Synthesis and structure-activity relationships of benzyloxyphenyl derivatives as a novel class of NCX inhibitors: Effects on heart failure

Article abstract of DOI:10.1016/j.bmc.2004.10.048

In the context of heart failure and myocardial ischemia reperfusion, the activity of the sodium-calcium exchanger can lead to calcium overload, which in turn can lead to contractile dysfunction and arrhythmia. Therefore, NCX is an attractive target for treatment of heart failure and myocardial ischemia reperfusion. We have designed and synthesized a series of benzyloxyphenyl derivatives as potential NCX inhibitors, based on compound 4. These derivatives have been evaluated for their inhibitory activity against both the reverse and forward modes of NCX, and two novel potent NCX inhibitors (7i, 10a) were discovered. Compound 7i was evaluated for its efficacy on ouabain-induced tonotropy and arrhythmia in a heart-failure model.

Full text of DOI:10.1016/j.bmc.2004.10.048

Bioorganic & Medicinal Chemistry 13 (2005) 725–734
Synthesis and structure–activity relationships of
benzyloxyphenyl derivatives as a novel class of NCX
inhibitors: effects on heart failure
Takahiro Kuramochi,a,* Akio Kakefuda,a Hiroyoshi Yamada,a Takashi Ogiyama,a
Taku Taguchib and Shuichi Sakamotoc
aInstitute for Drug Discovery Research, Yamanouchi Pharmaceutical Co. Ltd, 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
bQA&RA Division, Yamanouchi Pharmaceutical Co. Ltd, 3-17-1 Hasune, Itabashi-Ku, Tokyo 174-8612, Japan
cInstitute for Technology Development, Yamanouchi Pharmaceutical Co. Ltd, 160-2 Akahama, Takahagi, Ibaraki 318-0001, Japan
Received 27 September 2004; revised 23 October 2004; accepted 23 October 2004
Available online 18 November 2004
Abstract—In the context of heart failure and myocardial ischemia reperfusion, the activity of the sodium–calcium exchanger can
lead to calcium overload, which in turn can lead to contractile dysfunction and arrhythmia. Therefore, NCX is an attractive target
for treatment of heart failure and myocardial ischemia reperfusion. We have designed and synthesized a series of benzyloxyphenyl
derivatives as potential NCX inhibitors, based on compound 4. These derivatives have been evaluated for their inhibitory activity
against both the reverse and forward modes of NCX, and two novel potent NCX inhibitors (7i, 10a) were discovered. Compound 7i
was evaluated for its efficacy on ouabain-induced tonotropy and arrhythmia in a heart-failure model.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
A number of compounds, including the quinazoline
derivative, 110 and several benzyloxyphenyl derivatives
(2–4),11–13 have been reported to be NCX inhibitors
(Fig. 1). Among these, KB-R7943 (2) has been shown
to be therapeutically effective for ischemia-reperfusion
injury. In a previous paper, we reported the synthesis
of a series of 6-{4-[(3-fluorobenzyl)oxy]phenoxy}nico-
tinamide derivatives, and studied the SAR of these
derivatives.13 N-Benzyl-6-{4-[(3-fluorobenzyl)oxy]phen-
oxy}nicotinamide (4) was found to be a highly potent
and selective inhibitor of reverse NCX activity. In the
present paper, we have designed further novel and more
potent reverse NCX inhibitors, based on compound 4,
and we describe the synthesis, biological activities and
SAR of a novel class of benzyloxyphenyl derivatives.
The sodium–calcium exchanger (NCX) is a transporter
that controls the Ca2+ concentration in myocytes.1 In
myocardial ischemia, the Na+ concentration in myo-
cytes is raised via the sodium–hydrogen exchanger
(NHE).2,3 This causes NCX-mediated efflux of Na+ ions
to the extracellular fluid and influx of Ca2+ ions into
cells, thus inducing calcium overload in ischemia reper-
fusion. Such calcium overload causes necrosis of myo-
cytes, and is responsible for contractile dysfunction
and arrhythmia.4–7 NCX typically functions in the for-
ward mode, but the reverse mode has a more important
role with regard to calcium overload. Consequently,
selective inhibition of reverse NCX function could pro-
vide a novel therapeutic approach to the prevention and
treatment of reperfusion arrhythmias, aberrant myocar-
dial contracture, and necrosis. Indeed, reverse mode
NCX inhibitors are currently considered to be beneficial
in treating these disease states.8,9
2. Chemistry
Syntheses of the novel NCX inhibitors are summarized in
Schemes 1 and 2. In Scheme 1, compound 6 was obtained
from 513 by hydrolysis of the cyano group. Compounds
7a and 7c–i were prepared from 6 by amidation with var-
ious amines. Compound 7b was afforded by condensa-
tion of 6 and (1-trityl-1H-imidazol-4-yl)methylamine,
Keywords: Sodium–calcium exchanger; NCX; Transporter; Anti-
arrhythmias.
*
Corresponding author. Tel.: +81 29 852 5111; fax: +81 29 852
5387; e-mail: kuramochi.takahiro@yamanouchi.co.jp
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.10.048

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T. Kuramochi et al. / Bioorg. Med. Chem. 13 (2005) 725–734
Figure 1. Several inhibitors of sodium–calcium exchanger. (1) SM-15811; (2) KB-R7943; (3) patented compound of JP11092454; (4) nicotinamide
derivative.
Scheme 1. Reagents and conditions: (a) 5M NaOH, EtOH, reflux; (b) WSCÆHCl, HOBt, RCH2NH2, THF; (c) WSCÆHCl, HOBt, (1-trityl-1H-
imidazol-4-yl)methylamine, THF; (d) AcOH, 70°C; (e) PPh3, t-butyl 4-hydroxypiperidine-1-carboxylate, DEAD, THF; (f) HCl, AcOEt–MeOH; (g)
bis(trichloromethyl)carbonate, Et3N, THF, then pyridin-4-ylmethylamine; (h) methyl chloroformate, Et3N, THF; (i) pyridin-4-ylmethanol, NaH,
toluene, reflux; (j) WSCÆHCl, HOBt, 3-(pyridin-4-yl)propanoic acid, THF; (k) WSCÆHCl, HOBt, (2E)-3-(pyridin-4-yl)acrylic acid, THF.
followed by removal of the trityl group. Mitsunobu con-
densation of the starting material 812 and t-butyl 4-
hydroxypiperidine-1-carboxylate, followed by deprotec-
tion of the t-butoxycarbonyl group with hydrochloric
acid, produced the intermediate amine 9, which on treat-
ment with bis(trichloromethyl)carbonate and pyridin-4-
ylmethylamine yielded the desired compound 10a. Com-
pound 10b was prepared from intermediate amine 9 by
condensation with methyl chloroformate, followed by a
substitution reaction with pyridin-4-ylmethanol. Amide
condensation of intermediate 9 with the corresponding
amines afforded compounds 10c and 10d, respectively.
Mitsunobu condensation of compound 8 and t-butyl 3-
hydroxypiperidine-1-carboxylate, followed by deprotec-
tion of the t-butoxycarbonyl group with hydrochloric
acid, afforded intermediate amine 11, as shown in
Scheme 2. Compound 12 was prepared from 8 by a pro-
cedure similar to that used for compound 11. Desired
compounds 13 and 14 were obtained from 11 and 12,

T. Kuramochi et al. / Bioorg. Med. Chem. 13 (2005) 725–734
727
Scheme 2. Reagents and conditions: (a) DEAD, PPh3, t-butyl 3-hydroxypiperidine-1-carboxylate, or t-butyl 3-hydroxymethylpiperidine-1-
carboxylate, THF; (b) HCl, AcOEt–MeOH; (c) bis(trichloromethyl)carbonate, Et3N, THF, then pyridin-4-ylmethylamine; (d) 1-(bromomethyl)-3-
fluorobenzene, t-BuOK, DMF; (e) t-butyl 4-oxopiperidine-1-carboxylate, NaBH(OAc)3, AcOH; (f) formaldehyde, NaBH(OAc)3, AcOH.
respectively, by amide condensation. O-alkylation of 15
with 1-(bromomethyl)-3-fluorobenzene, followed by
reductive alkylations with t-butyl 4-oxopiperidine-1-
carboxylate and formaldehyde, afforded 16. Compound
16 was converted to 17 by deprotection of the t-butoxy-
carbonyl moiety with hydrochloric acid. Urea condensa-
tion of 17 with pyridin-4-ylmethylamine gave desired
compound 18.
according to reported protocols, using 45Ca and
CCL39 cells that stably express NCX1.1.11,13 The inhibi-
tory effect on forward mode NCX activity was assayed
using a cell necrosis assay, in which NCX1.1-expressing
CCL39 cells were also used.13,14 The inhibitory poten-
cies of our novel compounds were thus evaluated in
both reverse and forward NCX assays. Results for these
compounds were then compared to those for KB-R7943
(2) and compounds 3 and 4. The effects of selected com-
pounds on the tonotropic effects of ouabain and the time
for ouabain-induced arrhythmia onset in isolated guinea
pig atria were also tested.
3. Results and discussion
In order to measure the inhibitory effect of the synthe-
sized compounds on reverse mode NCX activity, a
Na+-dependent Ca2+ influx assay was performed
The structure–activity relationships of our novel series
of NCX inhibitors are summarized in Tables 1 and 2.
Table 1. Inhibitory activity of benzyloxyphenyl derivatives against the sodium–calcium exchanger
Compound
R
45Ca influxa IC50 (lM)c
Cell necrosisb EC50 (lM)c
Selectivityd
7a
7b
2-Furyl
1.3
>100
35
64
>76
50
4-Imidazolyl
4-Thiazolyl
4-Pyrimidinyl
2-Pyrimidinyl
2-Pyrazinyl
2-Pyridyl
0.70
0.81
0.97
3.9
7c
7d
79
71
69
7e
NTe
NTe
NTe
>30
19
––
––
7f
7g
3.9
1.7
––
7h
7i
3-Pyridyl
4-Pyridyl
Phenyl
1.1
>27
86
0.22
0.79
5.1
4
KB-R7943 (2)
>100
24
34
>120
4.7
36
3
0.94
a Activity at the NCX1.1 expressed in CCL39 cells. 45Ca influx mean NCX inhibitory activity for reverse mode.
b Activity at the NCX1.1 expressed in CCL39 cells. Cell necrosis mean NCX inhibitory activity for forward mode.
c IC50 values and EC50 values were determined in a single experimental run in triplicate.
d Ratio of EC50 value of cell necrosis and IC50 value of 45Ca influx.
e Not tested.

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T. Kuramochi et al. / Bioorg. Med. Chem. 13 (2005) 725–734
Table 2. Inhibitory activity of benzyloxyphenyl derivatives against the sodium–calcium exchanger
Compound
Y
45Ca influxa IC50 (lM)c
Cell necrosisb EC50 (lM)c
Selectivityd
120
10a
0.22
27
18
4.7
NTe
24
—
63
72
19
10b
10c
10d
0.38
0.60
2.7
43
50
13
14
5.9
3.9
NTe
NTe
––
––
7i
4
0.22
0.79
19
86
>100
>120
a Activity at the NCX1.1 expressed in CCL39 cells. 45Ca influx mean NCX inhibitory activity for reverse mode.
b Activity at the NCX1.1 expressed in CCL39 cells. Cell necrosis mean NCX inhibitory activity for forward mode.
c IC50 values and EC50 values were determined in a single experimental run in triplicate.
d Ratio of EC50 value of cell necrosis and IC50 value of 45Ca influx.
e Not tested.
Initially, the phenyl ring of compound 4 was replaced
with several heterocyclic ring systems, such as furyl, thi-
azole, pyrimidine, and pyridine rings (7a–i) (Table 1).
Compounds with five-membered heterocycles (furyl,
imidazolyl, and thiazolyl derivatives) had similar inhibi-
tory activities against reverse NCX activity, compared
to 4. These results indicate that five-membered heterocy-
cles seem to be tolerated for inhibition of reverse NCX
activity. For six-membered heterocycles, the 4-pyrimid-
inyl derivative (7d) was as potent as compound 4, but
the inhibitory activities of the 2-pyrimidinyl (7e), and
2-pyrazinyl derivatives (7f) were five times less potent
than 4. The 2-pyridyl derivative (7g) showed an approx-
imately 2-fold loss of reverse NCX inhibitory activity,
but the 3-pyridyl derivative (7h) was equipotent to 4.
Furthermore, the 4-pyridyl derivative (7i) showed a 4-
fold increase in activity compared to 4. The results for
pyridine derivatives (7g–i) suggest that the position of
the hetero atom in the heterocyclic ring seems to be
important for inhibition of reverse NCX activity, and
also suggest that the N atom of the 4-pyridyl ring (7i)
may be behaving as a H-bond acceptor. Among the
heterocyclic derivatives, compound 7i was the most po-
tent against reverse NCX activity, with an IC50 value of
0.22lM. Hence, the 4-pyridyl ring system was found to
be the best ring system for inhibition of reverse NCX
function.
KB-R7943 (2) does not have a pyridine ring connected
to the benzyloxyphenyl moiety. This prompted us to
change the nicotinamide moiety in 7i into several ali-
phatic cyclic linkers, as shown in Table 2. Replacement
of the nicotinamide by a 4-piperidino ring (10a) main-
tained inhibition of reverse NCX function, compared
to 7i. In contrast, compound 18, in which the –O– link
connecting the phenoxy group to the piperidine ring

T. Kuramochi et al. / Bioorg. Med. Chem. 13 (2005) 725–734
729
Figure 2. Effects of compounds KB-R7943 (2), 3, and 7i on tonotropic effects of ouabain in guinea pig isolated atria. *P < 0.05, **P < 0.01 versus
control (Dunnettꢀs test). Each value is the mean s.e.mean of at least four experiments.
was changed into an –N(Me)– link, had reduced inhibi-
tory activity for reverse NCX, indicating that the basic
nature of the linker in 18 was not tolerated. The deriva-
tives with carbamate (10b) and amide (10c) linkages also
showed slightly weaker inhibitory activity. The acryl-
amide derivative (10d), which has a planar structure
from the amide to the pyridine, showed a 4-fold loss
of inhibitory activity, compared to compound 10c, sug-
gesting that such a planar structure is conformationally
unfavorable for inhibition. Compounds 13 and 14,
which both contain a 3-piperidino ring, rather than a
4-piperidino ring, also showed a dramatic decrease in
inhibitory potency. Collectively, these results indicate
that the structure of the linker is important for activity.
On the basis of the in vitro study described above, we se-
lected compound 7i15 and evaluated its efficacy in an
ouabain-induced tonotropic and arrhythmia model of
heart failure.16 The effects of compound 7i and reference
compounds KB-R7943 (2) and 3 on the tonotropic ef-
fects of ouabain and on the time for ouabain-induced
Figure 3. Effects of compounds KB-R7943 (2), 3, and 7i on the onset
of arrhythmia induced by ouabain in guinea pig isolated atria.
*P < 0.05, **P < 0.01 versus control (Dunnettꢀs test). Each value is the
mean s.e.mean of at least four experiments.
onset of arrhythmia in isolated guinea pig atria were
evaluated. The results are shown in Figures 2 and 3.
The tonotropic effect of ouabain was lowered in a con-
centration-dependent manner by compounds KB-
R7943 (2), 3, and 7i. The effect of 7i was approximately
10-times greater than that of KB-R7943 (2), and 7i was
also more potent than 3, as shown in Figure 2. The time
to the onset of arrhythmia following treatment with
compound 7i was delayed in a concentration-dependent
manner, and compound 7i was more effective than
KB-R7943 (2) and 3, as shown in Figure 3. Hence,
compound 7i was confirmed to have efficacy against
ouabain-induced tonotropy and arrhythmia onset, and
was found to be more potent than KB-R7943 (2) and
compound 3 in this particular heart failure model.
their design based on compound 4. We found that a
compound containing a 4-pyridyl ring (7i), rather than
a phenyl ring, had enhanced reverse NCX inhibitory
activity. Compound 10a, in which the pyridine ring
was changed into a piperidine ring, had the same inhibi-
tory activity as 7i, and both 7i and 10a were potent NCX
inhibitors, with IC50 values of 0.22lM against reverse
NCX activity. Compound 7i also showed efficacy
against tonotropic effect of ouabain and ouabain-
induced arrhythmia onset in a heart failure model using
isolated atria from guinea pigs. Further optimization
studies to discover orally active and potent inhibitors
of reverse NCX activity, based on compound 7i, will
be reported in future publications.
5. Experimental
5.1. Chemistry
4. Conclusion
A series of benzyloxyphenyl derivatives have been pre-
pared and evaluated for their inhibitory activity against
reverse and forward mode NCX functions, following
Melting points were determined with a Yanaco MP-
500D melting point apparatus or a Buchi B-545 melting
¨

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T. Kuramochi et al. / Bioorg. Med. Chem. 13 (2005) 725–734
1
point apparatus and are uncorrected. H NMR spectra
were recorded on a JEOL JNM-LA300 or a JNM-
EX400 spectrometer and the chemical shifts are ex-
pressed in d (ppm) values with tetramethylsilane as an
internal standard (in NMR description, s = singlet,
d = doublet, t = triplet, m = multiplet, and br = broad
peak). Mass spectra were recorded on a Hitachi M-80
or a JEOL JMS-LX2000 spectrometer. The elemental
analyses were performed with a Yanaco MT-5 micro-
analyzer (C, H, N) and were within 0.4% of theoretical
values. Drying of organic solutions during workup was
done over anhydrous Na2SO4.
(10mL). The mixture was stirred at 70°C for 1h. The
mixture was concentrated in vacuo. The residue was
partitioned between CHCl3 and aqueous NaOH. The
organic layer was dried and concentrated in vacuo.
The residue was recrystallized from CHCl3. This mate-
rial was converted to its hydrochloride salt by treating
it with 4M HCl/AcOEt (0.256mL). The crude salt was
crystallized from EtOH–AcOEt to give 7b as a colorless
powder (341mg, 68%): mp 207–221°C; 1H NMR
(400MHz, DMSO-d6): d 4.54 (2H, d, J = 4.9Hz), 5.15
(2H, s), 7.04–7.14 (5H, m), 7.14–7.22 (1H, m), 7.28–
7.35 (2H, m), 7.43–7.50 (1H, m), 7.51 (1H, s), 8.26
(1H, dd, J = 8.8, 2.2Hz), 8.67 (1H, d, J = 2.2Hz), 9.04
(1H, s), 9.30 (1H, t, J = 5.9Hz), 14.52 (2H, br s); MS
(FAB) m/z 419 (M+H)+. Anal. Calcd for C23H19N4O3S-
FÆHCl: C, 60.73; H, 4.43; N, 12.32; F, 4.18; Cl, 7.79.
Found: C, 60.59; H, 4.41; N, 12.36; F, 4.07; Cl, 7.90.
5.1.1. 6-{4-[(3-Fluorobenzyl)oxy]phenoxy}nicotinic acid
(6). To the mixture of 6-{4-[(3-fluorobenzyl)oxy]phen-
oxy}nicotinonitrile (5) (12.0g, 37.5mmol), EtOH
(80mL), and 5M NaOH (75mL, 375mmol) was stirred
at 100°C for 1.5h. After cooling at room temperature,
the mixture was concentrated in vacuo. To the mixture
was added 1M HCl at 0°C. The precipitate was filtered
and dried in vacuo to afford compound 6 as a beige
5.1.4. 6-{4-[(3-Fluorobenzyl)oxy]phenoxy}-N-(1,3-thiazol-
4-ylmethyl)nicotinamide hydrochloride (7c). Compound
7c was prepared from 6 by a procedure similar to that
described for 7a. Compound 7c was obtained as a color-
less amorphous (66%): 1H NMR (400MHz, DMSO-d6):
d 4.62 (2H, d, J = 5.9Hz), 5.15 (2H, s), 7.06–7.21 (6H,
m), 7.28–7.34 (2H, m), 7.43–7.51 (2H, m), 8.28 (1H,
dd, J = 8.8, 2.5Hz), 8.65 (1H, d, J = 2.5Hz), 9.08 (1H,
d, J = 2.0Hz), 9.16 (1H, t, J = 5.5Hz); MS (FAB) m/z
436 (M+H)+. Anal. Calcd for C23H18N4O3FÆ0.7HClÆ0.8-
H2O: C, 58.11; H, 4.30; N, 8.84; F, 4.00; Cl, 5.22; S,
6.74. Found: C, 57.95; H, 4.13; N, 8.84; F, 3.97; Cl,
5.42; S, 6.76.
1
powder (12.6g, 99%): H NMR (300MHz, DMSO-d6):
d 5.15 (2H, s), 7.04–7.20 (6H, m), 7.27–7.33 (2H, m),
7.42–7.49 (1H, m), 8.26 (1H, dd, J = 8.6, 2.4Hz), 8.65
(1H, d, J = 2.4Hz), 13.16 (1H, br s); MS (FAB) m/z
340 (M+H)+.
5.1.2. 6-{4-[(3-Fluorobenzyl)oxy]phenoxy}-N-(2-furylmeth-
yl)nicotinamide (7a). To the mixture of furfurylamine
(78mg, 0.80mmol), compound 6 (285mg, 0.84mmol),
HOBt (54mg, 0.40mmol), and THF (5mL) was added
WSCÆHCl (169mg, 0.880mmol) at 0°C. The mixture
was stirred at room temperature for 2h. The mixture
was partitioned between CHCl3 and aqueous NaOH.
The organic layer was dried, concentrated in vacuo.
The residue was purified by column chromatography
on silica gel (CHCl3/MeOH = 1:0–98:2) to give 7a as a
beige solid (316mg). This material was crystallized from
hexane–AcOEt to give 7a as a colorless powder (185mg,
5.1.5. 6-{4-[(3-Fluorobenzyl)oxy]phenoxy}-N-(pyrimidin-
4-ylmethyl)nicotinamide (7d). Compound 7d was pre-
pared from 6 by a procedure similar to that described
for 7a. Compound 7d was obtained as a colorless solid
(74%): mp 138–139°C; 1H NMR (400MHz, DMSO-
d6): d 4.56 (2H, d, J = 5.9Hz), 5.15 (2H, s), 7.05–7.21
(6H, m), 7.28–7.34 (2H, m), 7.43–7.49 (2H, m), 8.29
(1H, dd, J = 8.8, 2.4Hz), 8.67 (1H, d, J = 2.4Hz), 8.73
(1H, d, J = 5.4Hz), 9.11 (1H, s), 9.25 (1H, t,
J = 6.1Hz); MS (FAB) m/z 431 (M+H)+. Anal. Calcd
for C24H19N4O3F: C, 66.97; H, 4.45; N, 13.02; F, 4.41.
Found: C, 67.11; H, 4.57; N, 13.20; F, 4.24.
1
55%): mp 100–101°C; H NMR (400MHz, DMSO-d6):
d 4.47 (2H, d, J = 5.3Hz), 5.15 (2H, s), 6.28 (1H, d,
J = 2.9Hz), 6.38–6.41 (1H, m), 7.02–7.13 (5H, m),
7.14–7.20 (1H, m), 7.28–7.33 (2H, m), 7.42–7.49 (1H,
m), 7.57–7.59 (1H, m), 8.25 (1H, dd, J = 8.5, 2.5Hz),
8.60 (1H, d, J = 2.5Hz), 9.02 (1H, t, J = 5.9Hz); MS
(FAB) m/z 419 (M+H)+. Anal. Calcd for C24H19N2O4F:
C, 68.89; H, 4.58; N, 6.70; F, 4.54. Found: C, 68.75; H,
4.54; N, 6.67; F, 4.54.
5.1.6. 6-{4-[(3-Fluorobenzyl)oxy]phenoxy}-N-(pyrimidin-
2-ylmethyl)nicotinamide hydrobromide (7e). Compound
7e was prepared from 6 by a procedure similar to that
described for 7a. Compound 7e was obtained as an or-
ange powder (54%): mp 161–166°C; 1H NMR
(400MHz, DMSO-d6): d 4.67 (2H, d, J = 5.9Hz), 5.16
(2H, s), 7.04–7.21 (6H, m), 7.28–7.34 (2H, m), 7.41–
7.50 (2H, m), 8.29 (1H, dd, J = 8.5, 2.4Hz), 8.66 (1H,
d, J = 2.4Hz), 8.79 (2H, d, J = 4.9Hz), 9.16 (1H, t,
J = 5.8Hz); MS (FAB) m/z 431 (M+H)+. Anal. Calcd
for C24H19N4O3FÆ2HBrÆ0.1H2O: C, 48.52; H, 3.60; N,
9.43; F, 3.20; Br, 26.96. Found: C, 48.30; H, 3.43; N,
9.41; F, 3.24; Br, 26.71.
5.1.3. 6-{4-[(3-Fluorobenzyl)oxy]phenoxy}-N-(1H-imid-
azol-4-ylmethyl)nicotinamide hydrochloride (7b). To the
mixture
of
(1-trityl-1H-imidazol-4-yl)methylamine
(407mg, 1.20mmol), compound 6 (407mg, 1.20mmol),
HOBt (81mg, 0.60mmol) and THF (6mL), DMF
(1mL) was added WSCÆHCl (253mg, 1.32mmol) at
0°C. The mixture was stirred at room temperature for
3h, and was partitioned between CHCl3 and aqueous
NaOH. The organic layer was dried, concentrated in
vacuo. The residue was purified by column chromato-
graphy on silica gel (CHCl3/MeOH = 98:2–96:4) to give
6-{4-[(3-fluorobenzyl)oxy]phenoxy}-N-[(1-trityl-1H-imid-
azol-4-yl)methyl]nicotinamide as a beige form (746mg,
94%). The intermediate was dissolved in 90% AcOH
5.1.7. 6-{4-[(3-Fluorobenzyl)oxy]phenoxy}-N-(pyrazin-2-
ylmethyl)nicotinamide (7f). Compound 7f was prepared
from 6 by a procedure similar to that described for 7a.
Compound 7f was obtained as a colorless powder

T. Kuramochi et al. / Bioorg. Med. Chem. 13 (2005) 725–734
731
(69%): mp 135–136°C; 1H NMR (400MHz, DMSO-d6):
d 4.62 (2H, d, J = 5.9Hz), 5.15 (2H, s), 7.05–7.14 (5H,
m), 7.14–7.21 (1H, m), 7.28–7.33 (2H, m), 7.42–7.49
(1H, m), 8.28 (1H, dd, J = 8.8, 2.4Hz), 8.54 (1H, d,
J = 2.4Hz), 8.57–8.61 (1H, m), 8.63–8.66 (2H, m), 9.23
(1H, t, J = 5.8Hz); MS (FAB) m/z 431 (M+H)+. Anal.
Calcd for C24H19N4O3F: C, 66.97; H, 4.45; N, 13.02;
F, 4.41. Found: C, 67.10; H, 4.40; N, 12.93; F, 4.23.
silica gel (hexane/AcOEt = 1:0–4:1) to give t-butyl 4-{4-
[(3-fluorobenzyl)oxy]phenoxy}piperidine-1-carboxylate
as a colorless oil (1.37g, 97%). The mixture of the inter-
mediate (1.36g, 3.39mmol), 4M HCl in AcOEt
(4.23mL, 16.9mmol), and MeOH (10mL) was stirred
at room temperature. The mixture was partitioned be-
tween CHCl3 and aqueous NaOH. The organic layer
was dried and concentrated in vacuo to give a crude
beige solid (650mg). The residue was dissolved in
AcOEt (10mL). To the mixture was added 4M HCl in
AcOEt (0.650mL, 2.59mmol) at 0°C. The precipitate
was collected to give 9 as a colorless powder (640mg,
56%): mp 199–200°C; 1H NMR (400MHz, DMSO-
d6): d 1.75–1.86 (2H, m), 2.01–2.10 (2H, m), 2.95–3.07
(2H, m), 3.16–3.24 (2H, m), 4.46–4.54 (1H, m), 5.07
(2H, s), 6.95 (4H, s), 7.11–7.18 (1H, m), 7.23–7.29
(2H, m), 7.39–7.47 (1H, m), 8.98 (2H, br s); MS
(FAB) m/z 302 (M+H)+. Anal. Calcd for C18H20NO2FÆ
HCl: C, 64.00; H, 6.27; N, 4.15; F, 5.62; Cl, 10.49.
Found: C, 63.98; H, 6.28; N, 4.15; F, 5.61; Cl, 10.51.
5.1.8. 6-{4-[(3-Fluorobenzyl)oxy]phenoxy}-N-(pyridin-2-
ylmethyl)nicotinamide hydrochloride (7g). Compound
7g was prepared from 6 by a procedure similar to that
described for 7a. Compound 7g was obtained as a color-
less solid (167mg, 45%): mp 160–163°C; 1H NMR
(400MHz, DMSO-d6): d 4.78 (2H, d, J = 5.4Hz),
7.06–7.12 (5H, m), 7.12–7.21 (1H, s), 7.28–7.34 (2H,
m), 7.42–7.49 (1H, m), 7.76–7.87 (2H, m), 8.30–8.38
(2H, m), 8.70 (1H, d, J = 2.4Hz), 8.75–8.78 (1H, m),
9.53 (1H, t, J = 5.4Hz); MS (FAB) m/z 430 (M+H)+.
Anal. Calcd for C25H20N3O3FÆHCl: C, 64.45; H, 4.54;
N, 9.02; F, 4.08; Cl, 7.61. Found: C, 64.23; H, 4.46;
N, 9.00; F, 4.12; Cl, 7.59.
5.1.12. 4-{4-[(3-Fluorobenzyl)oxy]phenoxy}-N-(pyridin-4-
ylmethyl)piperidine-1-carboxamide hydrochloride (10a).
To the mixture of bis(trichloromethyl) carbonate
(119mg, 0.40mmol) and THF (5mL) was added a
THF (5mL) solution of 4-{4-[(3-fluorobenzyl)oxy]phen-
oxy}piperidine (301mg, 1.00mmol) and Et3N
(0.209mL, 1.50mmol) at 0°C. The mixture was stirred
at room temperature for 70min. To the mixture were
added a THF (5mL) solution of pyridin-4-ylmethylam-
ine (130mg, 1.20mmol) and Et3N (0.167mL, 1.20mmol)
at 0°C. The mixture was stirred at room temperature for
16h. The mixture was partitioned between CH3Cl and
aqueous NaOH. The organic layer was dried and con-
centrated in vacuo. The residue was purified by column
chromatography on silica gel (CHCl3/MeOH = 98:2–
96:4) to give the free base of 10a as a brown syrup
(290mg, 67%). This material was converted to its hydro-
chloride salt by treating it with hydrochloride in
AcOEt–MeOH. The mixture was concentrated in vacuo.
The residue was recrystallized from AcOEt–EtOH–
CH3CN to give 10a as a light brown powder (95mg,
20%): mp 175–183°C; 1H NMR (400MHz, DMSO-
d6): d 1.46–1.56 (2H, m), 1.84–1.94 (2H, m), 3.13–3.23
(2H, m), 3.68–3.77 (2H, m), 4.40–4.49 (3H, m), 5.07
(2H, m), 6.90–6.96 (4H, m), 7.12–7.18 (1H, m), 7.23–
7.30 (2H, m), 7.40–7.47 (1H, m), 7.50–7.55 (1H, m),
7.87 (2H, t, J = 6.9Hz), 8.81 (2H, d, J = 6.9Hz); MS
(FAB) m/z 436 (M+H)+. Anal. Calcd for C25H26N3O3FÆ
HClÆ0.3H2O: C, 62.90; H, 5.83; N, 8.80; F, 3.98; Cl,
7.43. Found: C, 62.82; H, 5.76; N, 8.94; F, 3.96; Cl, 7.30.
5.1.9. 6-{4-[(3-Fluorobenzyl)oxy]phenoxy}-N-(pyridin-3-
ylmethyl)nicotinamide hydrochloride (7h). Compound
7h was prepared from 6 by a procedure similar to that
described for 7a. Compound 7h was obtained as a color-
less powder (75%): mp 178–185°C; 1H NMR (400MHz,
DMSO-d6): d 4.64 (2H, d, J = 5.6Hz), 5.15 (2H, s), 7.05–
7.13 (5H, m), 7.14–7.21 (1H, m), 7.28–7.34 (2H, m),
7.42–7.49 (1H, m), 7.93–7.98 (1H, m), 8.30 (1H, dd,
J = 8.8, 2.5Hz), 8.44 (1H, d, J = 7.8Hz), 8.67 (1H, d,
J = 2.5Hz), 8.79 (1H, d, J = 5.3Hz), 8.87 (1H, s), 9.45
(1H, t, J = 5.6Hz); MS (FAB) m/z 430 (M+H)+. Anal.
Calcd for C25H20N3O3FÆHCl: C, 64.45; H, 4.54; N,
9.02; F, 4.08; Cl, 7.61. Found: C, 64.35; H, 4.51; N,
9.04; F, 3.88; Cl, 7.65.
5.1.10. 6-{4-[(3-Fluorobenzyl)oxy]phenoxy}-N-(pyridin-4-
ylmethyl)nicotinamide hydrochloride (7i). Compound 7i
was prepared from 6 by a procedure similar to that de-
scribed for 7a. Compound 7i was obtained as a colorless
powder (69%): mp 158–162°C; 1H NMR (400MHz,
DMSO-d6): d 4.74 (2H, d, J = 5.8Hz), 5.15 (2H, s),
7.06–7.14 (5H, m), 7.14–7.21 (1H, m), 7.28–7.34 (2H,
m), 7.43–7.49 (1H, m), 7.96 (2H, d, J = 6.5Hz), 8.34
(1H, dd, J = 8.8, 2.5Hz), 8.72 (1H, d, J = 2.5Hz), 8.84
(2H, d, J = 6.3Hz), 9.58 (1H, t, J = 5.8Hz); MS (FAB)
m/z 430 (M+H)+. Anal. Calcd for C25H20N3O3FÆHClÆ
0.25H2O: C, 63.83; H, 4.61; N, 8.93; F, 4.04; Cl, 7.54.
Found: C, 64.05; H, 4.47; N, 8.92; F, 4.74; Cl, 7.41.
5.1.11.
4-{4-[(3-Fluorobenzyl)oxy]phenoxy}piperidine
5.1.13. Pyridin-4-ylmethyl 4-{4-[(3-fluorobenzyl)oxy]phen-
oxy}piperidine-1-carboxylate hydrochloride (10b). To the
mixture of 4-{4-[(3-fluorobenzyl)oxy]phenoxy}piper-
idine hydrochloride (9) (406mg, 1.20mmol), Et3N
(0.201mL, 1.44mmol), and THF (5mL) was added the
THF (2mL) solution of methyl chloroformate
(0.102mL, 1.44mmol) at 0°C. The mixture was stirred
at room temperature for 30min. The mixture was parti-
tioned between AcOEt and aqueous HCl. The organic
layer was washed with aqueous NaOH, dried, and con-
centrated in vacuo. The residue was purified by column
hydrochloride (9). To the mixture of 4-[(3-fluorobenzyl)-
oxy]phenol (8) (770mg, 3.53mmol), PPh3 (1.20g,
4.59mmol), and THF (10mL) was added a mixture of
DEAD (0.723mL, 4.59mmol), t-butyl 4-hydroxypiper-
idine-1-carboxylate (853mg, 4.24mmol), and THF
(10mL) at 0°C. The mixture was stirred at room tem-
perature for 1day. The mixture was partitioned between
AcOEt and H2O. The organic layer was washed with
brine and dried over MgSO4 and concentrated in vacuo.
The residue was purified by column chromatography on

732
T. Kuramochi et al. / Bioorg. Med. Chem. 13 (2005) 725–734
chromatography on silica gel (CHCl3/MeOH = 99:1) to
give methyl 4-{4-[(3-fluorobenzyl)oxy]phenoxy}piper-
idine-1-carboxylate as a light yellow oil (445mg). The
material was added to the mixture of pyridin-4-ylmeth-
anol (313mg, 2.88mmol), 60% NaH (46mg, 1.16mmol)
and toluene (10mL) at room temperature. The mixture
was refluxed for 2days. After cooling at room tempera-
ture, the mixture was partitioned between CHCl3 and
brine. The organic layer was dried, concentrated in va-
cuo. The residue was purified by column chromatogra-
phy on silica gel (CHCl3/MeOH = 99:1) to give the
free base of 10b as a light yellow syrup (235mg). The
material was converted to its hydrochloride salt by treat-
ing it with hydrochloride in AcOEt. The mixture was
concentrated in vacuo. The residue was recrystallized
from AcOEt–CH3CN to give 10b as a colorless powder
(300MHz, DMSO-d6): d 1.34–1.50 (2H, m), 1.56–1.69
(1H, m), 1.90–2.04 (1H, m), 2.38–2.48 (2H, m), 2.62–
2.79 (1H, m), 2.97–3.03 (1H, m), 4.01–4.12 (1H, m),
5.05 (2H, s), 6.83–6.95 (4H, m), 7.09–7.19 (1H, m),
7.20–7.30 (2H, m), 7.37–7.47 (1H, m); MS (FAB) m/z
302 (M+H)+.
5.1.17. 3-({4-[(3-Fluorobenzyl)oxy]phenoxy}methyl)piper-
idine hydrochloride (12). Compound 12 was prepared
from 8 by a procedure similar to that described for 9.
Compound 12 was obtained as a white powder (76%
for two steps): 1H NMR (300MHz, DMSO-d6): d
1.24–1.40 (1H, m), 1.63–1.87 (3H, m), 2.14–2.30 (1H,
m), 2.63–2.85 (2H, m), 3.16–3.26 (1H, m), 3.27–3.35
(1H, m), 3.74–3.83 (1H, m), 3.84–3.91 (1H, m), 5.07
(2H, s), 6.87 (2H, d, J = 9.2Hz), 6.95 (2H, d,
J = 9.2Hz), 7.10–7.19 (1H, m), 7.22–7.29 (2H, m),
7.39–7.47 (1H, m), 8.91–9.25 (2H, m); MS (FAB) m/z
316 (M+H)+.
1
(100mg, 18% for two steps): mp 155–162°C; H NMR
(400MHz, DMSO-d6): d 1.51–1.69 (2H, m), 1.87–2.00
(2H, m), 3.22–3.47 (2H, m), 3.64–3.91 (2H, m), 4.45–
4.52 (1H, m), 5.07 (2H, s), 5.38 (2H, s), 6.91–6.97 (4H,
m), 7.12–7.18 (1H, m), 7.24–7.30 (2H, m), 7.40–7.47
(1H, m), 7.93 (2H, t, J = 6.9Hz), 8.87 (2H, t,
J = 6.9Hz); MS (FAB) m/z 437 (M+H)+. Anal. Calcd
for C25H25N2O4FÆHCl: C, 63.49; H, 5.54; N, 5.92; F,
4.02; Cl, 7.50. Found: C, 63.48; H, 5.53; N, 5.98; F,
3.97; Cl, 7.56.
5.1.18. 3-{4-[(3-Fluorobenzyl)oxy]phenoxy}-N-(pyridin-4-
ylmethyl)piperidine-1-carboxamide (13). Compound 13
was prepared from 11 by a procedure similar to that de-
scribed for 10a. Compound 13 was obtained as a color-
1
less solid (29%): mp 148–152°C; H NMR (400MHz,
DMSO-d6): d 1.40–1.53 (1H, m), 1.53–1.66 (1H, m),
1.65–1.78 (1H, m), 1.93–2.04 (1H, m), 3.05–3.18 (2H,
m), 3.52–3.60 (1H, m), 3.85–3.93 (1H, m), 4.13–4.21
(1H, m), 4.27 (2H, d, J = 5.8Hz), 5.07 (2H, s), 6.90
(2H, d, J = 9.3Hz), 6.93 (2H, d, J = 9.3Hz), 7.11–7.18
(1H, m), 7.19–7.32 (5H, m), 7.39–7.46 (1H, m), 8.47–
8.56 (2H, m); MS (FAB) m/z 436 (M+H)+. Anal. Calcd
for C25H26N3O3FÆC2H2O4: C, 61.71; H, 5.37; N, 8.00; F,
3.62. Found: C, 61.60; H, 5.25; N, 7.99; F, 3.66.
5.1.14.
idin-1-yl)-3-oxopropyl]pyridine
4-[3-(4-{4-[(3-Fluorobenzyl)oxy]phenoxy}piper-
hydrochloride (10c).
Compound 10c was prepared from 9 by a procedure
similar to that described for 7a. Compound 10c was ob-
tained as a beige powder (68%): mp 159–165°C; 1H
NMR (400MHz, DMSO-d6): d 1.39–1.50 (1H, m),
1.50–1.61 (1H, m), 1.78–1.96 (2H, m), 2.87 (2H, t,
J = 7.3Hz), 3.09 (2H, t, J = 7.3Hz), 3.17–3.26 (1H, m),
3.27–3.37 (1H, m), 3.65–3.76 (1H, m), 3.78–3.88 (1H,
m), 4.43–4.52 (1H, m), 5.07 (2H, s), 6.88–6.97 (4H, m),
7.12–7.18 (1H, m), 7.23–7.29 (2H, m), 7.39–7.47 (1H,
m), 7.96 (2H, d, J = 6.3Hz), 8.80 (2H, d, J = 6.3Hz);
MS (FAB) m/z 435 (M+H)+. Anal. Calcd for C26H27-
N2O3FÆHCl: C, 66.31; H, 5.99; N, 5.95; F, 4.03; Cl,
7.53. Found: C, 66.07; H, 5.87; N, 5.97; F, 4.07; Cl, 7.47.
5.1.19. 3-({4-[(3-Fluorobenzyl)oxy]phenoxy}methyl)-N-
(pyridin-4-ylmethyl)piperidine-1-carboxamide (14). Com-
pound 14 was prepared from 12 by a procedure similar
to that described for 10a. Compound 14 was obtained as
a colorless solid (24%): mp 129–134°C; 1H NMR
(400MHz, DMSO-d6): d 1.23–1.46 (2H, m), 1.59–1.68
(1H, m), 1.78–1.90 (2H, m), 2.63–2.71 (1H, m), 2.78–
2.86 (1H, m), 3.73–3.87 (3H, m), 4.00–4.07 (1H, m),
4.25 (2H, d, J = 5.9Hz), 5.06 (2H, s), 6.87 (2H, d,
J = 9.2Hz), 6.93 (2H, d, J = 9.2Hz), 7.11–7.18 (2H,
m), 7.23–7.30 (4H, m), 7.39–7.46 (1H, m), 8.40–8.60
(2H, m); MS (FAB) m/z 450 (M+H)+. HRMS calcd
for C26H28N3O3F 450.2153. Found: 450.2166.
5.1.15. 4-[(1E)-3-(4-{4-[(3-Fluorobenzyl)oxy]phenoxy}-
piperidin-1-yl)-3-oxoprop-1-en-1-yl]pyridine hydrochlo-
ride (10d). Compound 10d was prepared from 9 by a
procedure similar to that described for 7a. Compound
10d was obtained as a beige powder (52%): mp 185–
199°C; 1H NMR (400MHz, DMSO-d6): d 1.51–1.68
(2H, m), 1.88–2.02 (2H, m), 3.37–3.46 (1H, m), 3.55–
3.64 (1H, m), 3.90–4.05 (2H, m), 4.49–4.57 (1H, m),
5.07 (2H, s), 6.95 (4H, m), 7.12–7.18 (1H, m), 7.24–
7.30 (2H, m), 7.40–7.47 (1H, m), 7.60 (1H, d,
J = 15.6Hz), 7.85 (1H, d, J = 15.6Hz), 8.29 (2H, d,
J = 5.4Hz), 8.89 (2H, d, J = 6.9Hz); MS (FAB) m/z
433 (M+H)+. Anal. Calcd for C26H25N2O3FÆHCl: C,
66.59; H, 5.59; N, 5.97; F, 4.05; Cl, 7.56. Found: C,
66.40; H, 5.68; N, 5.98; F, 3.93; Cl, 7.56.
5.1.20. tert-Butyl 4-[{4-[(3-fluorobenzyl)oxy]phenyl}(meth-
yl)amino]piperidine-1-carboxylate (16). To the mixture of
4-aminophenol 15 (1.09g, 10.0mmol) and DMF (40mL)
were added t-BuOK (1.35g, 12.0mmol) and 1-(bromo-
methyl)-3-fluorobenzene (1.23mL, 10.0mmol) at 0°C.
The mixture was stirred at room temperature for 2h.
H2O was added to the mixture at 0°C. The mixture
was partitioned between AcOEt and brine. The organic
layer was dried and concentrated in vacuo. The residue
was purified by column chromatography on silica gel
(hexane/AcOEt = 1:0–1:1) to give {4-[(3-fluorobenzyl)-
oxy]phenyl}amine as a yellow oil (1.87g, 86%). To the
mixture of {4-[(3-fluorobenzyl)oxy]phenyl}amine (869
mg, 4.00mmol) and AcOH (10mL) were added t-butyl
5.1.16. 3-{4-[(3-Fluorobenzyl)oxy]phenoxy}piperidine (11).
Compound 11 was prepared from 8 by a procedure sim-
ilar to that described for 9. Compound 11 was obtained
as an orange liquid (44% for two steps): 1H NMR

T. Kuramochi et al. / Bioorg. Med. Chem. 13 (2005) 725–734
733
4-oxopiperidine-1-carboxylate (797mg, 4.00mmol) and
NaBH(OAc)3 (1.02g, 4.80mmol) at 10°C. The mixture
was stirred at room temperature for 4h. The mixture
was concentrated in vacuo. The residue was partitioned
between CHCl3 and 5M NaOH. The organic layer was
dried and concentrated in vacuo. The residue was puri-
fied by column chromatography on silica gel (hexane/
AcOEt = 1:0–4:1) to give t-butyl 4-({4-[(3-fluoro-
benzyl)oxy]phenyl}amino)piperidine-1-carboxylate as a
yellow syrup (1.62g). To the mixture of t-butyl 4-({4-
[(3-fluorobenzyl)oxy]phenyl}amino)piperidine-1-carbox-
ylate as a yellow syrup (1.62g) and AcOH (10mL) was
added 35% formaldehyde in H2O (1.72g, 20.0mmol)
and NaBH(OAc)3 (1.70g, 8.00mmol) at 10°C. The
mixture was stirred at room temperature for 18h. The
mixture was concentrated in vacuo. The residue was
partitioned between CHCl3 and 5M NaOH. The organ-
ic layer was dried and concentrated in vacuo. The resi-
due was purified by column chromatography on
silica gel (hexane/AcOEt = 1:0–4:1) to give 16 as a
yellow syrup (1.33g, 80% for two steps): 1H NMR
(300MHz, CDCl3): d 1.44–1.50 (3H, m), 1.46 (9H, s),
1.67–1.76 (2H, m), 2.70 (3H, s), 2.70–2.79 (2H, m),
3.42–3.55 (1H, m), 4.12–4.25 (1H, m), 5.00 (2H, s),
6.81 (2H, d, J = 9.2Hz), 6.89 (2H, d, J = 9.2Hz), 6.97–
7.03 (1H, m), 7.12–7.21 (2H, m), 7.29–7.37 (1H, m);
MS (FAB) m/z 414 M+.
6.1.1. Effects on tonotropic effects of ouabain and the
onset of arrhythmia induced by ouabain in guinea pig
isolated atria.16 Toxic effects of ouabain on hearts have
partly been explained by its action on the reverse mode of
Na+/Ca2+ exchangers playing in the cardiac myocytes.
The influx of Ca2+ induced by the reverse mode
of Na+/Ca2+ exchangers have been thought to produce
cardiac injuries triggering arrhythmia and/or diastolic
dysfunctions. These compounds were thus tested on the
cardiac diastolic dysfunctions and arrhythmia in the gui-
nea pig atria evoked by ouabain at its toxic dose (3lM).
Isolated whole atria of male guinea pigs (350–450g) were
bathed in warmed (37°C) and gassed (95% O2 + 5%
CO2) Tyrode solution containing (in mM): NaCl 137,
KCl 6, CaCl2 1.82, MgCl2 1.05, NaH2PO4 0.417, NaH-
CO3 11.5, and glucose 5.5. One end of the muscle was at-
tached to a rigid support and the other end was attached
to a force–displacement transducer (Nihon Kohden SB-
1T) via a silk suture in the 30mL bath. Each tissue was
placed under 0.5g tension. Following stabilization of
spontaneous beating of the preparations for about
45min with replacing the solution every 15min, com-
pounds were added. Ouabain (3lM) was added 45min
after the addition of the compounds. The concentration
of atria was recorded continuously for 90min after the
addition of ouabain. Beating rates were counted from
the signals of the contraction measured.
5.1.21. N-{4-[(3-Fluorobenzyl)oxy]phenyl}-N-methylpiper-
idin-4-amine hydrochloride (17). To the mixture of 16
(1.32g, 3.17mmol) and MeOH (10mL) was added 4M
AcOEt solution of HCl (3.97mL, 15.9mmol) at 0°C.
The mixture was stirred at room temperature for over-
night and concentrated in vacuo. The residue was crys-
tallized from solvent of AcOEt and CH3CN, MeOH
Acknowledgements
We wish to thank Mr. Takayuki Sato for evaluating the
inhibitory activity of compounds against the sodium–
calcium exchanger. We would like to express our grati-
tude to Dr. Minoru Okada, Dr. Toshihiro Watanabe,
and Dr. Tomo-Oki Satoh for helpful support in the
preparation of this manuscript and are also grateful to
Mr. Masashi Funatsu for measurements of experimental
data.
1
to afford 17 as a beige powder (1.03g, 84%): H NMR
(400MHz, DMSO-d6): d 1.69–2.07 (3H, m), 2.30–2.44
(1H, m), 2.78–2.92 (2H, m), 2.98–3.08 (3H, m), 3.27–
3.48 (2H, m), 3.77–3.90 (1H, m), 5.16 (2H, s), 7.11–
7.25 (3H, m), 7.30 (2H, d, J = 8.3Hz), 7.41–7.49 (1H,
m), 7.50–7.79 (2H, m), 8.69 (1H, br s), 9.26 (1H, br s),
13.43 (1H, br s); MS (FAB) m/z 315 (M+H)+.
References and notes
5.1.22. 4-[{4-[(3-Fluorobenzyl)oxy]phenyl}(methyl) amino]-
(18).
1. Ver Donck, L.; Marcal, B.; Verdonck, F. Cardiovasc. Res.
1993, 27, 349.
2. Karmazyn, M.; Gan, X. T.; Humphreys, R. A.; Yoshida,
H.; Kusumoto, K. Circ. Res. 1999, 85, 777.
3. Eigel, B. N.; Hadley, R. W. Am. J. Physiol. 1999, 277,
H1817.
4. Pogwizd, S. M.; Schlotthauer, K.; Li, L.; Yuan, W.; Bers,
D. M. Circ. Res. 2001, 88, 1159.
5. Ko¨ster, O. F.; Szigeti, G. P.; Beuckelmann, D. J. Cardio-
vasc. Res. 1999, 41, 175.
6. Tani, M.; Neely, J. R. Circ. Res. 1989, 65, 1045.
7. Haigney, M. C. P.; Miyata, H.; Lakatta, E. G.; Stern, M.
G.; Silverman, H. S. Circ. Res. 1992, 71, 547.
8. Kaczorowski, G. J.; Garcia, M. L.; King, R.; Slaughter, R.
S. In Sodium–Calcium Exchange; Allen, F. J., Noble, D.,
Reuter, H., Eds.; Oxford University Press: Oxford, 1989;
pp 66–101.
N-(pyridin-4-ylmethyl)piperidine-1-carboxamide
Compound 18 was prepared from 17 by a procedure
similar to that described for 10a. Compound 18 was ob-
1
tained as a light yellow powder (17%): mp 75–77°C; H
NMR (400MHz, DMSO-d6): d 1.42–1.55 (2H, m), 1.55–
1.63 (2H, m), 2.63 (3H, s), 2.73–2.83 (2H, m), 3.59–3.69
(1H, m), 4.03–4.12 (2H, m), 4.25 (2H, d, J = 5.3Hz),
5.04 (2H, s), 6.83 (2H, d, J = 8.8Hz), 6.89 (2H, d,
J = 8.8Hz), 7.10–7.21 (2H, m), 7.22–7.29 (4H, m),
7.39–7.46 (1H, m), 8.45–8.52 (2H, m); MS (FAB) m/z
449 (M+H)+. Anal. Calcd for C26H29N4O2FÆ1.4H2O:
C, 65.92; H, 6.77; N, 11.83; F, 4.01. Found: C, 65.83;
H, 6.40; N, 11.59; F, 3.92.
6. Pharmacology
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6.1. 45Ca influx assay and cell necrosis assay
The methods were described in previous report.13

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NCX mode. While compound 10a was slightly better than
compound 7i in terms of its selectivity for reverse NCX
activity versus forward NCX activity, compound 7i was
better than compound 10a with regard to oral activity.
Since our goal is to create orally-active NCX inhibitors,
compound 7i was selected as the test compound in the
ouabain-induced heart failure model.
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15. Both compounds 7i and 10a were found to be potent NCX
inhibitors with IC50 values of 0.22lM against the reverse
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