Journal of Medicinal Chemistry
ARTICLE
All animal studies described herein were approved by Merck Research
Laboratories Institutional Animal Care and Use Committee.
silica gel chromatography (0ꢀ20% ethyl acetate in hexanes) to provide
tert-butyl 4-cyano-4-pyridin-2-ylpiperidine-1-carboxylate as a colorless oil.
A solution of tert-butyl 4-cyano-4-pyridin-2-ylpiperidine-1-carboxy-
late (3.11 g, 10.8 mmol) in ethyl acetate (0.2 M, 54 mL) and cooled to
0 °C was saturated with HCl(g) for 5 min. The reaction was warmed to rt
and after 2 h, diluted with dichloromethane (100 mL), and neutralized
with saturated aqueous sodium bicarbonate. The mixture was parti-
tioned, and the aqueous layer was extracted 6 times with dichloro-
methane. The combined organic fractions were dried over sodium
sulfate, filtered, and concentrated in vacuo to provide 11i (1.01 g,
99.0%) as a beige solid. MS m/z = 188.3 (MHþ). 1H NMR (400 MHz,
CD3OD) δ 2.26 ꢀ 2.39 (m, 4H), 3.20 (ddd, J = 3.2 Hz, 12.9 Hz, 12.9 Hz,
2H), 3.37 ꢀ 3.41 (dt, J = 2.9 Hz, 2.9 Hz, 13.4 Hz, 2H), 7.39 (m, 1H),
7.67 (d, J = 8.1 Hz, 1H), 7.90 (t, J = 7.8 Hz, 1H), 8.61 (d, J = 4.9 Hz, 1H).
General Procedure for the Preparation of Compounds
4aꢀv: 1-[(4-Cyano-4-pyridin-2-ylpiperidin-1-yl)methyl]-4-
oxo-4H-quinolizine-3-carboxylic Acid (4i). To a 10ꢀ20 mL
Emrys process vial was added a solution of 8 (684 mg, 2.79 mmol),
11i (627 mg, 3.35 mmol), glacial acetic acid (0.96 mL, 16.7 mmol), and
dichloroethane (0.68 M, 4.1 mL). The mixture was stirred vigorously,
and resin-bound MP-cyanoborohydride (2.74 g, 5.58 mmol) was added.
The reaction was irradiated in a Emrys Optimizer microwave to 120 °C
for 30 min, then cooled to room temperature, filtered, and concentrated
in vacuo. The residue was subjected to silica gel chromatography
(55ꢀ75% ethyl acetate in hexanes) to provide ethyl 4-[(4-cyano-4-
pyridin-2-ylpiperidin-1-yl)methyl]-1-oxo-1,8a-dihydronaphthalene-2-
carboxylate as a yellow solid.
To a solution of ethyl 4-[(4-cyano-4-pyridin-2-ylpiperidin-1-yl)methyl]-
1-oxo-1,8a-dihydronaphthalene-2-carboxylate (705 mg, 1.69 mmol) in a
2:1 mixture of THF/ethanol (0.2 M, 8.4 mL) was added aqueous
sodium hydroxide NaOH (1.0 N, 1.78 mL, 1.78 mmol). The mixture was
warmed to 60 °C for 16 h, cooled to room temperature, and concen-
trated in vacuo. The residue was subjected to purification via preparative
reverse phase HPLC. The purified fractions were combined and con-
centrated in vacuo, then treated with aqueous sodium hydroxide (1.0 N,
2.74 mL, 2.74 mmol). After 1 h, the reaction was concentrated in vacuo.
The residue was washed with cold water (5 mL), redissolved in
methanol, and concentrated in vacuo to provide 4i (0.688 g, 80.0%)
as a yellow solid. 1H NMR (500 MHz, CD3OD) δ 2.13 (d, J = 11.7 Hz,
2H), 2.23 (t, J = 12.7 Hz, 2H), 2.55 (t, J = 12.1 Hz, 2H), 3.09 (d, J = 12.2
Hz, 2H), 3.82 (s, 2H), 7.29 (t, J = 7.0 Hz, 1H), 7.32ꢀ7.36 (m, 1H), 7.62
(d, J = 8.0 Hz, 1H), 7.69 (t, J = 6.3 Hz, 1H), 7.85 (t, J = 7.8 Hz, 1H), 8.30
(s, 1H), 8.18 (d, J = 8.9 Hz, 1H), 8.55 (d, J = 3.9 Hz, 1H), 9.37 (d, J = 7.2
Hz, 1H). MS m/z = 389.16 (MHþ).
Fluorometric Imaging Plate Reader (FLIPR) Assay. Chinese
hamster ovary (CHO) cells under control of the nuclear factor of
activated T cells (NFAT) promoter (CHONFAT) cells expressing M1,
M2, M3, M4, M5, rhesus M1, dog M1, mouse M1, and rat M1 (in CHOK1
from ATCC) receptors were plated (25,000 cells per well) in clear-
bottomed, poly D-lysine-coated 384-well plates in growth medium by
using a Labsystems (Chicago) Multidrop. The plated cells were grown
overnight at 37 °C in the presence of 6% CO2. The next day, the cells
were washed with 3 ꢁ 100 μL assay buffer (Hanks’ balanced salt solution
containing 20 mM Hepes, 2.5 mM probenecid, and 0.1% BSA). The cells
were incubated with 1 μM Fluo-4AM (Molecular Probes) for 1 h at
37 °C and 6% CO2. The extracellular dye was removed by washing as
describedabove. Ca2þ fluxwas measured by using aFLIPR384 fluorometric
imaging plate reader (Molecular Devices). Compounds were serially
diluted in 100% DMSO and then diluted in assay buffer to a 3ꢁ stock at
2% DMSO. This stock was then applied to the cells for a final DMSO
concentration of 0.67%. For potency determination, the cells were
preincubated with various concentrations of compound for 4 min and
then stimulated for 4 min with an EC20 concentration of agonist (i.e.,
ACh) for potentiation measurements.
Ethyl 4-Oxo-4H-quinolizine-3-carboxylate (7). To a heat-
dried flask under N2 was added a solution of diisopropylamine (19.1 mL,
0.134 mmol) in THF (1.0 M, 400 mL). The mixture was cooled to
ꢀ50 °C, and a solution of n-butyllithium in hexanes (2.5 M, 53.7 mL,
0.134 mol) was added. After 30 min, the reaction was cooled to ꢀ78 °C,
and a solution of 2-picoline (10.0 g, 0.107 mol) in THF (0.8 M, 80 mL)
was slowly added, keeping the temperature of the reaction below
ꢀ60 °C . After 30 min, a solution of diethyl ethoxymethylenemalonate
(24.5 mL, 0.121 mol) in THF (1.8 M, 67 mL) was slowly added, keeping
the temperature of the reaction below ꢀ60 °C. The mixture was slowly
warmed to ꢀ20 °C over 2 h, then poured into water, and extracted
3 times with dichloromethane. The combined organic fractions were
concentrated in vacuo, and the residue was dissolved in xylenes
(84 mL). The mixture was heated to reflux for 16 h, cooled to room
temperature, and filtered. The filtrate was concentrated in vacuo, and the
residue was triturated overnight with diethyl ether. The solid was
collected via filtration to provide 7 (19.2 g, 82.0%) as an orange brown
solid. 1H NMR (400 MHz, CDCl3) δ 1.43 (t, J = 7.1 Hz, 3H), 4.43 (q, J =
7.1 Hz, 2H), 6.66 (d, J = 8.5 Hz, 1H), 7.18ꢀ7.21 (m, 1H), 7.57ꢀ7.63
(m, 2H), 8.41 (d, J = 8.6 Hz, 1H), 9.41 (d, J = 7.1 Hz, 1H). MS m/z =
218.3 (MHþ).
Ethyl 1-Formyl-4-oxo-4H-quinolizine-3-carboxylate (8).
To a solution of compound 7 (10.4 g, 47.9 mmol) dissolved in DMF
(2.14 M, 22.4 mL) was added dropwise phosphorus oxytrichloride
(11.2 mL, 0.120 mol). After 1 h, the mixture was poured into water and
extracted 3 times with dichloromethane. The combined organic frac-
tions were dried over sodium sulfate, filtered, and concentrated in vacuo.
The residue was subjected to silica gel chromatography (0ꢀ2% metha-
nol in dichloromethane) to provide 8 (4.02 g, 33.9%) as a yellow solid.
1H NMR (400 MHz, CDCl3) δ 1.32 (t, J = 7.1 Hz, 3H), 4.31 (q, J = 7.1
Hz, 2H), 7.73ꢀ7.76 (m, 1H), 8.29ꢀ8.33 (m, 1H), 8.78 (s, 1H), 9.33 (d,
J = 8.6 Hz, 1H), 9.44 (d, J = 7.8 Hz, 1H), 9.96 (s, 1H). MS m/z = 246.08
(MHþ).
General Procedure for the Preparation of Compounds
11bꢀh, j: 4-(2-Fluorophenyl)piperidine-4-carbonitrile (11c).
To a solution of tert-butyl bis(2-chloroethyl)carbamate (0.524 g, 2.16
mmol) and 2-fluorophenylacetonitrile (0.292 g, 2.16 mmol) in DMSO
(0.4 M, 5.4 mL) cooled to 18 °C was added sodium hydride (0.190 g,
4.76 mmol). The mixture was slowly warmed to room temperature, and
after 1 h, was heated to 50 °C. After 17 h, the reaction was diluted with
dichloromethane, washed 3 times with water, dried over sodium sulfate,
filtered, and concentrated in vacuo. The residue was subjected to silica
gel chromatography (0ꢀ25% ethyl acetate in hexanes) to provide tert-
butyl-4-cyano-4-(2-fluorophenyl)piperidine-1-carboxylate as an orange oil.
A solution of tert-butyl-4-cyano-4-(2-fluorophenyl)piperidine-1-car-
boxylate (0.511 g, 1.68 mmol) in ethyl acetate (0.1 M, 16 mL) cooled to
0 °C was saturated with HCl(g) for 5 min. After 30 min, the reaction was
concentrated in vacuo to provide the hydrogen chloride salt of 11c
(0.408 g, >99%) as a white solid. MS m/z = 205.2 (MHþ). 1H NMR
(400 MHz, CD3OD) δ 2.41 (ddd, J = 3.8 Hz, 13.9 Hz, 13.9 Hz, 2H),
2.57 (m, 2H), 3.42 (ddd, J = 2.9 Hz, 13.5 Hz, 13.5 Hz, 2H), 3.63 (m,
2H), 7.24 ꢀ 7.34 (m, 2H), 7.50 (m, 1H), 7.57 (t, J = 1.6 Hz, 1H).
General Procedure for the Preparation of Compounds 11i,
kꢀr: 4-Pyridin-2-ylpiperidine-4-carbonitrile (11i). To a solu-
tion of tert-butyl 4-cyanopiperidine-1-carboxylate (220 mg, 1.05 mmol)
and 2-fluoropyridine (0.094 mL, 1.10 mmol) in THF (0.2 M, 5.2 mL)
cooled to ꢀ78 °C under an atmosphere of nitrogen was added lithium
bis(trimethylsilyl)amide(1.0 M in THF, 1.47 mL, 1.47 mmol). After 1 h,
the reaction was warmed to room temperature. After an additional 1 h,
the reaction was quenched with a saturated ammonium chloride
solution, extracted 3 times with dichloromethane, dried over sodium
sulfate, filtered, and concentrated in vacuo. The residue was subjected to
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dx.doi.org/10.1021/jm200400m |J. Med. Chem. 2011, 54, 4773–4780