Potent and Selective Blockers for ANO1
729
N-((4-Bromo)-2-Naphthyl)-5-Nitroanthranilic Acid (5ac).
pulled with a P-97 programmable pipette puller (Sutter Instrument Co.,
The final step yields 93.5% (yellow powder). 1H-NMR (400 MHz, Novato, CA). Microelectrodes had resistances of ∼1–3 MV. During
DMSO–d6) d 10.51 (bs, 1H), 8.74 (s, 1H), 8.22 (d, J 5 9.8 Hz, 1H), 8.11 recordings, oocytes were continuously perfused with oocyte recording
(d, J 5 8.0 Hz, 1H), 8.00–7.93 (m, 3H), 7.66–7.64 (m, 2H), 7.30 (d, J 5 solution. All recordings were obtained at a holding potential of –60 mV.
8.8 Hz, 1H); 13C-NMR (100 MHz, DMSO–d6) d 168.9, 152.2, 137.7, Synthesized drug derivatives were prepared in separate bottles and
136.8, 134.9, 129.8, 129.5, 128.7, 128.2, 127.9, 127.6, 126.6, 123.1,
applied by gravity. The flow of solutions was approximately 1 ml/min.
121.3, 114.4; HR-ESI-MS m/z 384.9839 [M–H]2.
N-((7-Bromo)-2-Naphthyl)-5-Nitroanthranilic Acid (5ad).
The final step yields 85% (yellow powder). 1H-NMR (400 MHz,
DMSO–d6) d 10.64 (bs, 1H), 8.73 (d, J 5 2.7, Hz, 1H), 8.20–8.17 (m,
2H), 8.01 (d, J 5 8.6 Hz, 1H), 7.89 (d, J 5 8.2 Hz, 2H), 7.60–7.58 (m, 1H),
7.53 (dd, J 5 8.6, 1.8 Hz, 1H), 7.33 (d, J 5 9.4 Hz, 1H); 13C-NMR (100
MHz, DMSO–d6) d 169.1, 152.0, 137.5, 135.3, 130.3, 130.0, 129.6, 128.8,
123.9, 120.6, 119.2, 114.0, 112.2; HR-ESI-MS m/z 334.9822 [M–H]2.
N-((8-Bromo)-2-Naphthyl)-5-Nitroanthranilic Acid (5ae).
The final step yields 92.4% (yellow powder). 1H-NMR (400 MHz,
DMSO–d6) d 10.69 (bs, 1H), 8.75 (d, J 5 2.8 Hz, 1H), 8.22 (dd, J 5 9.4,
2.8 Hz, 1H), 8.09 (d, J 5 8.8 Hz, 1H), 8.03 (s, 1H), 8.00 (d, J 5 8.2 Hz, 1H),
7.90 (d, J 5 7.4 Hz, 1H), 7.62 (dd, J 5 8.7, 1.9 Hz, 1H), 7.42 (d, J 5 7.8, 7.8
Hz, 1H), 7.33 (d, J 5 9.4 Hz, 1H); 13C-NMR (100 MHz, DMSO–d6) d 169.0,
152.1, 138.4, 137.7, 132.5, 132.4, 131.3, 131.0, 129.7, 128.8, 128.6, 126.7,
124.5, 121.4, 119.0, 114.1, 112.6; HR-ESI-MS m/z 384.9837 [M–H]2.
N-((4-Iodo)naphthyl)-5-Nitroanthranilic Acid (5af). The final
step yields 83.3% (yellow powder). 1H-NMR (400 MHz, DMSO–d6) d
10.70 (bs, 1H), 8.76 (s, 1H), 8.22 (d, J 5 7.7 Hz, 1H), 8.09 (dd, J 5 8.0,
7.7 Hz, 2H), 7.91 (d, J 5 8.2 Hz, 1H), 7.74 (dd, J 5 7.6, 6.9 Hz, 1H), 7.65
(dd, J 5 7.3, 7.2 Hz, 1H), 7.39 (d, J 5 7.7 Hz, 1H), 6.72 (d, J 5 9.3 Hz,
1H); 13C-NMR (100 MHz, DMSO–d6) d 169.3, 153.9, 137.9, 137.2,
135.9, 135.2, 132.8, 130.5, 129.7, 129.3, 128.8, 128.5, 124.9, 123.4,
114.2, 111.8, 98.1; HR-ESI-MS m/z 432.9682 [M–H]2.
N-((4-Iodo)-2-Naphthyl)-5-Nitroanthranilic Acid (5ag). The
final step yields 95.3% (yellow powder). 1H-NMR (400 MHz, DMSO–
d6) d 10.53 (bs, 1H), 8.74 (bs, 1H), 8.21 (d, J 5 8.0 Hz, 1H), 8.13 (bs,
1H), 8.00 (bs, 2H), 7.91 (d, J 5 5.9 Hz, 1H), 7.62 (bs, 2H), 7.28 (d, J 5
8.8 Hz, 1H); 13C-NMR (100 MHz, DMSO–d6) d 169.0, 152.4, 137.6,
137.1, 134.9, 134.7, 134.2, 132.2, 131.6, 129.8, 128.9, 128.8, 128.2,
114.4, 112.4, 101.1; HR-ESI-MS m/z 432.9653 [M–H]2.
N-((7-Iodo)-2-Naphthyl)-5-Nitroanthranilic Acid (5ah). The
final step yields 96.2% (yellow powder). 1H-NMR (400 MHz, DMSO–
d6) d 10.93 (bs, 1H), 8.75 (bs, 1H), 8.37 (bs, 1H), 8.18 (d, J 5 6.7 Hz,
1H), 7.99 (d, J 5 8.5 Hz, 1H), 7.86 (bs, 1H), 7.74 (bs, 2H), 7.51 (d, J 5
8.0 Hz, 1H), 7.34 (d, J 5 8.6 Hz, 1H); 13C-NMR (100 MHz, DMSO–d6) d
169.1, 152.2, 137.5, 137.2, 136.1, 135.8, 134.2, 130.1, 130.0, 129.9,
128.8, 124.2, 119.3, 114.2, 112.4, 93.8; HR-ESI-MS m/z 432.9686
[M–H]2. 1H- and 13C-NMR spectra were recorded on a spectrometer
operating at Bruker (Billerica, MA) 400 and 100 MHz, respectively.
HR-ESI-MS was carried out by the Advanced Analysis Center at the
Korea Institute of Science and Technology.
Electrophysiology of Cultured Astrocytes, Human
Embryonic Kidney 293 Cells
Mouse astrocyte cell cultures were prepared as previously described
(Park et al., 2009). Human embryonic kidney 293 (HEK293) cells were
transfected with cDNA [human ANO1 (hANO1), mouse ANO1, or
mouse chloride channel protein 2 (mCLC2)] expression plasmids
tagged with enhanced green fluorescent protein using Effectene
(Qiagen, Valencia, CA) or a blend of lipids (Fugene-6; Roche Molecular
Biochemicals, Indianapolis, IN) at 1 mg of DNA per 35-mm culture
dish. Single cells identified by enhanced green fluorescent protein
fluorescence were used for whole-cell patch clamp experiments within
72 hours. Transfected HEK293 cells were recorded using a conven-
tional whole-cell and an excised inside-out patch clamp. Fire-polished
borosilicate glass patch pipettes were ∼3–5 MV. Experiments were
conducted at room temperature (∼20–25°C). To activate the channels
directly, cultured astrocytes or HEK293 cells were patch clamped
with a high Ca21 intracellular solution that contained 146 mM CsCl,
5 mM Ca21/EGTA/N-methyl-D-glucamine, 2 mM MgCl2, 8 mM HEPES,
and 10 mM sucrose, at pH 7.3, adjusted with N-methyl-D-glucamine.
The free Ca21 concentration was estimated to be 4.5 mM (Hartzell
et al., 2005). The extracellular solution contained the following:
150 mM NaCl, 10 mM HEPES, 3 mM KCl, 2 mM CaCl2, 2 mM MgCl2,
and 5.5 mM glucose or 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM
MgCl2, 15 mM glucose, and 10 mM HEPES at pH 7.3 adjusted with
NaOH (300–320 mOsm). Current-voltage curves were established by
applying 500-millisecond duration voltage ramps from –100 to 1100 mV.
Data were acquired with an Axopatch 200A amplifier controlled by
Clampex 10.0 software via a Digidata 1322A data acquisition system
(Molecular Devices, Sunnyvale, CA). Mouse bestrophin-1 (mBest1)
current or hANO1 current was recorded after treatment with N-((4-
methoxy)-2-naphthyl)-5-nitroanthranilic acid (MONNA) for 10 minutes.
Cells were ruptured and calcium-induced currents were recorded in the
presence of MONNA. Current amplitude at one time point of 100–300
seconds after rupture was selected to be analyzed.
Chemicals
Chemical compounds, including the following reagents, were pur-
chased from Sigma-Aldrich (St. Louis, MO): collagenase type 1A,
ionomycin-Ca21 salt, thapsigargin, and chelerythrine. HEPES was
obtained from J. T. Baker (Mallinckrodt Baker, Inc., Phillipsburg, NJ).
Data Analysis
Electrophysiology of X. laevis Oocytes
Currents were digitally recorded with AxoScope software (Axon
Instruments, Burlingame, CA) and Clampex 10.0 software. Data anal-
ysis was performed with SigmaPlot 10.0 (Systat Software, Inc., San
Jose, CA). All current responses elicited during application of a blocker
were normalized to the average of a Ca21-induced Cl2 current applied
before application of the blocker. Normalized and average data were
fitted using the SigmaPlot three-parameter logistic function to de-
termine the dose-response relationship and the IC50. All data are
expressed as the mean 6 S.E.M. Statistical analyses were performed
using a 2-tailed t test.
To test the blocking effect of compounds on xANO1, xANO1 was
activated by permeabilizing the plasma membrane to Ca21 and
changing the extracellular Ca21 concentration. The oocyte plasma
membrane was made permeable to Ca21 by treatment with the Ca21
ionophore ionomycin. Ca21 sequestration by intracellular stores was
inhibited with thapsigargin. Oocytes were incubated in an oocyte
recording solution containing 96 mM NaCl, 2 mM KCl, 2 mM MgCl2,
0.5 mM EGTA, and 10 mM HEPES (pH 7.4), as well as 10 mM
ionomycin, for a 30-minute period. After 30 minutes, the ionomycin was
removed from the external solution by washing with oocyte recording
solution. Ionomycin-treated oocytes were subsequently incubated in the
oocyte recording solution containing 1 mM thapsigargin for 90 minutes.
Thapsigargin was then also removed by washing with the oocyte
recording solution. Two-electrode voltage-clamp recordings were made
using the Warner model OC725B two-electrode voltage clamp amplifier
(Warner Instruments, Inc., Hamden, CT) with 1 M KCl-filled micro-
Results
Potency of Synthesized Derivatives with a Phenyl
Group at the B Position. Synthesis of anthranilic acid
derivatives was accomplished as described in Fig. 1 (see
electrodes (1B150F-4; World Precision Instruments, Sarasota, FL) Materials and Methods and Supplemental Methods).