Synthesis and biological evaluation of 3-substituted-benzofuran-2- carboxylic esters as a novel class of ischemic cell death inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.09.102

A series of 3-substituted-benzofuran-2-carboxylic esters was synthesized and evaluated for biological activity as ischemic cell death inhibitors in H9c2 cells and rat primary cardiac myocytes under conditions of oxygen and glucose deprivation. The introduction of a sulfur atom at the three-position substituent of the benzofuran ring markedly improved ischemic cell death inhibitory potency. In particular, 3-[2-(4-nitro-phenylsulfanyl)-acetylamino]-benzofuran-2- carboxylic acid ester (10) (EC₅₀ = 0.532 μM, cell death = 6.18%) and 4-chloro-3-[3-(pyridin-2-ylsulfanyl)-propionylamino]-benzofuran-2-carboxylic ester (18) (EC₅₀ = 0.557 μM, cell death = 7.02%) were shown to be the most potent in this series of benzofuran analogs.

Full text of DOI:10.1016/j.bmcl.2010.09.102

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6362–6365
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 3-substituted-benzofuran-2-carboxylic
esters as a novel class of ischemic cell death inhibitors
Jeehee Suh a,c, , Kyu Yang Yi a, Yun-Suk Lee b, Eunhee Kim b, Eul Kgun Yum c, Sung-eun Yoo a
⇑
a Bio-organic Science Division, Korea Research Institute of Chemical Technology, Yuseong-gu, Daejeon 305-600, Republic of Korea
b Department of Bioscience and Biotechnology and BK21 Daedeok R&D Innopolis Bio Brain Center, Chungnam National University, Yuseong-gu, Daejeon 305-764, Republic of Korea
c Department of Chemistry, Chungnam National University, Yuseong-gu, Daejeon 305-764, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 July 2010
Revised 8 September 2010
Accepted 21 September 2010
Available online 27 September 2010
A series of 3-substituted-benzofuran-2-carboxylic esters was synthesized and evaluated for biological
activity as ischemic cell death inhibitors in H9c2 cells and rat primary cardiac myocytes under conditions
of oxygen and glucose deprivation. The introduction of a sulfur atom at the three-position substituent of
the benzofuran ring markedly improved ischemic cell death inhibitory potency. In particular, 3-[2-(4-
nitro-phenylsulfanyl)-acetylamino]-benzofuran-2-carboxylic acid ester (10) (EC50 = 0.532
lM, cell
death = 6.18%) and 4-chloro-3-[3-(pyridin-2-ylsulfanyl)-propionylamino]-benzofuran-2-carboxylic ester
Keywords:
(18) (EC50 = 0.557
lM, cell death = 7.02%) were shown to be the most potent in this series of benzofuran
Ischemic cell death inhibitors
3-Substituted-benzofuran-2-carboxylic acid
Anti-ischemic
analogs.
Ó 2010 Elsevier Ltd. All rights reserved.
H9c2 cells
Ischemia, a symptom of reduced blood supply to organs or tis-
sues, is caused by contraction or occlusion of the blood vessels.1
Once ischemia occurs, subsequent reperfusion causes various se-
quelae, due to damaged nerve cells.2 Ischemia is frequently associ-
ated with coronary artery diseases, cardiovascular diseases, angina
pectoris, headache, or other symptoms related to such reduced
blood supply, which ultimately leads to necrosis underlying the
cells or tissues involved.3 Although the mechanisms of cell death
after cerebral ischemia and myocardial ischemia are not yet fully
understood, Fas and FasL are known to be important factors in
the pathology of ischemic stroke4–7 and ischemic heart failure.8,9
Recent studies have shown that Fas contributes to cell death after
ischemic injury.10 The interaction of Fas with its ligand allows the
formation of a death-inducing signaling complex (DISC), which in-
cludes Fas, FADD,11 FAF1,12 and caspase-8.13 Following Fas-DISC
formation, caspase-8 is activated, leading to programmed cell
death. Inhibitors of the Fas-mediated cell death pathway have been
shown to be effective in animal models of ischemic stroke,10 trau-
matic brain injury,14 and myocardial hypertrophy.15 Herein, we
summarized 3-substituted-benzofuran-2-carboxylic esters found
by high-throughput screening assay as inhibitors of the Fas-medi-
ated cell death pathway. For lead optimization of inhibitors of
ischemic cell death, we synthesized 3-substituted-benzofuran-2-
carboxylic ester analogs. We describe the synthetic pathway and
biological activity of a series of 3-substituted-benzofuran-2-car-
boxylic ester analogs as inhibitors of ischemic cell death. The sim-
ple synthetic route of 3-substituted-benzofuran-2-carboxylic
esters analogs is shown in Scheme 1.16
The 3-amino-benzofuran analogs 1, as key intermediates, were
prepared by cyclization of corresponding 2-fluorobenzonitriles
with methyl glycolate. The 3-amino benzofuran analogs 2 were ob-
tained by reaction of 1 with each bromoacyl halide in the presence
of TEA in THF. Alternatively, these compound were also obtained
by a coupling reaction of the respective bromoalkyl acid with
N,N0-diisopropylcarbodiimide (DIC) in CH2Cl2. The 3-(2-substi-
tuted-acethylamino) benzofuran analogs 3–17 with variously
substituted aryl groups were synthesized through the two syn-
thetic pathways.
The benzofuran derivatives with S and NH groups at the Y-posi-
tion of 3–11, 13–14, and 16 were synthesized by nucleophilic sub-
stitution reaction of compound 2 with corresponding thiophenol,
phenol or aniline compounds. The compound 17 was prepared
by nucleophilic substitution reaction using 2-mercaptopyridine.
On the other hand, the benzofuran derivatives containing O and
CH2 groups at the Y-position of 12 and 15 were prepared from
3-amino-benzofuran analogs 1 with substituted carboxylic acids
by coupling reaction.
The 3-[3-(pyridin-2-ylsulfanyl)-propionylamino]-benzofuran
analogs 18–19 were prepared from the corresponding bromide
compounds 2 through a sequence of reactions including HBr
elimination and 1,4-Micheal addition of 2-mercaptopyridine to
a,b-unsaturated ketones.
The ischemic cell death inhibitory activities of the synthesized
compounds were determined by staining of nuclei with DAPI or
⇑
Corresponding author. Tel.: +82 42 860 7145; fax: +82 42 861 1291.
E-mail address: jhsuh@krict.re.kr (J. Suh).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.102

J. Suh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6362–6365
6363
N
O
W
+
S
O
Br
n
CN
F
W
W
W
NH
NH2
NH
O
O
O
O
O
O
n = 2
e, f
O
O
O
a
b
2a n = 1
2b n = 2
O
18 - 19
1
HO
O
c
d
n = 1
HY
O
Y
A
HO
Z
Z
O
Y
Z
A
W
NH
O
O
W = H, Cl
Y = S, O, NH, CH2
A = CH, N
O
3 - 17
Z = H, Br, OCH3, CH3, NO2, NH2
Scheme 1. (a) methyl glycolate, K2CO3, DMF, 100 °C, 40–50%; (b) (i) bromoacyl halide, NEt3, THF, rt or (ii) bromoalkyl acid, N,N0-diisopropylcarbodiimide (DIC), CH2Cl2, rt, 50–
90%; (c) NEt3, THF, 0 °C–rt, 20–91%; (d) N,N0-diisopropylcarbodiimide (DIC), CH2Cl2, rt, 25–90%; (e) NEt3, THF, rt, 90–95%; (f) 2-mercaptopyridine, NEt3, THF, 0 °C–rt, 80–90%.
Table 1
lactate dehydrogenase (LDH) release after oxygen and glucose
deprivation (OGD)-induced cell death in H9c2 cells17–20 (Fig. 1).
In vitro efficacy against ischemic cell death of H9c2 cells by the effects of substituent
on benzofuran and benzothiophene.
Initially, we compared benzofuran and benzothiophene analogs
for their inhibitory activity against ischemic cell death. The benzo-
furan compound 3 with oxygen at X showed stronger in vitro
ischemic cell death inhibitory activity than benzothiophene com-
pound 4 (Table 1).
From initial biological results, the 3-(3-substituted-propionyla-
mino)-benzofuran-2-carboxylic esters and 3-(2-substituted-aceth-
ylamino)-benzofuran-2-carboxylic esters were synthesized. The
biological inhibitory results are shown in Table 2.
O
S
Br
NH
O
O
X
Compounds
X
Cell death (%, 10 lM)
DAPI
LDH
3
4
O
S
10.59 0.14#
13.92 1.08#
32.39 5.44#
41.31 0.82#
The 4-substituted derivative 3 with a bromo group at Ar1
showed markedly increased ischemic cell death inhibitory activity,
compared with unsubstituted and 2- or 3-substituted derivatives
Data are means SEM of three independent experiments.
#
5–7. Remarkably, 10 (cell death = 6.18% at 10 lM) with an electron
P <0.001 versus control.
withdrawing NO2 group at the para position was much more po-
tent than most of the other analogs in this series and three-fold
more potent than compound 9 with 4-methyl group.
Generally, the introduction of an O, NH, or CH2 group at Y
reduced the inhibitory functions, compared with benzofuran
derivatives containing S at Y. The benzofuran derivatives 16–17
with Cl at W were more potent than the unsubstituted derivatives.
Replacement of the 4-bromophenyl group at Ar1 in 16 with a
Figure 1. Nuclear morphological changes upon ischemic insult in H9c2 cells. DAPI (400ꢀ) images were acquired using an inverted Zeiss LSM510 META confocal microscope.
Arrows indicate dead cells.

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J. Suh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6362–6365
Table 2
In vitro efficacy against ischemic cell death of H9c2 cells by the effects of substituent on 3-(3-substituted-propionylamino)-benzofuran-2-
carboxylic esters and 3-(2-substituted-acethylamino)-benzofuran-2-carboxylic esters
O
Y
Ar1
n
W
NH
O
O
O
Compounds
W
n
Y
Ar1
Cell death (%, 10 lM)
DAPI
LDH
Control
Vehicle
KR-31378
3
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
3.93 0.75
15.26 1.54
73.40 6.32#
43.33 2.06#
32.39 5.44*
40.93 3.58#
39.51 2.64#
41.01 0.74#
40.34 2.45#
56.39 5.54#
30.21 2.16#
40.15 0.61#
36.17 0.76#
60.32 3.33#
62.76 1.03#
38.72 3.66#
35.48 5.05*
25.59 3.80
29.46 1.37#
36.07 1.01
36.04 0.46#
15.54 0.77#
10.59 0.14#
15.97 0.83#
16.85 2.92#
13.63 0.54#
9.73 0.81#
21.57 1.85#
6.18 0.88*
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
H
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
S
S
S
S
S
S
S
S
O
NH
NH
CH2
S
S
S
S
4-Bromophenyl
phenyl
3-Bromophenyl
2-Bromophenyl
4-Methoxyphenyl
4-Methylphenyl
4-Nitrophenyl
4-Aminophenyl
4-Bromophenyl
4-Bromophenyl
Phenyl
Phenyl
4-Bromophenyl
2-Pyridyl
2-Pyridyl
2-Pyridyl
16.54 0.77#
13.17 0.17#
22.37 1.60#
21.04 1.67#
15.94 4.76*
8.94 2.10*
7.33 0.35#
7.02 0.75*
11.55 1.35#
*
Data are means SEM of three independent experiments. P <0.05, P <0.01, #P <0.001 versus control.
2-pyridyl group 17 resulted in further enhanced the potency to in-
hibit ischemic cell death. Thus, benzofuran analogs with a Cl group
at W and 2-pyridyl group at Ar1 showed very potent inhibitory ef-
fects on ischemic cell death. A longer carbon chain at the 3-posi-
tion in this series of benzofuran analogs influenced the inhibitory
activity. The 4-chloro-3-[3-(pyridin-2-ylsulfanyl)-propionylami-
no]-benzofuran-2-carboxylic ester 18 showed better activity than
17. However, 19 with H at W was much less potent than Cl substi-
tuted analogs. The median effective concentration (EC50) of com-
pounds for protection of cells from ischemic death was
determined by counting after staining of nuclei with DAPI after
OGD in rat primary cardiac myocytes. KR-31378 was used as a
standard to measure the EC50 of potent active compounds. KR-
31378 has been reported to show antiapoptotic activities against
ischemic injury21 and has been assessed in vivo.22
and 4-chloro-3-[3-(pyridin-2-ylsulfanyl)-propionylamino]-benzo-
furan-2-carboxylic ester 18 (EC50 = 0.557
lM, cell death = 7.02%
at 10 M) were shown to be the most potent in this series of ben-
l
zofuran analogs. However, compound 12 with oxygen at Y was
much less potent than most of the other analogs in this series.
These results indicated that sulfur at Y has an important effect
on ischemic cell death inhibitory activity.
In conclusion, we synthesized and evaluated a series of 3-
substituted-benzofuran-2-carboxylic esters as a novel class of
ischemic cell death inhibitors targeting Fas-mediated cell death
pathway through cell-based screening. In particular, 10 and 18
were exhibited to be the most potent in vitro efficacies, superior
to KR-31378, a potent KATP-channel opener. Additionally, the intro-
duction of a sulfur atom at the 3-position of the benzofuran ring
markedly improved ischemic cell death inhibitory potency. Further
studies including analysis of in vivo efficacy as well as pharmaco-
kinetic and metabolic studies are underway to identify as a novel
ischemic cell death inhibitor.
As shown in Table 3, the EC50 values for the benzofuran deriva-
tives indicated that they were generally more potent than KR-
31378.
3-[2-(4-Nitro-phenylsulfanyl)-acetylamino]-benzofuran-2-car-
boxylic acid ester 10 (EC50 = 0.532 lM, cell death = 6.18% at 10 lM)
Acknowledgments
Table 3
This research was primarily supported by grants (CBM33-
A2300-01-01-00) from the Center for Biological Modulators of
the 21st Century Frontier R&D program, the Ministry of Science
and Technology, Korea.
In vitro efficacy against ischemic cell death in rat
primary cardiac myocytes by the effects of substituent
on 3-(3-substituted-propionylamino)-benzofuran-2-
carboxylic esters and 3-(2-substituted-acethylamino)-
benzofuran-2-carboxylic esters
Compounds
EC50 (l
M)a
Supplementary data
KR-31378
3
8
10
12
16
17
18
19
1.109
0.778
0.752
0.532
1.177
0.630
0.653
0.557
0.929
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.09.102.
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