(5-Arylfuran-2-ylcarbonyl)guanidines as cardioprotectives through the inhibition of Na+/H+ exchanger isoform-1

Article abstract of DOI:10.1021/jm0492305

A series of (5-arylfuran-2-ylcarbonyl)guanidines was synthesized and evaluated for the NHE-1 inhibitory activity and cardiprotective efficacy against ischemia-reperfusion injury. Starting with (5-phenylfuran-2-ylcarbonyl) guanidine 47 with a moderate inhibitory effect on NHE-1, the compounds with various substituents at the phenyl ring were investigated with the aim to optimize the potency. In this study, the 2,5-disubstituted compounds appeared to have better activities than the other analogues, and the 2-methoxy-5- chlorophenyl compound 85 was found as a potent inhibitor of NHE-1 (IC ₅₀ = 0.081 μM). Furthermore, 85 showed a marked reduction of infarct size in the rat myocardial infarction model in vivo and significant improvement of cardiac contractile function in the isolated rat heart ischemia model in vitro.

Full text of DOI:10.1021/jm0492305

2882
J. Med. Chem. 2005, 48, 2882-2891
(5-Arylfuran-2-ylcarbonyl)guanidines as Cardioprotectives through the
Inhibition of Na+/H+ Exchanger Isoform-1
Sunkyung Lee, Kyu Yang Yi,* Sun Kyung Hwang, Byung Ho Lee, Sung-eun Yoo, and Kyunghee Lee†
Medicinal Science Division, Korea Research Institute of Chemical Technology, Yuseong-gu, Daejeon 305-600, Korea, and
Central Research Center of Yuyu Inc., Kyounggi-do 430-804, Korea
Received September 22, 2004
A series of (5-arylfuran-2-ylcarbonyl)guanidines was synthesized and evaluated for the NHE-1
inhibitory activity and cardiprotective efficacy against ischemia-reperfusion injury. Starting
with (5-phenylfuran-2-ylcarbonyl)guanidine 47 with a moderate inhibitory effect on NHE-1,
the compounds with various substituents at the phenyl ring were investigated with the aim to
optimize the potency. In this study, the 2,5-disubstituted compounds appeared to have better
activities than the other analogues, and the 2-methoxy-5-chlorophenyl compound 85 was found
as a potent inhibitor of NHE-1 (IC50 ) 0.081 µM). Furthermore, 85 showed a marked reduction
of infarct size in the rat myocardial infarction model in vivo and significant improvement of
cardiac contractile function in the isolated rat heart ischemia model in vitro.
Introduction
Even though early reperfusion has been the best
strategy so far to limit infarct size,1 morbidity and
mortality from acute myocardial infarction (AMI) still
remain significant. Therefore there is a need to provide
better cardioprotection therapy which slows the pro-
gression of myocardial ischemic injury and attenuates
the detrimental consequences of reperfusion.2,3 The Na+/
H+ exchanger type 1 (NHE-1) inhibitors have been
found to reduce infarct size, myocardial stunning,
arrhythmia, and endothelial dysfunction in animal
models of myocardial ischemia and reperfusion.4-6
The available experimental evidence suggests that the
cardioprotection of NHE-1 inhibitors is likely to arise
primarily from the attenuation of intracellular Na+
accumulation during ischemia, which would attenuate
intracellular Ca2+ accumulation during both ischemia
and subsequent reperfusion.7,8 Ischemic conditions in-
duce intracellular acidification, which strongly activates
the NHE-1.9 Under these conditions, the NHE-1 plays
the crucial role to restore physiological pH by the
extrusion of H+ and the influx of Na+ with the ischemia-
induced inhibition of Na+/K+ ATPase which is the
primary Na+ extrusion pathway from the cardiac myo-
cyte. Then intracellular Na+ is accumulated, which
alters the sarcolemmal Na+/Ca2+ exchanger (NCX) to
the reversal mode in a manner that inhibits Ca2+ efflux
and/or enhances Ca2+ influx, resulting in a pathologic
increase in intracellular Ca2+. This intracellular Ca2+
overload is involved in ischemic and reperfusion injuries
such as activation of contractile dysfunction, arrhyth-
mia, and cellular death.10 With a strategy to attenuate
the harmful consequences of excessive NHE activation,
several NHE-1 inhibitors have been developed as car-
dioprotective agents.11
Figure 1. Representative NHE-1 inhibitors.
NHE-1 inhibitors,12 with its bioisostere such as ami-
noguanidine13 and 2-aminoimidazole,14 but it was quite
limited. However, the studies on the template bearing
an acylguanidine moiety were diversely performed
including the six-membered monocycles such as the
phenyl of cariporide15 and the pyrazine of amiloride,16
the five-membered monocycles such as the pyrazole of
zoniporide (Figure 1),17 and the bicycles such as a
quinoline and an indole.18
From our efforts to identify a novel template, espe-
cially based on five-membered ring which has not been
investigated as intensively as six-membered ring, (5-
phenylfuran-2-ylcarbonyl)guanidine 47 was found as an
initial lead. Starting with 47, the activity of this series
of compounds was optimized through the introduction
of various one or two substituents on the phenyl ring
attached to the 5-position of the furan ring (Figure 2).
Herein, the synthesis, structure-activity relationships,
and pharmacological properties of this analogues are
described.
There have been several trials to replace the acylguani-
dine moiety known as a key pharmacophoric unit for
Chemistry. The synthesis of (5-arylfuran-2-ylcarbon-
yl)guanidines was accomplished by relatively simple
procedures. Starting with methyl 5-bromofuran-2-car-
boxylate, variously substituted phenyl ring was intro-
duced into the 5-position of furan ring by the Pd-
* To whom correspondence should be addressed. Phone: +82-42-
860-7143. Fax: +82-42-861-1291. E-mail: kyyi@krict.re.kr.
† Yuyu Inc.
10.1021/jm0492305 CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/30/2005

Inhibition of Na+/H+ Exchanger Isoform-1
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2883
Figure 2. Identification of 5-(arylfuran-2-ylcarbonyl)guanidines.
Scheme 1. Preparation of Methyl 5-Arylfuran-2-carboxylatea
a Reagents and conditions: (a) Na2CO3, Pd(PPh3)4, toluene, reflux; (b) Ba(OH)2, Pd(PPh3)4, DME, 80 °C; (c) H2, Pd/C, MeOH, rt; (d)
BBr3, CH2Cl2, rt; (e) RI, K2CO3, DMF, rt.
Scheme 2. Preparation of
in PS120 variant cells in which the human NHE-1 was
selectively expressed.20 The cardioprotective efficacy was
evaluated using a perfused rat heart model according
to Langendorff21 and an in vivo rat myocardial infarction
model.22 In the heart perfused model, each isolated rat
heart was stabilized for 15 min, treated with 10 µM
concentration of the compound for 10 min, and subjected
to 30 min global ischemia followed by 30 min reperfu-
sion. The % recovery of rate pressure product (RPP )
HR × LVDP, heart rate × left ventricular developing
pressure) at the end of reperfusion to the preischemic
value was measured as an index for cardiac contractile
function. Antiischemic in vivo potency was determined
by measuring a percentage ratio of myocardial infarction
size to the area at risk (IS/AAR, %), using an ischemic
myocardium-damaged rat model which was stabilized
for 20 min after a left thoracotomy operation and
subjected to coronary artery occlusion for 45 min,
followed by reperfusion for 90 min. The vehicle or
compounds were intravenously administered by bolus
injection at 5 min prior to onset of ischemia into the
femoral vein.
(5-Arylfuran-2-ylcarbonyl)guanidinesa
a Reagents and conditions: (a) guanidine, MeOH, reflux; (b)
guanidine, DMF, rt; (c) (i) CDI, THF, rt; (ii) guanidine, rt.
catalyzed Suzuki reaction (Scheme 1).19 Generally the
Suzuki reaction proceeded well using sodium carbonate
as a base in toluene in the presence of Pd(PPh3)4. In
some cases, barium hydroxide was used as a base in
1,2-dimethoxyethane (DME). The compounds substi-
tuted with amino group (41-43) were prepared from
the corresponding nitro compounds (15-17) by catalytic
hydrogenation. Demethylation of the methoxy group of
5-(2-methoxy-5-chlorophenyl)furan-2-carboxylate 28 us-
ing BBr3 gave the phenol 44, which was further diversi-
fied to the ethoxy compound 45 and isopropoxy com-
pound 46 by O-alkylation.
As outlined in Scheme 2, acylguanidines were ob-
tained either from esters by treating with excess guani-
dine in methanol (method A) or in DMF (method B), or
from the corresponding carboxylic acids, first activating
with stoichiometric amounts of 1,1′-carbonyldiimidazole
(CDI), followed by treating with guanidine (method C).
The final acylguanidine compounds were purified by
silica gel column chromatography or crystallization as
a methanesulfonic acid salt.
Results and Discussion
The starting (5-phenylfuran-2-ylcarbonyl)guanidine
47 showed a weaker NHE-1 inhibitory activity (IC50
)
3.1 µM) than cariporide (IC50 ) 1.2 µM). The effect of
various substituents at the ortho-, meta-, or para-
position of the phenyl ring was primarily investigated
(Table 1). Most of the para-substituted analogues (66-
72) were inactive. The activities of the meta-substituted
analogues varied in a wide range, from almost inactive
compounds (64, 65), to moderately active compounds
(61, 62, and 63), and to quite active compounds (59, 60).
The ortho-substituted analogues were generally more
active than 47 as shown in 49 (IC50 ) 1.1 µM), 50
(IC50 ) 2.3 µM), 52 (IC50 ) 2.0 µM), and 51 (IC50 ) 0.34
µM). The compounds with bulkier 2-alkyl or -alkoxy
groups such as 55 (IC50 ) 1.4 µM), 56 (IC50 ) 2.6 µM),
57 (IC50 ) 2.4 µM), and 58 (IC50 ) 1.1 µM) also exhibited
good activities.
Biology. The NHE-1 inhibitory activity was deter-
mined by measuring their ability to inhibit the sodium
dependent recovery of pH following an imposed acidosis,

2884 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Lee et al.
Table 1. Inhibitory Effect on NHE-1 and Cardioprotective Efficacy of Monosubstituted 5-(Arylfuran-2-ylcarbonyl)guanidines
a
a
IC50
RPPb (%)
10 µM
IS/AARc (%)
0.3 mg/kg
IC50
RPPb (%)
10 µM
IS/AARc (%)
0.3 mg/kg
compd
X
(µM)
compd
X
(µM)
control
13 ( 1
45 ( 4
49 ( 2
43 ( 11
44 ( 4
46 ( 11
93 ( 26
44 ( 4
40 ( 11
59 ( 1
38 ( 3
50 ( 2d
51 ( 3
37 ( 2d
33 ( 3d
46 ( 1d
46 ( 4
50 ( 3
59
60
61
62
63
64
65
66
67
68
69
70
71
72
3-F
2.4
78 ( 12
101 ( 5
38 ( 5
45 ( 0
cariporide
1.2
3.1
3.5
1.1
2.3
0.34
2.0
2.3
20
1.4
2.6
2.4
1.1
3-Cl
0.75
47
48
49
50
51
52
53
54
55
56
57
58
H
3-OMe
3-Me
3-CF3
3-NO2
3-NH2
4-F
7.4
6.0
9.4
2-F
75 ( 5
40 ( 2d
2-Cl
2-OMe
2-Me
2-CF3
2-NO2
2-NH2
2-Et
>30
> 30
39
4-Cl
27
4-OMe
4-Me
4-CF3
4-NO2
4-NH2
> 30
> 30
> 30
> 30
> 30
45 ( 9
45 ( 13
42 ( 21
38 ( 2d
41 ( 3d
43 ( 8d
2-OEt
2-OiPr
2-OPh
a IC50 values resulted from at least two experiments. b In vitro cardioprotective effect was evaluated measuring a % RPP (LVDP × HR)
at the end of reperfusion to preischemic value in the isolated ischemic rat heart model (10 µM). Value are means ( SEM of at least three
determinations. c In vivo anti-infarction effect was determined by measuring a % ratio of myocardial infarct size to area at risk (IS/AAR)
in rat myocardial infarction model (0.3 mg/kg), n ) 3 or higher. Values are means ( SEM. d Administration of 1.0 mg/kg
Table 2. Inhibitory Effect on NHE-1 and Cardioprotective Efficacy of Disubstituted 5-(Arylfuran-2-ylcarbonyl)guanidines
IC50
RPPb (%) IS/AARc (%)
IC50
RPPb (%) IS/AARc (%)
a
a
compd
X
Y
(µM)
10 µM
0.3 mg/kg
compd
X
Y
(µM)
10 µM
0.3 mg/kg
control
13 ( 1
45 ( 4
59 ( 2
38 ( 3
85
86
87
88
89
90
91
92
93
94
95
96
97
2-OMe 5-Cl
0.081
42 ( 6
34 ( 2
cariporide
1.2
7.9
1.7
2-Cl
5-CF3
>30
73
74
75
76
77
78
79
80
81
82
83
84
2-F
2-Cl
3-F
3-Cl
2-OMe 5-Br
0.093
>30
>30
6 ( 1
50 ( 5
40 ( 13
40 ( 4d
2-F
2-Cl
6-F
6-F
2-OMe 3-OMe >30
2-Me
2-F
2-F
2-Cl
3-Me
4-F
5-F
5-Cl
4.4
15
2-OMe 6-OMe >30
2-Me
3-F
6-Me
5-F
5-Cl
5-Me
5-Cl
5-Cl
5-Cl
3.7
1.3
0.73
3.0
0.14
0.14
0.13
38 ( 2
57 ( 6
27 ( 4
50 ( 13
81 ( 4
6 ( 3
1.3
0.12
4.4
0.65
1.7
0.23
0.48
64 ( 22
22 ( 2
39 ( 2d
28 ( 3d
36 ( 5
35 ( 0d
38 ( 4
42 ( 1
31 ( 3d
39 ( 3d
3-Cl
2-OMe 5-OMe
3-Me
2-OH
2-OEt
2-OiPr
2-Me
2-F
2-Me
5-Me
5-Me
5-F
31 ( 1
17 ( 1
41 ( 8
27 ( 4
35 ( 3d
36 ( 3d
4 ( 1
2-OMe 5-F
a IC50 values resulted from at least two experiments. b In vitro cardioprotective effect was evaluated measuring a % RPP (LVDP × HR)
at the end of reperfusion to preischemic value in the isolated ischemic rat heart model (10 µM). Values are means ( SEM of at least three
determinations. c In vivo anti-infarction effect was determined by measuring a % ratio of myocardial infarct size to area at risk (IS/AAR)
in ischemic myocardium damage rat model (0.3 mg/kg), n ) 3 or higher. Values are means ( SEM. d Administration of 1.0 mg/kg.
The compounds showing good NHE-1 inhibitory ac-
tivity were further investigated for their cardioprotec-
tive effects against ischemia-reperfusion injury. In the
perfused rat heart model, cariporide significantly im-
proved the recovery of cardiac contractile function (45%
RPP) compared with the control (13% RPP). In the in
vivo rat myocardial infarct model, cariporide also sig-
nificantly reduced the infarct size (38% IS/AAR vs 59%
of the control) at a dose of 0.3 mg/kg. Most compounds
with a good activity on NHE-1 exhibited a good cardio-
protective efficacy both in vitro and in vivo. In the
isolated rat heart ischemia model, the compounds 51
(93%), 59 (78%), 60 (101%), and 62 (75%) greatly
improved the recovery of contractile function against
ischemia-reperfusion injury.
except the dimethyl compound 91 (IC50 ) 3.7 µM). The
2,3-disubstituted compounds (73, 74, 76) showed mod-
erate activities except the 2,3-dimethoxy compound 75
(IC50 > 30 µM). The 3,5-difluoro 92 (IC50 ) 1.3 µM), 3,5-
dichloro 93 (IC50 ) 0.73 µM), and 3,5-dimethyl 94
(IC50 ) 3.0 µM) compounds exhibited similar or slightly
increased potency compared with the monosubstituted
derivatives. The 2,5-disubstitution appeared to be much
better for increasing the activity on NHE-1 than the
other substitution patterns investigated. Especially the
2-methoxy-5-chloro compound 85 (IC50 ) 0.081 µM) and
2-methoxy-5-bromo compound 87 (IC50 ) 0.093 µM)
showed highly improved potency on NHE-1, which were
10 times more potent than cariporide. While the excel-
lent NHE-1 inhibitory activity of 87 was not translated
into the efficacy for the improvement of cardiac con-
tractility nor in the reduction of infarct size, the
compound 85 greatly reduced the infarct size (34% IS/
Next the effect of disubstituion at the phenyl ring on
the activity was examined as shown in Table 2. The 2,6-
disubstituted compounds 88-90 were almost inactive

Inhibition of Na+/H+ Exchanger Isoform-1
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2885
Table 3. Dose-Dependent Cardioprotective Efficacies of Selected Compounds
RPP (%),b µM
IS/AAR (%),c mg/kg
a
IC50
compd
X
Y
(µM)
0.3
13
1
3
10
45
78
101
42
0.1
0.3
1.0
3.0
26
control
cariporide
59
60
85
92
94
59
41
41
53
35
40
43
1.2
2.4
15
44
64
27
28
37
33
54
67
32
41
49
38
38
45
34
36
38
35
32
40
27
30
3-F
3-Cl
2-OMe
3-F
3-Me
25
0.75
0.081
1.3
5-Cl
5-F
5-Me
57
50
3.0
15
a IC50 values resulted from at least two experiments. b In vitro cardioprotective effect was evaluated measuring a % RPP (LVDP × HR)
at the end of reperfusion to pre-ischemic value in the isolated ischemic rat heart model. Values are means ( SEM (0.3, 1, 3, and 10 µM)
of at least three determinations. c In vivo anti-infarction effect was determined by measuring a % ratio of myocardial infarct size to area
at risk (IS/AAR) in ischemic myocardium damage rat model, n ) 3 or higher (0.1, 0.3, 1.0, and 3.0 mg/kg iv administration). Values are
means ( SEM.
commercial quality were used from freshly opened containers
unless otherwise stated. Analytical TLC was performed on
Merck-glass-backed silica gel 60 F254 plates. Merck silica gel
60 was used for flash chromatography. 1H NMR spectra were
recorded on a Bruker AM-300 (300 MHz) with TMS as an
internal standard. Chemical shifts are reported in δ (ppm).
Mass spectra were obtained with a JEOL JMS-DX 303
instrument by using electron impact or chemical ionization
techniques. Elemental analyses were performed by Korea
Research Institute of Chemical Technology’s Analytical De-
partment and C, H, and N values are within (0.4% of the
calculated values.
General Procedures for the Preparation of the Com-
pounds 1-28. To a solution of methyl 5-bromo-2-furoate (300
mg, 1.46 mmol) in toluene (6 mL) were added arylboronic acid
(1.76 mmol) in CH3OH (0.5 mL), 2 M Na2CO3 (0.9 mL, 1.8
mmol), and catalytic amounts of Pd(PPh3)4. The reaction
mixture was heated at reflux for 6 h, diluted with water (20
mL), and extracted with ethyl acetate (20 mL × 2). The
extracts were washed with brine, dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (n-hexane:
ethyl acetate ) 6:1) to afford the corresponding 5-aryl-
substituted furans.
AAR) and significantly recovered the cardiac contractile
function (42% RPP). In general the potency on NHE-1
of this series of compounds was well correlated with
their cardioprotective efficacy both in vitro and in vivo.
Additionally the activity of the 2-hydroxy-5-chloro
compound 95, which was found as the major metabolite
of 85 in our preliminary study on metabolism, was
investigated. The compound 85 (50 µM) was incubated
with human or rat liver microsomes (1 mg/mL of protein
concentration) for 1 h and analyzed using LC/MS/MS
including its metabolites; the results indicate that 84-
88% of 85 remained, and 95 was identified as a major
metabolite. Compound 95 also showed a good NHE-1
inhibitory activity and cardioprotective effect.
The dose-dependent cardioprotective efficacy was
further studied for the selected compounds, cariporide,
59, 60, 85, 92, and 94, which exhibited the high potency
on NHE-1 and good cardioprotective efficacy (Table 3).
All tested compounds showed a dose dependency. The
most potent compound 85 in the NHE-1 inhibition assay
(IC50 ) 0.081 µM), greatly reduced infarct size dose-
dependently with a significant improvement of cardiac
contractile dysfunction from ischemia-reperfusion in-
jury, which was comparable to cariporide. Further
studies on 85 employing various models are in progress.
5-Phenylfuran-2-carboxylic acid methyl ester (1): 72%
1
yield (white solid); mp 90-91 °C; H NMR (CDCl3) δ 3.92 (s,
3H), 6.74 (dd, J ) 3.7 Hz, 1H), 7.25 (d, J ) 3.7 Hz, 1H), 7.35-
7.46 (m, 3H), 7.81 (m, 2H); MS 202 (M+).
5-(2-Fluorophenyl)furan-2-carboxylic acid methyl es-
ter (2): 90% yield (white sold); 1H NMR (CDCl3) δ 3.94 (s,
3H), 6.93 (dd, J ) 3.2, 3.3 Hz, 1H), 7.14-7.34 (m, 4H), 7.99
(m, 1H); MS 220 (M+).
5-(3-Fluorophenyl)furan-2-carboxylic acid methyl es-
ter (3): 79% yield (white solid); mp 82 °C; 1H NMR (CDCl3) δ
3.85 (s, 3H), 6.69 (d, J ) 3.6 Hz, 1H), 6.97 (m, 1H), 7.18 (d,
J ) 3.6 Hz, 1H), 7.31 (m, 1H), 7.39 (dd, J ) 2.1, 8.1 Hz, 1H),
7.48 (d, J ) 8.0 Hz, 1H); MS 220 (M+).
5-(4-Fluorophenyl)furan-2-carboxylic acid methyl es-
ter (4): 75% yield (white solid); mp 70 °C; 1H NMR (CDCl3) δ
3.92 (s, 3H), 6.68 (d, J ) 3.6 Hz, 1H), 7.12 (dd, J ) 8.7, 8.7
Hz, 2H), 7.24 (d, J ) 3.6 Hz, 1H), 7.76 (dd, J ) 8.7, 12.0 Hz,
2H); MS 220 (M+).
5-(2-Chlorophenyl)furan-2-carboxylic acid methyl es-
ter (5): 43% yield (white solid); mp 68 °C; 1H NMR (CDCl3) δ
3.95 (s, 3H), 7.21 (d, J ) 3.6 Hz, 1H), 7.28 (m, 2H), 7.34 (ddd,
J ) 7.2, 6.9, 1.2 Hz, 1H), 7.46 (dd, J ) 1.2, 7.8 Hz, 2H), 7.99
(dd, J ) 1.8, 7.8 Hz, 2H); MS 236 (M+).
Conclusion
A series of (5-arylfuran-2-ylcarbonyl)guanidine ana-
logues were synthesized and evaluated for their cardio-
protective efficacy against ischemia-reperfusion injury
as well as the NHE-1 inhibitory activity. With the aim
to optimize the activity of (5-phenylfuran-2-ylcarbonyl)-
guanidine 47 having a moderate activity on NHE-1,
various substituents were introduced at the phenyl ring.
From these studies, [5-(2-methoxy-5-chlorophenyl)furan-
2-ylcarbonyl]guanidine 85 was identified as a highly
potent NHE-1 inhibitor. In addition, 85 greatly reduced
the infarct size in the rat myocardial infarction model
in vivo and significantly improved the cardiac contrac-
tile dysfunction in the isolated ischemic rat heart model.
These findings suggest that 85 could be an attractive
therapeutic candidate for myocardial ischemic diseases.
5-(3-Chlorophenyl)furan-2-carboxylic acid methyl es-
ter (6): 83% yield (white solid); mp 76 °C; 1H NMR (CDCl3) δ
3.92 (s, 3H), 6.75 (d, J ) 3.3 Hz, 1H), 7.24 (d, J ) 3.2 Hz, 1H),
7.33 (m, 2H), 7.65 (dd, J ) 2.0, 7.1 Hz, 2H), 7.76 (d, J ) 2.0
Hz, 1H); MS 236 (M+).
Experimental Section
Chemistry. Melting points were determined on a capillary
melting point apparatus and are uncorrected. Anhydrous
solvents were dried by conventional methods. Reagents of

2886 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Lee et al.
5-(2-Methylphenyl)furan-2-carboxylic acid methyl es-
5-(3,5-Dichlorophenyl)furan-2-carboxylic acid methyl
ester (22): 67% yield (white solid); mp 136 °C; 1H NMR
(CDCl3) δ 3.93 (s, 3H), 6.78 (d, J ) 3.6 Hz, 1H), 7.25 (d, J )
3.3 Hz, 1H), 7.33 (dd, J ) 1.5, 1.8 Hz, 1H), 7.65 (m, 2H); MS
270 (M+).
5-(3,5-Dimethylphenyl)furan-2-carboxylic acid methyl
ester (23): 71% yield (white solid); mp 105-106 °C; 1H NMR
(CDCl3) δ 2.36 (s, 6H), 3.92 (s, 3H), 6.70 (d, J ) 3.6 Hz, 1H),
6.99 (s, 1H), 7.24 (d, J ) 3.6 Hz, 1H), 7.41(s, 2H); MS 230 (M+).
5-(2,5-Dimethoxyphenyl)furan-2-carboxylic acid meth-
yl ester (24): 47% yield (oil); 1H NMR (CDCl3) δ 3.85 (s, 3H),
3.90 (s, 3H), 3.91 (s, 3H), 6.87 (m, 2H), 7.05 (d, J ) 3.7 Hz,
1H), 7.25 (d, J ) 3.7 Hz, 1H), 7.54 (d, J ) 2.7 Hz, 1H); MS
262 (M+).
1
ter (7): 90% yield (oil); H NMR (CDCl3) δ 2.52 (s, 3H), 3.91
(s, 3H), 6.63 (d, J ) 3.6 Hz, 1H), 7.25-7.29 (m, 4H), 7.76 (m,
1H); MS 216 (M+).
5-(3-Methylphenyl)furan-2-carboxylic acid methyl es-
ter (8): 78% yield (white solid); mp 85-86 °C; 1H NMR
(CDCl3) δ 2.42 (s, 3H), 3.94 (s, 3H), 6.74 (d, J ) 3.4 Hz, 1H),
7.18 (d, J ) 7.7 Hz, 1H), 7.26 (d, J ) 3.4 Hz, 1H), 7.33 (dd,
J ) 7.7, 7.7 Hz, 1H), 7.60 (d, J ) 7.7 Hz, 1H), 7.64 (s, 1H); MS
216 (M+).
5-(4-Methylphenyl)furan-2-carboxylic acid methyl es-
ter (9): 83% yield (white solid); mp 71-72 °C; 1H NMR
(CDCl3) δ 2.31 (s, 3H), 3.84 (s, 3H), 6.61 (d, J ) 3.5 Hz, 1H),
7.15-7.17 (m, 3H), 7.60 (d, J ) 8.2 Hz, 1H); MS 216 (M+).
5-[2-(Trifluoromethyl)phenyl]furan-2-carboxylic acid
5-(2-Fluoro-5-methylphenyl)furan-2-carboxylic acid
1
1
methyl ester (10): 94% yield (oil); H NMR (CDCl3) δ 3.92
methyl ester (25): 52% yield (oil); H NMR (CDCl3) δ 2.38
(s, 3H), 6.79 (d, J ) 3.6 Hz, 1H), 7.26 (d, J ) 3.6 Hz, 1H), 7.50
(dd, J ) 8.7, 8.7 Hz, 1H), 7.62 (dd, J ) 8.7, 9.0 Hz, 1H), 7.78
(d, J ) 9.0 Hz, 1H), 7.83 (d, J ) 8.7 Hz, 1H); MS 270 (M+).
5-[4-(Trifluoromethyl)phenyl]furan-2-carboxylic acid
methyl ester (11): 76% yield (white solid); mp 86-87 °C; 1H
NMR (CDCl3) δ 3.93 (s, 3H), 6.85 (d, J ) 3.6 Hz, 1H), 7.27 (d,
J ) 3.6 Hz, 1H), 7.67 (d, J ) 8.4 Hz, 2H), 7.89 (d, J ) 8.4 Hz,
1H); MS 270 (M+).
5-(2-Methoxyphenyl)furan-2-carboxylic acid methyl
ester (12): 67% yield (white solid); mp 63 °C; 1H NMR (CDCl3)
δ 3.91 (s, 3H), 3.95 (s, 3H), 6.97 (d, J ) 8.1 Hz, 1H), 7.03 (d,
J ) 3.9 Hz, 1H), 7.06 (d, J ) 7.5 Hz, 1H), 7.26 (d, J ) 3.6 Hz,
1H), 7.32 (ddd, J ) 1.8, 7.5, 8.1 Hz, 1H), 8.01 (dd, J ) 1.8, 7.5
Hz, 1H); MS 232 (M+).
(s, 3H), 3.93 (s, 3H), 6.91 (dd, J ) 3.6, 3.6 Hz, 1H), 7.02 (dd,
J ) 8.4, 10.9 1H), 7.11 (m, 1H), 7.27 (d, J ) 3.6 Hz 1H), 7.78
(d, J ) 5.9 Hz, 1H); MS 234 (M+).
5-(2-Methyl-5-fluorophenyl)furan-2-carboxylic acid
1
methyl ester (26): 91% yield (oil); H NMR (CDCl3) δ 2.48
(s, 3H), 3.92 (s, 3H), 6.67 (d, J ) 3.6 Hz, 1H), 6.94 (ddd, J )
2.9, 8.1, 8.3 Hz, 1H), 7.20 (m, 1H), 7.27 (d, J ) 3.6 Hz, 1H),
7.49(dd, J ) 2.9, 9.8 Hz, 1H); MS 234 (M+).
5-(2-Methoxy-5-fluorophenyl)furan-2-carboxylic acid
methyl ester (27): 63% yield (white solid); mp 94 °C; 1H NMR
(CDCl3) δ 3.92 (s, 3H), 3.93 (s, 3H), 6.90 (dd, J ) 4.0, 9.0 Hz,
1H), 7.00 (m, 1H), 7.07 (d, J ) 3.6 Hz, 1H), 7.25(d, J ) 3.6 Hz,
1H), 7.71 (dd, J ) 3.0, 9.3 Hz, 1H); MS 250 (M+).
5-(2-Methoxy-5-chlorophenyl)furan-2-carboxylic acid
methyl ester (28): 54% yield (white solid); mp 128 °C; 1H
NMR (CDCl3) δ 3.93 (s, 3H), 3.94 (s, 3H), 6.90 (d, J ) 9.0 Hz,
1H), 7.05 (d, J ) 3.7 Hz, 1H), 7.26 (m, 2H), 7.98(d, J ) 2.8 Hz,
1H); MS 266 (M+).
5-(3-Methoxyphenyl)furan-2-carboxylic acid methyl
ester (13): 96% yield (white solid); mp 62 °C; 1H NMR (CDCl3)
δ 3.86 (s, 3H), 3.91 (s, 3H), 6.73 (d, J ) 3.6 Hz, 1H), 6.91 (ddd,
J ) 1.5, 1.8, 9.0 Hz, 1H), 7.24 (d, J ) 3.6 Hz, 1H), 7.29-7.36
(m, 3H); MS 232 (M+).
General Procedures for the Preparation of the Com-
pounds 29-40. To a solution of methyl 5-bromo-2-furoate (300
mg, 1.46 mmol) and arylboronic acid (1.76 mmol) in 1,2-
dimethoxyethane (8 mL) was added a 2 M aqueous solution
of Ba(OH)2‚H2O (0.9 mL, 1.8 mmol). After the addition of
catalytic amounts of Pd(PPh3)4, the reaction mixture was
stirred at 80 °C for 12 h, diluted with water (20 mL), and
extracted with ethyl acetate (20 mL × 2). The extracts were
washed with brine, dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (n-hexane:ethyl acetate
) 20:1).
5-(4-Methoxyphenyl)furan-2-carboxylic acid methyl
ester (14): 97% yield (white solid); mp 83-83.5 °C; 1H NMR
(CDCl3) δ 3.85 (s, 3H), 3.91 (s, 3H), 6.61 (d, J ) 3.6 Hz, 1H),
6.94 (d, J ) 9.0 Hz, 1H), 7.24 (d, J ) 3.6 Hz, 1H), 7.71 (d, J )
9.0 Hz, 1H); MS 232 (M+).
5-(2-Nitrophenyl)furan-2-carboxylic acid methyl ester
(15): 94% yield (yellow solid); 1H NMR (CDCl3) δ 3.91 (s, 3H),
6.70 (d, J ) 3.6 Hz, 1H), 7.25 (d, J ) 3.6 Hz, 1H), 7.52 (ddd,
J ) 1.2, 7.5, 8.1 Hz, 1H), 7.65 (ddd, J ) 1.2, 7.5, 7.8 Hz, 1H),
7.81 (m, 2H); MS 247 (M+).
5-(3-Nitrophenyl)furan-2-carboxylic acid methyl ester
(16): 51% yield (yellow solid); mp 146 °C; 1H NMR (CDCl3) δ
3.95 (s, 3H), 6.91 (d, J ) 3.6 Hz, 1H), 7.29 (d, J ) 3.6 Hz, 1H),
7.71 (dd, J ) 7.8, 8.4 Hz, 1H), 7.98 (dd, J ) 0.9, 7.8 Hz, 1H),
8.31 (dd, J ) 2.1, 8.4 Hz, 1H), 8.51 (d, J ) 2.1 Hz, 1H); MS
247 (M+).
5-(4-Nitrophenyl)furan-2-carboxylic acid methyl ester
(17): 99% yield (yellow solid); 1H NMR (CDCl3) δ 3.95 (s, 3H),
6.95 (d, J ) 3.6 Hz, 1H), 7.29 (d, J ) 3.6 Hz, 1H), 7.93 (d, J )
8.7 Hz, 2H), 8.29 (d, J ) 8.7 Hz, 2H); MS 247 (M+).
5-(2-Ethoxyphenyl)furan-2-carboxylic acid methyl es-
ter (18): 67% yield (white solid); mp 74-75 °C; 1H NMR
(CDCl3) δ 1.53 (t, J ) 7.0 Hz, 3H), 3.91 (s, 3H), 4.17 (q, J )
7.0 Hz, 2H), 6.95 (d, J ) 8.3 Hz, 1H), 7.03 (dd, J ) 7.5, 7.6 Hz,
1H), 7.07 (d, J ) 3.7 Hz, 1H), 7.29 (m, 2H), 8.02 (dd, J ) 1.6,
7.8 Hz, 1H); MS 246 (M+).
5-(2-Isopropoxyphenyl)furan-2-carboxylic acid methyl
ester (19): 61% yield (oil); 1H NMR (CDCl3) δ 1.43 (d, J )
7.2 Hz, 6H), 3.91 (s, 3H), 4.71 (m, 1H), 6.99 (m, 2H), 7.08 (d,
J ) 3.6 Hz, 1H), 7.25 (d, J ) 3.6 Hz, 1H), 7.29 (dd, J ) 1.7,
8.3 Hz, 1H), 8.02 (dd, J ) 1.7, 7.8 Hz, 1H); MS 260 (M+).
5-(2-Phenoxyphenyl)furan-2-carboxylic acid methyl
ester (20): 70% yield (white solid); mp 86 °C; 1H NMR (CDCl3)
δ 3.91 (s, 3H), 6.93 (d, J ) 7.7 Hz, 1H), 7.00 (m, 3H), 7.13-
7.38 (m, 6H), 8.10 (dd, J ) 1.9, 7.7 Hz, 1H).
5-(2,3-Dichlorophenyl)furan-2-carboxylic acid methyl
ester (21): 39% yield (white solid); mp 108 °C; 1H NMR
(CDCl3) δ 3.93 (s, 3H), 7.23-7.33 (m, 3H), 7.47 (dd, J ) 1.5,
8.1 Hz, 1H), 7.95 (dd, J ) 1.5, 7.8 Hz, 1H); MS 270 (M+).
5-(2,3-Difluorophenyl)furan-2-carboxylic acid methyl
1
ester (29): 35% yield (white solid); mp 84-85 °C; H NMR
(CDCl3) δ 3.92 (s, 3H), 6.74 (dd, J ) 3.6, 3.7 Hz, 1H), 7.18 (m,
2H), 7.28 (m, 1H), 7.75 (m, 1H); MS 238 (M+).
5-(2,4-Difluorophenyl)furan-2-carboxylic acid methyl
1
ester (30): 71% yield (white solid); H NMR (CDCl3) δ 3.92
(s, 3H), 6.87 (dd, J ) 3.6, 3.9 Hz, 1H), 6.90-7.01 (m, 2H), 7.26
(d, J ) 3.6 Hz, 1H), 7.97 (m, 1H); MS 238 (M+).
5-(2,5-Difluorophenyl)furan-2-carboxylic acid methyl
ester (31): 65% yield (white solid); mp 73 °C; 1H NMR (CDCl3)
δ 3.93 (s, 3H), 6.95-7.17 (m, 3H), 7.27 (d, J ) 3.6 Hz, 1H),
7.68 (m, 1H); MS 238 (M+).
5-(3,5-Difluorophenyl)furan-2-carboxylic acid methyl
ester (32): 79% yield (white solid); mp 145-147 °C; 1H NMR
(CDCl3) δ 3.93 (s, 3H), 6.75-6.83 (m, 2H), 7.24-7.32 (m, 3H);
MS 238 (M+).
5-(2,5-Dichlorophenyl)furan-2-carboxylic acid methyl
1
ester (33): 42% yield (white solid); mp 85-86 °C; H NMR
(CDCl3) δ 3.92 (s, 3H), 7.21-7.28 (m, 3H), 7.39 (d, J ) 8.7 Hz,
1H), 7.98 (d, J ) 1.8 Hz, 1H); MS 270 (M+).
5-(2,6-Dimethylphenyl)furan-2-carboxylic acid methyl
1
ester (34): 57% yield (oil); H NMR (CDCl3) δ 2.35 (s, 3H),
2.37 (s, 3H), 3.91 (s, 3H), 6.56 (d, J ) 3.5 Hz, 1H), 7.18 (m,
2H), 7.48 (dd, J ) 1.8, 7.1 Hz, 1H); MS 230 (M+).
5-(2,3-Dimethylphenyl)furan-2-carboxylic acid methyl
1
ester (35): 60% yield (oil); H NMR (CDCl3) δ 2.34 (s, 3H),
2.37 (s, 3H), 3.91 (s, 3H), 6.55 (d, J ) 3.6 Hz, 1H), 7.13-7.21

Inhibition of Na+/H+ Exchanger Isoform-1
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2887
1
(m, 2H), 7.27 (d, J ) 3.6 Hz, 1H), 7.48 (dd, J ) 1.7, 7.3 Hz,
1H); MS 230 (M+).
45 (84 mg, 75%): mp 118 °C; H NMR (CDCl3) δ 1.54 (t, J )
7.0 Hz, 3H), 3.93 (s, 3H), 4.15 (q, J ) 7.0 Hz, 2H), 6.88 (d, J )
8.7 Hz, 1H), 7.09 (d, J ) 3.6 Hz, 1H), 7.24 (m, 2H), 7.98 (d,
J ) 2.7 Hz, 1H); MS 280 (M+).
5-(2,5-Dimethylphenyl)furan-2-carboxylic acid methyl
1
ester (36): 68% yield (oil); H NMR (CDCl3) δ 2.36 (s, 3H),
2.47 (s, 3H), 3.92 (s, 3H), 6.61 (d, J ) 3.6 Hz, 1H), 7.08 (d,
J ) 7.9 Hz, 1H), 7.15 (d, J ) 7.9 Hz, 1H), 7.27 (d, J ) 3.6 Hz,
1H), 7.59 (s, 1H); MS 230 (M+).
5-(2-Isopropoxy-5-chlorophenyl)furan-2-carboxylic Acid
Methyl Ester (46). The compound 46 was prepared by the
same procedure to synthesize the compound 45, except using
isopropyl iodide as an alkylating agent: mp 68-69 °C; 1H
NMR (CDCl3) δ 1.42 (d, J ) 6.0 Hz, 6H), 3.92 (s, 3H), 4.67 (m,
1H), 6.89 (d, J ) 9.0 Hz, 1H), 7.09 (d, J ) 3.6 Hz, 1H), 7.22
(dd, J ) 2.7, 9.0 Hz, 1H), 7.25 (d, J ) 3.6 Hz, 1H), 7.89 (d,
J ) 2.7 Hz, 1H); MS 294 (M+).
5-(2,6-Dimethoxyphenyl)furan-2-carboxylic acid meth-
yl ester (37): 53% yield (white solid); 1H NMR (CDCl3) δ 3.80
(s, 6H), 3.89 (s, 3H), 6.60 (m, 3H), 7.29 (m, 2H); MS 262 (M+).
5-(2,3-Dimethoxyphenyl)furan-2-carboxylic acid meth-
yl ester (38): 95% yield (oil); 1H NMR (CDCl3) δ 3.87 (s, 3H),
3.90 (s, 3H), 3.91 (s, 3H), 6.92 (dd, J ) 1.5, 6.8 Hz, 1H), 7.08
(d, J ) 3.6 Hz, 1H), 7.13 (dd, J ) 6.8, 8.1 Hz, 1H), 7.27 (d,
J ) 3.6 Hz, 1H), 7.57 (dd, J ) 1.5, 8.1 Hz, 1H); MS 262 (M+).
5-(2-Chloro-6-fluorophenyl)furan-2-carboxylic acid
methyl ester (39): 10% yield (oil) 1H NMR (CDCl3) δ 3.93 (s,
3H), 6.79 (m, 1H), 7.10 (m, 1H), 7.25-7.34 (m, 3H); MS 254
(M+).
5-(2-Methoxy-5-bromophenyl)furan-2-carboxylic acid
methyl ester (40): 38% yield (white solid) mp 140 °C; 1H
NMR (CDCl3) δ 3.93 (s, 3H), 3.94 (s, 3H), 6.85 (d, J ) 8.8 Hz,
1H), 7.04 (d, J ) 3.6 Hz, 1H), 7.25 (d, J ) 3.6 Hz, 1H), 7.39
(dd, J ) 2.4, 8.8 Hz, 1H), 8.11 (d, J ) 2.4 Hz, 1H); MS 311
(M+).
5-(2-Aminophenyl)furan-2-carboxylic Acid Methyl Es-
ter (41). To a solution of the nitro compound 15 (290 mg, 1.17
mmol) in CH3OH (10 mL) was added 10% Pd/C (29 mg). The
resulting mixture was stirred for 2 h under H2 (3 atm) at room
temperature. After the reaction was completed, the mixture
was filtered over a pad of Celite, and the filtrate was
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (n-hexane:ethyl acetate
) 4:1) to yield a pale yellow solid (239 mg, 94%): 1H NMR
(CDCl3) δ 3.90 (s, 3H), 6.67 (d, J ) 3.6 Hz, 1H), 6.73-6.80 (m,
2H), 7.15 (ddd, J ) 1.5, 7.2, 7.2 Hz, 1H), 7.26 (d, J ) 3.6 Hz,
1H), 7.50 (dd, J ) 1.5, 7.8 Hz, 1H); MS 217 (M+).
5-(3-Aminophenyl)furan-2-carboxylic Acid Methyl Es-
ter (42). The compound 42 (101 mg, 51%) was prepared by
the same procedure to prepare the compound 41 except using
the compound 16 (222 mg, 0. 9 mmol) as a starting material:
mp 99-101 °C; 1H NMR (CDCl3) δ 3.91 (s, 3H), 6.62-6.70 (m,
2H), 7.15 (m, 2H), 7.25 (m, 2H); MS 217 (M+).
General Procedures for the Preparation of (5-Arylfu-
ran-2-ylcarbonyl)guanidines 47-97. Method A. Small
pieces of sodium (4.6 g, 0.2 mol) were slowly added to dry CH3-
OH (100 mL) with stirring. After the complete dissolution of
sodium in CH3OH, guanidine hydrochloride (19.1 g, 0.2 mol)
was added, and the resulting mixture was stirred at room
temperature for 1 h. White precipitates were filtered, resulting
a 2 M solution of free guanidine in methanol had been stored
in a refrigerator for several months. The corresponding ester
compound (1.0 mmol) was dissolved CH3OH (5 mL), and a 2
M methanol solution of guanidine (3.0 mL, 6.0 mmol) was
added. The reaction mixture was heated at reflux with stirring
for 12 h. Brine (25 mL) was added, and the mixture was
extracted with ethyl acetate (30 mL × 3). The organic layer
was washed with brine twice, dried (MgSO4), filtered, and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (5% methanol in CH2-
Cl2) to yield the (furan-2-ylcarbonyl)guanidines. Otherwise the
compounds were purified as mathanesulfonic acid salts. The
residue was dissolved in acetone (5 mL), and methanesulfonic
acid was added with stirring and cooled to 0 °C. The precipi-
tates were filtered to give the methanesulfonate.
(5-Phenylfuran-2-ylcarbonyl)guanidine methanesul-
fonate (47): 46% yield (white solid); mp 205 °C; 1H NMR (D2O)
δ 2.81 (s, 3H), 6.97 (d, J ) 3.7 Hz, 1H), 7.45 (d, J ) 3.7 Hz,
1H), 7.46-7.52 (m, 3H), 7.80-7.85 (m, 2H); MS 229 (M+); Anal.
(C13H15N3O5S) C, H, N, S.
[5-(2-Fluorophenyl)furan-2-ylcarbonyl]guanidine meth-
anesulfonate (48): 54% yield (white solid); mp 220 °C; 1H
NMR (DMSO-d6) δ 2.39 (s, 3H), 7.14 (dd, J ) 3.3, 3.6 Hz, 1H),
7.54 (m, 1H), 7.40-7.46 (m, 2H), 7.69 (d, J ) 3.6 Hz, 1H), 8.11
(dd, J ) 8.7, 9.0 Hz, 1H), 8.41 (brs, 4H), 11.19 (brs, 1H); M+
247;); MS 247 (M+); Anal. (C13H14FN3O5S) C, H, N, S.
5-(4-Aminophenyl)furan-2-carboxylic Acid Methyl Es-
ter (43). The compound 43 (84 mg, 97%) was prepared by the
same procedure to prepare the compound 41 except using the
compound 17 (100 mg, 0. 40 mmol) as a starting material: mp
[5-(2-Chlorophenyl)furan-2-ylcarbonyl]guanidine (49):
1
94% yield (white solid); mp 201-202 °C; H NMR (CD3OD) δ
1
125-126 °C; H NMR (CDCl3) δ 3.89 (s, 3H), 6.53 (d, J ) 3.6
7.22 (m, 2H), 7.32 (ddd, J ) 1.7, 7.6, 7.9 Hz, 1H), 7.42 (ddd,
J ) 1.4, 7.6, 7.9 Hz, 2H), 7.50 (dd, J ) 1.4, 7.9 Hz, 1H), 8.13
(dd, J ) 1.7, 7.9 Hz, 2H); MS 263 (M+); Anal. (C12H10ClN3O2)
C, H, N.
Hz, 1H), 6.69 (d, J ) 8.8 Hz, 2H), 7.22 (d, J ) 3.6 Hz, 1H),
7.59 (d, J ) 8.5 Hz, 1H); MS 217 (M+).
5-(2-Hydroxy-5-chlorophenyl)furan-2-carboxylic Acid
Methyl Ester (44). To a solution of the compound 28 (200
mg, 0.75 mmol) in CH2Cl2 was added an 1 M solution of BBr3
in CH2Cl2 (1.65 mmol) at 0 °C. The reaction mixture was
stirred at room temperature for 3 h, treated with saturated
NaHCO3 solution (20 mL), and extracted with ethyl acetate
(30 mL × 2). The combined organic layers were washed with
brine, dried over anhydrous MgSO4, filtered, and concentrated
under reduced pressure. The residue was purified by silica gel
column chromatography (n-hexane:ethyl acetate ) 20:1) to give
[5-(2-Methoxyphenyl)furan-2-ylcarbonyl]guanidine (50):
49% yield (pale yellow solid); mp 121-123 °C; 1H NMR (CD3-
OD) δ 3.97 (s, 3H), 7.03-7.11 (m, 3H), 7.23 (d, J ) 3.5 Hz,
1H), 7.33 (ddd, J ) 1.8, 7.0, 7.5 Hz, 2H), 8.10 (d, J ) 1.8, 7.6
Hz, 1H); MS 259 (M+); Anal. (C13H13N3O3) C, H, N.
[5-(2-Ethylphenyl)furan-2-ylcarbonyl]guanidine (55):
1
32% yield (white solid); mp 151-153 °C; H NMR (CD3OD) δ
1.22 (t, J ) 7.5 Hz, 3H), 2.90 (q, J ) 7.5 Hz, 2H), 6.68 (d, J )
3.6 Hz, 1H), 7.23 (d, J ) 3.6 Hz, 1H), 7.29 (m, 3H), 7.73 (d,
J ) 7.3 Hz, 1H); MS 257 (M+); Anal. (C14H15N3O2) C, H, N.
1
the compound 44 (121 mg, 64%): mp 176-177 °C; H NMR
(CDCl3) δ 3.93 (s, 3H), 6.91 (m, 2H), 7.03 (brs, 1H), 7.18 (dd,
J ) 2.7, 8.7 Hz, 1H), 7.29 (d, J ) 3.3 Hz, 1H), 7.68 (d, J ) 2.7
Hz, 1H); MS 252 (M+).
[5-(2-Ethoxyphenyl)furan-2-ylcarbonyl]guanidine (56):
40% yield (yellow solid); mp 174-175 °C; 1H NMR (CD3OD) δ
1.53 (t, J ) 7.0 Hz, 3H), 4.20 (q, J ) 7.0 Hz, 2H), 7.01-7.09
(m, 3H), 7.21 (d, J ) 3.5 Hz, 1H), 7.29 (dd, J ) 1.5, 7.4 Hz,
1H), 8.11 (dd, J ) 1.7, 7.7 Hz, 1H); MS 273 (M+); Anal.
(C14H15N3O3) C, H, N.
[5-(2-Isopropoxyphenyl)furan-2-ylcarbonyl]guani-
dine (57): 95% yield (white solid); mp 178 °C; 1H NMR (CD3-
OD) δ 1.42 (d, J ) 6.0 Hz, 6H), 4.79 (m, 1H), 7.04-7.09 (m,
3H), 7.20 (d, J ) 3.5 Hz, 1H), 7.28 (ddd, J ) 1.3, 7.0, 7.4 Hz,
1H), 8.11 (dd, J ) 1.7, 6.9 Hz, 1H); MS 287 (M+); Anal.
(C15H17N3O3) C, H, N.
5-(2-Ethoxy-5-chlorophenyl)furan-2-carboxylic Acid
Methyl Ester (45). To a solution of the compound 44 (100
mg, 0.4 mmol) in DMF (1.5 mL) were added K2CO3 (82 mg,
0.59 mmol) and iodoethane (38 µL, 0.47 mmol). The reaction
mixture was stirred at room temperature for 3 h, diluted with
water (20 mL), and extracted with ethyl acetate (20 mL × 2).
The organic layer was washed with brine, dried over anhy-
drous MgSO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by silica gel column chroma-
tography (n-hexane:ethyl acetate ) 10:1) to afford the compound

2888 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Lee et al.
[5-(2-Phenoxyphenyl)furan-2-ylcarbonyl]guanidine
(58): 88% yield (white solid); mp 191-193 °C; 1H NMR (CD3-
OD) δ 6.94-7.02 (m, 4H), 7.13 (m, 2H), 7.26-7.38 (m, 4H),
8.22 (dd, J ) 1.9, 7.6 Hz, 1H); MS 321 (M+); Anal. (C18H15N3O3)
C, H, N.
5-(2,5-Dimethoxyphenyl)furan-2-ylcarbonyl]guani-
dine (80): 76% yield (white solid); mp 131 °C; 1H NMR (CD3-
OD) δ 3.84 (s, 3H), 3.89 (s, 3H), 6.87 (dd, J ) 3.0, 9.0 Hz, 1H),
7.00 (d, J ) 9.0 Hz, 1H), 7.04 (d, J ) 3.6 Hz, 1H), 7.18 (d, J )
3.6 Hz, 1H), 7.71 (d, J ) 3.0 Hz, 1H); MS 289 (M+); Anal.
(C14H15N3O4) C, H, N.
[5-(2,5-Dimethylphenyl)furan-2-ylcarbonyl]guani-
dine (81): 94% yield (white solid); mp 176-177 °C; 1H NMR
(CD3OD) δ 2.35 (s, 3H), 2.46 (s, 3H), 6.68 (d, J ) 3.5 Hz, 1H),
7.07 (d, J ) 7.6 Hz, 1H), 7.15 (d, J ) 7.6 Hz, 1H), 7.19 (d, J )
3.5 Hz, 1H), 7.71 (s, 1H); MS 257 (M+); Anal. (C14H15N3O2) C,
H, N.
[5-(2-Fluoro-5-methylphenyl)furan-2-ylcarbonyl]guani-
dine (82): 87% yield (white solid); mp 194 °C; 1H NMR (CD3-
OD) δ 2.38 (s, 3H), 6.87 (m, 1H), 7.02-7.14 (m, 2H), 7.19 (d,
J ) 3.6 Hz, 1H), 7.94 (dd, J ) 2.1, 7.5 Hz, 1H); MS 261 (M+);
Anal. (C13H12FN3O2) C, H, N.
[5-(2-Methyl-5-fluorophenyl)furan-2-ylcarbonyl]guani-
dine (83): 91% yield (white solid); mp 176 °C; 1H NMR (CD3-
OD) δ 2.48 (s, 3H), 6.79 (d, J ) 3.5 Hz, 1H), 6.98 (ddd, J )
2.7, 8.3, 8.3 Hz, 1H), 7.20 (d, J ) 3.5 Hz, 1H), 7.27 (dd, J )
5.9, 8.3 Hz), 7.68 (dd, J ) 2.7, 10.4 Hz); MS 261 (M+); Anal.
(C13H12FN3O2) C, H, N.
[5-(2-Methoxy-5-fluorophenyl)furan-2-ylcarbonyl]guani-
dine (84): 87% yield (white solid); mp 195-197 °C; 1H NMR
(CD3OD) δ 3.94 (s, 3H), 7.04 (m, 2H), 7.08 (d, J ) 3.5 Hz, 1H),
7.17 (d, J ) 3.5 Hz, 1H), 7.87 (dd, J ) 2.6, 9.7 Hz, 1H); MS
281 (M+); Anal. (C13H12FN3O3) C, H, N.
[5-(2-Methoxy-5-chlorophenyl)furan-2-ylcarbonyl]gua-
nidine (85): 92% yield (white solid); mp 190 °C; 1H NMR
(CD3OD) δ 3.95 (s, 3H), 7.06 (m, 2H), 7.16 (d, J ) 3.6 Hz, 1H),
7.27 (dd, J ) 2.6, 8.8 Hz, 1H), 8.13 (d, J ) 2.6 Hz, 2H); MS
293 (M+); Anal. (C13H12ClN3O3) C, H, N.
[5-(2-Methoxy-5-bromophenyl)furan-2-ylcarbonyl]gua-
nidine (87): 89% yield (white solid); mp 145 °C; 1H NMR
(CD3OD) δ 3.96 (s, 3H), 7.04 (m, 2H), 7.16 (d, J ) 3.5 Hz, 1H),
7.41 (d, J ) 8.8 Hz, 1H), 8.27 (s, 1H); MS 337 (M+); Anal.
(C13H12BrN3O3) C, H, N.
[5-(2,6-Difluorophenyl)furan-2-ylcarbonyl]guanidine
(88): 79% yield (white solid); mp 220 °C; 1H NMR (CD3OD) δ
6.86 (brs, 1H), 7.09 (m, 2H), 7.21 (brs, 1H), 7.39 (m, 1H); MS
265 (M+); Anal. (C12H9F2N3O2) C, H, N.
[5-(2-Chloro-6-fluorophenyl)furan-2-ylcarbonyl]guani-
dine (89): 77% yield (white solid); mp 198 °C; 1H NMR (CD3-
OD) δ 6.75 (brs, 1H), 7.20 (m, 2H), 7.42 (m, 2H); MS 281 (M+);
Anal. (C12H9ClFN3O2) C, H, N.
[5-(2,6-Dimethoxyphenyl)furan-2-ylcarbonyl]guani-
dine (90): 53% yield (white solid); mp 122 °C; 1H NMR (CD3-
OD) δ 3.77 (s, 6H), 6.49 (d, J ) 3.6 Hz, 1H), 6.69 (d, J ) 8.4
Hz, 2H), 7.19 (d, J ) 3.6 Hz, 1H), 7.35 (t, J ) 8.4 Hz, 1H); MS
289 (M+); Anal. (C14H15N3O4) C, H, N.
[5-(2,6-Dimethylphenyl)furan-2-ylcarbonyl]guani-
dine (91): 88% yield (white solid); mp 189 °C; 1H NMR (CD3-
OD) δ 2.34 (s, 3H), 2.37 (s, 3H), 6.60 (d, J ) 3.5 Hz, 1H), 7.14
(m, 2H), 7.19 (d, J ) 3.5 Hz, 1H), 7.52 (t, J ) 7.3 Hz, 1H); MS
257 (M+); Anal. (C14H15N3O2) C, H, N.
[5-(3,5-Difluorophenyl)furan-2-ylcarbonyl]guanidine
(92): 89% yield (white solid); mp 220 °C; 1H NMR (DMSO-d6)
δ 7.04 (d, J ) 3.5 Hz, 1H), 7.21 (m, 2H), 7.49 (m, 2H); MS 265
(M+); Anal. (C12H9F2N3O2) C, H, N.
[5-(3,5-Dichlorophenyl)furan-2-ylcarbonyl]guani-
dine (93): 75% yield (white solid); mp 216 °C; 1H NMR
(DMSO-d6) δ 7.05 (d, J ) 3.5 Hz, 1H), 7.23 (d, J ) 3.5 Hz,
1H), 7.52 (d, J ) 1.7 Hz, 1H), 7.80 (d, J ) 1.8 Hz, 2H); MS
297 (M+); Anal. (C12H9Cl2N3O2) C, H, N.
[5-(3,5-Dimethylphenyl)furan-2-ylcarbonyl]guani-
dine (94): 92% yield (white solid); mp 189 °C; 1H NMR (CD3-
OD) δ 2.32 (s, 6H), 6.80 (d, J ) 3.3 Hz, 1H), 6.96 (s, 1H), 7.14
(d, J ) 3.3 Hz, 1H), 7.47 (s, 2H); MS 257 (M+); Anal.
(C14H15N3O2) C, H, N.
[5-(3-Chlorophenyl)furan-2-ylcarbonyl]guanidine (60):
1
47% yield (white solid); mp 128 °C; H NMR (CD3OD) δ 6.89
(d, J ) 3.6 Hz, 1H), 7.15 (d, J ) 3.6 Hz, 1H), 7.22-7.34 (m,
2H), 7.68 (d, J ) 7.6 Hz, 1H), 7.83 (s, 1H); MS 263 (M+); Anal.
(C12H10ClN3O2) C, H, N.
[5-(3-Methoxyphenyl)furan-2-ylcarbonyl]guanidine
methanesulfonate (61): 4% yield (yellow solid); mp 194-195
1
°C; H NMR (DMSO-d6) δ 2.34 (s, 3H), 3.85 (s, 3H), 7.06 (d,
J ) 9.0 Hz, 1H), 7.36 (d, J ) 3.6 Hz, 1H), 7.43-7.56 (m, 3H),
7.66 (d, J ) 3.6 Hz, 1H), 8.36 (brs, 4H), 11.07 (brs, 1H); MS
259 (M+); Anal. (C14H17N3O6S) C, H, N, S.
[5-(3-Nitrophenyl)furan-2-ylcarbonyl]guanidine (64):
63% yield (yellow solid); mp 270 °C; 1H NMR (DMSO-d6) δ
7.38 (d, J ) 3.6 Hz, 1H), 7.45 (d, J ) 3.6 Hz, 1H), 7.79 (m,
1H), 8.24 (m, 2H), 8.55 (s, 1H); MS 274 (M+); Anal. (C12H10N4O4)
C, H, N.
[5-(4-Fluorophenyl)furan-2-ylcarbonyl]guanidine (66):
51% yield (white solid); mp 191 °C; 1H NMR (DMSO-d6) δ 6.99
(d, J ) 3.6 Hz, 1H), 7.07 (d, J ) 3.6 Hz, 1H), 7.43 (dd, J ) 8.6,
8.9 Hz, 2H), 7.67 (dd, J ) 5.5, 8.6 Hz, 2H); MS 247 (M+); Anal.
(C12H10FN3O2) C, H, N.
[5-(4-Methylphenyl)furan-2-ylcarbonyl]guanidine meth-
anesulfonate (69): 14% yield (white solid); mp 245 °C; 1H
NMR (DMSO-d6) δ 2.54 (s, 6H), 7.41 (d, J ) 3.6 Hz, 1H), 7.52
(d, J ) 8.1 Hz, 2H), 7.80 (d, J ) 3.6 Hz, 1H), 8.03 (d, J ) 8.1
Hz, 2H), 8.53 (brs, 4H); MS 243 (M+); Anal. (C14H17N3O5S) C,
H, N, S.
[5-[4-(Trifluoromethyl)phenyl]furan-2-ylcarbonyl]gua-
nidine (70): 35% yield (white solid); mp 183-185 °C; 1H NMR
(DMSO-d6) δ 7.22 (d, J ) 3.5 Hz, 1H), 7.27 (d, J ) 3.5 Hz,
1H), 7.82 (d, J ) 8.3 Hz, 2H), 8.03 (d, J ) 8.3 Hz, 2H); MS
297 (M+); Anal. (C13H10F3N3O2) C, H, N.
[5-(2,3-Difluorophenyl)furan-2-ylcarbonyl]guanidine
(73): 93% yield (white solid); mp 209-210 °C; 1H NMR (CD3-
OD) δ 6.96 (dd, J ) 3.6, 3.6 Hz, 1H), 7.24 (m, 3H), 7.89 (m,
1H); MS 265 (M+); Anal. (C12H9F2N3O2) C, H, N.
[5-(2,3-Dichlorophenyl)furan-2-ylcarbonyl]guani-
dine (74): 84% yield (white solid); mp 227 °C; 1H NMR
(DMSO-d6) δ 7.20 (d, J ) 3.6 Hz, 1H), 7.34 (d, J ) 3.6 Hz,
1H), 7.59 (dd, J ) 7.9, 8.0 Hz, 1H), 7.75 (dd, J ) 1.3, 8.0 Hz,
1H), 7.98 (dd, J ) 1.3, 7.9 Hz, 1H); MS 297 (M+); Anal. (C12H9-
Cl2N3O2) C, H, N.
[5-(2,3-Dimethoxyphenyl)furan-2-ylcarbonyl]guani-
dine (75): 83% yield (white solid); mp 185 °C; 1H NMR (CD3-
OD) δ 3.84 (s, 3H), 3.88 (s, 3H), 7.00 (dd, J ) 1.4, 8.2 Hz, 1H),
7.05 (d, J ) 3.6 Hz, 1H), 7.13 (dd, J ) 8.2, 8.2 Hz, 1H), 7.19
(d, J ) 3.6 Hz, 1H), 7.66 (dd, J ) 1.4, 8.2 Hz, 1H); MS 289
(M+); Anal. (C14H15N3O4) C, H, N.
[5-(2,3-Dimethylphenyl)furan-2-ylcarbonyl]guani-
1
dine (76): 92% yield (white solid); mp 192-194 °C; H NMR
(CD3OD) δ 2.34 (s, 3H), 2.38 (s, 3H), 6.60 (d, J ) 3.5 Hz, 1H),
7.11-7.20 (m, 3H), 7.52 (dd, J ) 1.7, 9.0 Hz, 1H); MS 257 (M+);
Anal. (C14H15N3O2) C, H, N.
[5-(2,4-Difluorophenyl)furan-2-ylcarbonyl]guanidine
methanesulfonate (77): 81% yield (yellow solid); mp 240 °C;
1H NMR (DMSO-d6) δ 2.37 (s, 3H), 7.11 (dd, J ) 3.6, 3.9 Hz,
1H), 7.34 (m, 1H) 7.51 (ddd, J ) 2.1, 8.1, 8.4 Hz, 1H), 7.67 (d,
J ) 3.9 Hz, 1H), 8.14 (m, 1H), 8.37 (brs, 4H), 11.17 (brs, 1H);
MS 265 (M+); Anal. (C13H13F2N3O5S) C, H, N, S.
[5-(2,5-Difluorophenyl)furan-2-ylcarbonyl]guanidine
(78): 85% yield (white solid); mp 209 °C; 1H NMR (CD3OD) δ
6.96 (dd, J ) 3.6, 4.2 Hz, 1H), 7.07 (m, 1H), 7.20 (m, 2H), 7.88
(m, 1H); MS 265 (M+); Anal. (C12H9F2N3O2) C, H, N.
[5-(2,5-Dichlorophenyl)furan-2-ylcarbonyl]guani-
1
dine (79): 84% yield (white solid); mp 198-200 °C; H NMR
(DMSO-d6) δ 7.09(d, J ) 3.5 Hz, 1H), 7.27 (d, J ) 3.5 Hz, 1H),
7.43 (dd, J ) 2.6, 8.6 Hz, 1H), 7.60 (d, J ) 8.6 Hz, 1H), 7.94
(d, J ) 2.6 Hz, 1H); MS 297 (M+); Anal. (C12H9Cl2N3O2) C, H,
N.
[5-(2-Hydroxy-5-chlorophenyl)furan-2-ylcarbonyl]gua-
nidine methanesulfonate (95): 30% yield (white solid); mp
1
223 °C; H NMR (CD3OD) δ 2.68 (s, 3H), 6.91 (d, J ) 8.7 Hz,
1H), 7.20 (dd, J ) 2.6, 8.7 Hz, 1H), 7.27 (d, J ) 3.8 Hz, 1H),

Inhibition of Na+/H+ Exchanger Isoform-1
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2889
7.55 (d, J ) 3.8 Hz, 1H), 7.99 (d, J ) 2.6 Hz, 1H); MS 279
(M+); Anal. (C13H14ClN3O6S) C, H, N, S.
1H), 7.87 (dd, J ) 8.7, 9.0 Hz, 1H), 7.94 (m, 2H), 8.34 (brs,
4H), 11.25 (brs, 1H); MS 297 (M+); Anal. (C14H14F3N3O5S) C,
H, N, S.
[5-(2-Ethoxy-5-chlorophenyl)furan-2-ylcarbonyl]guani-
dine (96): 93% yield (white solid); mp 175-177 °C; 1H NMR
(CD3OD) δ 1.54 (t, J ) 7.0 Hz, 3H), 4.20 (q, J ) 7.0 Hz, 2H)
7.06 (d, J ) 8.9 Hz, 1H), 7.12 (d, J ) 3.6 Hz, 1H), 7.19 (d, J )
3.6 Hz, 1H), 7.27 (dd, J ) 2.6, 8.9 Hz, 1H), 8.15 (d, J ) 2.6 Hz,
1H); MS 307 (M+); Anal. (C14H14ClN3O3) C, H, N.
[5-(2-Isopropoxy-5-chlorophenyl)furan-2-ylcarbonyl]-
guanidine (97): 91% yield (white solid); mp 206-207 °C; 1H
NMR (CD3OD) δ 1.43 (d, J ) 6.0 Hz, 6H), 4.78 (m, 1H) 7.07
(d, J ) 9.0 Hz, 1H), 7.12 (d, J ) 3.5 Hz, 1H), 7.19 (d, J ) 3.5
Hz, 1H), 7.26 (dd, J ) 2.7, 9.0 Hz, 1H), 8.15 (d, J ) 2.7 Hz,
1H); MS 321 (M+); Anal. (C15H16ClN3O3) C, H, N.
Method B. To a solution of the corresponding ester (1.0
mmol) in DMF (3 mL) was added a 2 M methanol solution of
guanidine (3 mL, 6 mmol), and the resulting mixture was
stirred at room temperature for 2 h. Brine (20 mL) was added,
and the mixture was extracted with ethyl acetate (30 mL ×
3). The organic layer was washed with 10% NaCl, dried
(MgSO4), filtered, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography
(5% methanol in CH2Cl2) to yield the (furan-2-ylcarbonyl)-
guanidine.
[5-(2-Aminophenyl)furan-2-ylcarbonyl]guanidine (54):
94% yield (pale yellow solid); mp 181 °C; 1H NMR (CD3OD) δ
6.70-6.75 (m, 2H), 6.83 (dd, J ) 0.9, 8.1 Hz, 1H), 7.09 (m,
1H), 7.21 (d, J ) 3.6 Hz, 1H), 7.56 (dd, J ) 1.5, 7.8 Hz, 1H);
MS 244 (M+); Anal. (C12H12N4O2) C, H, N.
[5-(2-Nitrophenyl)furan-2-ylcarbonyl]guanidine meth-
anesulfonate (53): 94% yield (yellow solid); mp 229-231 °C;
1H NMR (DMSO-d6) δ 2.38 (s, 3H), 7.13 (d, J ) 3.6 Hz, 2H),
7.70 (d, J ) 3.6 Hz, 1H), 7.79 (dd, J ) 8.7, 9.0 Hz, 1H), 7.96
(m, 2H), 7.99 (d, J ) 9.0 Hz, 1H), 8.36 (brs, 4H), 11.84 (brs,
1H); MS 274 (M+); Anal. (C13H14N4O7S) C, H, N, S.
[5-[3-(Trifluoromethyl)phenyl]furan-2-ylcarbonyl]gua-
nidine methanesulfonate (63): 69% yield (yellow solid); mp
1
238 °C; H NMR (DMSO-d6) δ 2.34 (s, 3H), 7.54 (d, J ) 3.6
Hz, 1H), 7.67 (d, J ) 3.6 Hz, 1H), 7.76-7.84 (m, 2H), 8.27 (m,
2H), 8.34 (brs, 4H); MS 297 (M+); Anal. (C14H14F3N3O5S) C,
H, N, S.
[5-(4-Chlorophenyl)furan-2-ylcarbonyl]guanidine meth-
anesulfonate (67): 70% yield (off white solid); mp 252-253
°C; 1H NMR (DMSO-d6) δ 2.38 (s, 3H), 6.81(d, J ) 3.6 Hz,
1H), 7.08 (d, J ) 3.6 Hz, 1H), 7.33 (d, J ) 8.2 Hz, 2H), 7.75 (d,
J ) 8.2 Hz, 2H), 8.36 (brs, 3H), 11.01 (brs, 1H); MS 263 (M+);
Anal. (C13H14ClN3O5S) C, H, N, S.
[5-(4-Methoxyphenyl)furan-2-ylcarbonyl]guanidine
methanesulfonate (68): 71% yield (yellow solid); mp 212 °C;
1H NMR (DMSO-d6) δ 2.34 (s, 3H), 3.84 (s, 3H), 7.10 (d, J )
9.0 Hz, 2H), 7.17 (d, J ) 3.6 Hz, 1H), 7.62 (d, J ) 3.6 Hz, 1H),
7.91 (d, J ) 9.0 Hz, 2H), 8.33 (brs, 4H), 11.01 (brs, 1H); M+
259; Anal. (C14H17N3O6S) C, H, N, S.
[5-(4-Nitrophenyl)furan-2-ylcarbonyl]guanidine (71):
99% yield (yellow solid); mp 193-195 °C; 1H NMR (DMSO-d6)
δ 7.08 (d, J ) 3.6 Hz, 1H), 7.33 (d, J ) 3.6 Hz, 1H), 8.01 (dd,
J ) 2.1, 7.0 Hz, 1H), 7.96 (dd, J ) 2.1, 7.0 Hz, 2H); MS 274
(M+); Anal. (C12H10N4O4) C, H, N.
[5-(3-Fluorophenyl)furan-2-ylcarbonyl]guanidine (59):
1
89% yield (white solid); mp 211 °C; H NMR (CD3OD) δ 1.43
(d, J ) 6.0 Hz, 6H), 4.78 (m, 1H) 7.07 (d, J ) 9.0 Hz, 1H), 7.12
(d, J ) 3.5 Hz, 1H), 7.19 (d, J ) 3.5 Hz, 1H), 7.26 (dd, J ) 2.7,
9.0 Hz, 1H), 8.15 (d, J ) 2.7 Hz, 1H); MS 247 (M+); Anal.
(C12H10FN3O2) C, H, N.
[5-[2-Chloro-5-(trifluoromethyl)phenyl]furan-2-ylcar-
bonyl]guanidine methanesulfonate (86): 90% yield (white
1
solid); mp 243 °C; H NMR (D2O) δ 2.74 (s, 3H), 7.50 (d, J )
[5-(3-Methylphenyl)furan-2-ylcarbonyl]guanidine (62):
3.7 Hz, 1H), 7.64 (d, J ) 3.7 Hz, 1H), 7.72-7.74 (m, 2H), 8.38
(s, 1H); MS 331 (M+); Anal. (C14H13ClF3N3O5S) C, H, N, S.
Biology. Inhibitory Effect on NHE-1. Na+/H+-exchanger
(NHE-1) inhibitory activity was used as our primary screen
and measured by the rate of NHE-1-mediated recovery of
intracellular pH (pHi) in a 96-well microplate using a pH
sensitive fluorescent dye, 2′,7′-bis-2-carboxyethyl-5(6)-carboxy-
fluorescein acetoxymethyl ester (BCECF-AM, Sigma-Aldrich
Co., MO).23,24 PS120 Fibroblast cells expressing human NHE-1
were obtained from Professor J. Pouyssegur (Nice, France)20
and maintained in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 1% penicillin/streptomycin (100X solution),
1% l-glutamine (200 mM solution), and 10% fetal bovine serum
at 37 °C, in a humidified atmosphere of 5% CO2 and 95% air.
Cells were grown to confluency and then harvested from 100
mm culture dishes using trypsin-EDTA solution. Cells were
washed twice with Na-free buffer (138.2 mM choline chloride,
4.9 mM KCl, 1.5 mM CaCl2‚2H2O, 1.2 mM MgSO4‚7H2O, 1.2
mM KH2PO4, 15 mM D-glucose, 20 mM HEPES, at pH 7.4)
and incubated with 10 µM of BCECF-AM and 20 mM NH4Cl
at 37 °C for 30 min. The BCECF- and NH4Cl-loaded cells were
then washed, resuspended in Na-free buffer, and kept on ice.
The 10 µL of the BCECF- and acid-loaded cells (2.5 × 104
cells) was added to 180 µL of HBS buffer containing NaCl (137
mM NaCl, 4.9 mM KCl, 1.5 mM CaCl2‚2H2O, 1.2 mM MgSO4‚
7H2O, 1.2 mM KH2PO4, 15 mM D-glucose, 20 mM HEPES, pH
7.4) with 10 µL of DMSO or compounds in 96-well microplate.
NHE activity was initiated by NaCl, and fluorescence readings
were taken 4 min after the addition of acidified cell to each
microplate containing NaCl, at 444 nm excitation/535 nm
emission and also at 485 nm excitation/535 nm emission using
a spectrofluorometer (GEMINI-XS, Molecular Device, CA) at
room temperature. Activity in the absence of NaCl was
subtracted from the activity in the presence of NaCl. The
initial increase in pHi in response to NaCl was taken as an
estimate of Na+/H+-exchange activity, and the inhibitory effect
of the compounds was evaluated as IC50 value (concentration
required to inhibit pHi recovery by 50%). Calibration of pHi
was accomplished using the high K+-nigericin method.25 The
1
39% yield (white solid); mp 131-133 °C; H NMR (CD3OD) δ
2.41 (s, 3H), 6.86 (d, J ) 3.5 Hz, 1H), 7.15 (m, 1H), 7.18 (d,
J ) 3.5 Hz, 1H), 7.31 (dd, J ) 7.5, 7.9 Hz, 1H), 7.66 (d, J )
7.5 Hz, 1H), 7.73 (s, 1H); MS 243 (M+); Anal. (C13H13N3O2) C,
H, N.
[5-(3-Aminophenyl)furan-2-ylcarbonyl]guanidine (65):
81% yield (yellow solid); mp 200 °C; 1H NMR (CD3OD) δ 6.69
(m, 1H), 6.78 (d, J ) 3.6 Hz, 1H), 6.78 (d, J ) 3.6 Hz, 1H),
7.11-7.22 (m, 4H); MS 244 (M+); Anal. (C12H12N4O2) C, H, N.
[5-(4-Aminophenyl)furan-2-ylcarbonyl]guanidine (72):
84% yield (yellow solid); mp 208 °C; 1H NMR (CD3OD) δ 6.59
(d, J ) 3.6 Hz, 1H), 6.73 (dd, J ) 2.0, 6.7 Hz, 2H), 7.15 (d,
J ) 3.6 Hz, 1H), 7.60 (dd, J ) 2.0, 6.7 Hz, 2H); MS 244 (M+);
Anal. (C12H12N4O2) C, H, N.
Method C. To a solution of the corresponding furan-2-
carboxylic acid (1.0 mmol) in THF (5 mL) was added 1,1′-
carbonyldiimidazole (182 mg, 1.12 mmol), and the reaction
mixture was stirred at room temperature for 30 min, followed
by an addition of a 2 M methanol solution of guanidine (3.0
mL, 6.0 mmol) and continuous stirring at room temperature
for 12 h. Brine (20 mL) was added, and the mixture was
extracted with ethyl acetate (30 mL × 3). The organic layer
was washed with 10% NaCl twice, dried (MgSO4), filtered, and
concentrated under reduced pressure. The residue was dis-
solved in acetone (4 mL), and methanesulfonic acid (0.2 mL)
was added. The resulting solution was cooled to 0 °C, and the
precipitates were filtered to yield the acylguanidine methane-
sulfonate.
[5-(2-Methylphenyl)furan-2-ylcarbonyl]guanidine meth-
anesulfonate (51): 94% yield (white solid); mp 190 °C; 1H
NMR (DMSO-d6) δ 2.32 (s, 3H), 2.51 (s, 3H), 7.05 (d, J ) 3.9
Hz, 1H), 7.38 (m, 3H), 7.65 (m, 1H), 7.84 (m, 1H), 8.31 (brs,
4H), 11.01 (brs, 1H); MS 243 (M+); Anal. (C14H17N3O5S) C, H,
N, S.
[5-[2-(Trifluoromethyl)phenyl]furan-2-ylcarbonyl]gua-
nidine methanesulfonate (52): 62% yield (yellow solid); mp
1
229 °C; H NMR (DMSO-d6) δ 2.36 (s, 3H), 7.07 (d, J ) 3.6
Hz, 1H), 7.69 (d, J ) 3.6 Hz, 1H), 7.75 (dd, J ) 8.7, 9.0 Hz,

2890 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Lee et al.
(5) Avkiran, M. Rational basis for use of sodium-hydrogen exchange
inhibitors in myocardial ischemia. Am J. Cardiol. 1999, 83, 10G-
18G.
BCECF fluorescence ratio (485/444) was plotted against pHi
and fitted by linear regression.
Cardioprotective Activity in Isolated Ischemic Rat
Heart Model. The anti-ischemic effects of the compounds on
isolated rat hearts were determined, according to our previ-
ously reported paper.26 Briefly male Sprague-Dawley rats
(400-450 g) were anesthetized with sodium pentobarbital, and
their hearts were then excised and quickly moved to a
Langendorff apparatus (H.S.E., Germany), where they were
perfused with oxygenated modified Krebs-Henseleit bicarbon-
ate buffer at a constant perfusion pressure (85 mmHg). After
stabilization for 15 min, the hearts were pretreated for 10 min
with compounds (10 µM, 0.04% DMSO) before onset of global
ischemia. Then the hearts were subjected to global ischemia
by completely shutting off the perfusate for 30 min, followed
by reperfusion. Contractile function (rate pressure product,
RPP) was measured as LVDP (left ventricular developing
pressure) × HR (heart rate) at 30 min after reperfusion.
(6) Karmazyn, M.; Gan, X. T.; Humphreys, R. A.; Yoshida, H.;
Kusumoto, K. The myocardial Na+-H+ exchange: structure,
regulation, and its role in heart disease. Circ. Res. 1999, 85,
777-786.
(7) Avkiran, M. Protection of the ischemic myocardium by Na+/H+
exchange inhibitors: potential mechanisms of action. Basic Res.
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In Vivo Cardioprotective Activity in Ischemic Myo-
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heart was removed. The left ventricle was cut horizontally to
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image analyzing program (Image Pro Plus). The area at risk
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the total AAR was divided by the total weight of the left
ventricle to yield % AAR. The heart slices were then stained
with 2,3,5-triphenyltetrazolium chloride (TTC). The viable
portion of the slice is stained red by TTC, whereas the TTC
stain is absent in the area of infarct. The infarct size of each
slice was summed for all slices and the resulting summed
infarct sized was divided by total AAR weight or total left
ventricle weight to yield % IS.
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Acknowledgment. This research was supported by
a grant (CBM-01-A100-001-1-0-0) from the Center for
Biological Modulators of the 21st Century Frontier R
& D program, the Ministry of Science and Technology,
Korea.
Supporting Information Available: Elemental analysis
data. These materials are available free of charge via the
Internet at http://pubs.acs.org.
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