Journal of Medicinal Chemistry
Drug Annotation
were used as received. All reactions were carried out under an argon
atmosphere unless noted otherwise. Reaction mixtures were
concentrated under reduced pressure at 40−65 °C on a rotary
evaporator. Analytical thin layer chromatography (TLC) was
performed on silica gel 60 F254 plates from Merck. Flash column
chromatography separations were preformed on RediSep normal
phase silica flash columns (230−400 mesh, 60 Å, Teledyne ISCO).
LC−MS measurements were obtained using either a Shimadzu HPLC
system with a Phenomenex Luna C18 column (5 μm, 100 Å, 4.6 mm
× 30 mm), operated at 40 °C with 0.1% TFA modified ACN/water
mobile phases or a Waters Acquity UPLC system with an Acquity
UPLC BEH C18 (1.7 μm, 130 Å, 2.1 mm × 50 mm) column operated
at 50 °C with 0.05% TFA modified ACN/water mobile phases. UV
detection was carried out with a Shimadzu SPD-10AV at 220 nm, and
mass detection was carried out with Waters ZQ single quadrupole
mass spectrometer hybrid system using positive electrospray
ionization.
Final HPLC purity determination was made with a Shimadzu
HPLC system, with a Waters Sunfire C18 (3.5 μm, 100 Å, 3.0 mm ×
150 mm) column and a Waters Xbridge phenyl (3.5 μm, 130 Å, 3.0
mm × 150 mm) column with 0.05% TFA modified ACN/water
mobile phases with gradient run from 10% to 90% ACN. UV detection
was carried out with a Shimadzu SPD-20AV at 220 and 254 nm. All
final compounds achieved a minimum of 95% purity. All
chromatography was carried out with HPLC grade organic solvents.
1H NMR spectra were obtained on a Bruker 400 MHz spectrometer
ppm 7.82−7.91 (m, 2 H), 7.62 (dd, J = 6.78, 8.53 Hz, 1 H), 7.32 (s, 1
H), 6.03 (s, 1 H), 4.57−4.64 (m, 1 H), 3.59−3.68 (m, 2 H), 3.46−3.55
(m, 2 H), 3.11 (s, 3 H), 1.84−2.02 (m, 4 H), 1.48 (s, 9 H). LC−MS:
m/z = 501.2 [M + H].
5-Chloro-1-(2-fluoro-4-(methylsulfonyl)phenyl)-4-(piperi-
din-4-yloxy)pyridin-2(1H)-one, HCl (45). A mixture of tert-butyl 4-
(5-chloro-1-(2-fluoro-4-(methylsulfonyl)phenyl)-2-oxo-1,2-dihydro-
pyridin-4-yloxy)piperidine-1-carboxylate (44) (1.31 g, 2.62 mmol) and
HCl (37% aq, 5.00 mL, 60.9 mmol) was stirred at room temperature
for 15 min. The reaction mixture was concentrated in vacuo to give 45
(1.15 g, 2.62 mmol, 100% yield) as a white solid. 1H NMR (400 MHz,
DMSO-d6) δ 8.96 (br s, 2H), 8.12 (s, 1H), 8.02 (dd, J = 1.76, 9.24 Hz,
1H), 7.88−7.94 (m, 1H), 7.79−7.87 (m, 1H), 6.31 (s, 1H), 4.80−4.92
(m, 1H), 3.35 (s, 3H), 3.16−3.26 (m, 2H), 3.03−3.16 (m, 2H), 2.10−
2.23 (m, 2H), 1.86−1.99 (m, 2H). LC−MS: m/z = 401.1 [M + H].
5-Chloro-4-(1-(5-chloropyrimidin-2-yl)piperidin-4-yloxy)-1-
(2-fluoro-4-(methylsulfonyl)phenyl)pyridin-2(1H)-one (42). To
a solution of 5-chloro-1-(2-fluoro-4-(methylsulfonyl)phenyl)-4-(piper-
idin-4-yloxy)pyridin-2(1H)-one, HCl (45) (1.15 g, 2.62 mmol) and
DIEA (1.37 mL, 7.85 mmol) in DMF (10 mL) was added 5-chloro-2-
iodopyrimidine (0.755 g, 3.14 mmol). The reaction mixture was
heated at 60 °C for 4 h. The reaction mixture was concentrated in
vacuo to give a yellow oil. The oil was dissolved in EtOAc (200 mL)
and washed with saturated NaHCO3 (200 mL), and the organic layer
was concentrated in vacuo. The residue was purified by flash column
chromatography (0−100%, EtOAc in DCM) to give the title
compound as an off-white solid. To increase the purity of the isolated
solid, the solid was added to MeOH (250 mL) and refluxed for 6 h.
The slurry was cooled to 4 °C, filtered, washed with MeOH (100 mL),
and dried in vacuo giving 42 (0.934 g, 1.82 mmol, 69.5% yield) as a
using the indicated solvent. Chemical (δ) shifts are reported in ppm
from tetramethylsilane with the residual solvent signal as the internal
standard; signals are expressed as s = singlet, d = doublet, t = triplet, q
= quartet, m = multiplet, and br = broad. Coupling constants (J) are in
hertz (Hz).
All animals were treated according to the standards of the BMS
Animal Care and Use Committee (ACUC). Animals were maintained
at the Bristol-Myers Squibb animal facilities (Hopewell, NJ) with 12 h
light/dark cycle and free access to food and water. The animals were
fed ad libitum normal chow.
1
white solid. H NMR (400 MHz, CDCl3) δ 8.25 (s, 2H), 7.82−7.94
(m, 2H), 7.63 (dd, J = 6.65, 8.41 Hz, 1H), 7.33 (d, J = 1.00 Hz, 1H),
6.07 (s, 1H), 4.70 (tt, J = 3.39, 6.53 Hz, 1H), 3.98−4.08 (m, 2H),
3.87−3.96 (m, 2H), 3.12 (s, 3H), 2.02−2.11 (m, 2H), 1.92−2.01 (m,
2H). LC−MS: m/z = 513.0 [M + H].
21-Day db/db Mouse Study. A cohort of 90 male BKS Cg-m+/+
Lepr db/db/J mice (5 weeks of age at arrival) purchased from Jackson
Laboratories (Bar Harbor, ME) was acclimated to the vivarium for 1
week prior to study start. Two days before the study date, mice were
randomized to day 1 or day 2 oGTT based on fed blood glucose and
HbA1c levels, measured by glucometer (Accu-Check) and COBAS
Mira, respectively, via tail nick. Following an overnight fast, mice were
randomized to treatment based on their blood glucose and body
weights. After 40 min in adaptation to the new group, mice were bled
again for blood glucose level at the −60 min time point, and 5 μL of
whole blood was collected into a COBAS tube containing 250 μL of
hemolysate reagent for determination of HbA1c. After the blood
collection, mice received vehicle (40% PEG 400, 10% Cremophor, and
50% water) or compound 29 orally (5 mL/kg). At 60 min after dosing,
an additional glucose measurement was taken. Immediately after the 0
min time point collection, mice were given an oral glucose challenge
with 50% dextrose (4 mL/kg). Plasma glucose was measured at 30, 60,
90, and 120 min after the glucose challenge. Mice were then returned
to home cages and given free access to food and water. From day 2 to
day 21, mice were orally dosed with the vehicle or compound once
daily at 9:00 a.m. After day 21 of dosing, mice were fasted overnight
and fasting plasma glucose and HbA1c were measured at 24 h after
dose, followed by a day 22 dosing in which trunk blood from three
mice of each group was collected for PK analysis by decapitation under
anesthesia with CO2 3.5 h after compound administration. All blood
samples (except the 5 μL blood for HbA1c) were centrifuged at 8000
rpm at 4 °C for 10 min. Remaining mice (seven mice in each group)
were returned to home cage and given access to food and water. The
24 h postdose plasma levels of compounds from trunk blood were
determined as well.
tert-Butyl 4-((5-Chloro-2-oxo-1,2-dihydropyridin-4-yl)oxy)-
piperidine-1-carboxylate (43). To an ice-cooled mixture of 5-
chloro-4-hydroxypyridin-2(1H)-one (11.86 g, 81 mmol), tert-butyl 4-
hydroxypiperidine-1-carboxylate (16.40 g, 81 mmol), and triphenyl-
phosphine (23.51 g, 90 mmol) in a mixture of DMF (55 mL) and
THF (55 mL) was added dropwise a solution of (E)-diisopropyl
diazene-1,2-dicarboxylate (17.65 mL, 90 mmol) in THF (50 mL) over
30 min. The mixture was allowed to warm to room temperature and
stirred for 2 days. The reaction mixture was concentrated in vacuo to
give a yellow oil. The oil was dissolved in EtOAc (300 mL), washed
with saturated NaHCO3 (150 mL), and the organic layer was
concentrated in vacuo. The residue was purified by flash column
chromatography. The column was initially eluted with EtOAc in DCM
(0−100%) until the impurities were off the column, and the mobile
phase was switched to MeOH in DCM (0−10%) to collect 43 (14.7 g,
1
55%) as a white solid. H NMR (400 MHz, CDCl3) δ ppm 12.98 (br
s, 1 H), 7.35 (s, 1 H), 5.92 (s, 1 H), 4.51−4.63 (m, 1 H), 3.57−3.68
(m, 2 H), 3.43−3.54 (m, 2 H), 1.79−2.00 (m, 4 H), 1.48 (s, 9 H).
LC−MS: m/z = 329.0 [M + H].
tert-Butyl 4-(5-Chloro-1-(2-fluoro-4-(methylsulfonyl)-
phenyl)-2-oxo-1,2-dihydropyridin-4-yloxy)piperidine-1-car-
boxylate (44). To a solution of tert-butyl 4-(5-chloro-2-oxo-1,2-
dihydropyridin-4-yloxy)piperidine-1-carboxylate (43) (1.93 g, 5.87
mmol) in DMF (15 mL) was added portionwise sodium hydride (60%
w/w in mineral oil, 0.282 g, 7.04 mmol) over a period of 5 min. The
mixture was stirred at room temperature for 30 min, followed by the
addition of 1,2-difluoro-4-(methylsulfonyl)benzene (1.47 g, 7.63
mmol). The reaction mixture was heated at 130 °C for 2 h, cooled
to room temperature, and quenched by the addition of H2O (200
mL). The quenched reaction mixture was extracted with EtOAc (200
mL). The organic layer was concentrated in vacuo. The residue was
purified by flash column chromatography (0−100% EtOAc in DCM),
with the unwanted O-isomer eluting before the desired N-isomer, to
give 44 (1.31 g, 45%) as a white solid. 1H NMR (400 MHz, CDCl3) δ
Mouse Acute Oral Glucose Tolerance Test Assay. Male
C57BL/6J mice (8 weeks of age at arrival) from Jackson Laboratories
(Bar Harbor, ME) were single-housed and had free access to normal
chow (Teklad 2018) and water ad libitum. They were allowed to
acclimate to the animal facilities for 2 weeks. The mice were fasted
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dx.doi.org/10.1021/jm501175v | J. Med. Chem. 2014, 57, 7499−7508