Design, synthesis, and biological evaluation of benzofuran derivatives as et receptor antagonists

Article abstract of DOI:10.1007/s00044-013-0542-3

A series of novel benzofuran carboxylic acid derivatives have been designed and synthesized, with their antagonism effect screened on ET-1-induced contraction in the rat thoracic aortic ring. Some target compounds demonstrated significant inhibitory activ

Full text of DOI:10.1007/s00044-013-0542-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0542-3
ORIGINAL RESEARCH
Design, synthesis, and biological evaluation of benzofuran
derivatives as ET receptor antagonists
•
Jin Cai Junqing Chen Meng Cao Peng Wang
•
•
•
•
Chengliang Feng Min Ji
Received: 23 November 2012 / Accepted: 15 February 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of novel benzofuran carboxylic acid
derivatives have been designed and synthesized, with their
antagonism effect screened on ET-1-induced contraction
in the rat thoracic aortic ring. Some target compounds
demonstrated significant inhibitory activity, especially
benzo[c]thiadiazole and benzo[c]oxadiazole compounds 29
and 30 showed potent inhibition percentage higher than the
contrast compound BQ123. Further affinity and selectivity
for ET binding assay showed that 29 demonstrated a dual
ETA/ETB antagonism activity in nanomole level. More-
over, 30 was effective in relieving hypoxia-induced pul-
monary arterial hypertension.
levels are associated with several cardiovascular disease
conditions, including systemic and pulmonary arterial
hypertension (Stewart et al., 1991), congestive heart failure
(Van Beneden et al., 2004), renal failure (Lariviere and
Lebel, 2003), prostate cancer (Zhou et al., 2007; Roh and
Abdulkadir, 2010) and atherosclerosis (Minamino et al.,
1997; Skalska and Grodzicki, 2010). The ETs function by
binding to transmembrane G-protein-coupled receptors of
which two major subtypes, ETA and ETB, have been
identified (Jacobs et al., 2006; Masaki et al., 1994). The
ETA subtype, which is selective for ET-1 and ET-2 over
ET-3, is found principally in peripheral tissues such as
vascular smooth muscle (Arai et al., 1990). The ETA
receptor mediates vasoconstriction and vascular smooth
muscle proliferation. However, the binding of ET to ETB
receptors located on the vascular endothelium causes
vasodilation through the production of nitric oxide and
prostacyclin (Seo et al., 1994). Antagonists of ET receptors
can be classified pharmacologically as selective and non-
selective antagonists or chemically as peptide and non-
peptide compounds. Bosentan (TracleersÒ), a non-selective
ET receptor antagonist, and Ambrisentan (letairiseÒ), a
selective ETA receptor antagonist have been approved for
the treatment of pulmonary arterial hypertension by the US
Food and Drug Administration and they are non-peptide
compounds with high bioavailability (Li et al., 2008; Patel
et al., 2010).
Keywords Benzofuran ꢀ Endothelin ꢀ
Pulmonary arterial hypertension ꢀ SAR ꢀ Synthesis
Introduction
Endothelin (ET), a peptide of 21-amino acids, was initially
identified from endothelial cells as a potent vasoconstrictor
(Yanagisawa et al., 1988). There are three isoforms (ET-1,
ET-2, ET-3) (Inoue et al., 1989). ET-1 is the predominant
component of the three ET-isopeptides largely because of
its ability to constrict vascular and nonvascular smooth
muscle (Prasanna et al., 2003). ET is a small peptide
hormone that is believed to play a critical role in the
control of blood flow and cell growth. Elevated ET blood
Screening of the Parke–Davis compound library identi-
fied two benzofuran carboxylic acid derivatives 1 and 2
(Fig. 1) that showed potent selectivity for the ETA receptor
(IC50 = 11 lM for 1, IC50 = 3.8 lM for 2) (Kaltenbronn
et al., 1997). The previous report of structure and activity
relationship of benzofuran carboxylic acid derivatives
indicated that 3-methyl group and the carboxyl group were
essential for ETA binding affinity. Therefore, we introduced
J. Cai ꢀ J. Chen ꢀ M. Cao ꢀ P. Wang ꢀ C. Feng ꢀ M. Ji (&)
School of Chemistry and Chemical Engineering, Institute
of Pharmaceutical Engineering, Southeast University,
87 Dingjiaqiao, Nanjing 210096, Jiangsu,
People’s Republic of China
e-mail: jimin@seu.edu.cn
123

Med Chem Res
OCH3
Biological evaluation
CH3
O
N
HOOC
O
HN
COOH
Functional assessment of antagonism on ET-1-induced
contraction of rat thoracic aortic ring
O
O
O
NH
O
1
NH
N
H
The synthesized compounds were evaluated for their antag-
onism of ET-1-induced contraction in the rat thoracic aortic
ring. BQ123 was taken as a positive control and DMSO was
taken as a blank control in the assays. The results reported in
Table 2 showed that ethyl and propyl group substituted
compounds 20 and 21 demonstrated significant inhibition
activity, but with prolongation of the alkyl chain, the activity
of22 and23 becamelower. Amongthealkylbenzolethers24–
27, substitution with either electron-donating or electron-
withdrawing groups gave moderate inhibition, it was notice-
able for compound 26 that percentage of ET-1 inhibition was
-39 %, we speculated that 26 may show some ETA agonist
activity or it could potentiate effect of ET-1-induced con-
traction with an unknown mechanism. These results indicated
thatthe increase ofR groupsvolumes would not benefitfor the
inhibition activity of the compounds. However, it was worth
pointing out that significant inhibition was achieved for
benzo[c]thiadiazole and benzo[c]oxadiazole compounds 29
and 30 (P \ 0.01) with potent inhibition percentage higher
than the contrast compound BQ123, which confirmed our
preliminary design idea.
O
CH3
O
NH
COOH
2
BQ123
Fig. 1 Chemical structure of 1, 2 and BQ123
some other substituents in 4-position, such as longer ali-
phatic chains, substituted benzene rings and benzothiadiaz-
oles, which were pharmacophores of some known ETA
antagonists in former report (Yuan et al., 2006). This paper
described the synthesis and SAR of a series of derivatives
where R function was varied sequentially.
Results and discussion
Chemistry
Compounds 20–32 were prepared as illustrated in Scheme 1.
1-(2,6-dihydroxyphenyl)-ethanone 3 as starting material
was monoalkylated with ethyl 2-bromoacetate to give 4 in
61 % yield. Cyclization of 4 with NaOMe in MeOH afforded
5 in low yield due to partial hydrolysis of the ester under the
alkali conditions in this step. Alkylation of 4-phenolic
hydroxyl group permitted easy access to the intermediate
ethers 6–19, which was hydrolyzed to produce 2, 20–32 in
yields of 38–84 %. As summarized in Table 1, up to four-
teen target compounds (2, 20–32) were synthesized.
Selective potency of compounds on the ETA and ETB
subtype of rat myocardium
Therefore, the further test of radio-receptor assay of com-
pound 29 and 30 were done for purpose of quantitative
evaluation for selectivity of effect on ETA and ETB
(Table 3). Compared with BQ123, 29 demonstrated more
potent binding affinity on ETA in nanomole level (23 nM),
and the ratio of ETA/ETB was 40. So we could infer that 29
was a non-selective dual antagonist of ETA and ETB, and
had potential for further development in cardiovascular
diseases. The next study would evaluate the ability of
orally administered 29 to relieve hypoxia-induced pul-
monary arterial hypertension in rats.
OH
OH
O
OH
O
O
CH3
a
b
CH3
OH
CH3
CO2
Et
CO2
O
Et
Relief activity of compound 29 on pulmonary arterial
hypertension
4
5
3
OR
OR
CH3
CH3
COOH
c
d
After hypoxic exposure for 28 days, the right ventricular
systolic pressure (RVSP) and central vein pressure (CVP) of
rats were elevated by ?118 % (P \ 0.01) and ?1,071 %
(P \ 0.05), respectively, relative to control, indicating
hypoxic PAH was established. Nifedipine p.o. was used as a
positive reference in this study due to its wide use in treating
PAH. After treatment with compound 29 (40, 80, 160
mg/kg) and nifedipine (10 mg/kg), the elevated RVSP and
Et
CO2
O
O
6-19
20-32
Scheme 1 Synthetic route for the preparation of the target com-
pounds 20–32. Reagents and conditions a BrCH2CO2Et, K2CO3,
acetone, 3 h, reflux, 61 %; b NaOEt, EtOH, 9 h, reflux, 36 %; c RX,
KI, K2CO3, DMF, 1 h, 60 °C, 84 %; d NaOH, EtOH, H2O, 1 h,
reflux, 38–84 %
123

Med Chem Res
Table 1 Structure of compounds 20–32
OR
CH3
COOH
O
Compound
R
Yield (%)
Mp (°C)
Formula
20
52
78
63
81
62
226–228
C12H12O4
21
22
23
2
188–190
182–183
186–188
183–184
C13H14O4
C14H16O4
C15H18O4
C17H14O4
24
25
26
27
28
29
30
31
67
60
38
63
75
81
66
84
191–193
239–241
250–252
208–210
217–218
256–258
224–226
213–214
C19H16O4
C19H18O5
C18H15ClO5
C18H15ClO5
C16H19NO4
C17H12N2O4S
C17H12N2O5
C18H14O6
123

Med Chem Res
Table 1 continued
Compound
R
Yield (%)
79
Mp (°C)
Formula
32
230–232
C20H18O6
Table 2 In vitro functional vascular ET-1 antagonism of test compounds in concentration 10 lM; inhibition of the ET-1 (2 nM)-induced
contraction of rat thoracic aortic ring
Compound
Percentage of inhibition SD (%)c
Pd
Cpd.
Percentage of inhibition SD (%)
Pd
DMSO
BQ123a
29.9 18.3 (8)
68.1 31.3 (3)
61.0 13.7 (3)
69.7 26.4 (3)
56.7 28.2 (4)
46.8 38.4 (3)
40.7 24.2 (4)
50 44.1 (3)
–
25
26
27
28
29
30
31
32
40.8 37.7 (3)
-39 26.0 (3)
21.7 22.2 (3)
34.1 22.5 (3)
82.2 12.6 (3)
70.0 15.5 (4)
29.0 26.8 (3)
44.0 42.6 (3)
0.5181
0.02985*
0.02715*
0.01807*
0.0723
0.00073**
0.54744
0.75313
0.00141**
0.0039**
0.95234
0.43836
20
21
22
23
2b
24
a
0.33102
0.40349
0.28848
BQ123 (Petersen et al., 2008): IC50 = 37 nM for hETA
b
c
d
Reported else where
values are the means of the indicated number of experiments (n)
P value of T test (* P \ 0.05; ** P \ 0.01)
Table 3 ETA and ETB receptor binding affinities for 29, 30 and BQ123
Compound
IC50 (nM)
ETA
Selectivity for ETA
ETB
930
29
23
39
28
40
30
1,070
–
27
BQ123
Specificity
CVP decreased individually (P \ 0.01), respectively,
compared with the untreated group (Fig. 2). The response to
29 was dose dependent with the lowest dose demonstrating
potent effect on relieving hypoxia-induced pulmonary arterial
hypertension.
million (ppm) relative to tetramethylsilane (TMS), used as
an internal standard. Mass spectra (MS) were obtained
from Agilent 1100LC/MS Spectrometry Services. Ele-
mental analyses were performed on Elementar Vario ELI
instrument. All compounds were routinely checked by TLC
with silica gel GF-254 glass plates and viewed under UV
light at 254 nm.
Experimental section
Synthesis
General
Ethyl 2-(2-acetyl-3-hydroxyphenoxy)acetate (4)
All reagents were purchased from commercial sources and
used without further purification. Melting points were
measured in open capillaries and are uncorrected. 1H-NMR
spectra were recorded in CDCl3 on a Bruker-ACF 300
spectrometer; chemical shifts (d) are reported in parts per
1-(2,6-dihydroxyphenyl)-ethanone 3 (15.2 g, 0.1 mol) and
K2CO3 (41.4 g, 0.3 mol) were dissolved in acetone (200 mL).
Ethyl 2-bromoacetate (20 g, 0.12 mol) was added to this
solution. The mixture was heated to reflux for 3 h. After
123

Med Chem Res
Fig. 2 Reversion of an elevation in RVSP and CVP in hypoxic pulmonary arterial hypertensive (PAH) rats by a dual ET receptor antagonist 29.
N = 10
Alkylation of the 4-hydroxyl group to give 6–19. A general
procedure illustrated with the preparation of ethyl
4-ethoxy-3-methylbenzofuran-2-carboxylate (6)
cooling to room temperature, the precipitate was filtered
and washed with acetone, about 2/3 of the filtrate was
evaporated under reduced pressure. Cold water (300 mL)
was added, and the precipitate obtained was filtered, washed
with water, and dried to yield 4 as a white tabular crystal
(14.6 g, 61 %). M.p. 80–82 °C. IR (KBr, cm-1): 3320,
2918, 1708, 1509, 1443, 1276; 1H-NMR(CDCl3), d: 1.38 (t,
J = 7.0 Hz, 3H, CO2CH2CH3), 2.63 (s, 3H, –CH3), 4.35 (q,
2H, J = 7.0 Hz, CO2CH2CH3), 4.98 (s, 2H, –CH2), 6.55 (d,
1H, J = 7.5 Hz, PhH), 7.04 (d, 1H, J = 8.1 Hz, PhH), 7.26
(t, 1H, J = 8.0 Hz, PhH); 13C-NMR, d: 13.9, 33.9, 61.5,
66.9, 106.7, 106.8, 113.1, 136.1, 160.5, 163.6, 170.3, 203.9;
MS (EI) m/e: 238 (M?); Anal. C12H14O5. Calcd: C, 60.50;
H, 5.92; Found: C, 60.63; H, 6.05.
5 (0.9 g, 3.6 mmol) was dissolved in DMF (20 mL), K2CO3
(3.5 g), KI (0.5 g) and bromoethane (1 mL) were added to
this solution. This mixture was heated to 60 °C for 2 h. After
cooling to room temperature, the solution was poured into
the cold water. The precipitate obtained was filtered and
recrystallized by methanol to yield 6 as colorless granular
crystal (0.76 g, 84 %). M.p. 94–95 °C. IR (KBr, cm-1):
1
2992, 1675, 1587, 1447, 1268, 1165, 1083, 739; H-NMR
(CDCl3), d: 1.36–1.52 (m, 6H, OCH2CH3), 2.70 (s, 3H,
3-CH3), 4.06 (q, 2H, J = 6.9 Hz, OCH2CH3), 4.41 (q, 2H,
J = 7.0 Hz, CO2CH2CH3), 6.58 (d, 1H, J = 8.0 Hz, 5-H),
7.03 (d, 1H, J = 8.0 Hz, 7-H), 7.29 (t, 1H, J = 7.9 Hz,
6-H); 13C-NMR, d: 10.1, 14.1, 15.3, 61.5, 66.8, 103.5, 112.3,
113.9, 126.7, 128.5, 139.8, 145.7, 161.3, 167.9; MS (EI) m/e:
248 (M?); Anal. C14H16O4. Calcd: C, 67.73; H, 6.50; Found:
C, 67.92; H, 6.63.
Ethyl 4-hydroxy-3-methylbenzofuran-2-carboxylate (5)
To absolute ethanol (180 mL) was added in portions (2.7 g,
0.118 mol) of Na. When all had reacted, 4 (13.6 g,
0.057 mol) was added, and the solution heated at reflux for
9 h. The solution was poured into the cold water and the pH
adjusted to 3 with dilute HCl. The precipitate obtained was
filtered, washed with water and dried. The crude product was
purified by silica gel flash chromatography (AcOEt/
pet = 1:2) to yield 5 as a white powder (4.5 g, 36 %). M.p.
158–159 °C. IR (KBr, cm-1): 3328, 2925, 1715, 1521,
1442, 1279; 1H-NMR (CDCl3), d: 1.43 (t, 3H, J = 7.1 Hz,
CO2CH2CH3), 2.72 (s, 3H, 3-CH3), 4.43 (q, 2H, J = 7.1 Hz,
CO2CH2CH3), 6.57 (d, 1H, J = 7.6 Hz, 5-H), 7.08 (d, 1H,
J = 8.3 Hz, 7-H), 7.27 (t, 1H, J = 8.1 Hz, 6-H); 13C-NMR,
d: 10.0, 14.0, 61.8, 106.1, 113.2, 113.9, 126.1, 126.3, 142.5,
152.2, 159.9, 160.8; MS (EI) m/e: 220 (M?); Anal.
C12H12O4. Calcd: C, 65.45; H, 5.49; Found: C, 65.57; H,
5.65.
Hydrolysis of 6–19 to give 2, 20–32. A general procedure
illustrated with the preparation of 4-ethoxy-3-
methylbenzofuran-2-carboxylic acid (20)
6 (0.47 g, 3 mmol) was dissolved in ethanol (30 mL), 10 %
NaOH (10 mL) was added to this solution. The mixture was
heated to reflux for 1 h. After cooling to room temperature,
the pH adjusted to 3 with dilute HCl. The precipitate
obtained was filtered, washed with water, and dried to get
crude product. Recrystallization from 75 % ethanol gave 20
as colorless needle crystal (0.38 g, 52 %). M.p. 226–228 °C.
IR (KBr, cm-1): 2980, 1677, 1582, 1442, 1273, 1163,
1
1087, 732; H-NMR(CDCl3), d: 1.45 (t, 3H, J = 7.1 Hz,
123

Med Chem Res
1
OCH2CH3), 2.69 (s, 3H, 3-CH3), 4.06 (q, 2H, J = 7.1 Hz,
OCH2CH3), 6.56 (d, 1H, J = 8.0 Hz, 5-H), 7.02 (d, 1H,
J = 8.2 Hz, 7-H), 7.27 (t, 1H, J = 8.0 Hz, 6-H), 10.37 (1H,
br, 2-CO2H); 13C-NMR, d: 10.2, 15.2, 66.5, 103.8, 112.5,
113.7, 126.3, 128.1, 139.4, 145.3, 161.6, 167.8; MS (EI) m/e:
220 (M?); Anal. C12H12O4. Calcd: C, 65.45; H, 5.49; Found:
C, 65.36; H, 5.59.
1446, 1358, 1258, 1163, 1090, 739; H-NMR (CDCl3), d:
2.78 (s, 3H, 3-CH3), 5.17 (s, 2H, 4-OCH2Ph), 6.69 (d, 1H,
J = 7.9 Hz, 5-H), 7.08–7.49 (m, 7H, PhH and 6-H, 7-H),
11.55 (br, 1H, 2-CO2H); 13C-NMR, d: 10.1, 72.3, 104.2,
112.1, 112.9, 126.3, 127.3, 127.4, 127.8, 128.0, 129.1, 129.2,
137.4, 140.7, 150.2, 162.3, 166.3; MS (EI) m/e: 282 (M?);
Anal. C17H14O4. Calcd: C, 72.34; H, 4.96. Found: C, 72.38;
H, 4.95.
3-Methyl-4-propoxybenzofuran-2-carboxylic acid (21)
Yield: 78 %. M.p. 188–190 °C. IR (KBr, cm-1): 2965,
1674, 1582, 1451, 1357, 1266, 1162, 1091, 740; H-NMR
4-(Cinnamyloxy)-3-methylbenzofuran-2-carboxylic
acid (24)
1
Yield: 67 %. M.p. 191–193 °C. IR (KBr, cm-1): 2862,
1681, 1582, 1497, 1446, 1358, 1254, 1166, 1071, 964, 784,
(CDCl3), d: 1.08 (t, 3H, J = 7.4 Hz, 4-OCH2CH2CH3),
1.81–1.89 (m, 2H, 4-OCH2CH2CH3), 2.69 (s, 3H, 3-CH3),
4.07 (t, 2H, J = 6.5 Hz, 4-OCH2CH2CH3), 6.64 (d, 1H,
J = 7.7 Hz, 5-H), 7.06 (d, 1H, J = 7.7 Hz, 7-H), 7.31 (t,
1H, J = 7.8 Hz, 6-H); 13C-NMR, d: 10.0, 10.5, 22.3, 69.3,
103.6, 112.3, 113.9, 126.0, 127.9, 140.1, 145.7, 161.9,
167.4; MS (EI) m/e: 234 (M?); Anal. C13H14O4. Calcd: C,
66.67; H, 5.98; Found: C, 66.65; H, 6.01.
1
744, 717; H-NMR (CDCl3 ? DMSO-d6), d: 2.74 (s, 3H,
3-CH3), 4.81 (d, 2H, J = 4.9 Hz, 4-OCH2–), 6.47 (dt, 1H,
J = 4.9 Hz and 16.0 Hz, 4-OCH2CH=CH–), 6.82 (d, 1H,
J = 16.0 Hz, 4-OCH2CH=CH–), 6.85 (d, 1H, J = 8.0 Hz,
5-H), 7.05–7.42 (m, 7H, PhH and 6-H, 7-H); 13C-NMR, d:
10.0, 63.2, 103.9, 112.0, 112.8, 123.9, 125.6, 126.9, 127.5,
128.0, 128.5, 128.6, 128.8, 130.0, 137.1, 140.5, 150.1,
162.1, 166.3; MS (EI) m/e: 308 (M?); Anal. C19H16O4.
Calcd: C, 74.51; H, 5.23. Found: C, 74.81; H, 5.36.
4-Butoxy-3-methylbenzofuran-2-carboxylic acid (22)
Yield: 63 %. M.p. 182–183 °C. IR (KBr, cm-1): 2960, 1671,
1581, 1499, 1449, 1359, 1259, 1162, 1090, 784, 740; H-
1
3-Methyl-4-(2-(p-tolyloxy)ethoxy)benzofuran-2-
carboxylic acid (25)
NMR (CDCl3 ? DMSO-d6), d: 0.99 (t, 3H, J = 6.9 Hz,
4-O(CH2)3CH3), 1.33–1.90 (m, 4H, 4-OCH2(CH2)2CH3),
2.69 (s, 3H, 3-CH3), 4.05 (t, 2H, J = 6.9 Hz, 4-OCH2
(CH2)2CH3), 6.64 (d, 1H, J = 7.3 Hz, 5-H), 7.04 (d, 1H,
J = 7.5 Hz, 7-H), 7.30 (t, 1H, J = 7.8 Hz, 6-H); 13C-NMR,
d: 10.1, 15.2, 19.9, 33.5, 69.1, 103.5, 112.7, 113.5, 126.1,
127.6, 140.5, 145.8, 161.7, 167.9; MS (EI) m/e: 248 (M?);
Anal. C14H16O4. Calcd: C, 67.74; H, 6.52; Found: C, 67.66;
H, 6.47.
Yield: 60 %. M.p. 239–241 °C. IR (KBr, cm-1): 2928,
1674, 1585, 1513, 1455, 1356, 1274, 1239, 1163, 1100,
1
933, 800, 741; H-NMR (CDCl3 ? DMSO-d6), d: 2.03 (s,
3H, Ph–CH3), 2.66 (s, 3H, 3-CH3), 4.37 (s, 4H, 4-OCH2-
CH2O–), 6.65–7.23 (m, 6H, PhH and 5-H, 7-H), 7.32 (t,
J = 7.9 Hz, 1H, 6-H); 13C-NMR, d: 10.2, 22.5, 68.8, 69.1,
103.8, 111.9, 112.7, 114.5, 114.6, 125.5, 127.5, 129.6,
129.8, 130.1, 140.5, 146.8, 156.5, 161.9, 166.2; MS (EI) m/
e: 326 (M?); Anal. C19H18O5. Calcd: C, 69.94; H, 5.52.
Found: C, 70.10; H, 5.74.
3-Methyl-4-(pentyloxy)benzofuran-2-carboxylic acid (23)
Yield: 81 %. M.p. 186–188 °C. IR (KBr, cm-1): 2987,
1671, 1581, 1501, 1452, 1350, 1259, 1162, 1090, 785, 741;
1H-NMR (CDCl3 ? DMSO-d6), d: 0.94 (t, 3H, J = 7.1 Hz,
4-O(CH2)4CH3), 1.20–2.10 (m, 6H, 4-OCH2(CH2)3CH3),
2.72 (s, 3H, 3-CH3), 4.06 (t, 2H, J = 6.7 Hz, 4-OCH2
(CH2)3CH3), 6.62 (d, 1H, J = 7.8 Hz, 5-H), 7.04 (d, 1H,
J = 7.8 Hz, 7-H), 7.30 (t, 1H, J = 8.0 Hz, 6-H); 13C-NMR,
d: 10.0, 14.7, 22.8, 28.3, 30.3, 69.5, 103.1, 112.8, 113.3,
126.4, 127.8, 140.3, 145.9, 161.6, 167.5; MS (EI) m/e: 262
(M?); Anal. C15H18O4. Calcd: C, 68.70; H, 6.87; Found: C,
68.50; H, 6.87.
4-(2-(4-Chlorophenoxy)ethoxy)-3-methylbenzofuran-2-
carboxylic acid (26)
Yield: 38 %. M.p. 250–252 °C. IR (KBr, cm-1): 2935,
1678, 1581, 1494, 1454, 1358, 1270, 1242, 1162, 1101,
1
932, 823, 786; H-NMR (CDCl3 ? DMSO-d6), d: 2.67 (s,
3H, 3-CH3), 4.41 (s, 4H, 4-OCH2CH2O–), 6.71 (d, 1H,
J = 7.7 Hz, 5-H), 7.10 (d, 1H, J = 8.1 Hz, 7-H), 6.86–
7.25 (m, 4H, PhH), 7.28 (t, 1H, J = 7.8 Hz, 6-H); 13C-
NMR, d: 10.1, 68.9, 69.4, 103.7, 111.8, 112.6, 117.7,
117.8, 125.8, 126.0, 127.5, 130.6, 130.8, 140.6, 146.8,
157.5, 161.8, 166.3; MS (EI) m/e: 346 (M?); Anal.
C18H15ClO5. Calcd: C, 62.34; H, 4.33. Found: C, 62.40; H,
4.53.
4-(Benzyloxy)-3-methylbenzofuran-2-carboxylic acid (2)
Yield: 62 %. M.p. 183–184 °C. [(Kaltenbronn et al., 1997)
M.p. 183–185 °C]. IR (KBr, cm-1): 2991, 1682, 1584, 1500,
123

Med Chem Res
9.3 Hz, Ar-60-H), 7.99 (d, 1H, J = 9.4 Hz, Ar-70-H), 8.03
(s, 1H, Ar-40-H); 13C-NMR, d: 10.1, 71.7, 103.6, 110.2,
111.5, 112.4, 116.5, 125.8, 126.3, 127.6, 140.1, 140.5,
147.5, 148.9, 150.2, 161.3, 166.1; MS (EI) m/e: 324 (M?);
Anal. C17H12N2O5. Calcd: C, 62.96; H, 3.70; N, 8.64.
Found: C, 62.98; H, 4.04; N, 8.41.
4-(2-(3-Chlorophenoxy)ethoxy)-3-methylbenzofuran-2-
carboxylic acid (27)
Yield: 63 %. M.p. 208–210 °C. IR (KBr, cm-1): 2930,
1680, 1595, 1452, 1357, 1271, 1161, 1101, 952, 786; H-
1
NMR (CDCl3 ? DMSO-d6), d: 2.66 (s, 3H, 3-CH3),
4.34–4.51 (m, 4H, 4-OCH2CH2O–), 6.70 (d, 1H, J =
7.6 Hz, 5-H), 6.65–7.23 (m, 6H, PhH and 6-H, 7-H); 13C-
NMR, d: 10.0, 68.8, 69.4, 103.6, 111.7, 112.4, 112.5,
115.3, 120.5, 125.7, 127.3, 130.7, 136.5, 140.5, 146.8,
158.7, 161.7, 166.2; MS (EI) m/e: 346 (M?); Anal.
C18H15ClO5. Calcd: C, 62.34; H, 4.33. Found: C, 62.17; H,
4.40.
4-(Carboxy(phenyl)methoxy)-3-methylbenzofuran-2-
carboxylic acid (31)
Yield: 84 %. M.p. 213–214 °C. IR (KBr, cm-1): 3034,
1682, 1586, 1497, 1357, 1255, 1100, 781, 737; H-NMR
1
(CDCl3), d: 2.82 (s, 3H, 3-CH3), 5.72 (s, 1H, 4-OCH–),
6.62 (d, 1H, J = 8.0 Hz, 5-H), 7.11 (d, 1H, J = 8.2 Hz,
7-H), 7.27 (t, 1H, J = 8.2 Hz, 6-H), 7.36–7.67 (m, 5H,
4-OCHPhH); 13C-NMR, d: 10.2, 93.6, 103.8, 111.2, 112.1,
125.8, 127.5, 127.6, 129.3, 129.4, 129.8, 129.9, 135.8,
140.4, 150.1, 161.3, 166.2, 178.5; MS (EI) m/e: 326 (M?);
Anal. C18H14O6ꢀ0.2H2O. Calcd: C, 65.53; H, 4.37. Found:
C, 65.77; H, 4.67.
3-Methyl-4-(2-(pyrrolidin-1-yl)ethoxy)benzofuran-2-
carboxylic acid (28)
Yield: 75 %. M.p. 217–218 °C. IR (KBr, cm-1): 3487,
3393, 2415, 1614, 1572, 1390, 1256, 1094, 786, 728; H-
1
NMR (CDCl3 ? DMSO-d6), d: 2.03–2.29 (m, 4H, –NCH2
(CH2)2–), 2.58 (s, 3H, 3-CH3), 3.31–3.69 (m, 6H,
N(CH2)3), 4.42 (t, 2H, J = 7.1 Hz, 4-OCH2–), 6.60 (d, 1H,
J = 6.8 Hz, 5-H), 6.88 (d, 1H, J = 7.0 Hz, 7-H), 7.07 (t,
1H, J = 6.8 Hz, 6-H); 13C-NMR, d: 10.1, 23.8, 23.9, 56.7,
56.8, 57.1, 67.5, 103.4, 111.5, 112.5, 125.6, 127.4, 140.6,
146.9, 161.5, 166.3; MS (EI) m/e: 346 (M?); MS (EI) m/e:
289 (M?); Anal. C16H19NO4ꢀ2H2O. Calcd: C, 59.08; H,
7.08; N, 4.31. Found: C, 59.55; H, 7.23; N, 4.44.
4-(3-Carboxy-1-phenylpropoxy)-3-methylbenzofuran-2-
carboxylic acid (32)
Yield: 79 %. M.p. 230–232 °C. IR (KBr, cm-1): 3028,
2928, 1674, 1587, 1445, 1353, 1270, 1178, 1089, 738;
1H-NMR (CDCl3), d: 2.21–2.43 (m, 2H, 4-OCHCH2–),
2.47–2.60 (m, 2H, –CH2CO2H), 2.87 (s, 3H, 3-CH3), 5.41
(t, 1H, J = 6.9 Hz, 4-OCH–), 6.45 (d, 1H, J = 7.9 Hz,
5-H), 7.01 (d, 1H, J = 8.1 Hz, 7-H), 7.12 (t, 1H,
J = 8.1 Hz, 6-H), 7.24–7.40 (m, 5H, 4-OCHPhH); 13C-
NMR, d: 10.1, 30.1, 31.6, 84,5, 103.6, 111.3, 112.3, 125.7,
126.3, 127.3, 128.2, 128.3, 130.0, 130.1, 140.3, 141.8,
146.9, 161.2, 166.3, 178.3; MS (EI) m/e: 354 (M?); Anal.
C20H18O6. Calcd: C, 67.80; H, 5.08. Found: C, 67.93; H,
5.07.
4-(Benzo[c][1,2,5]thiadiazol-5-ylmethoxy)-3-
methylbenzofuran-2-carboxylic acid (29)
Yield: 81 %. M.p. 256–258 °C. IR (KBr, cm-1): 2932,
1710, 1600, 1497, 1358, 1257, 1153, 1097, 867, 809, 776;
1H-NMR (CDCl3), d: 2.79 (s, 3H, 3-CH3), 5.41 (s, 2H,
4-OCH2–), 6.80 (d, 1H, J = 8.1 Hz, 5-H), 7.14 (d, 1H,
J = 8.2 Hz, 7-H), 7.34 (t, 1H, J = 8.2 Hz, 6-H), 7.75 (d,
1H, J = 9.0 Hz, Ar-60-H), 8.06 (d, 1H, J = 8.9 Hz, Ar-70-
H), 8.12 (s, 1H, Ar-40-H); 13C-NMR, d: 10.1, 71.8, 103.5,
111.4, 112.3, 120.5, 122.6, 125.9, 127.7, 127.9, 140.5,
141.3, 150.2, 154.4, 155.6, 161.4, 166.2; MS (EI) m/e: 340
(M?); Anal.C17H12N2O4S. Calcd: C, 60.00; H, 3.53; N,
8.24. Found: C, 59.85; H, 3.72; N, 8.16.
Biology
SD rats (200–220 g) were used through the studies. They
were housed in room temperature of 25 2 °C and
humidity of 60 5 %. Animals were free access to feed
and water ad libitum during the experimental period. The
animal experiments were carried out according to (the
Committee for the purpose of Control and Supervision of
Experimentation on Animals guideline) the Regulation of
Experimental Animal Administration issued by State
Committee of Science and Technology of PR China on 14
November, 1988 and Institutional Animal Ethics Commit-
tee approved all the procedures for the investigating
experimental pain in conscious animals.
4-(Benzo[c][1,2,5]oxadiazol-5-ylmethoxy)-3-
methylbenzofuran-2-carboxylic acid (30)
Yield: 66 %. M.p. 224–226 °C. IR (KBr, cm-1): 3117,
1717, 1601, 1499, 1359, 1238, 1147, 1096, 778; H-NMR
1
(CDCl3), d: 2.75 (s, 3H, 3-CH3), 5.35 (s, 2H, 4-OCH2–),
6.85 (d, 1H, J = 8.0 Hz, 5-H), 7.14 (d, 1H, J = 8.0 Hz,
7-H), 7.36 (t, 1H, J = 8.1 Hz, 6-H), 7.64 (d, 1H, J =
123

Med Chem Res
Inhibition of the ET-1 induced contraction of rat
thoracic aortic ring
Two kinds of radio-receptor assay were performed. A
primary screening test of the target compounds were con-
ducted to pick up the potent compound to inhibit the ET-1
binding sites on the rat myocardium. The IC50 of com-
pounds was computed according to the linear equation
individually. The second test of radio-receptor assay was
done for purpose of quantitative evaluation for selectivity
of effect on ETA and ETB in the presence of PD156707 and
IC50 of each and the ratio of IC50 (ETA)/IC50 (ETB) were
calculated.
>Male rats were killed by decapitation and the thoracic
aorta was quickly removed and placed in a Krebs–Hense-
leit buffer (120 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2,
25 mM NaHCO3, 1.2 mM KH2PO4, 2.5 mM CaCl2, 11 mM
glucose). The endothelium was removed by gentle rubbing
of the intimal surface using a small cotton ball. The aorta
was cut into rings of 2.5 mm in length and each ring was
immersed in a 15 mL organ chamber filled with 37 °C
Krebs–Henseleit buffer and constantly gassed with 95 %
O2 and 5 % CO2. Vessel segments were attached to an
isometric force transducer linked to a physiographic
recorder for monitoring tension change. The ring was
stretched by a resting force of 1 g and allowed to be
equilibrated for 1 h. During this period, the ring was
washed every 20 min with fresh 37 °C Krebs–Henseleit
buffer. The tissues were contracted with phenylephrine
(1 lM) followed by challenge with acetylcholine (1 lM).
A negative relaxant response to acetylcholine confirmed
the absence of endothelium. The rings were stimulated to
contract with 60 mM KCl repeatedly until the contractile
response to KCl became stable before starting the experi-
ments. To examine the effect of test compound on ET-1-
induced contraction, each individual ring was incubated
with ET-1 (2 nM) for 20 min before exposure to test
compound. Cumulative concentration–response curves to
ET-1 were performed in the presence or absence of test
compounds after a 30-min pretreatment period. Contractile
responses were expressed as a percentage of the response
(Li et al., 2008).
Hypoxic pulmonary arterial hypertension and treatment
Rats were divided into control, hypoxic PAH model
(untreated), and hypoxic PAH, and orally medicated with
29 (40, 80, and 160 mg/kg) or nifedipine (10 mg/kg).
Pulmonary arterial hypertension was induced by hypoxia as
described (Pauvert et al., 2004). In brief, the rats, except
the control group, were housed in a hypoxic chamber for
8 h per day for 28 days. The oxygen percentage was
monitored by a real-time oxygen detector and sustained at
10 0.5 % by flowing N2. Calcium chloride (30 g) and
soda lime (20 g) were placed in the chamber to absorb CO2
and moisture. Compound treatment started on the first day
of hypoxic exposure and lasted for 28 days. All the
chemicals were dissolved or suspended in 0.5 CMC
(0.4 mL per rat) and the control group given vehicle only.
Hemodynamic measurements
On the 29th day, the rats were anesthetized with urethane
(1.5 g/kg, i.p.) and subjected to surgical catheterization to
measure hemodynamics. In brief, the jugular vein and
carotid artery were exposed after a middle incision on the
neck, from which polyethylene catheters connected with a
pressure transducer (PE50, ID 0.58 mm, OD 0.965 mm)
were inserted into the right jugular vein and then the vena
cava and the right ventricle for measuring the CVP and
RVSP, respectively. Signals were recorded by a real-time
hemodynamic analyzer.
Radio-receptor assay of endothelin receptors
Protein concentration of the pellet was determined by
Lowry method and diluted to 1 mg/mL and reacted in a
volume of 150 lL on 48 pore plate. [125I] ET-1 was added
to reach final concentration in 7 levels from 12.5–
300 pmol/L. The nonspecific binding of the membrane
preparation was determined in the presence of 1 lmol/L
ET-1 with 0.2 mg/mL membrane protein and 50 lL [125I]
ET-1. The solution containing membrane protein was
reacted for 1 h under incubation at 37 °C and rapid filtra-
tion proceeded in 12 pore cell collector though No. 49 glass
fiber filter. The filters were rinsed with cold Tris–HCl for
six times and calculated in automatic c-calculator. The
specific binding (SB) was obtained by total binding (TB)
subtracting the nonspecific binding (NSB). The competi-
tive receptor ligand binding test was conducted in tripli-
cate. Scatchard analysis and Hill coefficient was calculated
in assays.
Conclusion
In conclusion, we have synthesized a series of novel ben-
zofuran carboxylic acid derivatives and tested for their
antagonism activity of ET-1 induced contraction in the rat
thoracic aortic ring. Preliminary screening results showed
that compounds 20, 21, 29, and 30 demonstrated significant
inhibition activities, especially 29 and 30 showed potent
inhibition percentage higher than the contrast compound
BQ123. Further affinity and selectivity for ET binding
123

Med Chem Res
oxo-1,4-dihydroquinoline-2-carboxylic acids as selective
ET(A) antagonists. Bioorg Med Chem Lett 20(22):6840–6844
Pauvert O, Bonnet S, Rousseau E, Marthan R, Savineau JP (2004)
Sildenafil alters calcium signaling and vascular tone in pulmon-
ary arteries from chronically hypoxic rats. Am J Physiol Lung
Cell Mol Physiol 287(3):L577–L583
assay showed that 29 demonstrated a dual ETA/ETB
antagonism activity in nanomole level. Moreover, 29 was
effective in relieving hypoxia-induced pulmonary arterial
hypertension.
Petersen B, Deja M, Bartholdy R, Donaubauer B, Laudi S, Francis
RCE, Boemke W, Kaisers U, Busch T (2008) Inhalation of the
ETA receptor antagonist LU-135252 selectively attenuates
hypoxic pulmonary vasoconstriction. Am J Physiol-Regul Integr
Comp Physiol 294(2):R601–R605
Acknowledgments This work is supported by National Basic
Research Program of China (No. 2011CB933503) and Technology
Supporting Program of Jiangsu province (BE2009639, BE2012657).
Prasanna G, Narayan S, Krishnamoorthy RR, Yorio T (2003) Eyeing
endothelins: a cellular perspective. Mol Cell Biochem 253(1–2):
71–88
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