Production of new amilorides as potent inhibitors of mitochondrial respiratory complex I

Article abstract of DOI:10.1080/09168451.2015.1010479

Amilorides, well-known inhibitors of Na⁺/H⁺ antiporters, have also shown to inhibit bacterial and mitochondrial NADH-quinone oxidoreductase (complex I). Since the membrane subunits ND2, ND4, and ND5 of bovine mitochondrial complex I

Full text of DOI:10.1080/09168451.2015.1010479

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Production of new amilorides as potent inhibitors of
mitochondrial respiratory complex I
Masatoshi Muraia, Sayako Habua, Sonomi Murakamia, Takeshi Itoa & Hideto Miyoshia
a Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,
Kyoto, Japan
Published online: 03 Mar 2015.
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To cite this article: Masatoshi Murai, Sayako Habu, Sonomi Murakami, Takeshi Ito & Hideto Miyoshi (2015): Production of
new amilorides as potent inhibitors of mitochondrial respiratory complex I, Bioscience, Biotechnology, and Biochemistry,
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Bioscience, Biotechnology, and Biochemistry, 2015
Production of new amilorides as potent inhibitors of mitochondrial respiratory
complex I
Masatoshi Murai, Sayako Habu1, Sonomi Murakami, Takeshi Ito and Hideto Miyoshi*
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
Received November 27, 2014; accepted January 10, 2015
http://dx.doi.org/10.1080/09168451.2015.1010479
Amilorides, well-known inhibitors of Na+/H+ anti-
porters, have also shown to inhibit bacterial and
mitochondrial NADH-quinone oxidoreductase (com-
plex I). Since the membrane subunits ND2, ND4, and
ND5 of bovine mitochondrial complex I are homolo-
gous to Na+/H+ antiporters, amilorides have been
thought to bind to any or all of the antiporter-like
subunits; however, there is no direct experimental
evidence in support of this notion. Photoaffinity label-
ing is a powerful technique to identify the binding
site of amilorides in bovine complex I. Commercially
available amilorides such as 5-(N-ethyl-N-isopropyl)
amiloride are not suitable as design templates to
synthesize photoreactive amilorides because of their
low binding affinities to bovine complex I. Thereby,
we attempted to modify the structures of commer-
cially available amilorides in order to obtain more
potent derivatives. We successfully produced two
photoreactive amilorides (PRA1 and PRA2) with a
photolabile azido group at opposite ends of the
molecule.
subunit(s) needs to be examined in more detail because
if this is the case, they may become useful molecular
tools to study the mechanism of proton translocation
through the antiporter-like subunits.
Photoaffinity labeling is a powerful technique to
identify the binding site of amilorides in bovine com-
plex I.8,9) In the photoaffinity labeling technique, less
nonspecific binding occurs at higher binding affinities
of photoreactive ligands; therefore, using a ligand with
high binding affinity as possible allows for the accurate
interpretation of data obtained in photoaffinity labeling
experiments. Commercially available amilorides are not
suitable as design templates for the synthesis of photo-
reactive amilorides possessing high binding affinities to
complex I because of their low binding affinities
to bovine complex I. Therefore, we herein attempted to
modify the structures of commercially available amilo-
rides and investigated the structure–activity relationship
(SAR) of these synthetic derivatives to obtain more
potent derivatives.
Key words: amilorides; structure–activity relation-
ship; NADH-quinone oxidoreductase
(complex I); mitochondria
Materials and methods
Materials.
5-(N-ethyl-N-isopropyl)amiloride
(EIPA), 5-(N-methyl-N-isobutyl)amiloride (MIA), and
benzamil were purchased from Sigma-Aldrich (St.
Louis, MO, USA). Ubiquinone-1 (Q1) was generously
provided by Eisai Co., Ltd. (Tokyo, Japan). Other
reagents were of analytical grade.
Amilorides are well-known inhibitors of Na+/H+ and
Na+/Ca2+ antiporters and Na+ channels.1,2) The struc-
tural factors of amilorides that are critical to the inhibi-
tory action greatly vary depending on the different ion
transporters and species.1) Amilorides have also shown
to inhibit bacterial and mitochondrial NADH-quinone
oxidoreductase (complex I),3,4) but their inhibitory
potencies, in terms of IC50 values, are much weaker
than those of traditional inhibitors such as rotenone,
piericidin A, and acetogenins. As the membrane sub-
units ND2, ND4, and ND5 of bovine mitochondrial
complex I are homologous to Na+/H+ antiporters,5–7)
amilorides have been thought to bind to any or all of
the antiporter-like subunits; however, there is currently
no direct experimental evidence in support of this
notion. This binding of amilorides to antiporter-like
General synthetic methods. 1H NMR spectra were
recorded at 500 or 400 MHz with Bruker AVANCE
III 500 or AVANCE III 400 spectrometers, respec-
tively, using tetramethylsilane (TMS) as the internal
standard. 13C NMR spectra were recorded at 125 or
100 MHz. Chemical shifts (δ) are given in ppm rela-
tive to TMS with coupling constants (J) in Hz. HPLC
purification was carried out with a Shimadzu LC-10
AS using a Wakosil-II 5C18 HG prep column. Elution
profiles were detected at 254 nm with a Shimadzu
SPD-10A.
*Corresponding author. Email: miyoshi@kais.kyoto-u.ac.jp
1Present address: Chemical Development Center, CMC Development Laboratories, Shionogi & Co., Ltd., Amagasaki, Hyogo 660 0813, Japan.
© 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
M. Murai et al.
General procedures for the preparation of series
To a solution of methyl 3-amino-5,6-dichloro-2-
General procedures for the preparation of series
4. To a solution of an appropriate primary amine
1.
pyrazinecarboxylate (1.0 eq.) in 2-propanol (5 mL/
mmol), an appropriate primary or secondary amine
(3–6 eq.) was added, and the reaction mixture was
refluxed for 2 h.10) The solution was cooled to room
temperature, concentrated under reduced pressure, and
the residue was purified by silica-gel column chroma-
tography (Wako gel® C-200, 30–40% ethyl acetate/
n-hexane) to afford the series 1 intermediate (yield:
32–95%). The regioselectivity of the nucleophilic
substitution at the 5-position of the pyrazine ring was
confirmed by an X-ray structural analysis (Fig. S1).
(1 eq.) in water (0.25 mL/mmol), S-methylisothiourea
sulfate (1 eq.) in ethanol (0.25 mL/mmol) was added,
and the resulting mixture was refluxed for 2 h.13) The
solution was cooled to room temperature, concentrated
under reduced pressure, and the residue was then
washed with diethyl ether. The guanidine compound
was recrystallized from water containing 5–30%
ethanol. The crystal was collected by filtration as an
alkylguanidine sulfate salt.
Under N2 atmosphere, the guanidine sulfate salt was
treated with sodium methoxide (1.1 eq.) in anhydrous
methanol (1 mL/mmol). The mixture was stirred for
30 min at room temperature, and the precipitate was
removed by filtration. The filtrate was concentrated
under reduced pressure, yielding the guanidine base 4
(yield: 59–85%).
General procedures for the preparation of series
2. Guanidine hydrochloride (5 eq.) was dissolved in
anhydrous methanol (1 mL/mmol) and stirred with
sodium methoxide (5.5 eq.) for 30 min at room temper-
ature under N2 atmosphere. The precipitate was
removed by filtration, and the resulting methanolic
solution was concentrated under reduced pressure to
give the guanidine base.10,11) The guanidine base was
further dissolved in anhydrous methanol (1 mL/mmol)
with an appropriate pyrazine unit 1 (1 eq.), and the
mixture was refluxed for 12–18 h under N2 atmosphere.
The solution was cooled to room temperature, acidified
with 1.0 M aqueous HCl, and concentrated. Excess
guanidine was removed from the residue using
reversed-phase column chromatography (YMC gel
ODS-A-HG, 10–100% methanol/0.1% aqueous HCl).
The crude product was further purified by silica-gel
column chromatography (Wako gel® C-200, 10% meth-
anol/chloroform) to afford the series 2 derivatives
(yield: 29–67%). The spectral data of 2c–2 k were
listed in the Supporting Information.
General procedure for the preparation of series 5.
A solution of an appropriate pyrazine 1 (1 eq. typically
~1 mmol) and guanidine base 4 (2.5–5 equiv) in anhy-
drous methanol (1.5 mL) was refluxed for 4 h. The
reaction mixture was cooled to room temperature, acid-
ified with 1.0 M aqueous HCl, and concentrated under
reduced pressure. The residue was purified by reversed-
phase column chromatography (YMC gel ODS-A-HG,
10–100% methanol/0.1% aqueous HCl) to remove
excess guanidine. The crude product was further puri-
fied by silica-gel column chromatography (Wako gel®
C-200, 10% methanol/chloroform). Final purification
was carried out using preparative HPLC (Wakosil-II
5C18 HG prep, 5 mL/min, 50–90% methanol/water
containing 0.1% TFA) to provide 5 (yield: 12–61%).
The spectral data of 5a–5s, PRA1, and PRA2 were
listed in the Supporting Information.
General procedures for the preparation of series
3.
To a solution of an appropriate primary amine
(2.5 eq.) in a mixture of anhydrous DMF and diisopro-
pylethylamine [3:1 (v/v), 5 mL/mmol], 1H-pyrazol-1-
carboxamidine hydrochloride (3.5 eq.) was added under
N2 atmosphere, and the mixture was stirred at room
temperature for 12–18 h, followed by the addition of
diethyl ether (10–20 mL).12) The formed oil containing
the guanidine salt was washed twice with ether and
dried under the reduced pressure. The resulting oil was
further dissolved in anhydrous methanol (2–3 mL/
mmol). Then, sodium methoxide (5 eq.) was added and
the solution was stirred for 30 min at room tempera-
ture, followed by filtration of the precipitate. The fil-
trate was concentrated under reduced pressure to
provide guanidine bases, which were used in the next
reaction without further purification.
The guanidine base was dissolved in anhydrous
methanol (2–3 mL/mmol) with methyl 3,5-diamino-6-
chloropyrazine-2-carboxylate (1 eq.) and refluxed for
2 h under N2 atmosphere. The mixture was cooled to
room temperature, concentrated under reduced pressure,
and purified by silica-gel column chromatography
(Wako gel® C-200, 10% methanol/chloroform) to
afford series 3 (yield: 23–28%). The spectral data of
3a–3c were listed in the Supporting Information.
Fig. 1. Structures of EIPA, MIA, benzamil, and photoreactive
amilorides (PRA1 and PRA2).

Amilorides as complex I inhibitors
3
General procedure for the preparation of 6. To a
solution of an appropriate series 1 (1 eq.) in a mixture
of THF and methanol [3:1 (v/v), 8 mL/mmol], 1.0 M
aqueous LiOH (2 eq.) was added, and the mixture was
stirred for 4 h at room temperature. The reaction was
quenched with saturated aqueous NH4Cl, extracted with
dichloromethane, and the organic layer was dried over
MgSO4. The crude product was purified by silica-gel
column chromatography (Wako gel® C-200, 10% meth-
anol/chloroform) to give 6 (yield: 75%).
Table 1. Inhibitory activities of series 2 and 3 derivativesa.
General procedures for the preparation of series
7.
To a solution of series 6 (1 eq.) in anhydrous
.
DMF (18 mL/mmol), an appropriate primary amine
(1.2 eq.), 4-methylmorphorine (5 eq.), and HATU (1.5
equiv) were added, and the mixture was stirred for 3 h
at room temperature.14) The reaction was quenched
with 0.1 M aqueous NaOH. The mixture was extracted
with dichloromethane, and the organic layer was dried
over MgSO4. The crude product was purified by silica-
gel column chromatography (Wako gel® C-200, 10–
30% ethyl acetate/n-hexane) to give 7 (yield: 64–68%).
The spectral data of 7a and 7b were listed in the
Supporting Information.
Compd.
R1
IC50 (µM)
15 ( 2.4)
2a (EIPA)
2b (MIA)
10 ( 0.75)
81 ( 2.5)
2c
Measurement of complex I activity in bovine heart
2d
2e
80 ( 15)
>100
submitochondrial particles (SMP).
SMP were pre-
pared from isolated bovine heart mitochondria by the
method of Matsuno-Yagi and Hatefi.15) and stored in
buffer containing 250 mM sucrose and 10 mM
Tris-HCl (pH 7.4) at −80 °C before being used. The
NADH oxidase activity in SMP was followed
spectrometrically with a Shimadzu UV-3000 (340 nm,
2f
>100
>100
2g
2h
2i
48 ( 5.3)
70 ( 14)
40 ( 2.2)
2j
2k
>100
Compd.
R2
IC50 (µM)
21 ( 0.5)
3a (benzamil)
3b
20 ( 1.0)
Scheme 1. Synthesis of amiloride derivatives (series 2, 3, 5, and 7).
Reagents and conditions: (a) primary or secondary amine, 2-propanol,
reflux, 2 h; (b) guanidine hydrochloride, NaOMe, MeOH, reflux, 12–
18 h; (c) (i) R2–NH2, 1H-pyrazole-1-carboxamidine hydrochloride,
DMF/(i-Pr)2EtN (3:1), rt, 12–18 h, (ii) NaOMe, MeOH, rt, 30 min,
(iii) R2–guanidine base, reflux, 2 h; (d) (i) R2–NH2, EtOH/H2O (1:1),
reflux, 4 h, (ii) NaOMe, MeOH, rt, 30 min; (e) MeOH, reflux, 2 h; (f)
aq. LiOH, THF/MeOH (1:1), rt, 4 h; (g) R3–NH2, 4-methylmorpho-
rine, HATU, DMF, rt, 3 h.
3c
12 ( 3.3)
aThe IC50 value is the molar concentration needed to reduce the control
NADH oxidase activity (approx. 0.55 µmol NADH/min/mg of protein) in
SMP by half. Values are means
measurements.
standard error of three independent

4
M. Murai et al.
ε = 6.2 mM−1 cm−1) at 30 °C. The reaction medium
(2.5 mL) contained 0.25 M sucrose, 1 mM MgCl2, and
50 mM phosphate buffer (pH 7.5). The final mitochon-
drial protein concentration was 30 μg of protein/mL.
The reaction was started by adding 50 µM NADH
after the equilibration of SMP with an inhibitor for
4 min. The NADH-Q1 oxidoreductase activity was also
measured under the same experimental conditions,
except that the reaction medium contained 50 μM Q1,
0.2 μM antimycin A, and 4 mM KCN.16)
Fig. 1. The inhibitory activities of these amilorides
were determined by the NADH oxidase assay using
bovine heart SMP, and their IC50 values were approxi-
mately 10–20 μM (Table 1); being comparable to those
reported.3) We also confirmed that the IC50 values of
these amilorides determined with the NADH-Q1 oxido-
reductase assay matched those determined with the
NADH oxidase assay, indicating that the inhibition of
other respiratory complexes is negligible. Since the
IC50 values of the photoreactive inhibitors used in ear-
lier photoaffinity labeling studies with complex I, such
as quinazoline,8) fenpyroximate,9) and acetogenins,17)
were all at one digit nm levels, the commercially avail-
able amilorides are not potent enough to be used as
templates for designing photoreactive amilorides.
Therefore, we needed to produce more potent amiloride
derivatives (Scheme 1).
Results and discussion
The structures of commercially available amilorides
[EIPA (2a), MIA (2b), and benzamil (3a)], which have
been used in the research on complex I, are shown in
Table 2. Inhibitory activities of series 5 and 7 derivatives.
Compd.
R1
R2
IC50 (µM)
10 ( 1.5)
Compd.
R1
R2
IC50 (µM)
5a
5l
7.0 ( 0.56)
5b
5c
5d
5e
5f
3.3 ( 0.31)
4.3 ( 0.53)
2.7 ( 0.51)
2.7 ( 0.19)
0.88 ( 0.01)
0.62 ( 0.09)
1.2 ( 0.09)
0.84 ( 0.13)
1.3 ( 0.12)
0.57 ( 0.02)
5m
5n
0.44 ( 0.045)
0.41 ( 0.059)
0.49 ( 0.016)
1.52 ( 0.032)
0.57 ( 0.037)
0.89 ( 0.072)
0.25 ( 0.04)
0.37 ( 0.05)
17.2 ( 3.3)
5o
5p
5q
5g
5h
5i
5r
RPA1
RPA2
7a
5j
5k
7b
96 ( 14)

Amilorides as complex I inhibitors
5
In the series 2 derivatives (Table 1), we modified the
Supplemental material
5-amino group on the pyrazine ring without modifying
the guanidine moiety. In spite of wide modifications,
we did not obtain any derivative eliciting more potent
activity than those of EIPA and MIA. The hydrogen-
bond donating –NH was not critical to the inhibitory
activity (2b vs. 2d, 2 h vs. 2 k).
The supplemental material for this paper is available
at http://dx.doi.org/10.1080/09168451.2015.1010479.
Acknowledgments
The SAR for series 3 revealed that the activity does
not significantly increase with elevation in the hydro-
phobicity of R2 unless the 5-position is fixed with the
primary amino group. However, this was not the case
when R1 and R2 were modified simultaneously, as
described below.
We thank Dr. Kiyosei Takasu and Dr. Ken-ichi Yam-
ada (Graduate School of Pharmaceutical Sciences,
Kyoto University) for the X-ray structural analysis.
In the series 5 derivatives (Table 2), we modified R1
and R2 simultaneously. A comparison of 5c ~ 5f
showed that the mono-benzyl secondary amine at the
5-position is sufficient to elicit potent activity; hence,
by taking the synthetic facility into consideration, we
fixed this position by nonsubstituted or substituted ben-
zyl secondary amine for this series of derivatives. The
SAR of this series indicated that the activity increases
with elevation in the hydrophobicity of R2 regardless
of R1 (5b vs. 5f, 5j vs. 5k, 5p vs. 5q). However, this
increase in the activity was saturated at a certain level
in spite of further increase in the hydrophobicity of R1
(5m vs. 5n). The electronic properties of the substituent
on the benzyl moiety had no critical effect on the activ-
ity (e.g. 5f vs. 5k, 5q vs. 5r). Replacing the pyrazinoyl
guanidine skeleton to the amide type resulted in a
marked loss in the activity (5m vs. 7a, 5o vs. 7b), indi-
cating the critical role of the guanidine skeleton. This
is probably because the positive charge formed at the
guanidine moiety (this moiety is protonated at physio-
logical pHs,1) is important for tightly binding to the
enzyme. Furthermore, deletion of the chloride atom
from the 6-position of 5k resulted in a significant
decrease in the activity (IC50 = 1.6 μM), indicating the
important role of the 6-chloride group.
Funding
This work was supported by the Grant-in-Aid for Scientific
Research [grant 23380064 to H. M.], [grant 26292060 to
H. M.]; Grant-in-Aid for Young Scientists [grant 23780116 to
M. M.] from the Japan Society for the Promotion of Science.
Conflict of interest
The authors declare that they have no conflict of interest.
References
[1] Kleyman TR, Cragoe EJ Jr. Amiloride and its analogs as tools
in the study of ion transport. J. Membr. Biol. 1988;105:1–21.
[2] Putney LK, Denker SP, Barber DL. The changing face of the
Na+/H+ exchanger, NHE: structure, regulation, and cellular
actions. Annu. Rev. Pharmacol. Toxicol. 2002;42:527–552.
[3] Nakamaru-Ogiso E, Seo BB, Yagi T, Matsuno-Yagi A. Amilo-
ride inhibition of the proton-translocating NADH-quinone oxido-
reductase of mammals and bacteria. FEBS Lett. 2003;549:43–46.
[4] Stolpe S, Friedrich T. The Escherichia coli NADH: ubiquinone
oxidoreductase (complex I) is a primary proton pump but may
be capable of secondary sodium antiport. J. Biol. Chem.
2004;279:18377–18383.
[5] Efremov RG, Baradaran R, Sazanov LA. The architecture of
respiratory complex I. Nature. 2010;465:441–445.
Using potent synthetic amilorides (such as 5m and
5q) as templates, we designed and synthesized two
photoreactive amilorides (PRA1 and PRA2, Fig. 1). An
azido (–N3) group and terminal acetylene (–C≡CH)
were incorporated into the two compounds as a phot-
olabile group and “footing” for sequential Cu+1–cata-
lyzed click chemistry, respectively, at opposite ends of
the molecules. The use of two amilorides possessing a
photolabile azido group in different positions may
make characterization of the binding site more reliable.
Some molecular tags, such as fluorophore and biotin,
can be covalently attached to the terminal acetylene via
Cu+1-catalyzed click chemistry following photoaffinity
labeling, which enables purification and/or detection of
the labeled peptides.18) We confirmed that PRA1 and
PRA2 maintain the potent inhibitory activities (Table 2),
and are the most potent class in this study. Photo-
affinity labeling experiments are currently under way in
our laboratory using these photoreactive amilorides.
[6] Efremov RG, Sazanov LA. Structure of the membrane domain
of respiratory complex I. Nature. 2011;476:414–420.
[7] Baradaran R, Berrisford JM, Minhas GS, Sazanov LA. Crystal
structure of the entire respiratory complex I. Nature.
2013;494:443–448.
[8] Murai M, Mashimo Y, Hirst J, Miyoshi H. Exploring interac-
tions between the 49 kDa and ND1 subunits in mitochondrial
NADH-ubiquinone oxidoreductase (complex I) by photoaffinity
labeling. Biochemistry. 2011;50:6901–6908.
[9] Shiraishi Y, Murai M, Sakiyama N, Ifuku K, Miyoshi H. Fenpy-
roximate binds to the interface between PSST and 49 kDa sub-
units in mitochondrial NADH-ubiquinone oxidoreductase.
Biochemistry. 2012;51:1953–1963.
[10] Cragoe EJ Jr, Woltersdorf OW Jr, Bicking JB, Kwong SF, Jones
JH. Pyrazine diuretics II. N-amidino-3-amino-5-substituted
6-halopyrazinecarboxamides. J. Med. Chem. 1967; 10: 66–75.
[11] Matthews H, Ranson M, Tyndall JDA, Kelso MJ. Synthesis and
preliminary evaluation of amiloride analogs as inhibitors of the
urokinase-type plasminogen activator (uPA). Bioorg. Med.
Chem. Lett. 2011;21:6760–6766.
[12] Hirsh AJ, Molino BF, Zhang J, Astakhova N, Geiss WB, Sargent
BJ, Swenson BD, Usyatinsky A, Wyle MJ, Boucher RC, Smith
RT, Zamurs A, Johnson MR. Design, synthesis, and struc-
ture−activity relationships of novel 2-substituted pyrazinoylguani-
dine epithelial sodium channel blockers: drugs for cystic fibrosis
and chronic bronchitis. J. Med. Chem. 2006;49:4098–4115.
[13] Secondo A, Pannaccione A, Molinaro P, Ambrosino P, Lippiello
P, Esposito A, Cantile M, Khatri PR, Melisi D, Di Renzo GD,
Annunziato L. Molecular pharmacology of the amiloride analog
Supporting Information
Fig. S1 (X-ray crystallographic data) and the spectral
data of compounds 2c–2k, 3a–3c, 5a–5s, 7a, 7b,
PRA1, and PRA2.

6
M. Murai et al.
3-amino-6- chloro-5-[(4-chloro-benzyl)amino]-N-[[(2,4-dimethyl-
[16] Murai M, Ichimaru N, Abe M, Nishioka T, Miyoshi H. Mode of
inhibitory action of Δlac-acetogenins, a new class of inhibitors
of bovine heart mitochondrial complex I. Biochemistry.
2006;45:9778–9787.
[17] Nakanishi S, Abe M, Yamamoto S, Murai M, Miyoshi H.
Bis-THF motif of acetogenin binds to the third matrix-side loop
of ND1 subunit in mitochondrial NADH-ubiquinone oxidoreduc-
tase. Biochim. Biophys. Acta. 2011;1807:1170–1176.
[18] Masuya T, Murai M, Ifuku K, Morisaka H, Miyoshi H. Site-specific
chemical labeling of mitochondrial respiratory complex I through
ligand-directed tosylate chemistry. Biochemistry. 2014;53:
2307–2317.
benzyl)-amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) as
a pan inhibitor of the Na+-Ca2+ exchanger isoforms NCX1,
NCX2, and NCX3 in stably transfected cells. J. Pharmacol. Exp.
Ther. 2009;331:212–221.
[14] Hunt, T, Atherton-Watson HC, Axford J, Collingwood SP,
Coote KJ, Cox B, Czarnecki S, Danahay H, Devereux Na,
Howsham C, Hunt P, Paddock V, Paisley D, Young A. Discov-
ery of a novel chemotype of potent human ENaC blockers using
a bioisostere approach. Part 1: quaternary amines. Bioorg. Med.
Chem. Lett. 2012;22:929–932.
[15] Matsuno-Yagi A, Hatefi Y. Studies on the mechanism of oxida-
tive phosphorylation. J Biol. Chem. 1985;260:11424–11427.