopentyl, cyclooctyl, bicyclo[2.2.1]heptyl, 2-methythiazolyl,
4-piperidinyl, and 4-hydroxycyclohexyl analogues were inac-
tive (data not shown). Thus, these studies indicated a clear
preference for nonpolar, nonplanar alicyclic groups in the R2
region of the molecule (Table 1).
Results and Discussion
The R1 aromatic group of the hit molecule 1 was explored
extensively. The SAR indicated the importance ofanaromatic
ring with one or two electron withdrawing groups to improve
potency and revealed a tolerance for a wide variety of
The SAR was explored with the conceptual division of the
acylurea hit molecule 1 into three regions around the chiral
center: R1, the substituted aromatic ring; R2, the alkyl group;
R3, the N-acylurea group (Figure 1). Initial studies were

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3621
Table 1. Structure-Activity Relationships of N-Acylureas
compd
R1
R2
R3
SC1.5 (μM)a
1
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3-Cl
isopropyl
NHCONH2
NHCONH2
NHCONH2
NHCONH2
NHCONH2
NHCONH2
29 ( 7.9 (4)
40 ( 6.5 (2)
10 ( 0.45 (2)
4.4 ( 0.35 (2)
8.9 ( 2.6 (3)
12 ( 6.5 (2)
5a
5b
5c
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
cycloheptyl
isopropyl
5d
5e
5f
NHCONHCH3
NHCONHCH3
6
5g
5h
5i
cyclohexyl
cyclopentyl
cyclopentyl-CH2
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cycloopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
isopropyl
1.4 ( 0.0 (2)
1 ( 0.19 (3)
na
NHCONHCH3
NHCONHCH3
5j
NHCONHCH2CH3
NHCONH(CH2)2CH3
NHCONHCH2CHdCH2
NHCONHCH2CH(CH3)2
NHCONH(CH2)3CH3
NHCONHCH2COOC2H5
NHCONHCH2COOH
NHCONHCH2COOCH3
NHCONH(CH2)2COOC2H5
NHCONH(CH2)2COOH
NHCONH(CH2)2COOCH3
NHCONH(CH2)2OH
NHCONHCH3
0.53 ( 0.13 (2)
1.3
5k
5l
1.3 ( 0.22 (2)
6.3
na
5m
5n
5o
5p
5q
5r
1.3 ( 0.25 (2)
na
0.55 ( 0.078 (2)
8.4
5s
na
3.2
5t
5u
5v
3.4
3.7
5w
5x
5y
5z
4-Cl
H
NHCONHCH3
NHCONHCH3
5.4
28.2
3,4-diF
4-Br
4-NO2
NHCONHCH3
NHCONHCH3
8.5
3.8
5za
5zb
5zc
5zd
5ze
5zf
16
17
18
19
20
9ab
9bc
NHCONHCH3
NHCONHCH3
5.4
3.2
4-SCH3
4-SCF3
4-SO2CH3
4-SO2CF3
3-SO2CF3
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
NHCONHCH3
NHCONHCH3
1.4
1.5
NHCONHCH3
NHCONHCH3
0.3
1
na
imidazoline-2-one
NHC(=N)NH2
na
na
N(CH3)CONH(CH3)
NHCON(CH3)2
na
3.8
cyclopentyl
cyclopentyl
cyclopentyl
NHC(dS)NH(CH3)
NHCONHCH3
NHCONHCH3
0.41 ( 0.049 (6)
na
a Data reported as mean ( SEM (n). The average CV of the assay was 15%, based on an analysis of 452 compounds (SC1.5 < 2 μM) assayed in
duplicate. na: not active up to 30 μM. bR-isomer. cS-isomer.
substituents at the para-position. For example, the mono-
chlorophenyl analogues 5v (SC1.5 = 3.7 μM) and 5w (SC1.5
potency was observed upon introducing an N-methyl group at
the terminal amide of 1. Thus, analogue 5f was about 5-fold
more active than 1 (6 μM vs 29 μM). Combining one of the
optimized groups at R2, cyclopentyl, with an N-ethyl group at
R3 gave 5j (SC1.5 = 0.53 μM), one of the first GK activators
with submicromolar potency. Further examination of the
SAR of the terminal amide by introduction of larger N-alkyl
groups, such as n-propyl, allyl, isopropyl, and n-butyl, led to a
gradual decrease to complete loss of GKA potency (e.g. 5k, 5l,
5m, and 5n), reflecting the size limitations at R3. For example,
the acetate analogues 5o and 5q were about 6- to 8- fold more
potent than the corresponding propionates 5r and 5t. The car-
boxylic acid analogues 5p and 5s were not active, while intro-
duction of the hydroxyethyl group, 5u, was well tolerated.
The optically pure R- and S-stereoisomers, 9a and 9b
of racemic 5h, were prepared as described in Scheme 2. The
=
5.4 μM) were about 6-fold more potent than the unsubstitued
phenyl analogue 5x (SC1.5 = 28 μM), and the 3,4-dichloro-
phenyl analogue 5h was about 30-fold more potent. The
4-trifluoromethylsulfonyl derivative 5ze (SC1.5 = 0.3 μM)
was among the most active analogues in this series and nearly
100-fold more potent than the unsubstituted 5x. In general
meta- or para-substitutions were well tolerated, while ortho-
substitutions, suchaso-chloro, o-methyl, o-methoxy, o-piperi-
dinyl, dramatically reduced or completely eliminated GKA
potency (data not shown).
Investigation of the R3 modifications of 1 resulted in
improvements in potency, identified areas to introduce func-
tionality, and provided clues regarding the active conforma-
tion required for GK activation. A significant increase in

3622 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Haynes et al.
Figure 2. Intramolecular H-bond and cis-amide characteristics of 1
and the resulting 2-amino heteroaromatics hypothesis.
Table 2. Structure-Activity Relationships of 2-Aminothiazoleamides
compd
R1
R2
R3
SC1.5 (μM)a
Figure 3. Effects of 9a on GK kinetics: (A) rate versus glucose plot
in the absence and presence of 9a; (B) apparent Vmax versus [9a]; (C)
apparent S0.5 versus [9a] plots.
8a
8b
8c
8d
8e
8f
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
4-Cl
cyclopentyl
phenyl
H
H
0.94 ( 0.16 (2)
na
cyclopentyl 4-methyl
cyclopentyl 5-methyl
cyclopentyl 4,5-dimethyl
cyclopentyl 4-COOC2H5
cyclopentyl 4-COOCH3
cyclopentyl 4-CH2OH
cyclopentyl 4-COOH
cyclopentyl 5-COOC2H5
cyclopentyl 5-COOH
cyclopentyl 5-COOCH3
cyclopentyl 5-CH2OH
1.2
0.64
1.6
2.7
0.5
0.99
29
5h, the cyclic urea 16, the N-methylacylurea 18, and the
terminal N,N0-dimethyl analogue 19, all of which lack the
ability to make the intramolecular H-bond, failed to activate
GK. The guanidine analogue 17, which potentially could have
an intramolecular H-bond but lacks the urea carbonyl that
may accept a hydrogen bond from glucokinase, also failed to
activate GK. The thiourea analogue 20 (SC1.5 = 3.8 μM) lost
about 4-fold in potency relative to the urea 5h possibly as a
reflection of the lower capacity of the sulfur atom to accept a
hydrogen bond. The CR-methylated analogue 15 (1-[3-cyclo-
pentyl-2-(3,4-dichlorophenyl)-2-methylpropionyl]-3-methylurea)
was found to be inactive, which suggested a potential steric
effect between the CR-methyl and the N-acylurea moiety,
preventing it from making an optimal interaction with GK.
Collectively, these observations suggested that the cyclic,
intramolecularly hydrogen-bonded conformation adopted
by the N-acylureas was necessary to support donor-acceptor
hydrogen bonding with GK.
Later, the molecular modeling experiments with the N-acylurea
GK activators using the X-ray co-crystal structures of other
GK activator-GK identified key H-bond interactions with the
backbone NH and carbonyl of Arg63 residue in GK.11 These
observations led to a hypothesis that amides derived from
heterocyclic amines of formula I could mimic the H-bond
donor-acceptor interactions of the N-acylurea’s cis-amide
bond (as shown in Figure 2). Several phenylacetamides of
various 2-amino-nitrogen heteroaromatic systems were eval-
uated as GKAs and found to be active. In the following
section, the SAR related to 2-aminothiazolephenylacetamide
GKAs is described in detail and the data are summarized in
Table 2.
8g
8h
8i
8j
8
26
8k
8l
0.92
0.75
8m
8n
8o
8p
8q
8r
8s
8t
cyclopentyl 4-CH2COOC2H5 0.61
cyclopentyl 4-CH2COOH
cyclopentyl 4-CH2COOCH3 0.82
8
cyclopentyl 4-CH2CH2OH
0.84
3.1
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
H
H
H
H
H
H
H
H
H
H
H
3-Cl
4-NO2
4-SCH3
1.7
0.84
8u
8v
8w
8x
8y
8z
2.2
4-S(O)CH3 cyclopentyl
4-SO2CH3 cyclopentyl
2.2 ( 0.44 (2)
0.35 ( 0.022 (5)
1.8
4-SCF3
cyclopentyl
4-SO2CF3 cyclopentyl
3-SO2CF3 cyclopentyl
0.19 ( 0.057 (3)
1.4
21ab 4-SO2CH3 cyclopentyl
21bc 4-SO2CH3 cyclopentyl
a Data reported as mean ( SEM (n). The average CV of the assay was
15%, based on an analysis of 452 compounds (SC1.5 < 2 μM) assayed in
duplicate. na: not active up to 30 μM. bR-isomer. cS-isomer.
0.24 ( 0.019 (25)
na
absolute stereochemistry of 9a was determined to be R on the
basis of a single crystal X-ray structure (Figure 1 of Support-
ing Information). The R-isomer 9a has an SC1.5 of 0.41 μM,
while the corresponding S isomer 9b has no effect on GK
activity at concentrations up to 10 μM. The crystal structure
of 9a further revealed that the N-acylurea moiety exists in a
six-membered cyclic conformation characterized by an intra-
molecular H-bond, between the carbonyl of the acyl oxygen
atom and the terminal NH of the urea, resulting in a cisoid
conformation of the amide bond.
In general, the SAR at R1 and R2 parallels the N-acylureas.
Thus, the cyclopentyl analogue 8a was very potent while the
corresponding aryl derivative 8b was significantly less active.
To explore the thiazole SAR, 8a was used as a positive
comparator. Several 4- and 5-substituted thiazole analogue
pairs were prepared to explore the optimal position to
introduce functionality. As the data in Table 2 illustrates,
A variety of compounds were prepared to explore the
significance of the N-acylurea conformation. In relation to

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3623
Figure 4. Compound 9a (cpd A) lowers the threshold for glucose stimulated insulin release: effects of 9a (cpd A) in isolated mouse islets, cultured
for 4-5 days in 10 mM glucose. Following a preperifusion phase with a balanced salt solution in the absence of glucose, a glucose ramp was
applied from 0 to 20 mM (0.8 mM/min) in the presence of the test agent. There were two to four replicates for each condition. Adapted from ref 14.
Table 3. In Vitro GK Enzyme Kinetics Data for Compounds 9a and 21a
9a
21a
Parameter: Vmax (μmol/min/mg protein)
18.0
control
19.1
23.7 (1.2)
26.1 (1.4)
1 μM activator (fold change vs control)
20 μM activator (fold change vs control)
24.7 (1.4)
31.2 (1.7)
Parameter: S0.5 (mmol/L)
control
1 μM activator (fold change vs control)
20 μM activator (fold change vs control)
8.0
5.2 (0.66)
1.8 (0.22)
8.7
3.3 (0.38)
0.84 (0.10)
Table 4. Rat Pharmacokinetics Data for Compounds 9a and 21a
dose Cl ((mL/ Vss AUC0-inf Cmax Tmax Fpo
compd (mg/kg) route min)/kg) (L/kg) (ng h/mL) (μg/mL) (h) (%)
3
9a
5
iva
43.48
4.21
1877
455
10
30
5
pob
pob
iva
133
471
1.4 12
3.3 13
1464
2774
5150
51748
21a
30
2.16
10
30
pob
pob
1140
4302
3.3 92.8
4.7 311
a Formulation: 10% PEG-400. b Formulation: gelucire.
introduction of a methyl group atposition 5 (8d) or at position
4 (8c) and 4,5-dimethyl (8e) thiazole analogues were not
significantly different compared to 8a. Although the presence
of an ethoxycarbonyl at the 4- or 5-position (8f and 8j)
resulted in significant reduction in potency, the corresponding
methoxycarbonyl derivatives 8g and 8l were equipotent to 8a.
The 4-acetic acid esters 8n and 8p and the hydroxyethyl
analogue 8q were all equipotent versus 8a. All carboxylic acid
containing analogues (8i and 8k) were inactive. Thus, these
observations identified areas to introduce polarity into the
GK activator molecule to improve their solubility (solubility
in water: 8a, 5 μg/mL; 8g, 24 μg/mL) and other “drug-like”
properties.
Figure 5. Acute effects of orally administered 9a (50 mg/kg)
and 21a (50 mg/kg) on an oral glucose tolerance test in male
C57BL/6J mice. All results are reported as the mean ( STD (n =
6/time point). A Student’s t test was used to test for statistical
significance: (/) P < 0.05 compared to vehicle; (#) P < 0.05 between
9a and 21a.
analogues clearly suggested preference for electron withdrawing
group(s) at R1. Similar to N-acylureas, the R stereoisomer 21a
activated GK with a SC1.5 of 0.24 μM while the corresponding S
isomer 21b did not activate GK up to 10 μM.
The potency of R1 analogues 4-nitro 8t, 4-methylsulfonyl 8w,
and 4-trifluoromethylsulfonyl 8y and the gradual potency im-
provement observed when comparing the thioether 8u, methyl
sulfoxide 8v, and methylsulfone 8w (SC1.5 of 2.2-0.35 μM)

3624 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Haynes et al.
On the basis of SAR studies, the structural elements
required in a phenylacetamide based GK activator can be
summarized as follows: (i) an electron withdrawing substitu-
ent is necessary at R1; (ii) a five- or six-membered alicyclic ring
is optimal at R2; (iii) a hydrogen bond donor-acceptor pair as
part of an N-acylurea or an amide of 2-amino heterocycle with
a H-bond acceptor capability (Figure 2) is required at R3;
(iv) the R-stereochemistry at the chiral center is needed.
In Vitro and in Vivo Activity Profile of Compounds 9a and
21a. Extensive evaluation of several analogues for their
in vitro potency and in vitro safety assessment assays led to
the selection of 9a and 21a as advanced lead molecules.
Consistent with the pancreatic β-cell hypothesis, these glu-
cokinase activators have been shown to augment insulin
release in studies conducted in freshly isolated rodent pan-
creatic islets and in pancreatic cell lines.5,11 In general,
glucokinase activators shift the glucose stimulated insulin
release (GSIR) threshold to the left in a concentration-depen-
dent and glucose-dependent manner. Glucokinase activator
concentrations associated with cellular activity ranged from
submicromolar to low micromolar range and can vary
depending on the assay conditions and glucose levels (vide
supra). Compound 9a dose-dependently increased the enzy-
matic activity of GK across a wide range of glucose con-
centrations (Figure 3), whereas the corresponding S-
enantiomer was inactive (not shown). Activation of GK
was driven by dual effects of increasing the enzyme’s Vmax
(Figure 3B) and lowering its S0.5 (concentration of glucose
at half-maximal velocity) for glucose (Figure 3C). The
magnitude of these effects at 1 and 20 μM activator con-
centration was determined by fitting the data to the Hill
equation (Table 3). Compound 9a exerted a relatively larger
increase in Vmax (1.7-fold change for 9a versus 1.4-fold
change for 21a at 20 μM), whereas 21a showed a greater
effect on reducing the enzyme’s S0.5 for glucose (1.8 mmol/L
for 9a versus 0.84 mmol/L for 21a at 20 μM activator
concentration).
trials in healthy volunteers. Following a single oral dose, 21a
reduced fasting and postprandial glucose levels following an
OGTT, was well tolerated, and displayed no adverse effects
related to drug administration other than hypoglycemia at
the maximum dose (400 mg), which defined the maximum
tolerated dose.13
Conclusion
In summary, we have discovered two potent allosteric GK
activator molecules, 9a and 21a, based on the optimization of
a unique N-acylurea hit molecule 1. The results of the SAR
work established a useful pharmacophore model that we
exploitedinthe design of severalpotent glucokinaseactivators
including 21a, the one that we used to demonstrate the clinical
relevance of targeting GK for the treatment of T2D.
Acknowledgment. The authors thank Dr. Jefferson Tilley
for helpful discussions. The authors are grateful to Dr.
Anthony Greway for DMPK support, Linda Marcus for
the GK activity assays, and Dr. Amy Sarjeant for the
compound 9a X-ray structure determination. The authors
are also grateful to all members of the Department of
Physical Chemistry, Hoffman-La Roche, for the character-
ization of the compounds.
Supporting Information Available: Experimental procedures
and analytical data for all intermediate and final compounds.
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) De Fronzo, R. A. From the triumvirate to the ominous octet: a new
paradigm for the treatment of type 2 diabetes mellitus diabetes.
Diabetes 2009, 58, 773–795.
(2) Matschinsky, F. M. Assessing the potential of glucokinase
activators in diabetes therapy. Nat. Rev. Drug Discovery 2009, 8,
399–416.
Consistent with the role of pancreatic β-cell GK, treat-
ment of rodent islets with 9a14 (Figure 4) or 21a5 shifted the
glucose-stimulated insulin release curve to the left as demon-
strated in perifusion studies using isolated pancreatic islets.
To better discriminate between the two GK activators, head-to-
head in vivo efficacy studies were performed. Oral adminis-
tration of 9a and 21a (50 mg/kg) to male C57Bl/6J mice
caused a statistically significant reduction in fasting glucose
levels and improvement in glucose tolerance relative to the
vehicle treated animals (Figure 5A). Compound 21a showed
a statistically significant superior effect during the oral
glucose tolerance test (OGTT) relative to 9a (Figure 5B).
Comparison of rat PK parameters indicated that 21a displayed
lower clearance and higher oral bioavailability compared to 9a
(Table 4). Oral bioavailability of the 30 mg/kg 21a dose
exceeded 100% probably as a result of precipitation at the
injection site or insufficient sampling following iv administra-
tion and the consequent failure to fully capture the distribution
phase. Other possibilities (saturable hepatic first-pass clear-
ance or gut wall metabolism) cannot be excluded. The PK
properties of orally administered 21a were similar between the
geluciresolution and klucel suspension. Oralbioavailabilityin
dog and monkey was 109% and 67%, respectively, using
Klucel (hydroxypropylcellulose)12 suspension.
(3) Grimsby, J.; Berthel, S. J.; Sarabu, R. Glucokinase activators for
the potential treatment of type 2 diabetes. Curr. Top. Med. Chem.
2008, 8, 1524–1532.
(4) Coghlan, M.; Leighton, B. Glucokinase activators in diabetes
management. Expert Opin. Invest. Drugs 2008, 17, 145–167.
(5) Grimsby, J.; Sarabu, R.; Corbett, W. L.; Haynes, N. E.; Bizzaro,
F. T.; Coffey, J. W.; Guertin, K. R.; Hilliard, D. H.; Kester,
R. F. K.; 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.
(6) (a) Bertram, L. S.; Black, D.; Biner, 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-yl-acetamides: discovery of PSN-GK1. J. Med. Chem.
2008, 51, 4340–4345. (b) Efanov, A. M.; Barrett, D. G.; Brenner, M. B.;
Briggs, S. L.; Delaunois, A.; Durbin, J. D.; Giese, U.; Guo, H.; Radloff,
M.; Gil, G. S.; Sewing, S.; Wang, Y.; Weichert, A.; Zaliani, A.; Gromada,
J. A novel glucokinase activator modulates pancreatic islet and hepato-
cyte function. Endocrinology 2005, 146, 3696–3701. (c) Castelhano,
A. L.; Dong, H.; Fyfe, M. C. T.; Gardner, L. S.; Kamikozawa, Y.;
Kurabayashi, S.; Nawano, M.; Ohashi, R.; Procter, M. J.; Qiu, L.;
Rasamison, C. M.; Schofield, K. L.; Shah, V. K.; Ueta, K.; Williams,
G. M.; Witter, D.; Yasuda, K. Glucokinase-activating ureas. Bioorg.
Med. Chem. Lett. 2005, 15, 1501–1504.
(7) 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.
(8) Karmouta, M. G.; Miocque, M.; Derdour, A.; Gayral, P.; Lafont,
O. Synthse de cyanacetylurees en vue d’essais vis-a-vis d’hymeno-
lepisnana. Eur. J. Med. Chem. 1989, 24, 547–549.
On the basis of the outcome of a 5-day repeat-dose toxicity
studies conducted in rats, 21a was chosen over 9a for further
development and advancement into single ascending dose

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3625
(9) Wiley, P. F. The reaction of amides with isocyanates. J. Am. Chem.
Soc. 1949, 71, 1310–1311.
(10) Froyen, P. The conversion of carboxylic acids into amides
via NCS/triphenylphosphine. Synth. Commun. 1995, 25, 959–
968.
(11) Dunten, P.; Swain, A.; Kammlott, U.; Crowther, R.; Lukacs,
C. M.; Levin, W.; Reik, L.; Grimsby, J.; Corbett, W. L.; Magnuson,
M. A.; Matschinsky, F. M. Crystal Structure of Human Liver
Glucokinase Bound to Small Molecule Allosteric Activator. In
Glucokinase and Glycimic Disease. From Basics to Therapeutics;
Matschinsky, F. M., Magnuson, M. A., Eds.; Karger AG: Basel, Switzerland,
2004; pp 145-154.
(12) Guo, J.-H.; Skinner, G. W.; Harcum, W. W.; Barnum, P. E.
Pharmaceutical applications of naturally occuring water-soluble
polymers. Pharm. Sci. Technol. Today 1998, 1, 254–250.
(13) Grimsby, J.; Zhi, J.; Mulligan, M. E.; Arbet-Engels, C.; Taub, R.;
Balena, R. Antidiabetic Effects of Glucokinase Activators: From
Benchside to Bedside. Presented at the Keystone Symposia: Dia-
betes Mellitus, Insulin Action and Resistance, Breckenridge, CO,
January 22-27, 2008; Poster No. 151.
(14) Grimsby,J.;Mastschinsky,F.M.;Grippo,J.F.DiscoveryandActions
of Glucokinase Activators. In Glucokinase and Glycemic Disease: From
Basics to Novel Therapeutics; Matschinsky, F. M., Magnuson, M. A., Eds.;
Karger AG: Basel, Switzerland, 2004; pp 360-378.