4-Substituted (benzo[b]thiophene-2-carbonyl)guanidines as novel Na +/H+ exchanger isoform-1 (NHE-1) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.04.046

A series of 4-substituted (benzo[b]thiophene-2-carbonyl)guanidines was synthesized and evaluated for the NHE-1 inhibitory activity and cardioprotective efficacy both in vitro and in vivo. Several analogs exhibited a strong inhibition on NHE-1, and which was generally well correlated with their cardioprotective efficacy. Especially the 4-nitro 20 and cyano 50 compounds excellently improved the cardiac function and reduced infarct size against ischemia/reperfusion injury.

Full text of DOI:10.1016/j.bmcl.2005.04.046

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2998–3001
4-Substituted (benzo[b]thiophene-2-carbonyl)guanidines as novel
Na+/H+ exchanger isoform-1 (NHE-1) inhibitors
Sunkyung Lee,a Hyunsuk Lee,b Kyu Yang Yi,a,* Byung Ho Lee,a Sung-eun Yoo,a
Kyunghee Leec and Nam Sook Chob
aMedicinal Science Division, Korea Research Institute of Chemical Technology, Yuseong-gu, Daejeon 305-600, Republic of Korea
bDepartment of Chemistry, Chungnam National University, Daejeon 305-764, Republic of Korea
cCentral Research Center of Yuyu Inc., Kyounggi-do 430-804, Republic of Korea
Received 21 February 2005; revised 15 April 2005; accepted 22 April 2005
Abstract—A series of 4-substituted (benzo[b]thiophene-2-carbonyl)guanidines was synthesized and evaluated for the NHE-1 inhib-
itory activity and cardioprotective efficacy both in vitro and in vivo. Several analogs exhibited a strong inhibition on NHE-1, and
which was generally well correlated with their cardioprotective efficacy. Especially the 4-nitro 20 and cyano 50 compounds excel-
lently improved the cardiac function and reduced infarct size against ischemia/reperfusion injury.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Since the excessive activation of Na+/H+ exchanger iso-
form-1 (NHE-1) has been known to play an important
role in the progression of ischemia/reperfusion injury,1,2
many efforts have been devoted to develop a potent and
selective NHE-1 inhibitor as cardioprotective drug.
Through the replacement of a pyrazine ring of amilo-
ride3 known as a non-selective NHE-1 inhibitor, several
monocyclic acylguanidines such as cariporide,4 enipo-
ride, and zoniporide5 have been identified as the selec-
tive and potent NHE-1 inhibitors. Later, several
compounds based on a bicyclic template including a
quinoline, indole, benzoxazinone, tetrahydronaph-
thalene, or tetrahydrocyclohepta-pyridine have been
designed with the aim to discover more potent and
highly water-soluble inhibitor.6,7 In our preliminary
study to identify a novel NHE-1 inhibitor, a variety
of bicyclic acylguanidines were investigated, and
As a stating point, 4-substituted benzo[b]thiophene-2-
carboxylates 6–10 were prepared by a simple 2-step
procedure (Scheme 1).8 A regioselective lithiation9 of 3-
substituted fluorobenzene using lithium diisopropylamide
(LDA), followed by formylation with dimethylformam-
ide (DMF) provided 6-substituted 2-fluorobenzaldehydes
1–5. Subsequent treatment of 1–5 with thioglycolate and
K2CO3 in DMF gave 6–10 through the nucleophilic dis-
placement of aromatic fluorine and base-induced ring-
closure in a single step. Based on the rate of nucleophilic
displacement (F > NO2 > Cl > Br)10 and an enhanced ef-
fect of carboxaldehyde function on the displacement
lability, the fluorine of 1–5 was displaced with thioglyco-
late in mild condition. The 4-nitro 11 and chloro 12 com-
pounds were similarly prepared from 2,6-dinitro- and
2-chloro-6-nitrobenzaldehyde, respectively. The 4-amino
compound 13 was obtained by Raney-nickel catalyzed
hydrogenation of the nitro compound 11. In this case,
Pd-catalyzed reduction did not proceed, presumably
due to sulfur of benzothiophene. Commercially available
4-trifluoromethyl compound 14 was also utilized.
(4-bromobenzo[b]thiophene-2-carbonyl)guanidine
15
was found as a potent NHE-1 inhibitor. Starting with
15, a series of (benzo[b]thiophene-2-carbonyl)guanidines
with various 4-substituents were synthesized, and evalu-
ated for the NHE-1 inhibitory activity and cardioprotec-
tive efficacy.
The 4-bromine of 6 was further converted to the alkyl,
cyano, and aryl functions (Scheme 2). The alkyl and aryl
substituted compounds were obtained by a Pd-catalyzed
cross coupling reaction. While the methyl 24 and aryl
compounds 29–45 were synthesized via the Suzuki reac-
tion,11 the vinyl compound 25 was prepared employing
the Stille condition.12 Raney-nickel catalyzed hydroge-
nation of 25 provided the 4-ethyl compound 26.
Keywords: Sodium hydrogen exchanger; (Benzo[b]thiophen-2-carbon-
yl)guanidine; Cardioprotective.
*
Corresponding author. Tel.: +82 42 860 7143; fax: +82 42 861
1291; e-mail: kyyi@krict.re.kr
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.046

S. Lee et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2998–3001
2999
The treatment of 6 with isopropylzinc slurry and
copper(I) catalyst as well as Pd(0) catalyst afforded the
isopropyl compound 27.13 The nitrile compound 28
was obtained by microwave-promoted nucleophilic sub-
stitution using CuCN in DMF from 6 in 20 min.
(Benzo[b]thiophene-2-carbonyl)guanidines (15–23, 46–
67) were prepared from the corresponding carboxylic
esters by treating with excess guanidine (free base) in
methanol or in DMF. The final acylguanidines were
purified by crystallization as a methanesulfonic acid salt.
The NHE-1 inhibitory activity of the synthesized com-
pounds was determined by measuring their ability to inhi-
bit the sodium dependent recovery of pH following an
imposed acidosis, in PS120 variant cells in which the hu-
man NHE-1 was selectively expressed.14 Primarily, the
IC50 values of 4-, 5-, and 6-bromobenzo[b]thiophene com-
pounds were determined as 0.20, 12, and 16 lM, respec-
tively. The 4-bromo compound 15 exhibited a higher
potency on NHE-1 than cariporide (IC50 = 0.68 lM).
Then various substituents were introduced at the 4-posi-
tion to optimize the activity of this series of compounds
Scheme 1. Reagents and conditions: (a) (i) LDA, THF, À78 ꢁC, (ii)
DMF, 60–95%; (b) methyl thioglycolate, K2CO3, DMF, 65–95%; (c)
40 psi H2, 50% Raney-Ni, CH3OH, rt, 98%; (d) (i) guanidine, DMF, rt,
(ii) CH3SO3H, acetone, 50–80%.
Table 1. Inhibitory effect on NHE-1 of compounds 15–23 and 46–67
Compds
X
IC50, lMa
Cariporide
15
16
0.68
0.20
12
Br
H
17
18
F
1.7
I
OMe
0.17
3.4
19
20
NO2
Cl
2.3
21
22
0.20
>30
0.40
0.48
0.31
0.27
0.85
2.0
NH2
CF3
23
46
Me
Vinyl
47
48
Et
i-Pr
49
50
CN
Ph
51
52
8.4
2-F–Ph
3-F–Ph
4-F–Ph
2-Cl–Ph
3-Cl–Ph
4-Cl–Ph
2-Me–Ph
3-Me–Ph
4-Me–Ph
3-OMe–Ph
3-CF3
2,3-diF–Ph
3,5-diF–Ph
2,5-diF–Ph
3,5-diCl–Ph
3-Cl-4-F–Ph
11
53
54
5.6
3.9
55
56
4.2
2.5
57
58
>30
7.7
3.4
59
60
>30
11
61
62
Scheme 2. Reagents and conditions: (a) trimethylboroxine, Pd(PPh3)4,
K2CO3, DMF, reflux, 92%; (b) tributyl(vinyl)tin, Pd(PPh3)4, toluene,
reflux, 61%; (c) 40 psi H2, 50% Raney-Ni, CH3OH, rt, 72%; (d) (i)
isopropylmagnesium chloride, ZnCl2, THF, 50 ꢁC, (ii) 6, CuI,
Pd(dppf)Cl2ÆCH2Cl2, THF, rt, 69%; (e) CuCN, microwave, DMF,
200 ꢁC, 20 min, 80%; (f) arylboronic acid, Pd(PPh3)4, Ba(OH)2ÆH2O, or
K2CO3, toluene or DME, reflux, 60–95%; (g) (i) guanidine, DMF, rt,
(ii) CH3SO3H, acetone, 50–90%.
4.4
8.9
63
64
5.1
8.6
65
66
0.68
2.5
67
a n = 3.

3000
S. Lee et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2998–3001
Table 2. Cardioprotective efficacy against ischemia–reperfusion injury
Compds
X
Langendorffa
In vivob IS/AAR (%)
RPP (%)
LVEDP (mmHg)
LDH (IU/g)
Control
Cariporide
13
44
1
4
55
44
34
38
19
11
2
3
2
7
3
4
28
12
nac
7.0
7.1
11
9
2
2
59
41
2
2
7
2
4
5
4
3
15
17
18
20
21
23
46
47
48
50
66
67
Br
F
55 13
47 12
66 13
46
45
3
3
2
1
3
2
1
3
2
I
NO2
49
36
73
8
Cl
CF3
61 17
81 17
25 11
48
6
38
42
38
19
64
68
4
3
9
9
6
8
9
5
45
Me
vinyl
40
4
17
13
7
nac
nac
52
51 23
43 13
Et
CN
2
4
63
19
6
6
7
1
13
nac
26
38
3,5-diCl–Ph
3-Cl–4-F–Ph
nac
nac
2
a In vitro cardioprotective effect was evaluated by measuring % RPP (LVDP · HR) to pre-ischemic value, LVEDP, and LDH release in the isolated
ischemic rat heart (10 lM). Values are means SEM, n = 3 or higher.
b In vivo cardioprotective effect was determined by measuring a ratio of myocardial infarct size to area at risk (IS/AAR) in rat myocardial infarction
model (0.1 mg/kg). Values are means SEM, n = 3 or higher.
c na: not assayed.
(Table 1). The unsubstituted analog 16 (IC50 = 12 lM)
represented far less activity than 15. The 4-iodo 18
(IC50 = 0.17 lM), chloro 21 (IC50 = 0.20 lM), and triflu-
oromethyl 23 (IC50 = 0.40 lM) compounds were highly
active, similarly to 15. While the 4-amino compound 22
was inactive, the fluoro 17, methoxy 19, nitro 20, and ni-
trile 50 compounds maintained an activity. The 4-alkyl
substituted compounds 46–49 demonstrated a good activ-
ity. The potency was increased from methyl 46, vinyl 47,
to ethyl 48, but decreased by the substitution with isopro-
pyl group 49. Presumably there may be an optimum size
for the activity. Additionally the 4-aryl substituted com-
pounds were investigated. The 4-phenyl compound 51
exhibited a moderate activity (IC50 = 8.4 lM). The meta
substituted phenyl analogs of 51 appeared to be more ac-
tive than the other substituted aryl compounds, but were
not as good as the halogen or alkyl substituted com-
pounds. The 3,5-dichlorophenyl compound 66 showed
an excellent activity (IC50 = 0.68 lM), which consistently
indicates that the meta substitution at the phenyl ring may
be beneficial to improve the activity compared with the
other substitution patterns.
tionally left ventricular end diastolic pressure (LVEDP)
as an indicator of cardiac contracture, and lactate dehy-
drogenase (LDH) released into the effluent perfusate
during reperfusion as an indicator of myocyte injury
were determined. In this model, cariporide significantly
improved the recovery of contractile function (44%
RPP), diminished LVEDP (44 mmHg), and reduced
the release of LDH (12 IU/g), compared with the vehicle
group (13% RPP, 55 mmHg LVEDP, and 28 IU/g LDH
release). Cardioprotective in vivo efficacy was deter-
mined by measuring a ratio of myocardial infarction size
to area at risk (IS/AAR), using rat myocardial infarction
model,16 which was stabilized for 20 min after a left
thoracotomy operation, subjected to 45 min coronary
artery occlusion, following 90 min reperfusion. The
compounds (0.1 mg/kg) were administered by bolus
intravenous injection at 5 min before onset of ischemia.
In this in vivo rat myocardial infarct model, cariporide
represented 41% of IS/AAR, which indicates the signif-
icant reduction of the infarct size compared with the
vehicle group (59% of IS/AAR).
The aryl substituted compounds 66, 67 did not show any
improvement of cardiac dysfunction in isolated rat heart
model regardless their strong inhibition on NHE-1.
However, the other potent analogs significantly im-
proved the cardiac function and reduced myocyte dam-
age against ischemia/reperfusion injury, which was
comparable to cariporide. Especially the iodo 18, nitro
20, chloro 21, trifluoromethyl 23, and cyano 50 com-
pounds, excellently recovered the cardiac contractility
above the 60% of basal RPP value as well as the signifi-
cant attenuation of cardiac contracture and protection of
myocyte damage in the isolated rat heart ischemia model.
Furthermore, the nitro 20 and cyano 50 compounds
Next, the compounds showing a good NHE-1 inhibitory
activity were evaluated for their cardioprotective efficacy
against ischemia/reperfusion injury both in vitro and in
vivo (Table 2). In the isolated rat heart model,15 each
isolated rat heart was treated with 10 lM concentration
of the compound for 10 min, and subjected to 30 min
global ischemia, followed by 30 min reperfusion. For
the evaluation of their cardioprotective effect, the %
recovery of rate pressure product (RPP, HR · LVDP,
heart rate · left ventricular developing pressure) at the
end of reperfusion to the pre-ischemic value was mea-
sured as an index of cardiac contractile function. Addi-

S. Lee et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2998–3001
3001
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In summary, a series of benzo[b]thiophene-2-carbonyl-
guanidine analogs with various 4-substituents including
halogen, nitrile, nitro, amine, alkyl, and aryl group, were
synthesized and evaluated for their in vitro and in vivo
cardioprotective efficacy in addition to the NHE-1
inhibitory activity. The 4-nitro 20 and 4-cyano 50 com-
pounds excellently improved the cardiac function and
reduced infarct size against ischemia/reperfusion injury
as well as strong inhibition on NHE-1. We will continu-
ously study on this series of compounds including their
advanced pharmacology, pharmacokinetic and safety
profiles.
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Acknowledgments
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.
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