Journal of Medicinal Chemistry
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(10 mL/mmol) and stirred at 70 °C until TLC monitoring (eluant
mixture: 6:4 n-hexane/EtOAc) showed reaction completion. Then,
TFA was removed under high vacuum with toluene stripping, and the
crude brown solid was diluted with DCM (5 mL). After centrifugation
(5000 RPM, 5 min), the solvent was then carefully removed with a
syringe. The process of DCM dilution (5 mL), centrifugation, and
DCM removal was repeated four times. The resulting, crude N1-
unsubstituted formyl pyrazoles 14a, 14d, and 14f were used without
further purification in the final reaction step.
Mono- and Bis-GHs, General Condensation Procedure F1:
Precipitation (3b, 4a′, 5d, 5e). AG.HCl (1.1 equiv) and 1 N aq HCl
(3−5 drops, catalytic) were sequentially added to a warm, vigorously
stirred suspension of 1 equiv of mono- (14d) or bis-formyl compounds
(8b, 11a′, or 14f) in absolute EtOH. After addition of catalytic acid, the
suspension became a solution. The reaction mixture was refluxed at 80
°C, with TLC monitoring (eluant mixtures: 100% EtOAc and 8:2
DCM/MeOH, with a few AcOH drops). Precipitation of a white solid
was observed after 4 h. Then, the reaction mixture was cooled to rt, and
the precipitate was filtered and washed with EtOH (15 mL) and 5:1
MeCN/H2O (15 mL). After drying in vacuum, the corresponding pure
mono- or bis-GH hydrochlorides 3b, 4a′, 5d, and 5e were obtained as
white solids.
Mono- and Bis-GHs, General Condensation Procedure F2:
Reverse-Phase Chromatography (3a, 5a, 5b′,″, 5c′,″, 5g, 5h).
AG.HCl (1.1 equiv) and 1 N aq HCl (3−5 drops, catalytic) were
sequentially added to a warm, vigorously stirred suspension of 1 equiv
of mono-formyl (8a, 14a, 14b′,″, 14c′,″) or di-formyl compounds (14g
or 14h) in absolute EtOH. After the addition of the catalytic acid, the
suspension became a solution. The reaction mixture was refluxed at 80
°C (2−8 h), with periodical TLC monitoring (eluant mixtures: 100%
EtOAc and 8:2 DCM/MeOH with a few AcOH drops). Then, the
reaction mixture was cooled to rt and concentrated under reduced
pressure. The crude solid was purified with reverse-phase chromatog-
raphy (eluant mixture: from 95/5 to 0/100 H2O:MeCN + 0.2% TFA),
obtaining the corresponding pure mono- or bis-GH trifluoroacetates as
white solids.
Electrophysiological and Fluorescence-Based Studies on
ASICs. HEK-293 Cell Culture. Human embryonic kidney (HEK-293)
cells endogenously expressing ASIC1a were used and cultured as
previously reported.57 For electrophysiological studies, HEK-293 cells
were seeded on glass coverslips (Thermo Fisher Scientific, Waltham,
MA) coated with poly-(L-lysine) (30 μg/mL) (Sigma-Aldrich) and
used at least 12 h after seeding.
Electrophysiology on HEK-293 Cells. ASIC1a currents were
recorded by patch-clamp electrophysiology in the whole-cell
configuration.36,59 HEK-293 cells were maintained in a physiological
extracellular solution (pH 7.4) containing 140 mM NaCl, 5 mM KCl, 2
mM CaCl2, 2 mM MgCl2, and 10 mM N-(2-hydroxyethyl)piperazine-
N′-ethanesulfonic acid (HEPES) and perfused with an acid
extracellular solution at pH 6.0 containing 10 mM glycine and 2-(N-
morpholino)ethanesulfonic acid (MES) instead of HEPES to elicit
ASIC currents. Pipettes were filled with 30 mM NaCl, 120 mM KCl, 2
mM MgCl2, and 10 mM HEPES with 300 mOsm (pH 7.3). A
multibarrel perfusion system (SF-77; Warner Instruments, Hamden,
CT) was used for rapid changes of extracellular solutions (from 7.4 to
6.0 and vice versa). Data were acquired by an Axopatch 200B amplifier
and analyzed using pClamp software (version 10.0, Molecular Devices).
Traces were filtered at 5 kHz and digitized using a Digidata 1322A
interface (Molecular Devices). Compounds were daily dissolved at the
final concentration reported. To obtain their effective concentration on
hASIC1a currents, data were fitted to the binding isotherm: y = max/{1
+(X/IC50)}n, where X is the inhibitor concentration and n is the Hill
coefficient.
starting from a cDNA sample extracted from mouse cortex. The primer
sequences used for cloning were FOR 5′-ATGGACCTCAAGGA-
GAGCCCCAG-3′ and REV 5′-TCAGCAGGCAATCTCCTC-
CAGGG-3′. The amplified product of mouse ASIC2a, bearing the
cutting sites for restriction enzymes, was subjected to enzymatic
digestion in parallel to the commercial vector chosen for cloning,
pcDNA3. Stable CHO-K1 cells expressing murine ASIC2a were
generated by transfecting cells with the pCDNA3.1/Hygro-ASIC2a
plasmid (10 μg) using the lipofectamine 2000 transfection kit
(ThermoFisher Scientific, Waltham, MA). Upon transfection, cells
were selected for 20 days by 400 μg/mL Hygromycin B (Thermo Fisher
Scientific; cat. #10687-010) and further cloned by limiting dilution.
Candidate cell clones were evaluated for ASIC2a expression by western
blot analysis.
CHO-K1 Cell Culture. CHO-K1 cells were cultured in a DMEM-F12
1:1 Mix with Ultraglutamine I (Lonza Group Ltd., Basel, Switzerland)
supplemented with 10% fetal bovine serum (FBS; EuroClone S.p.A.,
Pero, MI, Italy), 13 mM HEPES buffer (Lonza Group Ltd.), 0.375%
sodium bicarbonate solution (Lonza Group Ltd.), 1 mM sodium
pyruvate solution (Lonza Group Ltd.), and 1% Pen/Strep solution
(Lonza Group Ltd.) at 37 °C in a humidified atmosphere with 5% CO2.
ASIC1a-expressing cells were maintained in a complete medium
supplemented with a G418 disulfate salt solution (400 μg/mL, Sigma-
Aldrich, St. Louis, MO), while ASIC2a-expressing cells were
maintained in a medium supplemented with 400 μg/mL Hygromycin
B.
Electrochromic Fluorescence Voltage Sensor-Based Assay in
CHO-K1 Cells. Parental murine ASIC1a-expressing and ASIC2a-
expressing CHO-K1 cells were seeded in 96-well black microplates
(Greiner Bio-One GmbH, Kremsmunster, Austria) at 30 000 cells/well
density 48 h before the assay. Experiments were performed as described
previously,60 using di-4-ANEPPS (VSD, Sigma-Aldrich) as the dye.
Briefly, confluent cells were washed four times with a pH 7.4 Krebs−
Ringer solution buffered with HEPES (KRH) (120 mM NaCl, 5 mM
KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 6 mM glucose, 20
mM HEPES sodium salt, from Sigma-Aldrich), incubated with 10 μM
VSD for 15 min at rt and then washed twice with pH 7.4 KRH. These
steps were automated using a Multiwash III microplate washer machine
(TriContinent Scientific, Grass Valley, CA). DA 1 (Sigma-Aldrich),
mono-, and bis-GHs 3−5 were diluted at 10 mM in stock solutions with
sterile dimethyl sulfoxide (DMSO) and then diluted in KRH at the final
concentration of 10 μM. PcTx1 (Peptide Institute Inc., JP) was diluted
100 μM in water and then diluted in KRH at a final concentration of 300
nM. Such inhibitor solutions were added to pH 7.4 KRH into the wells
at the end of washing cycles to obtain a preincubation of 10 min before
starting the experimental procedure. Cells were stimulated by proton
injection using a KRH solution at pH 6.5 (135 mM NaCl, 5 mM KCl,
1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 6 mM glucose, 5 mM
HEPES sodium salt, all from Sigma-Aldrich) containing the final
concentration of each mono- and bis-GHs 3−5 and control inhibitors.
Acquisition and analysis of fluorescence signals were done as previously
published.60
Neuronal Cultures. Neuronal precursor cells (NPCs) derived from
healthy donors were provided by Dr. Vania Broccoli and differentiated
into neurons according to established protocols.40,41 We maintained
NPCs cultures in Neurobasal supplemented with 1× B27 (Thermo
Fisher Scientific Inc.), 1% penicillin/streptomycin (Merck Life Science
Srl, Milan, Italy), 1% glutamine (Merck Life Science Srl, Milan, Italy),
50 ng/mL sonic Hedgehog (R&D System, Inc., NM, Canada), and 0.8
μM CHIR99021(Miltenyi Biotech) at 37 °C with 5% CO2. When cells
reached ≈80% confluence, the medium was replaced by a differ-
entiation medium containing Neurobasal supplemented with 1× B27
(Thermo Fisher Scientific Inc.), 10 ng/mL BDNF (PeproTech EC,
Ltd., London, U.K.), 10 ng/mL GDNF (PeproTech EC, Ltd., London,
U.K.), 1 μM retinoic acid (Merck Life Science Srl, Milan, Italy), 5 μM
forskolin (Merck Life Science Srl, Milan, Italy), and 20 μM ascorbic
acid (Merck Life Science Srl, Milan, Italy). Half media was changed
every 2 days, and cells were kept at 37 °C with 5% CO2 until 30 DIV.
Electrophysiology on Neurons. Patch pipettes were pulled from
hard borosilicate glass (Sutter Instrument) on a two-step puller (P10,
Generation of Stable CHO-K1 Cell Lines Expressing Murine
ASIC1a. Stable CHO-K1 cells expressing murine ASIC1a were
generated by subcloning a CHO-K1 mASIC1a-expressing cell clone
previously generated in our lab.55
Generation of a Stable CHO-K1 Cell Line Expressing Mouse
ASIC2a. A polymerase chain reaction (PCR) was carried out to obtain
the encoding sequence of the mouse ASIC2a gene (NM_001034013.2)
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J. Med. Chem. 2021, 64, 8333−8353