Journal of Medicinal Chemistry
Article
and deoxygenated by vacuum-N2 fill cycles. The catalyst Pd(PPh3)4 (4
mg, 10% mol) was added, and the mixture was deoxygenated again
before heated in microwave reactor to 100 °C for 10 min. The mixture
was quenched with 4 mL of CH3CN−H2O (3:1, with 5% TFA) and
product separated through reverse phase preparative HPLC. The
collected product was treated with 1 mL of TFA−DCM (1:2) for 30
min and the residue lyophilized to give 30 mg (95%) of N-[4-((1S)-1-
{3-(3,5-dichlorophenyl)-5-[6-methoxy-2-naphthyl]-1H-pyrazol-1-yl}-
ethyl)benzoyl]-β-alanine (9m) as a fine powder. 1H NMR (500 MHz,
DMSO-d6) δ: 1.90 (d, J = 7.0 Hz, 3H), 2.47 (t, J = 7 Hz, 2H), 3.41 (q,
J = 7 Hz, 2H), 3.89 (s, 3H), 5.76 (q, J = 7.0 Hz, 1H), 7.16 (s, 1H),
7.20 (d, J = 8.4 Hz, 2H), 7.23 (dd, J = 2.6, 9.0 Hz, 1H), 7.39 (d, 2.6
Hz, 1H), 7.43 (dd, J = 1.7, 8.4 Hz, 1H), 7.56 (t, J = 1.9 Hz, 1H), 7.72
(d, J = 8.4 Hz, 2H), 7.83 (d, J = 9.0 Hz, 1H), 7.86 (d, J = 1.7 Hz, 1H),
7.91 (d, J = 8.4 Hz, 1H), 7.93 (d, J = 1.9 Hz, 2H), 8.44 (t, NH, J = 5.6
Hz, 1H). MS C32H27Cl2N3O4 calcd 587.14; obsd (M + 1) 588.24.
N-[(4-{(1S)-1-[3-(2,5-Dichlorophenyl)-5-(6-methoxynaphtha-
len-2-yl)-1H-pyrazol-1-yl]ethyl}phenyl)-carbonyl]-β-alanine
(15c). Step 1. A suspension of MgCl2 (3.5 g, 35 mmol), potassium
ethyl malonate (4.6 g, 30 mmol), and triethylamine (15 mL, 105
mmol) in dry ethyl acetate (100 mL) was stirred at 40 °C overnight. A
suspension of 6-methoxynaphthyl-2-acid chloride (4.9 g, 22.2 mmol)
in ethyl acetate (20 mL) was then added to the above mixture. The
reaction was stirred at room temperature for 2.5 h. The reaction was
quenched with 60 mL of 2N HCl, stirred for 5 min, and then washed
with 0.5 N HCl twice, 5% K2CO3 twice, and brine twice. Evaporation
of solvent and vacuum drying afforded 6.0 g (98%) of ethyl 3-(6-
Hz, 1H), 6.54 (s, 1H), 6.78 (br, 1H), 7.16 (br, 1H), 7.17 (dd, J = 2.6
Hz, 9 Hz, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 7.78
(d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.5 Hz, 1H), 7.93 (dd, J = 1.8 Hz, 8.5
Hz, 1H), 8.14 (d, J = 1.6 Hz, 1H). MS C31H32F3N3O7S calcd 647.19;
obsd (M + Na) 670.02. Compound 13: 1H NMR (500 MHz, CDCl3)
δ 1.46 (s, 9H), 1.85 (d, J = 7.1 Hz, 3H), 2.55 (t, J = 5.8 Hz, 2H), 3.68
(q, J = 5.8 Hz, 2H), 3.95 (s, 3H), 5.52 (q, J = 7.1 Hz, 1H), 6.23 (s,
1H), 6.85 (br, 1H), 7.16 (d, J = 2.6 Hz, 1H), 7.21 (dd, J = 2.6 Hz, 8.7
Hz, 1H), 7.22 (d, J = 8.4 Hz, 2H), 7.24 (dd, J = 1.5 Hz, 8.4 Hz, 1H),
7.62 (d, J = 1.5 Hz, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.71 (d, J = 8.3 Hz,
2H), 7.76 (d, J = 8.4 Hz, 1H). MS C31H32F3N3O7S calcd 647.19; obsd
(M + Na) 670.20.
Step 5 (Method B). Compound 13 (26 mg, 0.04 mmol), 2,5-
dichlorophenylboronic acid (15 mg, 0.08 mmol), and PdCl2(dppf) (12
mg, 0.014 mmol) were suspended in toluene (0.6 mL) in a glass tube.
A solution of Cs2CO3 (5 M, 25 ul) was added. The mixture was
deoxygenated by vacuum-N2 fill cycles and heated in a microwave
reactor to 140 °C for 10 min. The reaction mixture was filtered
through a glass-fiber plug and solvent removed under reduced
pressure. The residue was dissolved in CH3CN−H2O and purified by
reverse phase preparatory HPLC. The intermediate ester thus
obtained was deprotected by treatment with TFA−DCM (1:2, 1
mL) for 30 min. Evaporation of solvent and lyophilization from
CH3CN−H2O yielded 6.4 mg (27%) of N-1-(4-(2-hydroxycarbony-
lethylamino-carbonyl)phenyl)ethyl-3-(2,5-dichlorophenyl)-5-(6-me-
thoxynaphth-2-yl)pyrazole (15c( )) as a fine powder. 1H NMR (500
MHz, DMSO-d6) δ: 1.90 (d, J = 6.9 Hz, 3H), 2.47 (t, J = 7.1 Hz, 2H),
3.41 (q, J = 7.1 Hz, 2H), 3.89 (s, 3H), 5.79 (q, J = 6.9 Hz, 1H), 7.02
(s, 1H), 7.22 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 9.0 Hz, 1H), 7.39 (d, J =
2.6 Hz, 1H), 7.44 (dd, J = 1.7 Hz, 8.3 Hz, 1H), 7.47 (dd, J = 2.6 Hz,
8.6 Hz, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.74 (d, J = 8.4 Hz, 2H), 7.83
(d, J = 9.0 Hz, 1H), 7.88 (d, J = 1.7 Hz, 1H), 7.90 (d, J = 8.6 Hz, 1H),
7.92 (d, J = 2.6 Hz, 1H). MS C32H27Cl2N3O4 calcd (M + H)
588.1451; obsd 588.1444.
1
methoxy-2-naphthyl)-3-oxopropanoate (10) as an oil. H NMR (500
MHz, CDCl3) δ: 1.26 (t, J = 7.1 Hz, 3H), 3.95 (s, 3H), 4.09 (s, 2H),
4.23 (q, J = 7.1 Hz, 2H), 7.15 (d, J = 2.5 Hz, 1H), 7.21 (dd, J = 2,5 Hz,
9.0 Hz, 1H), 7.77 (d, J = 8.7 Hz, 1H), 7.85 (d, J = 9.0 Hz, 1H), 7.98
(dd, J = 1.8 Hz, 8.7 Hz, 1H), 8.38 (d, J = 1.8 Hz, 1H). About 10% of
10 existed in the enol and spectrum of the ketone form was reported.
Step 2. Compound 10 (5.0 g, 18.3 mmol) and anhydrous hydrazine
(0.63 mL 20 mmol) were refluxed in HOAc (100 mL) for 3 h. Solvent
was removed under reduced pressure, and the residue was washed with
DCM and collected by filtration to give 3.8 g (86%) of 5-(6-methoxy-
2-naphthyl)-2,4-dihydro-3H-pyrazol-3-one (11) as an off-white solid.
Racemic N-(4-{1-[3-(2,5-dichlorophenyl)-5-(6-methoxy-2-naph-
thyl)-1H-pyrazol-1-yl]ethyl}benzoyl)-β-alanine was separated into its
enantiomers by chromatography using a ChiralPak AS column (10
mm × 250 mm), eluting with 40% MeOH−CO2 (0.1%TFA).
Compound 15c is the slower eluting enantiomer.
1
This compound exists in the hydroxypyrazole form in DMSO. H
NMR (500 MHz, DMSO-d6) δ: 3.87 (s, 3H), 5.95 (s, 1H), 7.17 (dd, J
= 2.7 Hz, 9.0 Hz, 1H), 7.31 (d, J = 2.7 Hz, 1H), 7.74−7.84 (m, 3H),
8.11 (br s, 1H), 9.66 (br s, 1H), 12 (br, 1H). MS C14H12N2O2 calcd
240.09; obsd (M + 1) 241.08.
Glucagon Binding Assay. A CHO cell line expressing the human
glucagon receptor (CHO hGCGR) was maintained and membranes
prepared as described in Chicchi et al.42 Membranes (2−5 μg) were
incubated in buffer containing 50 mM Tris, pH 7.5, 5 mM MgCl2, 2
mM EDTA, 1% bovine serum albumin, 12% glycerol, 0.2 mg of wheat
germ agglutinin-coated polyvinyltoluene scintillation proximity assay
beads (Amersham Kit RPNP0001), increasing concentration of
compound (diluted in 100% DMSO and added to the assay at a
final concentration of 2.5%), and 50 pM 125I-glucagon. The assay was
incubated for 3 h at room temperature, and the total bound
radioactivity was measured with a Wallac-Microbeta counter. Non-
specific counts were determined using 1 μM unlabeled glucagon. Data
were analyzed using the nonlinear regression analysis software
GraphPad Prism, v4.
cAMP Assay. CHO hGCGR cells were grown in Iscove’s Modified
Dulbecco’s Medium (IMDM), 10% FBS, 1 mM L-glutamine,
penicillin−streptomycin (100 u/mL), and 500 ug G418/mL for 3−4
days before harvesting using Enzyme-Free Dissociation Media
(EFDM, Specialty Media). The cells were centrifuged at low speed
and resuspended in stimulation buffer (FlashPlate, Perkin-Elmer, Kit
SMP0004A). Compounds were diluted from DMSO stocks and added
to the assay at a final concentration of 5% DMSO. Cells were
preincubated with compound or DMSO controls for 30 min.
Glucagon (250 pM) was added, and the samples were incubated at
room temperature for an additional 30 min. The assay was terminated
with the addition of the FlashPlate kit detection buffer. The assay was
then incubated for an additional 3 h at room temperature, and bound
radioactivity was measured using a liquid scintillation counter
(TopCount-Packard Instruments). cAMP levels were determined as
per manufacturer’s instructions. For Schild Plot analysis, aliquots of
cells were preincubated with 56, 100, 178, 300, 560, and 1000 nM 9m
Step 3. Compound 11 (1.58 g, 6.58 mmol) and pyridine (1.62 mL,
20 mmol) were dissolved in THF (20 mL) at −78 °C. Triflic
anhydride (1.68 mL, 10 mmol) was added via syringe. The cooling
bath was removed, and the reaction mixture was stirred for 2 h. The
mixture was cooled down again to −78 °C and diluted with ethyl
acetate (50 mL) and 2N HCl (10 mL). The ethyl acetate layer was
washed with dilute HCl twice and brine twice. Evaporation of solvent
left a purple residue, which was purified by column chromatography
(SiO2, 0−2.5% ethyl acetate in DCM) to give 1.1 g (46%) of 3-(6-
methoxy-2-naphthyl)-1H-pyrazol-5-yl trifluoromethanesulfonate (12)
as a white solid. 1H NMR (500 MHz, DMSO-d6) δ: 3.89 (s, 3H), 6.93
(d, J = 2.2 Hz, 1H), 7.23 (dd, J = 2.7 Hz, 8.8 Hz, 1H), 7.37 (d, J = 2.7
Hz, 1H), 7.82−7.86 (m, 2H), 7.92 (d, J = 8.7 Hz, 1H), 8.28 (s, 1H).
MS C15H11F3N2O4S calcd 372.04; obsd (M + 1) 373.06.
Step 4. Compound 12 (1.36 g, 3.65 mmol), tert-butyl N-[4-(1-
hydroxyethyl)benzoyl]-β-alaninate (1.2 g, 4.02 mmol), and triphenyl-
phosphine (1.44 g, 5.48 mmol) were suspended in DCM (25 mL).
Diisopropyl azodicarboxylate (0.87 mL, 4.38 mmol) was added slowly.
The mixture was stirred for 2 h and then concentrated to ∼10 mL.
This residue was chromatographed (SiO2, 25−30% ethyl acetate
gradient) to give 0.73 g (31%) of tert-butyl N-{4-[1-(3-(6-methoxy-2-
naphthyl)-5-{[(trifluoromethyl)sulfonyl]oxy}-1H-pyrazol-1-yl)ethyl]-
benzoyl}-β-alaninate (14) and 1.17 g (49%) of tert-butyl N-{4-[1-(5-
(6-methoxy-2-naphthyl)-3-{[(trifluoromethyl)sulfonyl]oxy}-1H-pyra-
zol-1-yl)ethyl]benzoyl}-β-alaninate (13). Compound 14: 1H NMR
(500 MHz, CDCl3) δ 1.45 (s, 9H), 2.01 (d, J = 7.1 Hz, 3H), 2.53 (t, J
= 5.9 Hz, 2H), 3.67 (q, J = 5.9 Hz, 2H), 3.94 (s, 3H), 5.56 (q, J = 7.1
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dx.doi.org/10.1021/jm300579z | J. Med. Chem. 2012, 55, 6137−6148