Journal of Medicinal Chemistry
Article
(3-(2,6-Dichlorophenyl)-5-isopropylisoxazol-4-yl)methanol
(49). Methyl 3-(2,6-dichlorophenyl)-5-isopropylisoxazole-4-carboxy-
late (43, 0.27 g, 0.85 mmol, 1.0 equiv) was dissolved in THF (5 mL),
and the solution was cooled to 0 °C. A solution of diisobutylaluminum
hydride (1 M in toluene, 1.8 mL, 1.8 mmol, 2.1 equiv) was slowly
added. The reaction mixture was allowed to warm to rt and stirred for
24 h. The reaction mixture was then cooled to 0 °C again and quenched
with methanol (5 mL) and aqueous sodium hydroxide solution (2 M,
20 mL). The suspension was filtered through Celite, and the filtrate was
extracted with ethyl acetate (3 × 20 mL). The combined organic layers
were washed with saturated sodium chloride solution, dried over
MgSO4, and the solvents were evaporated in vacuo. The product was
purified by column chromatography using hexane/EtOAc (3:1) to
changed to Opti-MEM without supplements. Transient transfection
was carried out using Lipofectamine LTX reagent (Invitrogen)
according to the manufacturer’s protocol with pFR-Luc (Stratagene),
pRL-SV40 (Promega), and the respective pFA-CMV-hNR-LBD clone.
5 h after transfection, medium was changed to Opti-MEM
supplemented with penicillin (100 U/mL), streptomycin (100 μg/
mL), now additionally containing 0.1% DMSO and the respective test
compounds or 0.1% DMSO alone as untreated control. Each
concentration was tested in duplicates, and each experiment was
repeated independently at least twice. Following overnight (12−14 h)
incubation with the test compounds, cells were assayed for luciferase
activity using Dual-Glo luciferase assay system (Promega) according to
the manufacturer’s protocol. Luminescence was measured with a Tecan
Spark luminometer (Tecan Deutschland GmbH, Germany). Normal-
ization of transfection efficiency and cell growth was done by division of
firefly luciferase data by renilla luciferase data and multiplying the value
by 1000 resulting in relative light units (RLU). Fold activation was
obtained by dividing the mean RLU of test compounds at a respective
concentration by the mean RLU of untreated control. Relative
activation was obtained by dividing the fold activation of a test
compound at a respective concentration by the fold activation of a
respective reference agonist at 1 μM (RARα, tretinoin; RXRα,
bexarotene; PPARα, GW7647; PPARγ, pioglitazone; PPARδ,
L165,041; LXRα, T0901317; FXR, GW4064). All hybrid assays were
validated with the above-mentioned reference agonists which yielded
EC50 values in agreement with literature.
1
obtain 49 as a colorless solid (0.14 g, 58%). H NMR (500 MHz,
CDCl3) δ = 7.46−7.41 (m, 2H), 7.39−7.34 (m, 1H), 4.35−4.34 (m,
2H), 3.41−3.27 (m, 1H), 1.44 (d, J = 7.0 Hz, 6H). 13C NMR (126
MHz, CDCl3) δ = 176.47, 159.23, 135.84, 131.48, 128.33, 128.16,
112.71, 53.82, 27.13, 21.08. MS (ESI+): m/z 308.04 ([M + Na]+).
2-Chloro-4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-
yl)methoxy)benzaldehyde (108). 2-Chloro-4-hydroxybenzalde-
hyde (106, 0.10 g, 0.64 mmol, 1.0 equiv), (3-(2,6-dichlorophenyl)-5-
isopropylisoxazol-4-yl)methanol (49, 0.18 g, 0.64 mmol, 1.0 equiv),
and triphenylphosphine (0.17 g, 0.64 mmol, 1.0 equiv) were dissolved
in methylene chloride (13 mL). Diisopropyl azodicarboxylate (0.12
mL, 0.64 mmol, 1.0 equiv) was added dropwise. The reaction mixture
was stirred at rt for 4 h. The solvent was evaporated in vacuo and the
product was purified by column chromatography using hexane/EtOAc
(5:1) to obtain 108 as a yellow oil (0.21 g, 78%). 1H NMR (500 MHz,
CDCl3) δ = 10.30 (s, 1H), 7.82 (d, J = 8.7 Hz, 1H), 7.41 (d, J = 0.9 Hz,
1H), 7.40 (s, 1H), 7.35−7.32 (m, 1H), 6.79 (d, J = 2.4 Hz, 1H), 6.75
(dd, J = 8.7, 2.3 Hz, 1H), 4.80 (s, 2H), 3.32 (hept, J = 6.9 Hz, 1H), 1.44
(d, J = 7.0 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ = 188.59, 176.90,
163.15, 159.09, 139.77, 135.90, 131.61, 131.15, 128.34, 127.66, 126.56,
115.86, 114.21, 108.45, 59.99, 21.74, 20.97. MS (ESI+): m/z 424.07
([M + H]+).
Ethyl 2-(4-((2-Chloro-4-((3-(2,6-dichlorophenyl)-5-isopropy-
lisoxazol-4-yl)methoxy)benzyl)amino)phenyl)acetate (112). 2-
Chloro-4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)-
methoxy)benzaldehyde (108, 80 mg, 0.19 mmol, 1.0 equiv), ethyl 2-(4-
aminophenyl)acetate (109, 36 mg, 0.20 mmol, 1.0 equiv), and diethyl
1,4-dihydro-2,6-dimethylpyridine-3,5-dicarboxylate (Hantzsch ester,
0.15 g, 0.60 mmol, 3.0 equiv) were dissolved in toluene (20 mL),
molecular sieves (5 Å, 2 g) were added, and the reaction mixture was
stirred at 70 °C for 24 h. Aqueous hydrochloric acid (20 mL, 10%) was
then added, and the product was extracted with ethyl acetate (3 × 30
mL). The combined organic layers were dried over MgSO4, and the
solvents were evaporated in vacuo. The product was purified by column
chromatography using hexane/EtOAc (5:1) to obtain 112 as a yellow
oil (76 mg, 63%). 1H NMR (500 MHz, CDCl3) δ = 7.26 (s, 1H), 7.21
(d, J = 8.4 Hz, 1H), 7.09−7.06 (m, 3H), 6.90 (d, J = 2.5 Hz, 1H), 6.69−
6.66 (m, 1H), 6.64 (d, J = 8.4 Hz, 1H), 6.57 (d, J = 8.5 Hz, 2H), 5.21 (s,
2H), 4.31 (s, 2H), 4.20−4.14 (m, 2H), 3.48 (s, 2H), 2.75−2.66 (m,
1H), 1.29−1.22 (m, 9H). 13C NMR (126 MHz, CDCl3) δ = 172.60,
168.29, 156.16, 151.61, 145.01, 133.90, 130.30, 130.23, 130.18, 128.21,
123.32, 123.15, 116.81, 115.44, 114.36, 113.22, 99.68, 61.60, 60.87,
45.67, 40.68, 24.91, 19.33, 14.59. MS (ESI+): m/z 587.20 ([M + H]+).
Hybrid Reporter Gene Assays. Plasmids. The Gal4-fusion
receptor plasmids pFA-CMV-hPPARα-LBD,10 pFA-CMV-
hPPARγLBD,10 pFA-CMV-hPPARδ-LBD,10 pFA-CMV-hLXRα-
LBD,19 pFA-CMV-hRXRα-LBD,20 pFA-CMV-hRARα-LBD,21 and
pFA-CMV-hFXR-LBD11 coding for the hinge region and LBD of the
canonical isoform of the respective nuclear receptor have been reported
previously. pFR-Luc (Stratagene) was used as reporter plasmid and
pRL-SV40 (Promega) for normalization of transfection efficiency and
cell growth
FXRE and PPRE Reporter Assays. The effect of 25 on human
response elements for PPAR and FXR was studied using previously
described firefly reporter constructs comprising the human PPRE22 or
the human BSEP-promoter region containing FXRE.11 Renilla
luciferase (pRL-SV40) served for normalization purposes.
Assay Procedure. HEK293T cells were grown in DMEM high
glucose, supplemented with 10% FCS, sodium pyruvate (1 mM),
penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C and
5% CO2. The day before transfection, HEK293T cells were seeded in
96-well plates (3.0 × 104 cells/well). Before transfection, the medium
was changed to Opti-MEM without supplements. Transient trans-
fection was carried out using Lipofectamine LTX reagent (Invitrogen)
according to the manufacturer’s protocol with PPRE1-pGL322 or
BSEP-pGL311 and pRL-SV40. 5 h after transfection, the medium was
changed to Opti-MEM supplemented with penicillin (100 U/mL),
streptomycin (100 μg/mL), now additionally containing 0.1% DMSO
and the respective test compounds or 0.1% DMSO alone as untreated
control. Each concentration was tested in duplicates, and each
experiment was repeated independently three times. Following
overnight (12−14 h) incubation with the test compounds, cells were
assayed for luciferase activity using Dual-Glo luciferase assay system
(Promega) according to the manufacturer’s protocol. Luminescence
was measured with a Tecan Spark luminometer (Tecan Deutschland
GmbH, Germany). Normalization of transfection efficiency and cell
growth was done by division of firefly luciferase data by renilla luciferase
data and multiplying the value by 1000 resulting in relative light units
(RLU).
Quantification of FXR Regulated Gene Expression in HepG2
Cells. FXR target gene quantification was performed as described
previously.23 HepG2 cells were grown in DMEM high glucose,
supplemented with 10% FCS, 1 mM SP, penicillin (100 U/mL), and
streptomycin (100 μg/mL) at 37 °C and 5% CO2. Cells were seeded in
6-well plates (2 × 106 per well). 24 h after seeding, medium was
changed to MEM supplemented with 1% charcoal stripped FCS,
penicillin (100 U/mL), streptomycin (100 μg/mL), and 2 mM L-
glutamine. After an additional 24 h, cells were incubated with test
compound 25 (3 μM) or GW4064 (3, 1 μM) dissolved in the same
medium with 0.1% DMSO or medium with 0.1% DMSO alone as
untreated control for 8 h, harvested, washed with cold phosphate
buffered saline (PBS), and then directly used for RNA extraction. Total
RNA was extracted using the total RNA Mini Kit (R6834-02, Omega
Bio-Tek, Inc., Norcross, GA, USA). An amount of 2 μg of RNA was
then reverse-transcribed into cDNA using the high capacity cDNA
reverse transcription kit (4368814, Thermo Fischer Scientific, Inc.)
Assay Procedure. HEK293T cells were grown in DMEM high
glucose, supplemented with 10% FCS, sodium pyruvate (1 mM),
penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C and
5% CO2. The day before transfection, HEK293T cells were seeded in
96-well plates (3.0 × 104 cells/well). Before transfection, medium was
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J. Med. Chem. XXXX, XXX, XXX−XXX