Discovery of piragliatin-first glucokinase activator studied in type 2 diabetic patients

Article abstract of DOI:10.1021/jm3008689

Glucokinase (GK) activation as a potential strategy to treat type 2 diabetes (T2D) is well recognized. Compound 1, a glucokinase activator (GKA) lead that we have previously disclosed, caused reversible hepatic lipidosis in repeat-dose toxicology studies.

Full text of DOI:10.1021/jm3008689

Article
pubs.acs.org/jmc
Discovery of PiragliatinFirst Glucokinase Activator Studied in Type
2 Diabetic Patients
Ramakanth Sarabu,*,† Fred T. Bizzarro,† Wendy L. Corbett,† Mark T. Dvorozniak,† Wanping Geng,†
Joseph F. Grippo,† Nancy-Ellen Haynes,† Stanley Hutchings,† Lisa Garofalo,† Kevin R. Guertin,†
Darryl W. Hilliard,† Marek Kabat,† Robert F. Kester,† Wang Ka,† Zhenmin Liang,† Paige E. Mahaney,†
Linda Marcus,† Franz M. Matschinsky,‡ David Moore,† Jagdish Racha,† Roumen Radinov,† Yi Ren,†
Lida Qi,† Michael Pignatello,† Cheryl L. Spence,† Thomas Steele,† John Tengi,† and Joseph Grimsby†
†Hoffmann-La Roche Inc., pRED, Pharma Research & Early Development, DTA Metabolism, 340 Kingsland Street, Nutley, New
Jersey 07110, United States
‡Department of Biochemistry and Diabetes Center, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104,
United States
S
* Supporting Information
ABSTRACT: Glucokinase (GK) activation as a potential strategy
to treat type 2 diabetes (T2D) is well recognized. Compound 1, a
glucokinase activator (GKA) lead that we have previously
disclosed, caused reversible hepatic lipidosis in repeat-dose
toxicology studies. We hypothesized that the hepatic lipidosis
was due to the structure-based toxicity and later established that it
was due to the formation of a thiourea metabolite, 2. Subsequent
SAR studies of 1 led to the identification of a pyrazine-based lead
analogue 3, lacking the thiazole moiety. In vivo metabolite
identification studies, followed by the independent synthesis and
profiling of the cyclopentyl keto- and hydroxyl- metabolites of 3, led to the selection of piragliatin, 4, as the clinical lead.
Piragliatin was found to lower pre- and postprandial glucose levels, improve the insulin secretory profile, increase β-cell sensitivity
to glucose, and decrease hepatic glucose output in patients with T2D.
single GK allele causes persistent hyperinsulinemic hypoglyce-
mia of infancy (PHHI) due to excessive insulin release.6 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 liver,
allosteric activators of this enzyme are being actively pursued as
antidiabetic agents. We previously disclosed the discovery of
RO0281675, 1, an archetypal glucokinase activator (GKA), its
efficacy profile in mouse models of T2D, and its effect on
improving glycemic control in healthy volunteers administered
a single oral dose (25−400 mg) (Figure 1).7 However,
compound 1 was unsuitable as a full development candidate
due to its narrow safety margin in preclinical toxicology studies.
In this report, we disclose efforts to eliminate this liability
leading to the discovery of piragliatin,8 a potent and efficacious
GKA that was advanced to phase 2 clinical trials in T2D
patients.
INTRODUCTION
■
Glucokinase (GK) activation as a potential strategy to treat
type 2 diabetes (T2D) is now well recognized.1 GK, also called
hexokinase IV, is a glycolytic enzyme that catalyzes the
oxidative phosphorylation of glucose by adenosine triphosphate
(ATP). GK responds functionally to plasma glucose fluctua-
tions observed during pre- and postprandial periods. This
enables pancreatic β-cell GK to function as a molecular sensor2
that couples changes in blood glucose levels with insulin release
to maintain glucose homeostasis. As a sensor, GK determines
the rate and threshold concentration of glucose (∼5 mM in
healthy individuals) required to initiate the signaling cascade
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.3 GK is also expressed in pancreatic α-cells, entero-
endocrine 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.4
Compound 1 caused reversible hepatic lipidosis in repeat-
dose toxicology studies in rats and dogs. We hypothesized that
the hepatic lipidosis was due to structure-based toxicity and
investigated if the formation of a reactive metabolite from either
the parent molecule or its metabolite could be the causative
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.5 In contrast, a gain-of-function mutation in a
Received: January 25, 2012
© XXXX American Chemical Society
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comparison between chloro- and des-chloro (compounds 19−
26) are described in the Supporting Information.
Compound 3 was prepared in a seven-step stereoselective
synthesis outlined in Scheme 2. Hydrolysis of 37b afforded
pyruvate 60, which was then deoxygenated under hydrazine
hydrate/potassium hydroxide conditions, leading to the
formation of the arylacetic acid 61. The subsequent trans-
formations were (a) preparation of a chiral amide with (1R,
2R)-(−)-pseudoephedrine, (b) diastereoselective alkylation,9
and (c) hydrolysis of the amide to yield the carboxylic acid
intermediate 64. The thiomethyl of compound 64 was oxidized
to the key methylsulfone intermediate 65, which was then
coupled with 2-aminopyrazine to yield compound 3.
The synthesis of the oxidative metabolites of 3 required the
synthesis of protected 2- and 3-hydroxyl-cyclopentyl methyl
iodides, 69 and 72, respectively. The iodide 69 was accessible
from 2-hydoxycyclopentyl carboxylic acid ethyl ester, 66, in
three steps: (a) protection of hydroxyl group as tetrahydropyr-
anyl ether, (b) reduction of the ethyl ester to hydroxyl methyl
group, and (c) conversion of hydroxyl methyl to the
cyclopentylmethyl iodide analogue 69. The 3-cyclopenthyl-
methyl iodide, 72, was synthesized starting from the reported 3-
iodomethyl cyclopentanone.10 The two steps consists of (a)
reduction of the ketone followed by (b) the protection of the
resulting alcohol as a tetrahydropyranyl ether (Scheme 3).
The diasteromeric mixtures of cyclopentyl 2-hydroxy 27 and
2-ketone 29 metabolites were prepared in a synthetic sequence
outlined in Scheme 4. The sequence started with the
diastereoselctive alkylation of chiral acetamide 62 with the
protected cyclopentylmethyl iodide derivative, 69, to yield a
mixture of alkylated diastereomers, 73. These diastereomers
were transformed to the key 2-keto-carboxylic acid inter-
mediate, 76, in three steps consisting of (a) removal of the
tetrahydropyranyl- group, (b) oxidation of the resulting
cyclopentyl 2-hydroxy- derivatives 74 to ketones, 75, and
then, (c) removal of pseudoephridrine chiral auxiliary via acid
hydrolysis. The thiomethyl moiety in cyclopentyl 2-ketone
diastereomers 76 was oxidized to the corresponding sulfone 78
in a two-step procedure, which was then coupled with 2-
aminopyrazine to yield the 2-keto-metabolite diastereomeric
mixture 29. The corresponding mixture of diastereomeric
cyclopentyl 2-hydroxy metabolites 27 was readily accessible via
a sodium borohydride reduction of 29.
Figure 1. Structures of GK activators and the thiourea metabolite 2.
agent. These investigations provided strong evidence that the
hepatic lipidosis was due to the formation of a thiourea
metabolite, 2, of compound 1 and not due to the mechanism of
action. Further efforts to identify a 4,5- disubstituted or 4- or 5-
monosubstituted thiazole analogue of 1 unable to form a
thiourea metabolite, while maintaining the potency and
adequate pharmacokinetic properties, were unsuccessful.
Subsequent SAR studies of 1 led to the identification of a
pyrazine-based lead analogue 3 lacking the thiazole moiety. In
vivo metabolic identification studies revealed a number of
oxidative metabolites of compound 3 at the cyclopentyl ring.
Independent synthesis of the cyclopentyl keto- and hydroxyl-
metabolites and their in vitro and in vivo safety and efficacy
profiling led to the selection of piragliatin, 4, as the clinical lead
(Figure 1). Subchronic and chronic toxicology studies in rats
and canines with Piragliatin revealed no evidence of hepatic
lipidosis (unpublished results). Piragliatin was found to lower
pre- and postprandial glucose levels, improve the insulin
secretory profile, increase β-cell sensitivity to glucose, and
decrease hepatic glucose output in patients with T2D.
CHEMISTRY
■
The general synthetic strategy used to prepare the various aryl
acetamides is shown in Scheme 1, which consists of preparing
the corresponding arylpropionic acid or ester and subsequent
alkylation with cyclopentylmethyl iodide, followed by hydrol-
ysis of the ester group and coupling with the corresponding
aminoheterocycle.7b The syntheses of thiourea analogue 2, the
thiazole analogues of 1 shown in Table 3 (compounds 5−11),
the aryl substitution analogues shown in Table 4 (compounds
12−18), and the analogues shown in Table 5 for SAR
In an analogous manner, the 3-hydroxyl 28 and 3-ketone 30
diastereomeric mixtures were prepared starting from the chial
amide intermediate 62 and the 3-(2-tetrahydropyranyl)-oxy-
cyclopentyl derivative 72, in a six-step sequence (Scheme 5).
Both the R,S-31 and the R,R-4 diastereomers corresponding
to the C3-keto metabolites of 3 were available via
stereroselective syntheses starting from either S,S-84 or R,R-
91 hydrobenzoin ketals of cyclopent-2-enone. The key
a
Scheme 1. General Synthetic Strategy
a
Conditions: (i) alkylation, (ii) hydrolysis, and (iii) 2-amino-heterocycle coupling.
B
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a
Scheme 2. Synthesis of Compound 3
a
Reagents and conditions: (i) Aqueous NaOH, toluene, 60 °C, 2.5 h, 98%. (ii) N2H4, KOH, 16 h, 89%. (iii) K2CO3, (1R,2R)-(−)-pseudoephidrine,
Cl3COCl, acetone, −10 to 25 °C, ethyl acetate recrystallization, 78%. (iv) HMDS, n-BuLi, THF, cyclopentylmethyl iodide, −45 to 0 to 25 °C,
DMPU, 67%. (v) 9 N H2SO4, 98%. (vi) HCOOH, 30% H2O2, 16 h, 80%. (vii) (COCl)2, 1 drop DMF,CH2Cl2, 2-aminopyrazine, pyridine, 0−25 °C,
THF, 16 h, 70%.
a
Scheme 3. Synthesis of Oxygentated Cyclopentyl Methyl Iodides
a
Reagents and conditions for 69: (i) NaBH4, EtOH,84%. (ii) 3,4-Dihyro-(2H)-pyran, pyridinium PTS, CH2Cl2, 5 h, 88%. (iii) LiAlH4, THF, 0−25
°C, 18 h, 84%. (iv) Ph3P, imidazole, I2, CH2Cl2, 0−25 °C, 3 h, 75%. Reagents and conditions for 72: (i) NaBH4, CH3OH, 0 °C, 0.75 h, 59%. (ii) 3,4-
Dihydro-(2H)-pyran, pyrdinium PTS, CH2Cl2, 25 °C, 24 h, 75%.
transformations in the synthesis of piragliatin 4 were (a) the
high-yielding stereoselective Simmons−Smith cyclopropana-
tion reaction11 of the chiral ketal 91 to the corresponding
bicyclic intermediate 92, (b) trimethylsilyl iodide-catalyzed
opening of the cyclopropane12 92 to the alkyl iodide 93, and
(c) the diastereoselective alkylation of chiral amide 62 with
alkyl iodide 93 to yield the chiral alkylated phenyl acetamides
94. The remaining sequence of reactions was similar to that
described for compound 29 (Scheme 6).
formation of oxidative metabolites corresponding to the
cyclopentyl group [i.e, hydroxy (M2, M3) and ketone (M4)
products] and the thiourea metabolites (M1, M5, and M6)
corresponding to the parent, and the cyclopentyl hydroxy
metabolite (Table 1 and Figure 2) and an unidentified
metabolite M7.
The thiourea metabolites (M1, M5, and M6) were suspected
to be the causative agents of hepatic lipidosis, based on
literature reports on liver toxicity.14 We used the ent-1 (the S
enantiomer of 1), which does not activate GK or lower glucose
levels,2 and the racemic thiourea 2 (M6) (SC = 45 μM) as
RESULTS AND DISCUSSION
1.5
■
structural probes to investigate the lipidosis. We conducted 5
day and acute (single dose) toxicity studies with compounds 1,
ent-1, and 2 (Table 2). In the 5 day studies, glucose levels on
day 3 and hepatic lipidosis at the end of the study were
evaluated. All three compounds (1, ent-1, and 2) were found to
cause hepatic lipidosis as observed through Oil Red O staining,
and glucose lowering was observed only with compound 1
(Figures 1 and 2 in the Supporting Information). A single dose
The archetypal GKA 1 was found to cause reversible hepatic
lipidosis in rats and dogs and was unsuitable for chronic
therapeutic use. To determine whether lipidosis was due to GK
activation13 or the compound structure-based toxicity, we
explored if a putative reactive metabolite arising from either the
parent molecule or its metabolite could be the causative agent.
The metabolite profile of 1 in mouse, rat, dog, monkey, and
human liver microsomal incubation studies identified the
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a
Scheme 4. Synthesis of 2-Hydroxy (27) and 2-Keto (29) Metabolites of Compound 3
a
Reagents and conditions: (i) HMDS, n-BuLi, THF, 69, −45 to 0 to 25 °C, DMPU, 71%. (ii) Pyridinium-PTS, 50 °C, 2 h, EtOH, 93%. (iii) N-
Methyl-morpholine-N-oxide, tetrapropylammonium perrhuthenate, molecular sieves, CH2Cl2, 25 °C, 20 min, 77%. (iv) 18 M HCl, 120 °C, 18 h,
82%. (v) HCOOH, 30% H2O2, water, 0 °C, 1 h, 100%. (vi) KMnO4, water, CH3OH, 25 °C, 1 h, 43%. (vii) (COCl)2, 1 drop DMF, CH2Cl2, 2-
aminopyrazine, pyridine, 0−25 °C, THF, 1 h, 51%. (viii) NaBH4, 0 °C, 20 min, EtOH, 76%.
a
Scheme 5. Synthesis of 3-Hydroxy (29) and 3-Keto (31) Metabolites of 3
a
Reagents and conditions: (i) HMDS, n-BuLi, 72, −45 to 0 to 25 °C, 4 h, DMPU, THF, 91%. (ii) 9 N H2SO4, dioxane, reflux, 15 h, 44%. (iii)
HCOOH, 30% H2O2, 16 h, 103%. (iv) (COCl)2, DMF (2 drops), CH2Cl2, 2-aminopyrazine, toluene, THF, 82%. (v) HCOOH, NH3(gas), CH3OH,
0 °C, 0.25 h, 63%. (vi) Dess−Martin periodinane, CH2Cl2, 25 °C, 2 h, 75%.
rat acute toxicity study with compound 1 (300 mg/kg, po single
dose) and ent-1 (600 mg/kg single dose, po) was conducted. In
this experiment, a dramatic increase in serum triglycerides was
consistently noted with both compounds, with approximately
600 and 400% increases for ent-1- and 1-treated rats,
respectively. These data together demonstrate that both
D
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a
Scheme 6. Synthesis of Piragliatin 4
a
Reagents and conditions: (i) Pyridinium PTS, 2-cyclopetane-1-one, cyclohexane, reflux, 4 h, 92%. (ii) Et2Zn, ICH2Cl, 1,2-dichloroethane, −12 to
25 °C, 2.5 h, 68%. (iii) K2CO3, TMS-iodide, CH2Cl2, −8 to 25 °C, 2 h, 72%. (iv) HMDS, n-BuLi, THF, −20 to 0 °C, 62, 3.5 h, 65%. (v) 9 N H2SO4,
dioxane, reflux, 72%. (vi) 0.05 M dimethyl dioxirane, acetone, 25 °C, 1 h, 90%. (vii) (COCl)2, cat. DMF, 2-aminopyrazine, pyridine, toluene,
CH2Cl2, 25 °C, 18 h, 43%.
Table 1. Compound 1 Metabolite Profile
Table 2. Rat Hepatic Toxicity Studies
%
glucose-
lowering
effect
duration dose/s (mg/kg), route,
Oil Red O
stain result
liver microsomes
M1 M2/M3
M4
M5
M6
M7
compd
(days)
and frequency
a
CD 1 mouse
Wistar rat
1
8
ND
1.6
0
2.4
0.4
0.9
9.5
2.5
14
3.1
1.1
1
5
5
5
1
1
100, po, qd
positive
positive
positive
positive
positive
+
3
22.5
2.9
8.9
8
3.7
4.7
20.5
9.3
ent-1
2
250, po, qd
−
−
+
beagle dog
0.1
7.5
1.8
2.5
30, 100, 300, po, qd
300, po, qd
cynomolgus monkey
human
0.8
0.2
10.2
2.4
1
ent-1
600, po, qd
−
a
ND, not detected.
Figure 2. Metabolite profile of compound 1 in liver microsomes.
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Figure 3. Oil Red O stain of liver tissue following a single dose of vehicle or compound 1 (300 mg/kg) or ent-1 (600 mg/kg).
stereoisomers affected hepatobiliary function and caused
hypertriglyceridemia. The hypertriglyceridemia was not accom-
panied by hypercholesterolemia or an increase in free fatty
acids. In both compounds ent-1- and 1-treated rats, periportal
hepatocellular microvesicular lipidosis was noted in H&E-
stained sections (see Oil Red O staining comparison, Figure 3),
but only compound 1 lowered glucose levels. Taken together,
these findings strongly supported the hypothesis that the
thiourea metabolites formed via oxidative thiazole ring opening
were the primary cause for the observed hepatic lipidosis
associated with the GK activator 1.
not form a significant amount of the thiourea metabolite, but
their potency was markedly reduced. In view of suboptimal
properties of these thiazole analogues of 1, we turned our
attention to alternative heterocyclic ring systems as replace-
ments to the thiazole moiety.
Our SAR studies of the compound 1 suggested a significant
“cross-talk” between the aromatic ring and the heterocyclic ring
in modulating GK activity. Initially, we explored the SAR by
incorporating an electron-withdrawing or an electron-donating
group adjacent to the 4-methylsulfone in the aryl ring at
position 3 (Table 4). In these studies, 3-chloro 12 or 3-cyano
In an effort to identify thiazole analogues devoid of thiourea
metabolite formation, several substituted thiazole analogues of
1 (Table 3) were prepared and investigated for their propensity
Table 4. SAR of Compound 1 Analogues
Table 3. Metabolism of Thizole Analogues to the Thiourea 2
a
compd
R
SC (μM)
1.5
b
1a
H
0.34
12
13
14
15
16
17
18
Cl
0.119
0.111
0.292
0.068
0.065
0.188
0.579
thiourea 2 concn
Br
a
compd
substitution
SC1.5 (μM)
(ng/mL)
F
1
5
X, Y = H (R-isomer) 0.24 + 0.019 (25)
505.7
167.3
CF3
NO2
CN
NH2
X = CH2CH2OH, Y
= H
0.667
6
7
X = CH2OH, Y = H
X = H, Y = Cl
0.480
0.148
0.135
0.734
1.40
88.7
48.0
42.7
<10
a
Data reported as means SEMs. The average CV of the assay was
15%, based on an analysis of 452 compounds (SC1.5 < 2 μM) assayed
in duplicate. Racemate of 1
8
X = H, Y = Br
9
X = H, Y = CONH2
b
10
X = H, Y =
CONHCH3
<10
11
X = H, Y = NO2
4.17
<10
17 analogues were found to be about 2−3-fold more potent
than 1a, the racemate analogue corresponding to compound 1,
while the 3-fluoro 14 analogue was roughly equipotent. Other
analogues containing electron-withdrawing groups such as 3-
bromo 13, 3-trifluoromethyl 15, or 3-nitro 16 were 3−5-fold
more potent in comparison to 1a. The electron-donating 3-
amino 18 was about 2-fold less potent than 1a. Among these
aromatic ring three-substituents, we chose the 3-chloro 12
analogue as a comparator to explore the SAR of various six-
membered heterocycles due to its improved potency with only
a modest increase in the molecular weight vs 1a. The SAR
comparing the 3-chloro and 3-des chloro analogues is shown in
Table 5. Surprisingly, when the thiazole of 1a was replaced by a
six-membered heterocycle, such as a pyridine 19, a pyrazine 21,
a pyrimidine 25, or a quinoline 23, there was about 4−5-fold
loss of activity vs 1a (SC1.5 values 1.21, 1.78, 1.38, and 1.74 μM
vs 0.34 μM of 1a). In a striking contrast, the corresponding 3-
chloro-4-methylsulfone phenyl analogues 20, 22, 24, and 26
a
Data reported as means SEMs (n). The average CV of the assay
was 15%, based on an analysis of 452 compounds (SC1.5 < 2 μM)
assayed in duplicate.
to form the thiourea metabolite 2 in incubations with human
liver microsomes. Compounds 5 and 6, the hydroxyethyl and
hydroxymethyl analogues, respectively, were prepared to
introduce the polar hydroxyl moiety and potentially improve
the metabolic stability. These analogues were found to generate
only 1/3 and 1/5 as much of thiourea metabolite as 1 but were
less potent by about 3- and 2-fold, respectively, as compared to
1. While the chloro 7 and bromo 8 analogues had ∼2-fold
improved potency and only about 10% of the thiourea formed
as compared to 1, they were found to have poorer
phramacokinetics (PK) properties and in vivo activity (data
not shown) and therefore were abandoned. The carboxamide 9,
N-methylamine carboxamide 10, and nitro 11 analogues did
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similar to compound 1. More interestingly, in a rodent 14 day
range-finding toxicity study at doses up to 600 mg/kg, there
was no evidence for hepatic lipidosis (data not shown).
However, compound 3 was found to be a potent inhibitor of
the hERG potassium channel (IC20 < 3 μM; IC50 − 7.3 μM)
and increased the action duration potential in a rabbit Purkinje
fiber assay (EC50 at 1 Hz, 8.2 μM), suggesting a potential for
cardiovascular risk based on the low margin between the
plasma efficacious exposure levels and the hERG IC20 values. In
addition, compound 3 caused a time-dependent inhibition of
CYP3A4 (54% inhibition at 24 min, 10 μM incubation),
indicating a potential for drug−drug interactions and undesired
nonspecific covalent modification of proteins, which could lead
to unpredictable idiosyncratic toxicity. These findings posed an
unacceptable risk for further development, and consequently,
compound 3 was abandoned.
Detailed characterization of the metabolites of compound 3
from liver microsomal studies, in vivo rat PK, and safety studies
showed significant formation of alcohol (M1, M2, and M4) and
ketone (M3) metabolites resulting from oxidation of the
cyclopentyl ring at C2 and C3 (Figure 4 and Table 6). The
extent of metabolism in liver microsomal studies was found to
be 76% in human and rat, while in dog it was 56%. Initially, the
authentic diastereomeric mixtures of metabolites were synthe-
sized for potency and in vitro safety evaluations.
The diastereomeric mixtures of the C2-hydroxyl 27 and C3-
hydroxyl 28 metabolites were less potent GK activators as
compared to the parent 3 (Table 7). However, the diastereomic
mixtures of C2-(29) and C3-(30) ketone metabolites retained
the most potency. We chose to further evaluate the C3-ketone
metabolite diastereomeric mixture 30 over the C2-keto
metabolites 29 in view of the potential of the C2-ketone to
promote epimerization of the adjacent chiral center. Compar-
ison of both R,R-4 and R,S-31 diastereomers (Table 8) clearly
indicated the superior potency of 4 over 31. A review of the
compound profiles led to the selection of the R,R-diastereomer,
4, piragliatin as the clinical lead.
Table 5. Six-Membered Heteroaromatics SAR
a
compd
R1
R2
SC1.5 (μM)
19
20
21
22
23
24
25
26
H
2-aminopyridine
2-aminopyridine
2-aminopyrazine
2-aminopyrazine
2-aminoquinoline
2-aminoquinoline
3-aminopyrimidine
3-aminopyrimidine
1.21 0.49 (3)
Cl
H
0.083
1.78
0.18
1.38
0.12
1.74
0.19
Cl
H
Cl
H
Cl
a
Data reported as means SEMs (n). The average CV of the assay
was 15%, based on an analysis of 452 compounds (SC1.5 < 2 μM)
assayed in duplicate.
were found to have enhanced potency (SC1.5 values 0.083, 0.18,
0.12, and 0.15 μM, respectively) of about 9−15-fold vs the
corresponding des-chloro analogues (19, 21, 25, and 23)
(Table 5). These observations suggested significant cross-talk
between the aryl group and the heterocycle and a preference for
a combination of an electron-deficient aryl group with a five- or
a six-membered heterocycle to obtain potent GKA activity in
the phenyl acetamide series.
After extensive comparison of in vitro, in vivo potency, and
rodent pharmacokinetics, followed by rat exploratory toxicity
studies on select compounds, compound 3 (RO0505082) was
found to have the most desirable efficacy and toxicity profile in
rodents and was selected as a lead candidate for detailed
profiling. Compound 3 was found to be a potent GK activator
both in vitro (SC1.5, 0.14 μM) and in vivo. Compound 3 was
effective in reducing fasting and postprandial glucose levels,
Figure 4. Metabolite profile of compound 3.
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Table 6. Metabolite Profiles of Compound 3 in Rat, Mouse, Dog, Monkey, and Human Liver Microsomes
%
pooled liver microsomes
M1
M2
M3
M4
M5
3
total metabolism (%)
CD1 mouse
SD rat
38.4
44
16.4
17.9
37.9
28
12.3
13.2
27.7
6.2
1.1
1
0
31.9
23.9
7.8
68.1
76.1
92.2
55.5
76.4
0
cynomolgus monkey
beagle dog
human
24
2.5
2.2
1.9
0
19.1
32.4
0
44.5
23.6
26.9
12.5
2.6
Table 7. In Vitro Activity of Diastereomeric Mixtures of 2,3-
Hydroxyl and 2,3-Keto- Compound 3 Metabolites
a
compd
X
Y
SC1.5 (μM)
3
27
28
29
30
CH2
CH2
0.14 0.039 (276)
CH2
CHOH
CH2
2.73
CHOH
CH2
2.37
CO
0.19
CO
CH2
0.33 0.048 (5)
a
Figure 5. Effect of 4 on GK enzymatic activity.
Data reported as means SEMs (n). The average CV of the assay
was 15%, based on an analysis of 452 compounds (SC1.5 < 2 μM)
assayed in duplicate.
extensively evaluated in dog telemetry studies and found to be
devoid of QT prolongation (data not reported). Compound 4
did not show enzyme inhibition versus seven major CYPs,
suggesting a reduced potential for drug−drug interactions.
Thus, compound 4 was found to have acceptable in vitro safety
properties (Table 9).
Table 8. In Vitro Activity of 3-Keto Metabolite
Diastereomers of Compound 3
Table 9. Physical and in Vitro Safety Property Profile of
Compound 4
property
Log D at pH 7.4
value
1.45
a
compd
*
SC1.5 (μM)
solubility
0.8 mg/mL
31
4
S-
5.0 0.81 (2)
Caco-2 permeability (Papp
)
1.6 × 10−6 cm/s
2.2 mL/min/kg
50%
R-
0.18 0.039 (9)
human hepatocyte clearance (Clh)
human plasma protein binding
CYP inhibitionIC50
hERGIC20
a
Data reported as means SEMs (n). The average CV of the assay
was 15%, based on an analysis of 452 compounds (SC1.5 < 2 μM)
>50 μM vs 7 major CYPs
5.8 μM
assayed in duplicate.
hERGIC50
23.0 μM
Enzymatic assays conducted with recombinant human GK
illustrated that 4 increased the enzymatic activity of GK in a
dose-dependent manner at all glucose concentrations tested
(Figure 5). Compound 4 at a concentration of 1 μM caused an
increase in Vmax of GK from 10.6 to 17.9 μM/min and
decreased the S0.5 (also referred to as Km for noncooperative
enzyme kinetics) for glucose from 7.6 to 3.7 mM. Compound 4
increased the Vmax of GK and decreased its S0.5 for glucose in a
dose-dependent manner. These combined effects increased the
catalytic effectiveness of GK to metabolize glucose. Activation
of GK by 4 is consistent with a rate equation for a nonessential,
mixed-type activator.
Compound 4 was found to have good druglike properties
with good solubility, permeability, and low clearance in human
hepatocytes. Compound 4 was found to have 3-fold reduced
hERG IC50 inhibition vs compound 3, while in terms of IC20,
the improvement was only ∼2 fold. Compound 4 was also
Pharmacokinetic and drug metabolism studies in animals
showed that compound 4 was absorbed in its oral form, with
the following oral bioavailability, 39% in mice and rats, 144% in
dogs, and 35% in monkeys (Table 10), and volume of
distribution values range was Vss (L/kg) − 2.8−4.9. Plasma
protein binding was low in all species including humans
(∼50%). Thus, the PK of compound 4 was characterized by
high systemic plasma clearance, short to moderate elimination
half-lives, and large volume of distribution in preclinical animal
models.
Compound 4 was metabolized to at least five metabolites
(M1−M5) before elimination (Figure 6 and Table 11). Of
these, the metabolite M5 structure was not assigned. In
addition, reversible metabolism of 4 was observed. The major
circulating metabolite M4 was formed by a cytosolic reductase,
and CYP3A4 converted M4 back to 4. Seventy-two hours after
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Table 10. Pharmacokinetic Properties of Compound 4
species
dose (mg/kg)
route
Cl (mL/min/kg)
111.26
Vss(L/kg)
3.332
AUC0‑inf (ng h/mL)
Cmax (ng/mL)
Tmax (h)
Fpo (%)
10
30
60
10
30
60
10
30
5
iv
1501
1765
5804
1622
1885
8253
8830
38000
3416
1200
6460
1050
3750
4390
404
mouse
po
po
iv
0.5
1
39
65
103.85
4.975
rat
po
po
iv
2
1
39
2240
8750
5228
3328
254
79.5
19.66
24.47
2.15
dog
po
iv
2.75
144
2.872
monkey
5
po
0.875
35.3
Figure 6. Metabolite profile of compound 4.
Table 11. Metabolite Profile of C14-Labeled Compound 4 in
Liver Microsomes
postdose. As shown in Figure 7, at all doses, significant blunting
of the glucose excursion (p values <0.05 for all time points
between 0 and 120 min postdose) was observed. A parallel oral
PK exposure experiment with compound 4 at 5, 10, and 15 mg/
kg doses was conducted. In this experiment, exposure levels of
compound 4 at 60 min postdose ranged from 2.5 to 8 times
over its SC1.5 value (0.18 μM), explaining the robust response
in the OGTT experiment. These findings suggest an improved
glucose tolerance in the treatment group.
A dose−effect study in DIO mice at 5, 10, and 15 mg/kg
doses by oral route was conducted with compound 4. In this
study, dose-dependent decreased basal glucose levels were
observed (Figure 8), with a maximal potency value of an ED40
(effective dose for 40% glucose lowering vs vehicle control at 2
h postdose) value of 15.6 mg/kg and a minimal potency of
ED15 value at 4 h dose was 8.7 mg/kg.
Compound 4 was extensively evaluated for safety in rodent
and nonrodent species. In these studies, it was found to have an
acceptable therapeutic window. In a 4 week rat toxicity study
(120 mg/kg, po, qd) with compound 4, exposures as high as
43000 ng h/mL did not show lipidosis (data not shown),
whereas with compound 1, hepatic lipidosis was seen at all
doses in a 4 week rat GLP toxicity study. Therefore, a
therapeutic window for compound 1 was not established, due
to lack of a no adverse effect level (NOAEL) for hepatic
%
total
metabolism
a
c
species
M1
M2
M3 M4
M5 parent
(%)
beagle dog
0.3
0.6
1.3
7
1.1
2
0.2
1
90.4
89.4
80.6
9.6
10.6
19.4
b
human
6.1
10.6
1.1
1.7
0.8
1.1
cynolmogus
monkey
4.6
mouse
1.7
0.9
19.9
17
4.3
1.7
0.9
1.9
1.4
71.3
78.3
28.7
21.7
SD rat
0.8
a
b
c
Sex, male. Male and female pooled. Mixture of two diastereomers.
iv administration to rats, 74% of 4 was excreted in feces as
metabolites, while 22% was excreted in urine as parent drug and
metabolites. In vitro studies showed no significant inhibition of
the human liver CYP enzymes by 4.
On the basis of its overall potency, in vitro safety assessment,
and pharmacokinetic profile, compound 4 was evaluated for its
glucose-lowering effect in rodents. The effect of compound 4
on postprandial glucose was tested in an oral glucose tolerance
test (OGTT) study in diet-induced obese (DIO) male C57Bl/
6J mice. Overnight fasted mice were dosed orally (5, 10, and 15
mg/kg) and given an oral glucose challenge (1 g/kg) 1 h
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Figure 7. Compound 4 OGTT in DIO mice.
1090 PDA detector set at 215 nm with the MS detection performed
with a Micromass Platform II mass spectrometer with electrospray
ionization (ESI); Chromegabond WR C18 3 μm, 120 Å, 3.2 mm × 30
mm column; solvent A, H2O−0.02% TFA; solvent B, MeCN−0.02%
TFA; flow rate, 2 mL/min; start 2% B, final 98% B in 4 min, linear
gradient. Method 2: Waters 2795 pump and Waters 2996 photodiode
array detector set at 214 nm with the MS detection performed with a
Waters ZQ mass spectrometer (ESI); Epic Polar Hydrophilic 3 μm,
120 Å, 3.2 mm × 30 mm column; solvent A, H2O−0.03% HCO2H;
solvent B, MeCN−0.03% HCO2H; flow rate, 2 mL/min; start 10% B,
final 100% B in 3 min linear gradient, remaining for 1 min.
Compounds were purified using either of the following methods.
Flash column chromatography was performed on EM Science silica gel
60 (particle size of 32−63 μm, 60 Å). Preparative reverse-phase high-
pressure liquid chromatography (RP-HPLC) used a Waters Delta prep
4000 pump/controller, a 486 detector set at 215 nm, and a LKB
Ultrorac fraction collector. The sample was dissolved in a mixture of
MeCN/20 mM aqueous NH4Ac, applied on a C-18 20 mm × 100 mm
column, and eluted at 30 mL/min with a 20 min linear gradient of 10−
90% B, where solvent A was H2O with 20 mM NH4Ac and solvent B
was MeCN. The pooled fractions were concentrated under reduced
pressure and lyophilized to afford the desired compounds. All
compounds reported herein are analyzed in one of the two analytical
HPLC methods described above and possess purity of at least 95%. 1H
NMR spectra were recorded using a Varian Mercury 300 MHz or
Varian Inova 400 MHz spectrometer. The chemical shifts are in parts
per million (δ) referenced to Me4Si (0.00 ppm) or CHCl3 (7.26 ppm).
High-resolution mass spectra (HRMS) were recorded on a Bruker
Apex II FTICR mass spectrometers with a 4.7 T magnet (ES) or
Micromass AutoSpec (EI) mass spectrometers.
(3-Chloro-4-methylsulfanyl-phenyl)-oxo-acetic Acid Methyl Ester
(37b). A solution of anhydrous aluminum chloride (54.9 g, 412 mmol)
in chloroform (180 mL) under argon was cooled to 0 °C and then
treated dropwise with a solution of methyl chloroxoacetate (24.3 mL,
264 mmol) in chloroform (180 mL). The resulting reaction mixture
was stirred at 0 °C for an additional 30 min and then treated dropwise
with a solution of 2-chlorothioanisole 36 (39.4 g, 247 mmol) in
chloroform (180 mL). The solution turned red in color. The resulting
reaction mixture was allowed to warm to 25 °C, where it was stirred
for 4 h. The reaction mixture was then slowly poured onto ice (700
mL). The resulting yellow mixture was stirred for 15 min and filtered
through Celite to remove aluminum salts. The filtrate was extracted
with methylene chloride (3 × 50 mL). The combined organic layers
were washed with saturated aqueous sodium carbonate solution (1 ×
50 mL). The organic layer was dried over magnesium sulfate, filtered,
Figure 8. Compound 4 lowers basal glucose levels in DIO (*p < 0.05).
lipidosis. These studies strongly suggested that in rodents,
heptic lipidosis observed with compound 1 was due to its
structure and not due to GK activation.
Piragliatin 4 was advanced to clinical testing. In these trials,
compound 4 decreased fasting and post-OGTT glucose levels,
improved the insulin secretory profile, increased β-cell
sensitivity to glucose, and decreased hepatic glucose output.15
Piragliatin development was stopped because of its unfavorable
risk−benefit profile observed during the phase 2 program. A
third generation GKA was developed by Roche, called
RO5305552, and was recently out-licensed to Hua Medicine
for the treatment of T2D.16
EXPERIMENTAL SECTION
■
Chemistry. All nonaqueous reactions were carried out under an
argon or nitrogen atmosphere at 25 °C, unless otherwise noted. All
reagents and anhydrous solvents were used as obtained commercially
without further purification or distillation, unless otherwise stated.
Analytical thin-layer chromatography (TLC) was performed on EMD
Chemicals silica gel 60 F254 precoated plates (0.25 mm). Compounds
were visualized by UV light and/or stained with either p-anisaldehyde,
iodine, or phosphomolybdic acid or KMnO4 solutions followed by
heating. Analytical high-pressure liquid chromatography (HPLC) and
LC-MS analyses were conducted using the following two instruments
and conditions. Method 1: Hewlett-Packard HP-1090 pump and HP-
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and concentrated in vacuo to afford (3-chloro-4-methylsulfanyl-
phenyl)-oxo-acetic acid methyl ester 37b (4.32 g, 65.3%) as a light
yellow oil. EI-HRMS m/e calcd for C10H9ClSO3 (M+), 243.09961;
found, 243.9958. H NMR (300 MHz, chloroform-d): δ 8.02 (d, J =
1.83 Hz, 1H), 7.94 (dd, J = 1.65, 8.24 Hz, 1H), 7.23 (d, J = 8.42 Hz,
1H), 3.99 (s, 3H), 2.55 (s, 3H).
washed with water (2 × 40 mL). The combined aqueous layer was
back-extracted with ethyl acetate (2 × 50 mL). The combined organic
layer was dried over magnesium sulfate, filtered, and concentrated in
vacuo. The crude material was recrystallized from ethyl acetate (45
mL) and hexanes (80 mL) to afford (3-chloro-4-methylsulfanyl-
phenyl)-N-[2(R)-hydroxy-1(R)-methyl-2(R)-phenyl-ethyl]-N-methyl-
acetamide 62 (13.75 g, 78%) as a light yellow solid; mp 111.5−112.9
1
(3-Chloro-4-methylsulfanyl-phenyl)-oxo-acetic Acid (60). (3-
Chloro-4-methylsulfanyl-phenyl)-oxo-acetic acid methyl ester (61.7
g, 252 mmol) in toluene (120 mL) was heated at 50 °C. This heated
solution was treated dropwise with a 3 M aqueous sodium hydroxide
solution (105 mL, 313 mmol) via a dropping funnel, taking care to
keep temperature below 60 °C. After the addition was complete, the
reaction mixture was stirred at 50 °C for 1.5 h, during which time a
yellow precipitate began to form. After this time, the heat was
removed, and the warm solution was treated dropwise with
concentrated hydrochloric acid (10.6 mL, 290 mmol). The resulting
reaction mixture was cooled to 25 °C and then was stirred at 25 °C for
16 h. The solid was filtered and then washed with water (50 mL) and
toluene (50 mL). The solid was dried by suction for 1 h and then dried
in a high vacuum desiccator to afford (3-chloro-4-methylsulfanyl-
phenyl)-oxo-acetic acid 60 (57.22 g, 98%) as a white solid; mp 166°
(dec). FAB-HRMS m/e calcd for C9H7ClO3S (M + H)+, 252.9702;
23
°C; [α]589 = −97.2° (c 0.104, chloroform). FAB-HRMS m/e calcd
1
for C19H22ClNO2S (M + H)+, 364.1138; found, 364.1142. H NMR
(300 MHz, chloroform-d): δ 6.98−7.57 (m, 8H), 4.54−4.67 (m, 1H),
4.02, 4.50 (2s, 1H), 3.63, 3.75 (2s, 2H), 2.84, 2.97 (2m, 3H), 2.39−
2.54 (m, 3H), 0.92, 1.14 (2d, J = 6.6 Hz, 3H).
2(R)-(3-Chloro-4-methylsulfanyl-phenyl)-3-cyclopentyl-N-[2(R)-
hydroxy-1(R)-methyl-2(R)-phenyl-ethyl]-N-methyl-proprionamide
(63). A solution of 1,1,1,3,3,3-hexamethyldisilazane (17.9 mL, 85
mmol) in tetrahydrofuran (90 mL) was cooled to −78 °C and then
treated with a 2.34 M solution of n-butyl lithium in hexanes (33.9 mL,
79.3 mmol). The reaction mixture was stirred at −78 °C for 15 min
and then treated with (3-chloro-4-methylsulfanyl-phenyl)-N-[2(R)-
hydroxy-1(R)-methyl-2(R)-phenyl-ethyl]-N-methyl-acetamide 62
(13.75 g, 37.8 mmol) in tetrahydrofuran (90 mL), while maintaining
the temperature below −65 °C. The resulting yellow-orange solution
was stirred at −78 °C for 15 min and then allowed to warm up to 0
°C, where it was stirred for 20 min. The reaction mixture was then
recooled to −78 °C and treated with iodomethyl cyclopentane (11.9 g,
56.7 mmol) in 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone
(9.6 mL, 79.3 mmol). The resulting solution was stirred at −78 °C for
30 min and then allowed to warm to 25 °C, where it was stirred for 16
h. The reaction mixture was diluted with ethyl acetate (200 mL) and
then washed with aqueous ammonium chloride (1 × 100 mL). The
aqueous layer was extracted with ethyl acetate (2 × 50 mL). The
combined organic layer was washed with aqueous sodium chloride
solution, dried over sodium sulfate, filtered, and concentrated in vacuo.
The resulting material was redissolved in ethyl acetate and washed
with 10% sulfuric acid (2 × 100 mL) and saturated sodium
bicarbonate (2 × 100 mL), dried over magnesium sulfate, filtered,
and concentrated in vacuo. The crude material was recrystallized from
ethyl acetate/hexanes to afford 2(R)-(3-chloro-4-methylsulfanyl-
phenyl)-3-cyclopentyl-N-[2(R)-hydroxy-1(R)-methyl-2(R)-phenyl-
ethyl]-N-methyl-proprionamide 63 (11.36 g, 67%) as a light yellow
1
found, 252.9700. H NMR (300 MHz, DMSO-d6): δ 7.76−7.84 (m,
2H), 7.44 (d, J = 8.06 Hz, 1H), 2.57 (s, 3H).
(3-Chloro-4-methylsulfanyl-phenyl)-acetic Acid (61). A reaction
flask equipped with a mechanical stirrer was charged with hydrazine
hydrate (8.5 mL, 273 mmol). The hydrazine hydrate was cooled to
−50 °C and then treated with (3-chloro-4-methylsulfanyl-phenyl)-oxo-
acetic acid 60 (12.6 g, 54.6 mmol) in one portion. An exotherm
ensued that raised the temperature. The resulting white mixture was
then heated to 80 °C. After it reached 80 °C, the heating element was
removed, and the reaction mixture was treated with potassium
hydroxide (2.09 g, 31.7 mmol) in one portion. An exotherm was
observed. The reaction mixture was allowed to cool back to 80 °C.
This potassium hydroxide addition process was repeated three times
(3 × 2.09 g). At the end of the final potassium hydroxide portion
addition, the reaction mixture was heated to 100 °C for 16 h. The
resulting homogeneous reaction mixture was cooled to 25 °C and then
diluted with water (12 mL). The reaction mixture was then transferred
to separatory funnel, rinsing with additional water (12 mL) and diethyl
ether (40 mL). The layers were separated, and the aqueous layer was
transferred to a flask. The organic layer was extracted with water (2 ×
15 mL). The aqueous layers were combined and treated with heptane
(20 mL), and the resulting reaction mixture was vigorously stirred.
This stirred reaction mixture was treated with dropwise with
concentrated hydrochloric acid (26 mL) over 30 min, while the
temperature was kept under 50 °C with an ice bath. A cloudy
suspension was formed, and this suspension was stirred at 25 °C for 3
h. The solid that formed was filtered and washed sequentially 1 N
hydrochloric acid (2 × 6 mL), heptane (1 × 12 mL), and a solution of
heptane/diethyl ether (15 mL, 4:1). The resulting solid was dried
under high vacuum to afford (3-chloro-4-methylsulfanyl-phenyl)-acetic
acid 61 (10.48 g, 89%) as an off-white solid; mp 105.6−108.4 °C. EI-
HRMS m/e calcd for C9H9ClO2S (M+), 216.0012; found, 216.0022.
1H NMR (300 MHz, chloroform-d): δ 7.30 (d, J = 1.10 Hz, 1H),
7.16−7.21 (m, 1H), 7.11−7.16 (m, 1H), 3.61 (s, 2H), 2.48 (s, 3H).
(3-Chloro-4-methylsulfanyl-phenyl)-N-[2(R)-hydroxy-1(R)-meth-
yl-2(R)-phenyl-ethyl]-N-methyl-acetamide (62). A mixture of (3-
chloro-4-methylsulfanyl-phenyl)-acetic acid 61 (10.48 g, 48.4 mmol)
and potassium carbonate (20.1 g, 145.1 mmol) in acetone (65 mL)
was cooled to −10 °C. The pale yellow was then treated dropwise with
trimethyl acetylchloride (6.25 mL, 50.8 mmol) while maintaining the
temperature below −10 °C. The resulting reaction mixture was stirred
at −10 °C for 10 min and then allowed to warm to 0 °C, where it was
stirred for 10 min. The reaction mixture was recooled to −10 °C and
then treated with (1R,2R)-(−)-pseudoephedrine (11.99 g, 72.5
mmol), resulting in an exotherm. The reaction mixture was stirred
at −10 °C for 10 min and then warmed to 25 °C, where it was stirred
for 1 h. The reaction mixture was quenched with water (50 mL) and
then extracted with ethyl acetate (1 × 100 mL). The organic layer was
23
solid; mp 113.8−117.6 °C; [α]589 = −100.3° (c 0.049, chloroform).
FAB-HRMS m/e calcd for C25H32ClNO2S (M − H)+, 444.1764;
1
found, 444.1765. H NMR (300 MHz, chloroform-d): δ 6.98−7.49
(m, 8H), 4.57 (m, 1H), 4.37 (br. s., 1H), 3.57, 3.97 (2m, 1H), 2.74,
2.90 (2s, 3H), 2.37−2.52 (m, 3H), 1.97−2.14 (m, 1H), 1.37−1.88 (m,
8H), 0.62, 1.14 (2d, J = 6.6 Hz, 3H), 0.97−1.15 (m, 2H).
2(R)-(3-Chloro-4-methylsulfanyl-phenyl)-3-cyclopentyl-propionic
Acid (64). A solution of 2(R)-(3-chloro-4-methylsulfanyl-phenyl)-3-
cyclopentyl-N-[2(R)-hydroxy-1(R)-methyl-2(R)-phenyl-ethyl]-N-
methyl-proprionamide 63 (11.36 g, 25.5 mmol) in dioxane (45 mL)
was treated with 9 N aqueous sulfuric acid solution (28 mL). The
resulting reaction mixture was heated at 105 °C for 16 h. The reaction
was then cooled to 0 °C using an ice bath, and the product was then
precipitated using the addition of water (200 mL). The suspension was
stirred at 0 °C until the supernatant, which was turbid initially, became
clear and light yellow in color. The solid was filtered off and dried in
vacuo. The solid material was dissolved in hot glacial acetic acid (15
mL) and treated with water (10 mL) to initiate crystallization. The
mixture was cooled to 25 °C and then treated with an additional
amount of water (20 mL). After it was stirred at 25 °C for 1 h, the
solid was collected by filtration. The solid was dried in a high vacuum
desiccator with phosphorus pentoxide to afford 2(R)-(3-chloro-4-
methylsulfanyl-phenyl)-3-cyclopentyl-propionic acid 64 (7.46 g, 98%)
23
as a white solid; mp 116.9−119.2 °C; [α]589 = −55.8° (c 0.104,
chloroform). EI-HRMS m/e calcd for C15H19ClO2S (M)+, 298.0794;
1
found, 298.0804. H NMR (300 MHz, chloroform-d): δ 7.34 (d, J =
1.83 Hz, 1H), 7.18−7.26 (m, 1H), 7.07−7.16 (m, 1H), 3.55 (t, J =
7.69 Hz, 1H), 2.47 (s, 3H), 1.97−2.15 (m, 1H), 1.38−1.91 (m, 8H),
0.97−1.22 (m, 2H).
K
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2(R)-(3-Chloro-4-methylsulfonyl-phenyl)-3-cyclopentyl-propionic
Acid (65). A slurry of 2(R)-(3-chloro-4-methylsulfanyl-phenyl)-3-
cyclopentyl-propionic acid 64 (15.68 g, 52.5 mmol) in formic acid (10
mL) was cooled to 0 °C and then treated with 30% aqueous hydrogen
peroxide solution (30 mL). The resulting reaction mixture was allowed
to warm to 25 °C, where it was stirred for 16 h. The product was
precipitated by the addition of water (120 mL). The solid was filtered
off, washed with water, and dried by suction. Flash chromatography
(Merck silica gel 60, 230−400 mesh, 50/50 hexanes/ethyl acetate plus
1% acetic acid) afforded 2(R)-(3-chloro-4-methylsulfonyl-phenyl)-3-
cyclopentyl-propionic acid 65 (13.93 g, 80%) as a white solid; mp
123.9−126.2 °C; [α]58923 = −41.5° (c 0.176, chloroform). FAB-HRMS
m/e calcd for C15H19ClO4S (M + H)+, 331.0771; found, 331.0776. 1H
NMR (300 MHz, chloroform-d): δ 8.11 (d, J = 8.06 Hz, 1H), 7.55 (s,
1H), 7.44 (d, J = 8.42 Hz, 1H), 3.69 (t, J = 7.69 Hz, 1H), 3.28 (s, 3H),
2.02−2.19 (m, 1H), 1.41−1.93 (m, 8H), 1.01−1.22 (m, 2H).
(tetrahyro-pyran-2yloxy)-cyclopentanecarboxylic acid ethyl ester 67
(4.7 g, 19.59 mmol). The reaction mixture was stirred at 25 °C for 18
h. At this time, the reaction mixture was poured onto ice/water. The
mixture was filtered through a pad of Celite (methylene chloride as
eluent). The organics were washed with a saturated aqueous sodium
chloride solution (1 × 100 mL), dried over sodium sulfate, filtered,
and concentrated in vacuo to give [2-(tetrahydro-pyran-2yloxy)-
cyclopentyl]-methanol 68 (3.25 g, 83.6%) as a clear liquid. EI-HRMS
1
m/e calcd for C11H20O3 (M+), 200.1412; found, 200.1412. H NMR
(300 MHz, chloroform-d): δ 1.05−1.30 (m, 2 H), 1.34−2.01 (m, 12
H), 2.02−2.76 (m, 2 H), 3.37−4.09 (m, 3 H), 4.11−4.81 (m, 1 H).
2-(2-Iodomethyl-cyclopentyloxy)-tetrahydropyran (69). A solu-
tion of tripheylphosphine (1.70 g, 6.49 mmol) and imidazole (884 mg,
12.98 mmol) in methylene chloride (8.32 mL) cooled to 0 °C was
treated with iodine (1.64 g, 6.49 mmol). After iodine was completely
dissolved, a solution of 2-(tetrahydro-pyran-2yloxy)-cyclopentyl]-
methanol 68 (1.0 g, 4.99 mmol) was added to the reaction mixture.
The reaction was stirred at 0 °C for 1 h and 25 °C for 2 h. At this time,
the reaction mixture was poured into water (100 mL) and extracted
with methylene chloride (1 × 30 mL). The organics were washed with
aqueous sodium thiosulfate solution (1 × 50 mL), dried over sodium
sulfate, filtered, and concentrated in vacuo at 25 °C. Flash
chromatography (Merck silica gel 60, 230−400 mesh, 80/20
hexanes/ethyl acetate) afforded 2-(2-iodomethyl-cyclopentyloxy)-
tetrahydropyran 69 (1.17 g, 75.8%) as a clear liquid. EI-HRMS m/e
calcd for C11H19IO2 (M+), 309.0352; found, 309.0348. 1H NMR (300
MHz, chloroform-d): δ 1.08−2.17 (m, 12 H), 3.10−3.36 (m, 2 H),
3.47−3.57 (m, 1 H), 3.81−3.96 (m, 2 H), 4.56−4.79 (m, 2 H).
3-Iodomethyl-cyclopentanol (71). A solution of 3-iodomethyl-
cyclopentanone 70 (12.84 g, 57.31 mmol) (ref 10) in methanol (143
mL) was cooled to 0 °C and then slowly treated with sodium
borohydride powder (2.38 g, 63.04 mmol). The resulting reaction
mixture was stirred at 0 °C for 40 min and then slowly quenched with
water (100 mL). The reaction mixture was then concentrated in vacuo
to remove methanol. The resulting aqueous residue was extracted with
diethyl ether (3 × 100 mL). The combined organic extracts were dried
over magnesium sulfate, filtered, and concentrated in vacuo. Flash
chromatography (Merck silica gel 60, 70−230 mesh, 100% methylene
chloride) afforded 3-iodomethyl-cyclopentanol 71 (7.71 g, 59%) as a
green liquid. EI-HRMS m/e calcd for C6H11IO (M+), 225.9855; found,
225.9856. 1H NMR (400 MHz, chloroform-d): δ 1.20−2.56 (m, 7 H),
3.21−3.35 (m, 2 H), 4.34−4.51 (m, 1 H).
2(R)-(3-Chloro-4-methylsulfonyl-phenyl)-3-cyclopentyl-N-pyra-
zin-2-yl-propionamide (3). A solution of 2(R)-(3-chloro-4-methyl-
sulfonyl-phenyl)-3-cyclopentyl-propionic acid 65 (200 mg, 0.61
mmol) in methylene chloride (5 mL) was treated with N,N′-
dimethylformamide (1 drop) and then cooled to 0 °C. The reaction
mixture was then treated dropwise with a 2 M solution of oxalyl
chloride in methylene chloride (0.45 mL, 0.91 mmol) and stirred at 0
°C for 10 min. The reaction mixture was then treated with a solution
of 2-aminopyrazine (115 mg, 1.21 mmol) and pyridine (0.245 mL,
3.03 mmol) in anhydrous tetrahydrofuran (10 mL). The resulting
reaction mixture was allowed to warm to 25 °C, where it was stirred
for 16 h. The reaction mixture was diluted with water (10 mL) and
extracted with methylene chloride (3 × 15 mL). The combined
organic layers were dried over sodium sulfate, filtered, and
concentrated in vacuo. Biotage chromatography (FLASH 40S, Silica,
50/50 hexanes/ethyl acetate) afforded 2(R)-(3-chloro-4-methylsulfon-
yl-phenyl)-3-cyclopentyl-N-pyrazin-2-yl-propionamide 3 (172 mg,
70%) as a white foam. EI-HRMS m/e calcd for C19H22ClN3O3S
1
(M+), 407.1070; found, 407.1068. H NMR (300 MHz, chloroform-
d): δ 9.53 (br. s., 1H), 8.38 (br. s., 1H), 8.24 (br. s., 1H), 8.14 (d, J =
8.42 Hz, 1H), 7.92 (br. s., 1H), 7.63 (s, 1H), 7.50 (d, J = 8.42 Hz, 1H),
3.66 (t, J = 7.51 Hz, 1H), 3.28 (s, 3H), 2.24 (td, J = 7.19, 13.83 Hz,
1H), 1.39−2.02 (m, 8H), 1.16 (d, J = 3.30 Hz, 2H).
2-Hydroxy-cyclopentanecarboxylic Acid Ethyl Ester (66). A
solution of 2-oxo-cyclopentanecarboxylic acid ethyl ester (10 g, 64.0
mmol) in ethanol (106.7 mL) cooled to 0 °C was treated with 98%
sodium borohydride (686 mg, 17.78 mmol). The reaction mixture was
stirred at 0 °C for 30 min. At this time, the reaction mixture was
poured into water (53 mL) and was extracted into diethyl ether (3 ×
100 mL). The organics were dried over sodium sulfate, filtered, and
concentrated in vacuo. Flash chromatography (Merck silica gel 60,
230−400 mesh, 75/25 ethyl acetate/hexanes) afforded 2-hydroxy-
cyclopentanecarboxylic acid ethyl ester 66 (8.5 g, 83.9%) as a clear
2-(3-Iodomethy-cyclopentyloxy)-tetrahydropyran (72). A solution
of 3-iodomethyl-cyclopentanol 71 (7.71 g, 34.10 mmol) in methylene
chloride (171 mL) was treated with 3,4-dihydro-2H-pyran (4.7 mL,
51.16 mmol) and pyridinium p-toluenesulfonate (857.1 mg, 3.41
mmol). This solution was stirred at 25 °C for 24 h. The combined
organic extracts were dried over magnesium sulfate, filtered, and
concentrated in vacuo. Flash chromatography (Merck silica gel 60,
70−230 mesh, 19/1 petroleum ether/diethyl ether) afforded 2-(3-
iodomethy-cyclopentyloxy)-tetrahydropyran 72 (7.91 g, 75%) as a
yellow oil. EI-HRMS m/e calcd for C11H19IO2 (M+), 310.0430; found,
1
liquid. H NMR (300 MHz, chloroform-d): δ 1.21−1.30 (m, 3 H),
1.49−2.12 (m, 6 H), 2.52−2.79 (m, 1 H), 4.05−4.16 (m, 2 H), 4.29−
4.54 (m, 1 H).
1
2-(Tetrahyropyran-2-yloxy)-cyclopentanecarboxylic Acid Ethyl
Ester (67). A solution of 2-hydroxy-cyclopentanecarboxylic acid ethyl
ester 66 (3.5 g, 22.12 mmol) in methylene chloride (147.5 mL) was
treated with 3,4-dihydro-2H-pyran (3.03 mL, 33.1 mmol) and
pyridinium p-toluenesulfonate (556 mg, 2.21 mmol). This solution
was stirred at 25 °C for 5 h. The reaction was washed with half-
saturated aqueous sodium chloride solution (2 × 75 mL), dried over
sodium sulfate, filtered, and concentrated in vacuo. Flash chromatog-
raphy (Merck silica gel 60, 230−400 mesh, 90/10 hexanes/ethyl
acetate) afforded 2-(tetrahyro-pyran-2yloxy)-cyclopentanecarboxylic
acid ethyl ester 67 (4.7 g, 87.7%) as a clear liquid. EI-HRMS m/e
310.0433. H NMR (400 MHz, chloroform-d): δ 1.09−2.52 (m, 13
H), 3.13−3.37 (m, 2 H), 3.40−3.60 (m, 1 H), 3.80−3.99 (m, 1 H),
4.24−4.42 (m, 1 H), 4.60 (d, J = 3.30 Hz, 1 H).
2(R)-(3-Chloro-4-methylsulfanyl-phenyl)-N-[2(R)-hydroxy-1(R)-
methyl-2(R)-phenyl-ethyl]-N-methyl-3-[2-(tetrahydropyran-2yloxy)-
cyclopentyl]-proprionamide (73). A solution of 1,1,1,3,3,3-hexame-
thyldisilazane (14.5 mL, 68.7 mmol) in tetrahydrofuran (45.8 mL) was
cooled to −45 °C and then treated with 2.34 M solution of n-butyl
lithium in hexanes (25.8 mL, 63.2 mmol). The reaction mixture was
stirred at −45 °C for 30 min. At this time, it was treated with (3-
chloro-4-methylsulfanyl-phenyl)-N-[2(R)-hydroxy-1(R)-methyl-2(R)-
phenyl-ethyl]-N-methyl-acetamide 62 (10.0 g, 27.48 mmol) in
tetrahydrofuran (46 mL). Upon complete addition, the reaction
mixture was warmed to 0 °C and stirred at 0 °C for 30 min. At this
time, the reaction mixture was recooled to −45 °C and then was
treated with 2-(2-iodomethyl-cyclopentyloxy)-tetrahydropyran 69
(12.8, 41.22 mmol) in 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimi-
dinone (8.4 mL). The resulting solution was warmed to 0 °C, where it
1
calcd for C13H22O (M+), 242.1518; found, 242.1521. H NMR (300
MHz, chloroform-d): δ 1.11−1.39 (m, 5 H), 1.41−2.31 (m, 10 H),
2.63−2.98 (m, 1 H), 3.36−3.63 (m, 1 H), 3.75−3.97 (m, 1 H), 4.00−
4.29 (m, 2 H), 4.32−4.56 (m, 1 H), 4.59−4.83 (m, 1 H).
[2-(Tetrahydro-pyran-2yloxy)-cyclopentyl]-methanol (68). A
slurry of lithium aluminum hydride (883 mg, 23.27 mmol) in
tetrahydrofuran (19.4 mL) cooled to 0 °C was treated with 2-
L
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Journal of Medicinal Chemistry
Article
was stirred for 3 h. At this time, the reaction mixture was diluted with
aqueous sodium chloride solution (100 mL). The phases were
partitioned. The aqueous layer was extracted with ethyl acetate (3 ×
50 mL). The combined organics were washed with aqueous 10%
hydrochloric acid and sodium carbonate solution, dried over sodium
sulfate, filtered, and concentrated in vacuo. Flash chromatography
(Merck silica gel 60, 230−400 mesh, 75/25 ethyl acetate/hexanes)
afforded 2(R)-(3-chloro-4-methylsulfanyl-phenyl)-N-[2(R)-hydroxy-
1(R)-methyl-2(R)-phenyl-ethyl]-N-methyl-3-[2-(tetrahydropyran-
2yloxy)-cyclopentyl]-proprionamide 73 (10.6 g, 70.6%) as a yellow
foam. EI-HRMS m/e calcd for C30H40ClNO4S (M + Na)+, 568.2259;
mmol), formic acid (2.47 mL, 65.5 mmol), and water (0.41 mL)
cooled to 0 °C was treated with a 30% hydrogen peroxide solution
(1.11 mL, 10.9 mmol). The reaction mixture stirred at 0 °C for 1 h. At
this time, the reaction was treated with a saturated aqueous sodium
sulfite solution. The resulting solution was poured into water (50 mL)
and then extracted into methylene chloride (3 × 50 mL). The organics
were dried over sodium sulfate, filtered, and concentrated in vacuo to
afford 2(R)-(3-chloro-4-methylsulfinyl-phenyl)-3-(2-oxo-cyclopentyl)-
proprionic acid 77 (724 mg, 100%) as a white foam. EI-HRMS m/e
1
calcd for C15H17ClO4S (M + Na)+, 351.0428; found, 351.0433. H
NMR (300 MHz, chloroform-d): δ 1.37−2.58 (m, 9 H), 2.75−2.94
(m, 3 H), 3.77−4.09 (m, 1 H), 7.37−7.45 (m, 1 H), 7.49 (tdd, J =
6.50, 6.50, 3.30, 1.65 Hz, 1 H), 7.89 (d, J = 8.06 Hz, 1 H).
1
found, 568.2262. H NMR (300 MHz, chloroform-d): δ 0.44−1.21
(m, 4 H), 1.31−2.28 (m, 14 H), 2.30−2.56 (m, 3 H), 2.60−3.01 (m, 4
H), 3.28−5.00 (m, 7 H), 6.95−7.59 (m, 8 H).
2(R)-(3-Chloro-4-methylsulfonyl-phenyl)-3-(2-oxo-cyclopentyl)-
proprionic Acid (78). A solution of potassium permanganate (101 mg,
0.64 mmol) in water (1.83 mL) was treated with a solution of 2(R)-(3-
chloro-4-methylsulfinyl-phenyl)-3-(2-oxo-cyclopentyl)-proprionic acid
77 (191.5 mg, 0.58 mmol) in methanol (5.8 mL). The reaction
mixture was stirred at 25 °C for 1 h. At this time, the reaction mixture
was diluted with methanol and filtered through a pad of Celite. The
filtrate was concentrated and azeotroped with acetonitrile. Flash
chromatography (Merck silica gel 60, 230−400 mesh, 90/10
methylene chloride/methanol 1% glacial acetic acid) afforded 2(R)-
(3-chloro-4-methylsulfonyl-phenyl)-3-(2-oxo-cyclopentyl)-proprionic
acid 78 (87 mg, 43%) as an off white foam. EI-HRMS m/e calcd for
C15H17ClO5S (M − H2O)+, 326.0379; found, 326.0378. 1H NMR
(300 MHz, chloroform-d): δ 1.33−2.60 (m, 9 H) 3.27 (s, 3 H) 3.79−
4.21 (m, 1 H) 7.45 (ddd, J = 8.06, 4.76, 1.46 Hz, 1 H) 7.56 (dd, J =
4.76, 1.46 Hz, 1 H) 8.11 (d, J = 8.06 Hz, 1 H).
2(R)-(3-Chloro-4-methylsulfanyl-phenyl)-3-(2-hydroxy-cyclopen-
tyl)-N-[2(R)-hydroxy-1(R)-methyl-2(R)-phenyl-ethyl]-N-methyl-pro-
prionamide (74). A solution of 2(R)-(3-chloro-4-methylsulfanyl-
phenyl)-N-[2(R)-hydroxy-1(R)-methyl-2(R)-phenyl-ethyl]-N-methyl-
3-[2-(tetrahydropyran-2yloxy)-cyclopentyl]-proprionamide 73 (1.04 g,
1.91 mmol) in ethanol (19.1 mL) was treated with pyridinium p-
toluenesulfonate (48 mg, 0.19 mmol). The resulting solution was
heated to 55 °C for 2 h. At this time, the reaction mixture was
concentrated in vacuo. Flash chromatography (Merck silica gel 60,
230−400 mesh, 75/25 ethyl acetate/hexanes) afforded 2(R)-(3-
chloro-4-methylsulfanyl-phenyl)-3-(2-hydroxy-cyclopentyl)-N-[2(R)-
hydroxy-1(R)-methyl-2(R)-phenyl-ethyl]-N-methyl-proprionamide 74
(0.82 g, 93%) as a white foam. EI-HRMS m/e calcd for C25H32ClNO3S
(M + Na)+, 484.1684; found, 484.1674. 1H NMR (300 MHz,
chloroform-d): δ 0.51−2.38 (m, 12 H), 2.45 (d, J = 8.42 Hz, 3 H),
2.63−3.01 (m, 3 H), 3.30−4.87 (m, 6 H), 6.89−7.60 (m, 8 H).
2(R)-(3-Chloro-4-methylsulfanyl-phenyl)-N-methyl-N-[1(R)-meth-
yl-2-oxo-2(R)-phenyl-ethyl]-3-(2-oxo-cyclopentyl)-proprionamide
(75). A mixture of 2(R)-(3-chloro-4-methylsulfanyl-phenyl)-3-(2-
hydroxy-cyclopentyl)-N-[2(R)-hydroxy-1(R)-methyl-2(R)-phenyl-
ethyl]-N-methyl-proprionamide 74 (3.63 g, 7.85 mmol), N-methyl-
morpholine-N-oxide (2.76 g, 23.5 mmol), and powdered molecular
sieves (7.85 g) in methylene chloride (15.7 mL) at 25 °C treated with
tetrapropylammonium perruthenate (276 mg, 0.78 mmol). The
resulting reaction mixture was stirred at 25 °C for 20 min. At this
time, the reaction mixture was filtered through a pad of silica (ethyl
acetate as eluent). The filtrate was concentrated in vacuo. Flash
chromatography (Merck silica gel 60, 230−400 mesh, 75/25 ethyl
acetate/hexanes) afforded 2(R)-(3-chloro-4-methylsulfanyl-phenyl)-N-
methyl-N-(1(R)-methyl-2-oxo-2(R)-phenyl-ethyl)-3-(2-oxo-cyclopen-
tyl)-proprionamide 75 (2.75 g, 76.5%) as a white foam. EI-HRMS m/e
2(R)-(3-Chloro-4-methylsulfonyl-phenyl)-3-(2-oxo-cyclopentyl)-
N-pyrazin-2-yl-propionamide (29). A solution of 2(R)-(3-chloro-4-
methylsulfonyl-phenyl)-3-(2-oxo-cyclopentyl)-proprionic acid 78 (125
mg, 0.36 mmol) in methylene chloride (3.62 mL) was cooled to 0 °C
and was treated with a treated 2 M solution of oxalyl chloride in
methylene chloride (0.20 mL, 0.39 mmol) and with a few drops of
N,N′-dimethylformamide. The reaction mixture was then stirred at 0
°C for 10 min and at 25 °C for 20 min. The reaction mixture was then
treated with a solution of 2-aminopyrazine (76 mg, 0.8 mmol) and
pyridine (0.06 mL, 0.8 mmol) in anhydrous tetrahydrofuran (1.81
mL). The resulting reaction mixture was stirred at 25 °C for 1 h. At
this time, the reaction mixture was concentrated in vacuo. Flash
chromatography (Merck silica gel 60, 230−400 mesh, 75/25 ethyl
acetate/hexanes) afforded 2(R)-(3-chloro-4-methylsulfonyl-phenyl)-3-
(2-oxo-cyclopentyl)-N-pyrazin-2-yl-propionamide 29 (77.6 mg,
50.7%) as a white foam. EI-HRMS m/e calcd for C19H20ClN3O4S
1
calcd for C25H28ClNO3S (M)+, 457.1478; found, 457.1489. H NMR
1
(M)+, 421.0863; found, 421.0868. H NMR (300 MHz, chloroform-
(300 MHz, chloroform-d): δ 1.36 (dd, J = 6.96, 3.66 Hz, 3 H), 1.43−
2.56 (m, 11 H), 2.68−2.87 (m, 4 H), 4.14−4.32 (m, 1 H), 5.76−6.14
(m, 1 H), 6.89−6.97 (m, 1 H), 6.99−7.11 (m, 1 H), 7.24 (dd, J = 6.04,
2.01 Hz, 1 H), 7.27−7.35 (m, 2 H), 7.42−7.53 (m, 1 H), 7.69−7.86
(m, 2 H).
d): δ 1.42−2.49 (m, 9 H), 3.14−3.35 (m, 3 H), 4.21−4.41 (m, 1 H),
7.44−7.60 (m, 1 H), 7.67 (s, 1 H), 8.03−8.64 (m, 4 H), 9.50 (s, 1 H).
2(R)-(3-Chloro-4-methylsulfonyl-phenyl)-3-(2-hydroxy-cyclopen-
tyl)-N-pyrazin-2-yl-propionamide (27). A solution of 2(R)-(3-chloro-
4-methylsulfonyl-phenyl)-3-(2-oxo-cyclopentyl)-N-pyrazin-2-yl-pro-
pionamide 29 (13.5 mg, 0.03 mmol) in ethanol (0.32 mL) cooled to 0
°C was treated with sodium borohydride (1.2 mg, 0.3 mmol). The
reaction mixture was stirred at 0 °C for 20 min. At this time, the
reaction was diluted with water (25 mL). The mixture was extracted
with ethyl acetate (3 × 30 mL). The combined organics were dried
over sodium sulfate, filtered, and concentrated in vacuo. Flash
chromatography (Merck silica gel 60, 230−400 mesh, ethyl acetate)
afforded 2(R)-(3-chloro-4-methylsulfonyl-phenyl)-3-(2-hydroxy-cyclo-
pentyl)-N-pyrazin-2-yl-propionamide 27 (10.1 mg, 75.5%) as a white
foam. EI-HRMS m/e calcd for C19H22ClN3O4S (M + Na)+, 446.0912;
2(R)-(3-Chloro-4-methylsulfanyl-phenyl)-3-(2-oxo-cyclopentyl)-
proprionic Acid (76). A solution of 2(R)-(3-chloro-4-methylsulfanyl-
phenyl)-N-methyl-N-(1(R)-methyl-2-oxo-2(R)-phenyl-ethyl)-3-(2-
oxo-cyclopentyl)-proprionamide 75 (2.75 g, 6.0 mmol) in dioxane (9.4
mL) was treated with 18 M aqueous hydrochloric acid solution (9.4
mL). The reaction mixture was heated to 120 °C for 18 h. At this time,
the reaction mixture was cooled to 25 °C, diluted with water (100
mL), and extracted with 90/10 methylene chloride/methanol solution
(3 × 100 mL). The organics were dried over sodium sulfate, filtered,
and concentrated in vacuo. Flash chromatography (Merck silica gel 60,
230−400 mesh, ethyl acetate) afforded 2(R)-(3-chloro-4-methylsul-
fanyl-phenyl)-3-(2-oxo-cyclopentyl)-proprionic acid 76 (1.54 g, 82%)
as a yellow oil. EI-HRMS m/e calcd for C15H17ClO3S (M)+, 312.0587;
1
found, 446.0916. H NMR (300 MHz, chloroform-d): δ 1.10−2.58
(m, 9 H), 3.27 (s, 3 H), 3.74−4.08 (m, 2 H), 4.13 (m, J = 7.20, 7.20,
7.20 Hz, 1 H), 7.54 (d, J = 8.06 Hz, 1 H), 7.68 (d, J = 5.49 Hz, 1 H),
8.13 (d, J = 8.06 Hz, 1 H), 8.16−8.29 (m, 1 H), 8.36 (br. s., 1 H), 9.51
(br. s., 1 H).
1
found, 312.0581. H NMR (300 MHz, chloroform-d): δ 1.34−2.57
(m, 9 H), 2.47 (s, 3H), 3.63−3.99 (m, 1 H), 7.07−7.16 (m, 1 H),
7.19−7.26 (m, 1 H), 7.33 (dd, J = 6.23, 1.83 Hz, 1 H).
2(R)-(3-Chloro-4-methylsulfanyl-phenyl)-N-[2(R)-hydroxy-1(R)-
methyl-2(R)-phenyl-ethyl]-N-methyl-3-[3-(tetrahydro-pyran-2-
yloxy)-cyclopentyl]-propionamide (79). A solution of 1,1,1,3,3,3-
hexamethyldisilazane (3.75 mL, 17.24 mmol) in freshly distilled
2(R)-(3-Chloro-4-methylsulfinyl-phenyl)-3-(2-oxo-cyclopentyl)-
proprionic Acid (77). A solution of 2(R)-(3-chloro-4-methylsulfanyl-
phenyl)-3-(2-oxo-cyclopentyl)-proprionic acid 76 (683.2 mg, 2.18
M
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tetrahydrofuran (50 mL) was treated slowly with a 2.3 M solution of n-
butyllithium in hexanes (7.0 mL, 16.1 mmol) at −45 °C. The resulting
reaction solution was stirred at −40 °C for 45 min and then treated
slowly with a solution of 2-(3-chloro-4-methylsulfanyl-phenyl)-N-
[2(R)-hydroxy-1(R)-methyl-2(R)-phenyl-ethyl]-N-methyl-acetamide
62 (2.5 g, 6.87 mmol) in tetrahydrofuran (10 mL) via a cannula. A
yellow solution was obtained, and the reaction was allowed to warm to
0 °C, where it was stirred for 30 min. This reaction solution was
cooled to −50 °C and treated with 1,3-dimethyl-3,4,5,6-tetrahydro-
2(1H)-pyrimidinone (5 mL) followed by the dropwise addition of a
solution of 2-(3-iodomethyl-cyclopentyloxy)-tetrahydropyran (pre-
pared as in example 35, 3.2 g, 10.3 mmol) in tetrahydrofuran (10
mL). After the addition, the reaction solution was warmed to 0 °C,
where it was stirred for 2 h, and then warmed to 25 °C, where it was
stirred for an additional 2 h. The reaction solution was diluted with
methylene chloride (100 mL) and washed with a saturated aqueous
sodium chloride solution (100 mL). The organic layer was separated,
dried over magnesium sulfate, filtered, and concentrated in vacuo.
Flash chromatography (Merck Silica gel 60, 70−230 mesh, 40−60%
ethyl acetate/hexanes) afforded 2(R)-(3-chloro-4-methylsulfanyl-phe-
nyl)-N-(2(R)-hydroxy-1(R)-methyl-2(R)-phenyl-ethyl)-N-methyl-3-
[3-(tetrahydro-pyran-2-yloxy)-cyclopentyl]-propionamide 79 (3.4 g,
chloride (0.3 mL). The reaction mixture was stirred at 25 °C for an
additional 45 min and then concentrated in vacuo. The residue was
dissolved in dry tetrahydrofuran (5 mL) and cooled to 0 °C. This cold
solution was then treated with a solution of 2-aminopyrazine (142 mg,
1.5 mmol) and pyridine (0.121 mL, 1.5 mmol) in dry tetrahydrofuran
(5 mL) via a cannula. The resulting reaction mixture was stirred at 0
°C for 1 h and then diluted with methylene chloride (100 mL). The
organic layer was successively washed with a 1 M aqueous citric acid
solution (2 × 100 mL), a saturated sodium bicarbonate solution (1 ×
100 mL), and a saturated sodium chloride solution (1 × 100 mL). The
organics were dried over magnesium sulfate, filtered, and concentrated
in vacuo to afford formic acid 3-[2(R)-(3-chloro-4-methanesulfonyl-
phenyl)-2-(pyrazin-2-ylcarbamoyl)-ethyl]-cyclopentyl ester 82 (375
mg, 82%) as an off-white solid. 1H NMR (300 MHz, chloroform-d): δ
0.73−2.49 (m, 9 H), 3.12−3.40 (m, 3 H), 3.53−3.91 (m, 1 H), 4.13−
4.50 (m, 1 H), 5.41−6.22 (m, 1 H), 7.52 (d, J = 8.06 Hz, 1 H), 7.65 (s,
1 H), 8.12 (d, J = 8.06 Hz, 1 H), 8.17−8.45 (m, 2 H), 8.65−9.03 (m, 1
H), 9.54 (br. s., 1 H).
2(R)-(3-Chloro-4-methanesulfonyl-phenyl)-3-(3-hydroxy-cyclo-
pentyl)-N-pyrazin-2-yl-propionamide (28). To a solution of formic
acid 3-[2(R)-(3-chloro-4-methanesulfonyl-phenyl)-2-(pyrazin-2-ylcar-
bamoyl)-ethyl]-cyclopentyl ester 82 (375 mg, 0.83 mmol) in methanol
(50 mL) at 0 °C was bubbled ammonia gas for 15 min. The resulting
reaction solution was stirred at 0 °C for 15 min. The solvent was
removed in vacuo. Flash chromatography (Merck Silica gel 60, 70−
230 mesh, 1−5% methanol/methylene chloride) afforded 2(R)-(3-
chloro-4-methanesulfonyl-phenyl)-3-(3-hydroxy-cyclopentyl)-N-pyra-
1
90.7%) as a white solid. H NMR (300 MHz, chloroform-d): δ 0.47−
2.32 (m, 18 H), 2.44 (s, 3 H), 2.60−3.03 (m, 3 H), 3.27−4.76 (m, 7
H), 6.95−7.59 (m, 8 H).
2(R)-(3-Chloro-4-methylsulfanyl-phenyl)-3-(3-hydroxy-cyclopen-
tyl)-propionic Acid (80). A solution of 2(R)-(3-chloro-4-methylsul-
fanyl-phenyl)-N-(2(R)-hydroxy-1(R)-methyl-2(R)-phenyl-ethyl)-N-
methyl-3-[3-(tetrahydro-pyran-2-yloxy)-cyclopentyl]-propionamide 79
(0.96 g, 1.758 mmol) in dioxane (20 mL) was treated with a 9 N
aqueous sulfuric acid solution (1.5 mL). The resulting reaction
solution was heated under reflux for 15 h. In another flask, a solution
of 2(R)-(3-chloro-4-methylsulfanyl-phenyl)-N-(2(R)-hydroxy-1(R)-
methyl-2(R)-phenyl-ethyl)-N-methyl-3-[3-(tetrahydro-pyran-2-yloxy)-
cyclopentyl]-propionamide (1 g, 1.83 mmol) in dioxane (20 mL) and
a 9 N aqueous sulfuric acid solution (1.5 mL) was heated under reflux
for 7 h. The two reactions were combined, diluted with methylene
chloride (100 mL), and washed with a saturated aqueous sodium
chloride solution (100 mL). The organic layer was separated, dried
over magnesium sulfate, filtered, and concentrated in vacuo. Flash
chromatography (Merck Silica gel 60, 70−230 mesh, 8−10%
methanol/methylene chloride) afforded 2(R)-(3-chloro-4-methylsul-
fanyl-phenyl)-3-(3-hydroxy-cyclopentyl)-propionic acid 80 (496 mg
1
zin-2-yl-propionamide 28 (260 mg, 62.5%) as an off-white solid. H
NMR (300 MHz, methanol-d4): δ 1.11−2.48 (m, 9 H), 3.19−3.44 (m,
3 H), 3.92−4.12 (m, 1 H), 4.14−4.40 (m, 1 H), 7.60−7.74 (m, 1 H),
7.75−7.86 (m, 1 H), 8.05−8.19 (m, 1 H), 8.27−8.35 (m, 1 H), 8.36−
8.44 (m, 1 H), 9.41 (d, J = 4.03 Hz, 1 H).
2(R)-(3-Chloro-4-methanesulfonyl-phenyl)-3-(3-oxo-cyclopentyl)-
N-pyrazin-2-yl-propionamide (30). A solution of 2(R)-(3-chloro-4-
methanesulfonyl-phenyl)-3-(3-hydroxy-cyclopentyl)-N-pyrazin-2-yl-
propionamide 28 (60 mg, 0.142 mmol) in methylene chloride (10
mL) was treated with Dess−Martin periodinane (1,1,1-Tris-
(acetyloxy)-1,1-dihydro-1,2-benziodo-3-(1H)-one) (132.5 mg, 0.312
mmol). The reaction mixture was stirred at 25 °C for 2 h. The reaction
mixture was then diluted with methylene chloride (10 mL) and
washed with a 1 M aqueous citric acid solution (10 mL). The pH of
the aqueous layer was adjusted to 5. The aqueous layer was then
extracted with methylene chloride (20 mL). The combined organic
layers were dried over magnesium sulfate, filtered, and concentrated in
vacuo. Flash chromatography (Merck Silica gel 60, 70−230 mesh, 50−
100% ethyl acetate/hexanes) afforded 2(R)-(3-chloro-4-methanesul-
fonyl-phenyl)-3-(3-oxo-cyclopentyl)-N-pyrazin-2-yl-propionamide 30
1
43.9%) as an off-white foam. H NMR (300 MHz, chloroform-d): δ
0.57−2.24 (m, 9 H), 2.45 (s, 3 H), 3.51 (t, J = 7.51 Hz, 1 H), 4.18−
4.43 (m, 1 H), 6.94−7.55 (m, 3 H).
2(R)-(3-Chloro-4-methanesulfonyl-phenyl)-3-(3-formyloxy-cyclo-
pentyl)-propionic Acid (81). A solution of 2(R)-(3-chloro-4-
methylsulfanyl-phenyl)-3-(3-hydroxy-cyclopentyl)-propionic acid 80
(350 mg, 1.11 mmol) in formic acid (35 mL) was treated with a
30% aqueous hydrogen peroxide solution (1 mL, 7.89 mmol) at 25 °C.
The mixture was then allowed to stir at 25 °C overnight. The solvent
was evaporated in vacuo. The resulting residue was azeotroped with
toluene to remove water and then coevaporated with N,N-
dimethylformamide to remove formic acid to afford crude 2(R)-(3-
chloro-4-methanesulfonyl-phenyl)-3-(3-formyloxy-cyclopentyl)-pro-
1
(45 mg, 75%) as an off-white solid. H NMR (300 MHz, methanol-
d4): δ 1.46−2.55 (m, 8 H), 3.19−3.43 (m, 4 H), 4.02−4.11 (m, 1 H),
7.69 (dd, J = 8.42, 1.47 Hz, 1 H), 7.81 (d, J = 1.46 Hz, 1 H), 8.06−8.15
(m, 1 H), 8.32 (d, J = 2.56 Hz, 1 H), 8.37 (dd, J = 2.56, 1.46 Hz, 1 H),
9.40 (t, J = 1.28 Hz, 1 H).
2(R),3(R)-Diphenyl-1,4-dioxa-spiro[4.4]non-6-ene (91). 2(R),3-
(R)-Diphenyl-1,4-dioxa-spiro[4.4]non-6-ene 91 was obtained following
the similar procedure described for 84 from 2-cyclopentene-1-one and
(R,R)-hydrobenzoin 9017 in toluene in 91.5% yield as a white solid. 1H
NMR (300 MHz, chloroform-d): δ 2.32−2.46 (m, 2 H), 2.54 (td, J =
5.49, 2.93 Hz, 2 H), 4.76 (s, 2 H), 5.93−6.07 (m, 1 H), 6.17−6.31 (m,
1 H), 7.20−7.29 (m, 5 H), 7.29−7.41 (m, 5 H).
1(S),5(R)-Bicyclo[3.1.0]hexan-2-spiro-2′-[4(R),5(R)-diphenyl diox-
olane] (92). 1(S),5(R)-Bicyclo[3.1.0]hexan-2-spiro-2′-(4(R),5(R)-di-
phenyl dioxolane) 92 was obtained following the similar procedure
described for 85 from 2(R),3(R)-diphenyl-1,4-dioxa-spiro[4.4]non-6-
ene 91 in 68% yield after crystallization from pentane. HPLC (Zorbax
XDB-C8 150 mm × 5 mm, 5−100% acetonitrile/water + 0.1%
trifluoroacetic acid, Rt 18.0 min) indicated 96.5 area % purity. 1H
NMR (300 MHz, chloroform-d): δ 0.56−0.83 (m, 2 H), 1.40−2.24
(m, 6 H), 4.65−4.86 (m, 2 H), 7.21 (dd, J = 6.23, 2.93 Hz, 2 H),
7.28−7.42 (m, 8 H).
1
pionic acid 81 (430 mg, 103%) as an off-white solid. H NMR (300
MHz, chloroform-d): δ 0.70−2.53 (m, 9 H), 3.28 (s, 3 H), 3.58−3.90
(m, 1 H), 5.08−5.46 (m, 1 H), 7.51 (d, J = 8.06 Hz, 1 H), 7.63 (s, 1
H), 8.13 (d, J = 8.06 Hz, 1 H), 8.22 (br. s., 1 H), 9.53 (br. s., 1 H).
3-[2(R)-(3-Chloro-4-methanesulfonyl-phenyl)-2-(pyrazin-2-ylcar-
bamoyl)-ethyl]-cyclopentyl Ester (82). A solution of 2(R)-(3-chloro-
4-methanesulfonyl-phenyl)-3-(3-formyloxy-cyclopentyl)-propionic
acid 81 (380 mg, 1.01 mmol) in toluene (10 mL) at 0 °C was treated
with N,N-dimethylformamide (0.008 mL) followed by a 2.0 M
solution of oxalyl chloride in methylene chloride (0.75 mL). The
reaction mixture was allowed to stir at 0 °C for 30 min and at 25 °C
for 1.5 h. It appeared that a thick oil in the bottom of the reaction flask
never solubilized. Additional methylene chloride (10 mL) was added
at 25 °C followed by N,N-dimethylformamide (0.002 mL) and oxalyl
N
dx.doi.org/10.1021/jm3008689 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
7(S)-Iodomethyl-2(R),3(R)-diphenyl-1,4-dioxa-spiro[4.4]nonane
(93). 7(S)-Iodomethyl-2(R),3(R)-diphenyl-1,4-dioxa-spiro[4.4]nonane
93 was obtained following the similar procedure described for 86 from
1(S),5(R)-bicyclo[3.1.0]hexan-2-spiro-2′-(4(R),5(R)-diphenyl dioxo-
lane) 92 at 25 °C in 71.7% yield as a white solid.
2(R)-(3-Chloro-4-methylsulfanyl-phenyl)-3-(2(R),3(R)-diphenyl-
1,4-dioxa-spiro[4.4]non-7(R)-yl)-N-(2(R)-hydroxy-1(R)-methyl-2-
phenyl-ethyl)-N-methylpropionamide (94). 2(R)-(3-Chloro-4-meth-
ylsulfanyl-phenyl)-3-(2(R),3(R)-diphenyl-1,4-dioxa-spiro[4.4]non-
7(R)-yl)-N-(2(R)-hydroxy-1(R)-methyl-2-phenyl-ethyl)-N-methylpro-
pionamide 94 was obtained following the similar procedure described
for 87 from 7(S)-iodomethyl-2(R),3(R)-diphenyl-1,4-dioxa-spiro[4.4]-
nonane 93 in 65% yield.
2(R)-(3-Chloro-4-methylsulfanyl-phenyl)-3-[(R)-3-oxo-cyclopen-
yl]-propionic Acid (95). 2(R)-(3-Chloro-4-methylsulfanyl-phenyl)-3-
[(R)-3-oxo-cyclopenyl]-propionic acid 95 was obtained the similar
procedure described for 88 from 2(R)-(3-chloro-4-methylsulfanyl-
phenyl)-3-(2(R),3(R)-diphenyl-1,4-dioxa-spiro[4.4]non-7(R)-yl)-N-
(2(R)-hydroxy-1(R)-methyl-2-phenyl-ethyl)-N-methylpropionamide
94 in 72% yield as a yellow foam. 1H NMR (300 MHz, chloroform-d):
δ 1.48−2.57 (m, 9 H), 2.48 (s, 3H), 3.53−3.63 (m, 1 H), 7.09−7.17
(m, 1 H), 7.22 (dd, J = 8.42, 2.20 Hz, 1 H), 7.35 (d, J = 1.83 Hz, 1 H).
2(R)-(3-Chloro-4-methanesulfonyl-phenyl)-3-[(R)-3-oxo-cyclo-
pentyl]-propionic Acid (96). 2(R)-(3-Chloro-4-methanesulfonyl-phe-
nyl)-3-[(R)-3-oxo-cyclopentyl]-propionic acid 96 was obtained
following the similar procedure described for 89 from 2(R)-(3-
chloro-4-methylsulfanyl-phenyl)-3-[(R)-3-oxo-cyclopenyl]-propionic
acid 95 in 90% yield as a white solid. 1H NMR (300 MHz, chloroform-
d): δ 1.44−2.51 (m, 9 H), 3.28 (s, 3 H), 3.72 (t, J = 7.62 Hz, 1 H),
7.44 (dd, J = 8.31, 1.72 Hz, 1 H), 7.55 (d, J = 1.65 Hz, 1 H), 8.14 (d, J
= 8.24 Hz, 1 H).
min. The clear supernatant was used directly for HPLC radio-
chromatography and mass spectral analysis. The samples were
analyzed by Micromass Quattro Altima LCMS/MS using Water
symmetry C18 column, 5.0 μ, 2.1 cm × 15 cm.
In Vivo Studies in C57 Mice. Male C57Bl/6J mice (Jackson
Laboratories), 15 weeks of age, were fasted for 19 h prior to the
administration of oral dose of vehicle, 4 (5, 10, and 15 mg/kg). The
OGTT (2 g/kg) was initiated 2 h following administration of vehicle
or test compound. Blood obtained from a tail snip was collected in a
heparinized capillary tube and used immediately to measure glucose
levels using a YSI model 2700 Biochemistry Analyzer (YSI, Inc.,
Yellow Springs, OH). The formulation used for the in vivo study
consisted of Gelucire44/14:ethanol:PEG400 (4:66:30 v/w/v). Food
was withheld during the duration of the study, and mice had free
access to water. All results are reported as the means
standard
deviations (n = 6/time point). A Student's t test was used to test for
statistical significance (*, p < 0.05). All animal procedures were
approved by the Institutional Animal Care and Use Committee.
Toxicity Studies in Rats. Male Wistar rats were divided into three
groups of vehicle, compound 1, ent-1, with three animals per group.
Each rat in compound 1 treatment group received 300 mg/kg dose,
while in ent-1 treatment group rats received 600 mg/kg dose, via oral
administration. Blood was collected by cardiac puncture under
isoflurane/O2 conditions, before euthanization. After weighing, 2−3
sections of liver from the left lateral and medial lobes were fixed in
10% neutral-buffered formalin, processed in paraffin, sectioned, and
stained with hematoxylin and eosin (H&E). The remaining liver was
sliced, and systematic random samples were collected. Two to three
sections of liver were snap-frozen in OCT for Oil Red O staining.
Sections were examined microscopically.
2(R)-(3-Chloro-4-methanesulfonyl-phenyl)-3-[(R)-3-oxo-cyclo-
pentyl]-N-pyrazin-2-yl-propionamide (4). 2(R)-(3-Chloro-4-metha-
nesulfonyl-phenyl)-3-[(R)-3-oxo-cyclopentyl]-N-pyrazin-2-yl-propio-
namide 4 was obtained following the similar procedure described for
31 from 2(R)-(3-chloro-4-methanesulfonyl-phenyl)-3-[(R)-3-oxo-cy-
clopentyl]-propionic acid 96 in 43% yield as a white foam. HPLC
(Chiralpak AD-RH, 150 mm × 5 mm, 5 ethanol/5 methanol/4 water,
60 min run, 220 nm, 0.5 cc/min, Rt of 2(R),3(S) = 25.4 min;
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis of analogues 5−26, corresponding synthetic schemes
(1−9), and efficacy figures (1 and 2) related to 1, ent-1, and 2.
This material is available free of charge via the Internet at
http://pubs.acs.org.
1
2(R),3(R) = 38.9 min) indicated 95.9 area% purity and >99% de. H
AUTHOR INFORMATION
NMR (300 MHz, chloroform-d): δ 1.43−2.54 (m, 9 H), 3.29 (s, 3 H),
3.70 (t, J = 7.14 Hz, 1 H), 7.51 (dd, J = 8.17, 1.72 Hz, 1 H), 7.63 (d, J
= 1.65 Hz, 1 H), 8.00 (s, 1 H), 8.17 (d, J = 8.10 Hz, 1 H), 8.23 (dd, J =
2.61, 1.51 Hz, 1 H), 8.39 (d, J = 2.47 Hz, 1 H), 9.52 (s, 1 H). Anal.
(C19H20ClN3O4S) C, H, N, Cl, S.
■
Corresponding Author
*Tel: 973-235-3984. Fax: 973-235-2448. E-mail: ramakanth.
sarabu@roche.com.
Notes
GK in Vitro Enzyme Kinetics. The methods used to determine the
effects of GK activators on GK enzymatic activity (SC1.5 and enzyme
kinetic parameters Ka, α, and β) were previously described.2 Various
fixed concentrations of activators (ranging from ∼0.010 to 30 μM)
were assayed in duplicate in the presence of a single, fixed
concentration of glucose (5 mM) to determine the concentration of
activator that causes a 1.5-fold increase in enzymatic activity relative to
untreated GK. This term is called a stimulatory concentration (SC1.5).
To calculate an activator's effect of enzyme kinetic parameters, the
activator was assayed in the presence of various fixed concentrations of
glucose (0.8−50 mM). Apparent Vmax and S0.5 (referred to Km for an
enzyme showing Michaelis−Menten kinetics) values were determined
by fitting enzymatic rates to the Hill equation using GraphPad Prism
version 5.01 for Windows, GraphPad Software, San Diego, CA).
Typical Microsomal Incubations. Pooled liver microsomes from
mouse, rat, dog, monkey, and human (20 mg/mL) were purchased
from Xenotech Inc. Incubation reaction mixtures contained a final
concentration of 0.1 M potassium phosphate buffer (pH 7.4), 3 mM
MgCl2, 1 mM EDTA, 1 or 2 mg/mL microsomal protein, 20 μM C-14
GK activator compounds, and 1 mM NADPH in a total volume of 1.0
mL. The incubations were conducted for 30 min with 5 min of
preincubation after adding 10 μL of 2 mM GK compound in methanol
(total 1% methanol in incubation mixture), and reactions were
terminated with 2 mL of ice cold methanol or 100 μL of 50% aqueous
TFA, after vortexes for 3 min, centrifuged at 3000 rpm at 4 °C for 10
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge all members of the Physical
Chemistry Department for the characterization of the
compounds and Formulations group for their support of in
vivo experiments.
ABBREVIATIONS USED
■
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 that increases GK activity by
1.5 times over the untreated GK; T2D, type 2 diabetes
,
REFERENCES
■
(1) Matschinsky, F. M. Assessing the potential of glucokinase
activators in diabetes therapy. Nat. Rev. Drug Discovery 2009, 8, 399−
416.
O
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Journal of Medicinal Chemistry
Article
(2) Matschinsky, F. M.; Ellerman, J. E. Metabolism of glucose in islets
of Langerhans. J. Biol. Chem. 1968, 243, 2730−2736.
(15) 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.
(3) (a) Sharma, C.; Manjeshwar, R.; Weinhouse, S. Hormonal and
dietary regulation of hepatic glucokinase. Adv. Enzyme Regul. 1964, 2,
189−200. (b) Van Schaftingen, E. A protein from rat liver confers to
glucokinase the property of being antagonistically regulated by fructose
6-phosphate and fructose 1-phosphate. Eur. J. Biochem. 1989, 179,
179−184.
(16) http://www.scripintelligence.com/home/Roche-licenses-GKA-
diabetes-candidates-to-Chinas-Hua-Medicine-325220.
(17) Wang, Z. M.; Sharpless, K. B. A Solid-to-solid asymmetric
dihydroxylation procedure for kilogram-scale preparation of enantio-
pure hydrobenzoin. J. Org. Chem. 1994, 59, 8302−8303.
(4) Matschinsky, F. M.; Magnuson, M. A.; Zelent, D.; Jetton, T. L.;
Doliba, N.; Han, Y.; Taub, R.; Grimsby, J. The network of glucokinase-
expressing cells in glucose homeostasis and the potential of
glucokinase activators for diabetes therapy. Diabetes 2006, 55, 1−12.
(5) Edghill, E. L.; Hattersley, A. T. Genetic disorders of the
pancreatic beta cell and diabetes (Permanent neonatal diabetes and
maturity onset diabetes of the young). In Pancreatic Beta Cell in Health
and Disease; Seino, S., Bell, G. I., Eds.; Springer: Tokyo, 2008; pp
399−430.
(6) Glaser, B.; Kesavan, P.; Heyman, M.; Davis, E.; Cuesta, A.; Buchs,
A.; Stanley, C. A.; Thornton, P. S.; Permutt, M. A.; Matschinsky, F. M.;
Herold, K. C. Familial hyperinsulinism caused by an activating
glucokinase mutation. N. Engl. J. Med. 1998, 338, 226−230.
(7) (a) Grimsby, J.; Sarabu, R.; Corbett, W. L.; Haynes, N. E.;
Bizzarro, F. T.; Coffey, J. W.; Guertin, K. R.; Hilliard, D. H.; 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. (b) Grimsby, J.; Zhi, J.;
Mulligan, M. E.; Arbet-Engels, C.; Taub, R.; Balena, R. Presented at
the Keystone Symposia: Diabetes Mellitus, Insulin Action and
Resistance, January 2008, Breckenridge, CO, United States, poster
151. (c) Haynes, N. E.; Corbett, W. L.; Bizzarro, F. T.; Guertin, K. G.;
Hilliard, D. W.; Holland, G. W.; Kester, R. F. K.; Mahaney, P. E.; Qi,
L.; Spence, C. L.; Tengi, J.; Dvorozniak, M. T.; Railkar, A.;
Matschinsky, F. M.; Grippo, J. F.; Grimsby, J. G.; Sarabu, R.
Discovery, structure-activity relationships, pharmacokinetics, and
efficacy of glucokinase activator (2R)-3-cyclopentyl-2-(4-methanesul-
fonylphenyl)-N-thiazol-2-yl-propianamide. J. Med. Chem. 2010, 53,
3618−3625.
(8) (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.; Dvorozniak, M.;
Racha, J.; Wang, K.. Discovery of piragliatin, a small molecule activator
of GK (MEDI-005). 238th ACS National Meeting, Washington DC,
August 16−20, 2009. (b) Sarabu, R.; Tilley, J. W.; Grimsby, J. The
discovery of piragliatin, a glucokinase activator. RSC Drug Discovery
Ser. 2011, 4, 51−70.
(9) Myers, A. G.; Yang, H. B.; Chen, H.; Gleason, J. L. Use of
pseudoephedrine as a practical chiral auxiliary for symmetric synthesis.
J. Am. Chem. Soc. 1994, 116, 9361−9362.
(10) Miller, R. D.; McKean, D. R. Ring opening of cyclopropyl
ketones by trimethylsilyl iodide. J. Org. Chem. 1981, 46, 2412−2414.
(11) Mash, E. A; Torok, D. S. Homochiral ketals in organic
Synthesis. Diastereoselective cyclopropanation of α,β-unsaturated
ketals derived from (S,S)-(−)-hydrobenzoin. J. Org. Chem. 1989, 54,
250−253.
(12) Dieter, R. K.; Pounds, S. Ring-opening reactions of electrophilic
cyclopropanes. J. Org. Chem. 1982, 47, 3174−3177.
(13) O'Doherty, R. M.; Lehman, D. L.; Telemaque-Potts, S.;
Newgard, C. B. Metabolic impact of glucokinase overexpression in
liver: Lowering of blood glucose in fed rats is accompanied by
hyperlipidemia. Diabetes 1999, 48, 2022−2027.
(14) See, for example (a) Mizutani, T.; Yoshida, K.; Kawazoe, S.
Formation of toxic metabolites from thiabendazole and other thiazoles
in mice. Drug Metab. Dispos. 1994, 22, 750−755. (b) Stevens, G. J.;
Hitchcock, K.; Wang, Y. K.; Coppola, G. M.; Versace, R. W.; Chin, J.
A.; Shapiro, M.; Suwanrumpha, S.; Mangold, L. K. In vitro metabolism
of N-(5-chloro-2-methylphenyl)-N-(2-methylproyl)thiourea: Species
comparison and identification of a novel thiocarbamide-glutathione
adduct. Chem. Res. Toxicol. 1997, 10, 733−741.
P
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