Discovery of phenoxybutanoic acid derivatives as potent endothelin antagonists with antihypertensive activity

Article abstract of DOI:10.1016/j.bmc.2015.01.003

A series of phenoxybutanoic acid derivatives were synthesized and tested for their antagonistic activity on the contraction of the rat thoracic aortic ring induced by endothelin-1. Preliminary screening results showed that 6e and 6g with benzoheterocycles demonstrated significant antagonistic activities when compared to the reference compound BQ123. The results from additional assays for the binding affinity and selectivity for endothelin receptors showed that 6e was a selective ET_A antagonist with a nanomolar IC₅₀. Moreover, 6e was effective in relieving hypoxia-induced pulmonary arterial hypertension and right ventricular weight ratio. Therefore, 6e may have potential for further development as a therapeutic agent for the treatment of cardiovascular diseases.

Full text of DOI:10.1016/j.bmc.2015.01.003

Bioorganic & Medicinal Chemistry 23 (2015) 657–667
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of phenoxybutanoic acid derivatives as potent endothelin
antagonists with antihypertensive activity
Jin Cai a, Ligang Liu a, Kwon Ho Hong b, Peng Wang c, Lushen Li c, Meng Cao c, Chunlong Sun a,
Xiaoqing Wu d, Xi Zong a, Junqing Chen a, Min Ji a,c,
⇑
a School of Chemistry and Chemical Engineering, Institute of Pharmaceutical Engineering, Southeast University, 87 Dingjiaqiao, Nanjing, Jiangsu 210096, PR China
b Department of Medicinal Chemistry and the Institute for Therapeutics Discovery and Development, University of Minnesota, 717 Delaware Street SE, Minneapolis, MN 55414, USA
c School of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, PR China
d Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of phenoxybutanoic acid derivatives were synthesized and tested for their antagonistic activity
on the contraction of the rat thoracic aortic ring induced by endothelin-1. Preliminary screening results
showed that 6e and 6g with benzoheterocycles demonstrated significant antagonistic activities when
compared to the reference compound BQ123. The results from additional assays for the binding affinity
and selectivity for endothelin receptors showed that 6e was a selective ETA antagonist with a nanomolar
IC50. Moreover, 6e was effective in relieving hypoxia-induced pulmonary arterial hypertension and right
ventricular weight ratio. Therefore, 6e may have potential for further development as a therapeutic agent
for the treatment of cardiovascular diseases.
Received 18 November 2014
Revised 4 January 2015
Accepted 5 January 2015
Available online 8 January 2015
Keywords:
Endothelin
Phenoxybutanoic acid
PAH
Ó 2015 Elsevier Ltd. All rights reserved.
SAR
Synthesis
1. Introduction
hypertension (PAH), congestive heart failure, atherosclerosis,
myocardial infarction, cerebral and coronary vasospasm, and renal
Endothelin (ET) was first isolated from cultured porcine vascu-
lar endothelial cells as a highly potent and long-lasting vasocon-
strictor in 1988.1 Endothelins comprise three structurally similar
isoformes: endothelin-1, 2, and 3 (ET-1, ET-2, and ET-3, respec-
tively), which are members of 21 amino acid peptide families.2
Among these three isopeptides, ET-1 appears to be predominant
in the cardiovascular system. ET-2 and ET-3 are expressed specifi-
cally in the gastrointestinal tract and the brain, respectively.3,4
Endothelins exert their diverse biological effects by binding to
endothelin receptors type A (ETA) and/or type B (ETB), heptahelical
G-protein coupled receptors.5,6 The ETA subtype, selectively bind-
ing ET-1 and ET-2 over ET-3, is found principally in peripheral
tissues such as vascular smooth muscle.7 ETA mediates
vasoconstriction and vascular smooth muscle proliferation, while
ETB expressed on endothelial cells and smooth muscle cells is
responsible for vasodilation, vasoconstriction and ET-1 clearance.8
ET receptor antagonists, both selective ETA antagonist and
non-selective ETA/ETB dual antagonists, are novel therapeutic
candidates currently in the clinical development for various car-
diovascular diseases, including systemic and pulmonary arterial
failure.9,10 Bosentan (TracleerÒ, Fig. 1), a non-selective ET receptor
antagonist, and Ambrisentan (LetairisÒ, Fig. 1), a selective ETA
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.11–13
BQ123, a selective ETA antagonist, is a small cyclic peptide that
antagonizes the vasoconstriction induced by ET-1.14 BQ123 pos-
sesses an acid group and an aromatic ring. A combination of these
groups from BQ123 has been proposed to represent at least a por-
tion of a common pharmacophore. In Ashton’s work,15 a conforma-
tional analysis identified the range of 9.1–11.3 Å as the common
distance that established two-point pharmacophore. Using this
query model for 3D searching, a phenylpentanoic acid derivative
(IC50 = 730 nM) with a moderate antagonistic activity against the
ET receptor was identified (Fig. 2).
Consequently, on the basis of our previous work,16,17 we chose
phenylpentanoic acid as a lead compound for the development of
potent ET receptor antagonists. We devised, and synthesized a
series of phenoxybutanoic acid derivatives (Fig. 2): (1) Different
substituents such as acetyl, carboxyl, and cyan groups were intro-
duced in R1 position; (2) substituted benzene rings and benzohet-
erocycles were introduced in position 4. The entire structures
remained in accordance with the common two-point structural
⇑
Corresponding author. Tel.: +86 025 83272078.
E-mail address: jimin@seu.edu.cn (M. Ji).
http://dx.doi.org/10.1016/j.bmc.2015.01.003
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

658
J. Cai et al. / Bioorg. Med. Chem. 23 (2015) 657–667
dehyde 10e with potassium borohydride in methanol at room tem-
perature followed by the chlorination in the presence of thionyl
chloride and pyridine in dichloromethane in high yield.
N
N
O
O
O
N
OH
O
N
S
The synthesis of 2f is shown in Scheme 4. 4-Chlorobenzalde-
hyde 12 as a starting material was nitrated with HNO3 using
H2SO4 as a catalyst at 0 °C to give 13 in 97% yield. 4-Chloro-3-nitro-
benzaldehyde 13 was treated with sodium azide at room temper-
ature in dimethylsulfoxide to obtain 4-azido-3-nitrobenzaldehyde
14 in 94% yield. Cyclization of this azide in boiling toluene yielded
the 5-formylbenzofuroxan 15, which was reduced by triphenyl-
phosphine in dichloromethane at 0 °C to afford 16. Reduction of
aldehyde group of 16 with sodium borohydride in 83% yield fol-
lowed by chlorination with thionyl chloride gave 2f in 84% yield.
The synthesis of 2g is shown in Scheme 5. Starting from p-tolu-
idine 18, acetylation with acetic anhydride in benzene followed by
nitration with HNO3 gave 20 in 52% yield, which was hydrolyzed
using potassium hydroxide in satisfactory yield to afford 21.
Reduction of nitro group of 21 with sodium hydrosulfite as suitable
reducing agent in ethanol gave 4-methylbenzene-1,2-diamine 22
in 60% yield. Cyclization in the presence of thionyl chloride and tri-
ethylamine in refluxing benzene followed by bromination with
N-bromosuccinimide in tetrachloromethane gave 5-bromo-
methyl-benzothiadiazole 2g.
The syntheses of 4a and 4b are shown in Scheme 6. Friedel–
Crafts succinylation of benzene 24 with succinic anhydride in the
presence of anhydrous AlCl3 under a refluxing benzene condition
furnished the corresponding b-aroylpropionic acids 25 in good
yield. Reduction of 25 with sodium borohydride followed by cycli-
zation under an acidic condition gave 26 in 84% yield, which was
chlorinated with thionyl chloride and then esterified by ethanol
to obtain 4a in satisfactory yield. Starting from benzaldehyde 27,
treatment with acrylonitrile and sodium cyanide in DMF gave crys-
talline 4-oxo-4-phenylbutanenitrile 28. Reduction of 28 with
sodium borohydride followed by chlorination with thionyl chloride
gave 4b in satisfactory yield.
O
N
OH
N
O
NH
O
O
Ambrisentan
Figure 1. Chemical structure of Bosentan and Ambrisentan.
Bosentan
features of the most non-peptide ET receptor antagonists: a car-
boxylic acid group between two aromatic or hetero-aromatic ring
systems.18 This study aimed to determine whether these designed
compounds favored greater affinity activities for ET receptor.
2. Results and discussion
2.1. Chemistry
The syntheses of the target compounds described in this paper
are depicted in Scheme 1 and began from readily available 4-acetyl
resorcinol 1a, 4-methyl formate resorcinol 1b, and 4-cyano resor-
cinol 1c. Monoalkylation of 1a, 1b, and 1c with various kinds of
halogenated-methyl aromatics 2a–2g proceeded under standard
conditions and was facilitated in the presence of a phase-transfer
catalyst TBAB to easily afford 3a–3p, which were alkylated with
ethyl 4-chloro-4-phenylbutanoate 4a and 4-chloro-4-phenylbu-
tanenitrile 4b to yield 5a–5r. Compounds 6a–6r were achieved
by the hydrolysis of 5a–5r using 10% NaOH aqueous solution as
the most suitable catalyst in satisfactory yields. As summarized
in Table 1, up to eighteen target compounds were synthesized.
The synthesis of 1a is shown in Scheme 2. Starting from 2,4-
dihydroxybenzaldehyde 7, treatment with an aqueous solution of
hydroxylamine hydrochloride in EtOH gave the crystalline oxime
8 in 96% yield. Dehydration of 8 by refluxing in acetic anhydride
followed by the saponification of the aryl acetate intermediate
gave 2,4-dihydroxybenzonitrile 1a in 92% yield.
2.2. Functional assessment of antagonism on ET-1 induced
contraction of rat thoracic aortic ring
The synthesized 18 compounds were primarily evaluated for
their antagonistic activities on ET-1 induced vasocontraction in
the rat thoracic aortic ring. A plateau of contraction of a denuded
aorta was produced by ET-1 at 2 nM and the addition of tested
The syntheses of 2c–2e are shown in Scheme 3. Intermediates
2c–2e were achieved by the reduction of anisic aldehyde 10c,
3-methoxybenzaldehyde 10d and benzo[d][1,3]dioxole-5-carbal-
Figure 2. A design for phenoxybutanoic acid derivatives.

J. Cai et al. / Bioorg. Med. Chem. 23 (2015) 657–667
659
R1
O
R'1
O
R2
HO
HO
O
R'2
Ar
ii
iii
i
R'1
OH
R'1
O
2a-2g
4a-4b
O
1a-1c
3a-3p
Ar
6a-6r
Ar
5a-5r
O
O
N
Ar = 4-ClC6H4
C6H5
R'1 = COCH3
CO2CH3
CN
O
R'2 = CO2Et
CN
4-OCH3C6H4
3-OCH3C6H4
N
N
S
N
Scheme 1. Synthetic route for the preparation of the target compounds 6a–6r. Reagents and conditions: (i) ArCH2X, K2CO3, KI, TBAB, acetone, 2.5–12.5 h, reflux, 34–81%; (ii)
K2CO3, DMF, 24 h, 60 °C; (iii) NaOH, EtOH, H2O, then 10%HCl, 2 h, 30–93%.
Table 1
Structures and characteristics of the target compounds
R1
O
R2
6a-6r
O
Ar
Compd
R1
R2
Ar
Ph
4-ClPh
4-OCH3Ph
3-OCH3Ph
Yielda (%)
Mp (°C)
Formula
6a
6b
6c
6d
COCH3
COCH3
COCH3
COCH3
CO2H
CO2H
CO2H
CO2H
48
72
73
65
114–115
137–138
127–128
111–112
C25H24O5
C25H23ClO5
C26H26O6
C26H26O6
O
6e
6f
COCH3
COCH3
COCH3
CO2H
CO2H
CO2H
30
93
87
123–124
132–134
146–148
C26H24O70.5H2O
C25H22N2O6
O
N
O
S
N
N
6g
C25H22N2O5S
N
6h
6i
6j
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
Ph
4-ClPh
4-OCH3Ph
3-OCH3Ph
74
73
89
84
135–136
166–168
103–106
142–143
C24H22O6
C24H21ClO6
C25H24O7
C25H24O7
6k
O
O
6l
CO2H
CN
CO2H
CO2H
79
77
137–139
130–132
C25H22O8
6m
Ph
C24H21NO4
O
b
6n
6o
CN
CN
CO2H
CO2H
81
90
—
C25H21NO6
C24H19N3O5
O
N
O
S
132–134
N
N
6p
6q
CN
CO2H
CN
83
37
120–122
142–144
C24H19N3O4S
C24H21NO4
N
CO2H
Ph
O
O
6r
CO2H
CN
32
162–164
C25H21NO6
a
Yield for the last step.
Oil.
b
compounds caused a relaxation at a variation of amplitude by 18
compounds at 10 M. BQ123 was taken as a positive control in
the assays. As shown in Figure 3, most compounds exhibited mod-
erate to good antagonistic activities against the vasocontraction
induced by ET-1 in the rat thoracic aortic ring.
The compounds 6b–6d (À1.9% to 29.3%) and 6i–6k (24.6–
38.6%) substituted with either electron-donating or electron-with-
drawing groups at benzene of Ar exhibited decreased antagonism
compared to compounds 6a (58.2%) and 6h (42.9%) without any
substitution at the corresponding benzene. Although the introduc-
tion of substituted benzene groups at Ar dramatically decreased
the antagonistic activity, additional modification on R1 from acetyl
to carboxyl group slightly restored the antagonistic activity. Based
on the antagonistic activities of 6b–6d and 6i–6k, it seemed that
carboxyl on R1 plays a role in binding to the ET receptor at some
degree although it is not significant. It was noticeable for
l

660
J. Cai et al. / Bioorg. Med. Chem. 23 (2015) 657–667
AcO
NC
HO
NC
HO
HO
(ii)
(i)
(iii)
OAc
OH
OHC
OH
HON HC
OH
8
9
1a
7
Scheme 2. Synthetic route for the preparation of 1a. Reagents and conditions: (i) NH2OHÁHCl, KOH, EtOH, AcOH, 0.5 h, reflux, 96%; (ii) Ac2O, 8 h, reflux, 85%; (iii) NaOH, H2O,
then 10%HCl, 1.5 h, 92%.
CHO
CH2OH
R
CH2Cl
2c
: R = 4-OCH3
(i)
(ii)
R
R
2d: R = 3-OCH3
2e: R = 3,4-OCH2OC6H3
10c-10e
11c-11e
2c-2e
Scheme 3. Synthetic route for the preparation of 2c–2e. Reagents and conditions:
(i) KBH4, CH3OH, H2O, 2 h, rt, 85–98%; (ii) SOCl2, pyridine, CH2Cl2, 3 h, rt, 83–88%.
compound 6b that percentage of antagonism was À1.9%, one may
speculated that 6b show slight ETA agonist activity or it could
potentiate effect of ET-1 induced vasocontraction with an
unknown mechanism. It is worth pointing out that significant
antagonism was achieved for the compounds benzo[d]dioxole 6e
(81.8%) and benzo[c]thiadiazole 6g (85.2%), with potency higher
than the control compound BQ123 (68.1%). Since the benzohetero-
cycles of 6e and 6g are bioisosteres of the indole moiety in BQ123,
and they may occupy the pocket for the indole moiety of BQ123, it
is thought that they had potent interactions with residues of ET
Figure 3. In vitro functional vascular ET-1 antagonism of test compounds in
concentration 10 M; antagonistic activity of ET-1 (2 nM) induced contraction of
rat thoracic aortic ring; values are averages of at least three independent
l
experiments, SD <20%.
CHO
CHO
CHO
N+
CH2OH
N
CHO
N3
CHO
N
CH2Cl
(i)
(ii)
(iv)
(v)
(iii)
(vi)
NO2
NO2
N
O
N
O
N
O
N
O
Cl
Cl
N
-O
15
16
2f
14
17
12
13
Scheme 4. Synthetic route for the preparation of 2f. Reagents and conditions: (i) HNO3, H2SO4, 2 h, 0–5 °C, 97%; (ii) NaN3, DMSO, 1 h, rt, 94%; (iii) toluene, 10 h, reflux, 69%;
(iv) P(Ph)3, CH2Cl2, 0.5 h, 0 °C, 70%; (v) NaBH4, CH3OH, 1 h, rt, 83%; (vi) SOCl2, pyridine, CH2Cl2, 1.5 h, rt, 84%.
CH2Br
N
CH3
N
CH3
CH3
CH3
CH3
CH3
(vi)
(i)
(v)
(ii)
(iv)
(iii)
NO2
NH2
NO2
N
S
N
S
NH2
NH2
NH2
NHAc
NHAc
2g
23
18
20
21
22
19
Scheme 5. Synthetic route for the preparation of 2e. Reagents and conditions: (i) Ac2O, PhH, 1 h, reflux, 88%; (ii) HNO3, H2SO4, 1 h, 0–5 °C, 52%; (iii) KOH, EtOH, H2O, 3 h,
reflux, 92%; (iv) Na2S2O6, EtOH, H2O, 0.5 h, reflux, 60%; (v) SOCl2, Et3N, PhH, 3 h, reflux, 33%; (vi) NBS, CCl4, 20 h, reflux, 84%.
Cl
O
O
O
O
OH
(iii)
(i)
(ii)
O
O
26
4a
25
24
Cl
OH
O
CHO
(vi)
(iv)
(v)
CN
CN
CN
28
4b
29
27
Scheme 6. Synthetic route for the preparation of 4a and 4b. Reagents and conditions: (i) succinic anhydride, PhH, AlCl3, 45 min, reflux, 84%; (ii) NaBH4, EtOH, H2O, 12 h, rt,
84%; (iii) SOCl2, HCl satd EtOH, 3 h, rt, 89%; (iv) CH2@CHCN, NaCN, DMF, 2 h, 30 °C, 18%; (v) NaBH4, CH3OH, 5 h, rt, 90%; (vi) SOCl2, pyridine, CH2Cl2, 3 h, rt, 69%.

J. Cai et al. / Bioorg. Med. Chem. 23 (2015) 657–667
661
Figure 4. (A) Molecular modeling of 6e (wheat); (B) overlay of 6e and BQ123 (light blue); (C) common pharmacophore of 6e and BQ123.
2.3. Differential selectivity of compounds on the ETA and ETB
subtype of rat myocardium
Table 2
ETA and ETB receptor binding activity for the compounds
a
Compound
IC50 (nM)
Selectivity for ETA
The further evaluation of compounds 6a, 6e, 6g, 6o and 6q in a
radio receptor assay was carried out in order to quantitatively
determine their differential selectivity on ETA and ETB using rat
myocyte membrane (Table 2). 6e and 6g exerted higher binding
affinity for ETA (3.3 and 3.9 nM, respectively) than ETB (260 and
198 nM, respectively) demonstrating the higher selectivity than
Bosentan (Table 2). This result suggests that 6e may be a potential
candidate as a therapeutic agent for the treatment of cardiovascu-
lar diseases. Due to the result of antagonism of 6e against ET-1
induced vasocontraction via selectively antagonizing ETA, com-
pound 6e was chosen for the evaluation of its in vivo efficacy to
relieve hypoxia-induced pulmonary arterial hypertension and
right ventricular weight ratio in rats following oral administration.
ETA
ETB
6a
6e
6g
6o
6q
Bosentan
BQ123
12 1.5
3.3 0.6
3.9 1.1
5.5 1.2
19 2.6
4.7 1.8
28 3.9
453 38
260 23
198 17
183 21
340 52
95 27
—
38
79
51
33
18
20
Specificity
a
Values are averages of three independent experiments.
receptor. As shown in Figure 4, using Maestro and Pymol, com-
pound 6e was overlaid very well with BQ123, in particular, their
pharmacophore part.
In order to study the role of the acetyl group at R1, it was
replaced with cyano group. Modification of R1 of 6a, 6e and 6g
from acetyl to cyano group decreased the antagonistic activity
(6m, 6n and 6p). According to the bioassay results of 6e–6g, if
cyano group played the same role as acetyl on R1 and the binding
modes of the analogues are the same, it is expected that 6n and 6p
exert the better antagonistic activity than 6o. However, 6o (69.3%)
manifested the higher antagonistic activity than 6n (38.9%) and 6p
(15.9%). This indicated that analogues 6m–6p with cyano group
might have binding modes different from those of analogues 6a–
6g with acetyl group. Exchanging the positions of the cyano group
and the carboxylic acid group of compounds 6m (50.4%) and 6n
(38.9%) did not significantly alter the activities of the correspond-
ing compounds 6q (57.3%) and 6r (43.6%).
2.4. Potent effect of compound 6e on relieving pulmonary
arterial hypertension and right ventricular weight ratio
After hypoxic exposure for 28 days, the haemodynamic vari-
ables and heart weight index in the untreated PAH model group
were much higher than those in the normal control group. The
right ventricular systolic pressure (RVSP) and central vein pressure
(CVP) of rats were elevated by +102% (P <0.01) and +783% (P <0.05),
respectively, compare to the control group, indicating hypoxic PAH
was established. Nifedipine po was used as a positive reference in
this study due to its wide use in treating PAH. After treatment with
compound 6e (20, 40, 80 mg/kg) and nifedipine (10 mg/kg), the
elevated RVSP decreased by 38.0%, 47.3%, 54.3% and 43.0%, respec-
tively, compared to the untreated PAH model group; Similarly, the
Figure 5. Reversion of an elevation in RVSP and CVP in hypoxic PAH rats by 6e and nifedipine. N = 10.

662
J. Cai et al. / Bioorg. Med. Chem. 23 (2015) 657–667
Figure 6. Reversion of an increase in RV/BW ratio (mg/g) and RV/(LV + S) ratio (g/g) in hypoxic PAH rats by 6e and nifedipine. N = 10.
elevated CVP decreased by 56.3%, 77.1%, 85.8% and 83.6%, respec-
tively, compared to the untreated PAH model group (Fig. 5). The
response to 6e was dose-dependent with the lowest dose demon-
strating potent effect on relieving hypoxia-induced pulmonary
arterial hypertension.
RY-1 hot-stage microscope, and the thermometer was uncorrected.
1H NMR and 13C NMR spectra were recorded in CDCl3 on a Bruker-
ACF 300/500 spectrometer; chemical shifts (d) are reported in parts
per million (ppm) relative to tetramethylsilane (TMS), used as an
internal standard. Mass spectra (MS) were obtained from Agilent
1100LC/MS Spectrometry Services. IR spectra were obtained using
an FT-IR Spectrometer (Perkin–Elmer). Elemental analyses were
performed on Elementar Vario EL III instrument. All compounds
were routinely checked by TLC with silica gel GF-254 glass plates
and viewed under UV light at 254 nm.
A remodeling of the right ventricle (RV) occurred after chronic
hypoxic exposure for 28 days. Right ventricular hypertrophy was
assessed as the ratio of RV/bodyweight and RV/(LV + S), where
RV was the weight of the right ventricular free wall and (LV + S)
was the sum of the weight of left ventricular free wall plus the sep-
tum, in hypoxia-induced pulmonary arterial hypertensive rats. The
heart weight indices were used to evaluate the effects of 6e or
nifedipine intervention on the hypertrophy. RV/BW and RV/
(LV + S) were 85.7% and 66.6% higher in the untreated PAH model
group compared with the control group, respectively. 6e (20, 40,
80 mg/kg) and nifedipine (10 mg/kg) attenuated RV/BW by 30.9%,
37.7%, 42.5% and 32.1% compared to the untreated PAH model
group, respectively; RV/(LV + S) by 6.37%, 13.9%, 23.3% and 21.0%
compared to the untreated PAH model group, respectively
(Fig. 6). It seemed that 6e had an effect similar to nifedipine in
reversing the remodeling of the right ventricle. These results indi-
cate that the hypoxic PAH model was established successfully and
treatments of rats with low doses of nifedipine and 6e for a rela-
tively short duration were adequate to restore abnormalities in
haemodynamic variables and heart weight indices.
4.1.1. 2,4-Dihydroxybenzaldehyde oxime (8)
Hydroxylamine hydrochloride (28 g, 0.40 mol) was added to
water (20 mL). When all had dissolved, KOH (23 g, 0.41 mol) in
water (20 mL) was added dropwise at 0 °C. To this mixture was
added 2,4-dihydroxybenzaldehyde (50 g, 0.36 mol), then the reac-
tion mixture was heated to reflux for 0.5 h and cooled to room
temperature. The precipitate was filtered and the filtrate was evap-
orated under reduced pressure. The crude product was recrystal-
lized from water to afford product 8 (53.5 g, 96%) as white
acicular crystal. Mp: 191–192 °C (lit.19 mp: 194–196 °C).
4.1.2. 4-Cyano-1,3-phenylene diacetate (9)
A mixture of compound 8 (34 g, 0.22 mol) and acetic anhydride
(200 mL) was heated to reflux for 8 h. After cooling to room tem-
perature, the mixture was poured slowly to ice water (2 L) while
stirring constantly. The resulting precipitate was filtered and
recrystallized from ethanol/water to give product 9 (41 g, 85%) as
colorless acicular crystal. Mp: 73–75 °C (lit.19 mp: 72–73 °C).
3. Conclusion
A series of phenoxybutanoic acid derivatives were synthesized
and tested for their antagonistic activity on ET-1 induced contrac-
tion in the rat thoracic aortic ring. Preliminary screening results
showed that some compounds exhibited significant antagonistic
activity: in particular, 6e and 6g with benzoheterocycles exerted
potency compatible to or higher than the control compound
BQ123. Further assays showed that 6e was a highly potent and
selective ETA antagonist. Moreover, 6e was effective in relieving
hypoxia-induced pulmonary arterial hypertension and right ven-
tricular weight ratio, suggesting that 6e may have potential for fur-
ther development as a therapeutic agent for the treatment of
cardiovascular diseases.
4.1.3. 2,4-Dihydroxybenzonitrile (1a)
Compound 9 (60 g, 0.27 mol) was added to 2 N NaOH aqueous
solution (700 mL). The mixture was stirred at room temperature
for 1.5 h, the pH adjusted to 3 with 10% HCl. The aqueous phase
was extracted with ethyl ether (3 Â 300 mL) and the combined
organic phase was washed with saturated sodium bicarbonate
(2 Â 200 mL) and brine (2 Â 100 mL) and dried with MgSO4. Ethyl
ether was evaporated under reduced pressure to yield product 1a
(34 g, 92%) as white acicular crystal. Mp: 179–181 °C (lit.20 mp:
179 °C).
4.1.4. 1-(Chloromethyl)-4-methoxybenzene (2c)
4. Experimental protocols
4.1. Synthesis
4-Methoxybenzaldehyde (27 g, 0.2 mol) was dissolved in meth-
anol (80 mL) and water (20 mL). To this mixture was added KBH4
(5 g, 0.1 mol) in batches, then stirred at room temperature for
2 h. The pH was adjusted to 3 with 10% HCl to quench the reaction
and then the pH was adjusted to 8 with saturated sodium
All reagents were purchased from commercial sources and used
without further purification. Melting points were measured on an

J. Cai et al. / Bioorg. Med. Chem. 23 (2015) 657–667
663
hydrogen carbonate solution. Volatiles were removed under
reduced pressure, and then the residue was taken up with toluene
and washed with water and brine. The organic phase was dried
with MgSO4 and evaporated under reduced pressure. The crude
11c was added to thionyl chloride (50 mL) and heated to reflux
for 3 h. Volatiles were removed under reduced pressure to afford
2c (28.5 g, 89%) as colorless oil.
4.1.12. 5-(Chloromethyl)benzo[c][1,2,5]oxadiazole (2f)
Compound 17 (2.5 g, 17 mmol) was dissolved in dichlorometh-
ane (20 mL) and pyridine (1.6 mL). To this mixture was added thio-
nyl chloride (2.4 g, 20 mmol) dropwise at 0 °C and then stirred at
room temperature for 1.5 h. Volatiles were removed under reduced
pressure to afford 2f (2.4 g, 84%) as colorless oil.
4.1.13. N-(p-Tolyl)acetamide (19)
4.1.5. 1-(Chloromethyl)-4-methoxybenzene (2d)
Preparation of 2d is followed the procedure for 2c described
above. Colorless oil, yield: 79%.
p-Toluidine (32.2 g, 0.3 mol) was dissolved in benzene (60 mL).
To this solution was added acetic anhydride (31 g, 0.3 mol) drop-
wise at 100 °C and stirred for 30 min. When cooled to room tem-
perature, the resulting precipitate was filtered and washed with
petroleum ether, dried to give product 19 (39.5 g, 88%) as white
acicular crystal. Mp: 147–149 °C (lit.27 mp: 147–149 °C).
4.1.6. 5-(Chloromethyl)benzo[d][1,3]dioxole (2e)
Preparation of 2e is followed the procedure for 2c described
above. Colorless acicular crystal, mp: 23–24 °C (lit.21 mp: 23–
23.5 °C), yield: 85%.
4.1.14. N-(4-Methyl-2-nitrophenyl)acetamide (20)
To nitrosonitric acid (160 mL) was added 19 (39.5 g, 0.265 mol)
in batches at 5 °C and stirred for 2 h. Then the mixture was poured
slowly to ice water (1 L) while stirring constantly. The resulting
precipitate was filtered and recrystallized from ethanol to give
product 20 (27 g, 53%) as yellow acicular crystal. Mp: 91–92 °C
(lit.28 mp: 95–96 °C).
4.1.7. 4-Chloro-3-nitrobenzaldehyde (13)
To the mixture of nitrosonitric acid (55 mL) and sulfuric acid
(55 mL) was added 4-chlorobenzaldehyde (70 g, 0.5 mol) in
batches at 5 °C and stirred for 2 h. Then the mixture was poured
slowly to ice water (1 L) while stirring constantly. The resulting
precipitate was filtered and recrystallized from ethanol/water to
give product 13 (90 g, 97%) as white acicular crystal. Mp: 62–
63 °C (lit.22 mp: 60–63 °C).
4.1.15. 4-Methyl-2-nitroaniline (21)
Compound 20 (10 g, 0.052 mol) and KOH (6.8 g, 0.12 mol) were
added to the solution of ethanol (60 mL) and water (10 mL). The
mixture was heated to reflux for 3 h, and poured slowly to ice
water (500 mL) while stirring constantly. The resulting precipitate
was filtered and dried to give the product 21 (7.3 g, 92%) as red
acicular crystal. Mp: 114–115 °C (lit.29 mp: 114–115 °C).
4.1.8. 4-Azido-3-nitrobenzaldehyde (14)
Compound 13 (46.4 g, 0.25 mol) was dissolved in DMSO
(300 mL). To this solution was added NaN3 (18 g, 0.28 mol) in
batches at 10 °C and then stirred at room temperature for 1 h.
The mixture was poured slowly to ice water (800 mL) while stir-
ring constantly. The precipitate obtained was filtered and recrys-
tallized from ethanol to give product 14 (45 g, 94%) as yellow
crystal. Mp: 80–82 °C (lit.23 mp: 74–75 °C).
4.1.16. 4-Methylbenzene-1,2-diamine (22)
Compound 21 (10 g, 0.066 mol) and sodium hydrosulfite (40 g,
0.23 mol) were added to the solution of ethanol (50 mL) and water
(70 mL). The mixture was heated to reflux for 30 min. When cooled
to room temperature, 100 mL water was added and extracted with
ethyl acetate (3 Â 80 mL). The combined organic phase was
washed with brine (2 Â 50 mL) and dried with MgSO4. Ethyl ace-
tate was evaporated under reduced pressure to yield product 22
(4.8 g, 60%) as brown crystal.
4.1.9. 5-Formylbenzo[c][1,2,5]oxadiazole 1-oxide (15)
The solution of compound 14 (45 g, 0.23 mol) in 250 mL of
toluene was refluxed for 30 min. Following the removal of the
solvent under reduced pressure, the resulting crude product
was dissolved in boiling EtOAc (200 mL), and then petroleum
ether (900 mL) was added to the solution. The solution was
cooled to give 15 (26 g, 69%) as yellow acicular crystal. Mp:
69–71 °C (lit.24 mp: 69 °C).
4.1.17. 5-Methylbenzo[c][1,2,5]thiadiazole (23)
Compound 22 (4.8 g, 0.04 mol) was dissolved in the solution of
benzene (150 mL) and TEA (20 mL). To this mixture was added
thionyl chloride (30 mL) at 0 °C and heated to reflux for 3 h. When
cooled to room temperature, the mixture was washed with water
(100 mL), saturated sodium bicarbonate solution (100 mL) and
brine (100 mL), dried with MgSO4 to obtain the crude product,
which was purified by column chromatography on silica gel, elut-
ing with ethyl acetate/petroleum (1:8) to afford product 23 (2 g,
33%) as colorless granular crystal. Mp: 32–33 °C (lit.30 mp: 28–
30 °C).
4.1.10. Benzo[c][1,2,5]oxadiazole-5-carbaldehyde (16)
Compound 15 (10 g, 0.061 mol) was dissolved in dichlorometh-
ane (50 mL). The solution of triphenylphosphine (16 g, 0.061 mol)
in dichloromethane (50 mL) was added dropwise at 0 °C and then
stirred for 30 min. The solvent was removed under vacuum. The
crude product was purified by column chromatography on silica
gel, eluting with ethyl acetate/petroleum (1:20) to afford product
16 (6.3 g, 70%) as white acicular crystal. Mp: 55–56 °C (lit.25 mp:
55–56 °C).
4.1.18. 5-(Bromomethyl)benzo[c][1,2,5]thiadiazole (2g)
To CCl4 (50 mL) was added 23 (2 g, 0.013 mol), N-bromosuccin-
imide (2.4 g, 0.014 mol) and benzoperoxide (catalytic amount). The
mixture was heated to reflux for 20 h, when cooled to room tem-
perature, the resulting precipitate was filtered and the filtrate
was condensed to give the crude product, which was purified by
column chromatography on silica gel, eluting with ethyl acetate/
petroleum (1:20) to afford product 2g (2.5 g, 84%) as white acicular
crystal. Mp: 88–89 °C (lit.31 mp: 89–90 °C).
4.1.11. Benzo[c][1,2,5]oxadiazol-5-ylmethanol (17)
Compound 16 (3 g, 20 mmol) was dissolved in methanol
(30 mL). To this solution was added KBH4 (0.5 g, 9 mmol) in
batches and stirred at room temperature for 1 h. The pH was
adjusted to 4 with 10% HCl to quench the reaction and then
the pH was re-adjusted to 8 with saturated sodium hydrogen
carbonate solution. Volatiles were removed under reduced pres-
sure, and then the residue was taken up with ethyl acetate and
washed with water and brine. The organic phase was dried
with MgSO4 and evaporated under reduced pressure to yield
17 (2.5 g, 83%) as white acicular crystal. Mp: 51–52 °C (lit.26
mp: 52–53 °C).
4.1.19. 4-Oxo-4-phenylbutanoic acid (25)
AlCl3 (66 g, 0.5 mol) and succinic anhydride (20 g, 0.2 mol) were
added to anhydrous benzene (100 mL). The mixture was heated to

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J. Cai et al. / Bioorg. Med. Chem. 23 (2015) 657–667
reflux for 45 min. When cooled to room temperature, 100 mL ice
water was slowly added dropwise under ice bath. The excess ben-
zene was evaporated under atmospheric pressure, and then the
residue was added activated carbon and filtered. The filtrate was
acidified and the resulting precipitate was filtered to give the crude
product, which was recrystallized with 30% ethanol to get product
25 (30 g, 84%) as colorless acicular crystal. Mp: 117–119 °C (lit.32
mp: 117–119 °C).
this mixture was added 2a–2g (0.05 mol) dropwise for 0.5 h at
room temperature. The mixture was heated to reflux for 3 h. After
cooling to room temperature, the resulting precipitate was filtered
and the filtrate was evaporated under reduced pressure to yield
crude product. Recrystallization of the crude products from etha-
nol gave 3a–3p.
4.1.26. General process for the synthesis of the compounds 5a–
5r
4.1.20. 5-Phenyldihydrofuran-2(3H)-one (26)
Compounds 3a–3p (2.7 mmol), 4a–4b (4.5 mmol), K2CO3 (1 g,
7.2 mmol) and KI (0.5 g, 3.0 mmol) were added in DMF (25 mL).
The mixture was heated to 60 °C for 24 h. After cooling to room
temperature, the solution was poured into the cold water,
extracted with ethyl acetate (3 Â 30 mL) and dried with MgSO4
to give the crude product, which was purified by column chroma-
tography on silica gel, eluting with ethyl acetate/petroleum (1:10)
to afford products 5a–5r.
Compound 25 (9 g, 50 mmol) was dissolved in 95% ethanol
(40 mL). To this solution was added NaBH4 (1 g, 30 mmol) in
batches and stirred at room temperature overnight. The pH was
adjusted to 3 with 10% HCl to quench the reaction and then the
product was extracted with ethyl ether (3 Â 50 mL). The combined
organic phase was washed with saturated sodium bicarbonate
solution (2 Â 30 mL), brine (2 Â 30 mL) and dried with MgSO4.
Ethyl acetate was evaporated under reduced pressure to yield
product 26 (6.8 g, 84%) as colorless oil.
4.1.27. General process for the synthesis of the compounds 6a–6r
The mixture of 5a–5r (2.3 mmol) and 10% NaOH aqueous solu-
tion (4 mL) in ethanol (20 mL) was heated to reflux for 2 h. After
cooling to room temperature, the solvent was removed under
reduced pressure. The pH of the residue was adjusted to 3 with
10% HCl, the resulting precipitate was filtered and recrystallized
from methanol to give 6a–6r.
4.1.21. Ethyl 4-chloro-4-phenylbutanoate (4a)
The mixture of 26 (3.2 g, 20 mmol), thionyl chloride (4.5 mL)
and benzene (5 mL) was heated to reflux for 3 h. When cooled to
room temperature, the mixture was added to HCl saturated etha-
nol (20 mL) dropwise and stirred for another 3 h. Then the solution
was poured to ice water and extracted with ethyl acetate
(3 Â 50 mL). The combined organic phase was washed with water
(2 Â 30 mL), saturated sodium bicarbonate solution (2 Â 30 mL),
brine (2 Â 30 mL) and dried with MgSO4. Ethyl acetate was evapo-
rated under reduced pressure to yield product 4a (4.0 g, 89%) as
brown oil.
Compounds 6a–6r were characterized as follows:
4.1.28. 4-(2-Acetyl-5-(benzyloxy)phenoxy)-4-phenylbutanoic
acid (6a)
White acicular crystal, yield: 48%. Mp: 114–115 °C. IR (cmÀ1):
3085, 2967, 1731, 1634, 1592, 1276, 169, 1028, 838, 738; 1H
NMR (300 MHz, CDCl3) d: 2.17–2.23 (m, 2H, –CHCH2CH2–), 2.51–
2.57 (m, 2H, –CHCH2CH2CO2H), 2.73 (s, 3H, –COCH3), 4.93 (s, 2H,
–OCH2Ar), 5.29–5.33 (m, 1H, –OCH(Ar)CH2–), 6.36 (d, 1H,
J = 1.9 Hz, ArH), 6.53 (dd, 1H, J = 2.0 Hz and 8.7 Hz, ArH), 7.25–
7.40 (m, 10H, ArH), 7.79 (d, 1H, J = 8.7 Hz, ArH); 13C NMR
(75 MHz, CDCl3) d: 29.1, 30.5, 32.5, 69.3, 78.9, 101.4, 102.9,
108.3, 120.6, 126.0, 127.2, 127.3, 128.6, 128.8, 129.0, 129.1,
133.4, 133.6, 158.9, 163.5, 170.6, 198.8; MS (ESI(À)) m/e: 403
(MÀÀ1), 241 (100). Anal. Calcd for C25H24O5: C, 74.26; H, 5.94.
Found: C, 74.60; H, 5.83.
4.1.22. 4-Oxo-4-phenylbutanenitrile (28)
NaCN (1 g, 0.02 mol) and benzaldehyde (12 g, 0.2 mol) was
added to DMF (160 mL). The mixture was stirred for 15 min and
was added the solution of acrylonitrile (8 g, 0.15 mol) in DMF
(40 mL) dropwise, then stirred at 30 °C for 2 h. The mixture was
poured to water (400 mL) and extracted with dichloromethane,
dried with MgSO4 to get the crude product, which was purified
by column chromatography on silica gel, eluting with ethyl ace-
tate/petroleum (1:7) to afford the product 28 (4.1 g, 18%) as white
solid. Mp: 76–77 °C (lit.33 mp: 73.5–74 °C).
4.1.29. 4-(2-Acetyl-5-((4-chlorobenzyl)oxy)phenoxy)-4-phenyl-
butanoic acid (6b)
4.1.23. 4-Hydroxy-4-phenylbutanenitrile (29)
Compound 28 (1.6 g, 10 mmol) was dissolved in methanol
(20 mL). To this solution was added NaBH4 (0.4 g, 10 mmol) in
batches and stirred at room temperature for 4 h. The pH was
adjusted to 3 with 10% HCl to quench the reaction and then the
product was extracted with ethyl ether (3 Â 30 mL). The combined
organic phase was washed with saturated sodium bicarbonate
solution (2 Â 20 mL), brine (2 Â 20 mL) and dried with MgSO4.
Ethyl acetate was evaporated under reduced pressure to yield
product 29 (1.5 g, 90%) as colorless oil.
Light yellow acicular crystal, yield: 72%. Mp: 137–138 °C. IR
(cmÀ1): 3021, 2946, 1721, 1629, 1584, 1493, 1440, 1361, 1273,
1185, 1138, 1052, 835, 703, 592; 1H NMR (300 MHz, CDCl3) d:
2.05–2.31 (m, 4H, –CHCH2CH2–), 2.71 (s, 3H, –COCH3), 4.86 (s,
2H, –OCH2Ar), 5.27–5.31 (m, 1H, –OCH(Ar)CH2–), 6.32 (d, 1H,
J = 2.3 Hz, ArH), 6.47 (dd, 1H, J = 2.3 Hz and 8.8 Hz, ArH), 7.22–
7.32 (m, 9H, ArH), 7.77 (d, 1H, J = 8.6 Hz, ArH), 10.92 (br, 1H,
–CO2H); 13C NMR (75 MHz, CDCl3) d: 29.2, 30.4, 32.6, 69.9, 78.6,
101.7, 103.1, 108.5, 120.6, 128.2, 128.5, 128.7, 129.0, 129.1,
129.3, 131.8, 132.8, 133.2, 158.8, 163.9, 170.2, 198.5; MS (ESI(À))
m/e: 437 (MÀÀ1), 275 (100). Anal. Calcd for C25H23ClO5: C, 68.45;
H, 5.25. Found: C, 68.22; H, 5.17.
4.1.24. 4-Chloro-4-phenylbutanenitrile (4b)
Compound 29 (1.5 g, 9.3 mmol) was dissolved in dichlorometh-
ane (20 mL) and pyridine (1 mL). To this mixture was added thio-
nyl chloride (0.88 mL) dropwise at 0 °C and then stirred at room
temperature for 3 h. The mixture was washed with water
(100 mL), saturated sodium bicarbonate solution (100 mL) and
brine (100 mL), dried with MgSO4 to afford product 4b (1.2 g,
69%) as colorless oil.
4.1.30. 4-(2-Acetyl-5-((4-methoxybenzyl)oxy)phenoxy)-4-phenyl-
butanoic acid (6c)
Light yellow granular crystal, yield: 73%. Mp: 127–128 °C. IR
(cmÀ1): 3058, 3006, 2959, 2932, 1733, 1627, 1584, 1517, 1441,
1270, 1177, 1143, 1013, 843, 701; 1H NMR (300 MHz, CDCl3) d:
2.20–2.39 (m, 2H, –CHCH2CH2–), 2.48–2.56 (m, 2H, –CHCH2CH2
CO2H), 2.71 (s, 3H, –COCH3), 3.79 (s, 3H, –COCH3), 4.82 (s, 2H,
–OCH2Ar), 5.28–5.32 (m, 1H, –OCH(Ar)CH2–), 6.33 (d, 1H,
J = 2.1 Hz, ArH), 6.50 (dd, 1H, J = 2.2 Hz and 8.8 Hz, ArH), 6.88 (d,
4.1.25. General process for the synthesis of the compounds 3a–3p
Compounds 1a–1c (0.05 mol), K2CO3 (14 g, 0.1 mol), KI (3 g,
0.018 mol) and TBAB (0.1 g) were added in acetone (150 mL). To

J. Cai et al. / Bioorg. Med. Chem. 23 (2015) 657–667
665
2H, J = 8.6 Hz, ArH), 7.22 (d, 2H, J = 8.6 Hz, ArH), 7.25–7.38 (5H, m,
ArH), 7.78 (d, 1H, J = 8.8, ArH), 10.10 (br, 1H, –CO2H); 13C NMR
(75 MHz, CDCl3) d: 29.1, 30.3, 32.5, 56.9, 69.6, 78.8, 101.3, 102.9,
108.1, 112.5, 121.5, 126.3, 128.1, 128.5, 128.7, 128.9, 129.1,
129.3, 134.5, 156.7, 158.5, 163.8, 170.9, 198.5; MS (ESI(À)) m/e:
433 (MÀÀ1), 271 (100). Anal. Calcd for C26H26O6: C, 71.89; H,
6.00. Found: C, 72.03; H, 6.07.
7.78 (d, 1H, J = 8.7 Hz, ArH), 7.92 (s, 1H, ArH), 7.98 (d, 1H,
J = 9.0 Hz, ArH); 13C NMR (75 MHz, CDCl3) d: 29.1, 30.2, 32.5,
69.6, 78.6, 103.1, 107.9, 119.5, 120.7, 122.0, 125.3, 126.8,
127.5, 128.0, 128.9, 133.4, 134.1, 137.5, 154.5, 154.6, 159.9,
163.5, 170.1, 198.6; MS (ESI(À)) m/e: 461 (MÀÀ1), 299 (100).
Anal. Calcd for C25H22N2O5S: C, 64.93; H, 4.76; N, 6.06. Found:
C, 65.03; H, 4.95; N, 6.27.
4.1.31. 4-(2-Acetyl-5-((3-methoxybenzyl)oxy)phenoxy)-4-phenyl-
butanoic acid (6d)
4.1.35. 4-(Benzyloxy)-2-(3-carboxy-1-phenylpropoxy)benzoic
acid (6h)
Light yellow granular crystal, yield: 65%. Mp: 111–112 °C. IR
(cmÀ1): 3084, 2964, 1733, 1630, 1590, 1435, 1362, 1252, 1184,
1126, 1040, 825, 695; 1H NMR (300 MHz, CDCl3) d: 2.25–2.53 (m,
4H, –CHCH2CH2–), 2.73 (s, 3H, –COCH3), 3.80 (s, 3H, –COCH3),
4.91 (s, 2H, –OCH2Ar), 5.29–5.33 (m, 1H, –OCH(Ar)CH2–), 6.36 (d,
1H, J = 2.0 Hz, ArH), 6.53 (dd, 1H, J = 2.0 Hz and 8.5 Hz, ArH),
6.86–7.40 (m, 9H, ArH), 7.79 (d, 1H, J = 8.6 Hz, ArH), 9.63 (br, 1H,
–CO2H); 13C NMR (75 MHz, CDCl3) d: 29.2, 30.4, 32.5, 56.7, 70.4,
78.9, 101.5, 102.8, 108.1, 111.8, 112.0, 117.3, 121.6, 126.5, 128.2,
128.9, 129.1, 129.3, 134.6, 136.9, 157.3, 158.5, 163.7, 170.7,
198.3; MS (ESI(À)) m/e: 433 (MÀÀ1), 271 (100). Anal. Calcd for
White acicular crystal, yield: 74%. Mp: 135–136 °C. IR
(cmÀ1): 3470, 3216, 3032, 1707, 1659, 1608, 1565, 1501,
1432, 1252, 1186, 1165, 1097, 1024, 836, 704; 1H NMR
(300 MHz, CDCl3) d: 2.25–2.52 (m, 4H, –CHCH2CH2–), 4.97 (s,
2H, –OCH2Ar), 5.42–5.46 (m, 1H, –OCH(Ar)CH2–), 6.48 (d, 1H,
J = 2.1 Hz, ArH), 6.63 (dd, 1H, J = 2.1 Hz and 8.8 Hz, ArH), 7.28–
7.41 (m, 10H, ArH), 8.06 (d, 1H, J = 8.8 Hz, ArH); 13C NMR
(75 MHz, CDCl3) d: 30.6, 32.3, 69.5, 78.9, 101.4, 102.8, 108.5,
111.5, 126.1, 127.1, 127.3, 128.7, 128.9, 129.1, 130.4, 133.4,
133.7, 156.8, 163.9, 165.5, 170.8; MS (ESI(À)) m/e: 405
(MÀÀ1), 243 (100). Anal. Calcd for C24H22O6: C, 70.94; H,
5.42. Found: C, 70.63; H, 5.83.
C26H26O6: C, 71.89; H, 6.00. Found: C, 71.90; H, 5.86.
4.1.32. 4-(2-Acetyl-5-(benzo[d][1,3]dioxol-4-ylmethoxy)phenoxy)-
4-phenylbutanoic acid (6e)
4.1.36. 2-(3-Carboxy-1-phenylpropoxy)-4-((4-chlorobenzyl)oxy)-
benzoic acid (6i)
Lightgrey granular crystal, yield: 30%. Mp:123–124 °C. IR(cmÀ1):
3058, 2895, 1728, 1629, 1585, 1494, 1443, 1361, 1272, 1185, 1148,
1040, 1014, 933, 845, 699; 1H NMR (300 MHz, CDCl3) d: 2.22–2.26
(m, 1H, –CHCH2CH2–), 2.37–2.41 (m, 1H, –CHCH2CH2–), 2.52–2.58
(m, 2H, –CHCH2CH2CO2H), 2.71 (s, 3H, –COCH3), 4.80 (s, 2H, –OCH2
Ar), 5.28–5.32 (m, 1H, –OCH(Ar)CH2–), 5.94 (s, 2H, –OCH2O–), 6.32
(d, 1H, J = 2.0 Hz, ArH), 6.49 (dd, 1H, J = 2.1 Hz and 8.8 Hz, ArH),
6.73–6.79 (m, 3H, ArH), 7.27–7.38 (m, 5H, ArH), 7.77 (d, 1H, J = 8.7,
ArH), 10.10 (br, 1H, –CO2H); 13C NMR (75 MHz, CDCl3) d: 29.3,
30.3, 32.6, 70.3, 78.9, 101.2, 102.8, 108.0, 108.3, 108.5, 120.5,
121.3, 126.1, 128.5, 129.1, 129.2, 133.6, 134.8, 147.7, 148.0, 158.6,
163.8, 170.7, 198.7; MS (ESI(À)) m/e: 447 (MÀÀ1), 285 (100).
Anal. Calcd for C26H24O7.0.5H2O: C, 68.27; H, 5.47. Found: C, 68.21;
H, 5.41.
White acicular crystal, yield: 73%. Mp: 166–168 °C. IR (cmÀ1):
3082, 2923, 1691, 1601, 1573, 1495, 1444, 1296, 1261, 1244,
1189, 1090, 1013, 820, 704; 1H NMR (300 MHz, CDCl3) d: 2.25–
2.68 (m, 4H, –CHCH2CH2–), 4.95 (s, 2H, –OCH2Ar), 5.43–5.47 (m,
1H, –OCH(Ar)CH2–), 6.46 (d, 1H, J = 1.9 Hz, ArH), 6.60 (dd, 1H,
J = 2.0 Hz and 8.8 Hz, ArH), 7.24–7.39 (m, 9H, ArH), 8.06 (d, 1H,
J = 8.8 Hz, ArH); 13C NMR (75 MHz, CDCl3) d: 30.5, 32.9, 69.8,
78.7, 101.6, 103.1, 108.9, 111.9, 128.3, 128.5, 128.8, 129.0, 129.2,
129.3, 132.2, 132.9, 133.2, 156.9, 164.5, 165.3, 170.6; MS (ESI(À))
m/e: 439 (MÀÀ1), 277 (100). Anal. Calcd for C24H21ClO6: C, 65.45;
H, 4.77. Found: C, 65.91; H, 4.81.
4.1.37. 2-(3-Carboxy-1-phenylpropoxy)-4-((4-methoxybenzyl)-
oxy)benzoic acid (6j)
White acicular crystal, yield: 89%. Mp: 103–106 °C. IR (cmÀ1):
3371, 3169, 1706, 1656, 1608, 1563, 1519, 1444, 1299, 1250,
1183, 1020, 839, 771; 1H NMR (300 MHz, CDCl3) d: 2.25–2.67
(m, 4H, –CHCH2CH2–), 3.82 (s, 3H, –COCH3), 4.89 (s, 2H, –OCH2Ar),
5.42–5.46 (m, 1H, –OCH(Ar)CH2–), 6.46 (d, 1H, J = 2.0 Hz, ArH),
6.61 (dd, 1H, J = 2.0 Hz and 8.9 Hz, ArH), 6.89–7.41 (m, 9H,
ArH), 8.04 (d, 1H, J = 8.8 Hz, ArH); 13C NMR (75 MHz, CDCl3) d:
30.5, 32.7, 56.8, 69.9, 78.3, 101.5, 102.9, 108.5, 111.6, 112.8,
126.7, 128.3, 128.5, 128.7, 129.0, 129.1, 130.6, 134.7, 155.8,
156.3, 164.3, 165.9, 170.2; MS (ESI(À)) m/e: 435 (MÀÀ1), 273
(100). Anal. Calcd for C25H24O7: C, 68.81; H, 5.50. Found: C,
68.62; H, 5.65.
4.1.33. 4-(2-Acetyl-5-(benzo[c][1,2,5]oxadiazol-4-ylmethoxy)-
phenoxy)-4-phenylbutanoic acid (6f)
Light yellow acicular crystal, yield: 93%. Mp: 132–134 °C. IR
(cmÀ1): 3075, 2933, 1711, 1657, 1601, 1572, 1494, 1435, 1359,
1301, 1267, 1188, 1132, 1007, 806, 701; 1H NMR (300 MHz, CDCl3)
d: 2.26–2.30 (m, 1H, –CHCH2CH2–), 2.42–2.47 (m, 1H, –CHCH2CH2–),
2.51–2.58 (m, 2H, –CHCH2CH2CO2H), 2.73 (s, 3H, –COCH3), 5.02
(s, 2H, –OCH2Ar), 5.32–5.37 (m, 1H, –OCH(Ar)CH2–), 6.39 (d, 1H,
J = 2.2 Hz, ArH), 6.54 (dd, 1H, J = 2.2 Hz and 8.7 Hz, ArH), 7.25–
7.35 (m, 6H, ArH), 7.73 (s, 1H, ArH), 7.78 (d, 1H, J = 8.7 Hz, ArH),
7.83 (d, 1H, J = 9.8 Hz, ArH); 13C NMR (75 MHz, CDCl3) d: 29.2,
30.3, 32.5, 69.7, 78.8, 103.2, 108.0, 111.5, 117.5, 119.3, 125.4,
126.7, 127.1, 128.0, 128.8, 133.3, 134.3, 136.0, 147.4, 147.5,
159.8, 163.6, 170.0, 198.5; MS (ESI(À)) m/e: 445 (MÀÀ1), 283
(100). Anal. Calcd for C25H22N2O6: C, 67.26; H, 4.93; N, 6.28. Found:
C, 67.46; H, 4.95; N, 6.20.
4.1.38. 2-(3-Carboxy-1-phenylpropoxy)-4-((3-methoxybenzyl)-
oxy)benzoic acid (6k)
White acicular crystal, yield: 84%. Mp: 142–143 °C. IR (cmÀ1):
3030, 2945, 1698, 1677, 1603, 1571, 1491, 1460, 1282, 1188,
1109, 1042, 833, 774, 700; 1H NMR (300 MHz, CDCl3) d: 2.25–
2.65 (m, 4H, –CHCH2CH2–), 3.81 (s, 3H, –COCH3), 4.96 (s, 2H,
–OCH2Ar), 5.42–5.46 (m, 1H, –OCH(Ar)CH2–), 6.48 (d, 1H,
J = 2.2 Hz, ArH), 6.63 (dd, 1H, J = 2.2 Hz and 8.8 Hz, ArH), 6.88–
7.38 (m, 9H, ArH), 8.06 (d, 1H, J = 8.8 Hz, ArH); 13C NMR
(75 MHz, CDCl3) d: 30.5, 32.9, 56.8, 70.3, 78.8, 101.3, 102.8,
108.5, 111.6, 111.9, 112.3, 117.4, 126.6, 1283, 128.9, 129.5, 130.6,
134.5, 136.8, 156.5, 157.5, 164.3, 165.9, 170.5; MS (ESI(À)) m/e:
435 (MÀÀ1), 273 (100). Anal. Calcd for C25H24O7: C, 68.81; H,
5.50. Found: C, 68.74; H, 5.59.
4.1.34. 4-(2-Acetyl-5-(benzo[c][1,2,5]thiadiazol-4-ylmethoxy)-
phenoxy)-4-phenylbutanoic acid (6g)
Light yellow acicular crystal, yield: 87%. Mp: 146–148 °C. IR
(cmÀ1): 3078, 3026, 2933, 1723, 1627, 1586, 1436, 1268,
1239, 1183, 1129, 1024, 835; 1H NMR (300 MHz, CDCl3) d:
2.17–2.51 (m, 4H, –CHCH2CH2–), 2.71 (s, 3H, –COCH3), 5.14 (s,
2H, –OCH2Ar), 5.34–5.38 (m, 1H, –OCH(Ar)CH2–), 6.43 (d, 1H,
J = 2.2 Hz, ArH), 6.56 (dd, 1H, J = 2.2 Hz and 8.7 Hz, ArH), 7.18–
7.34 (m, 5H, ArH), 7.55 (dd, 1H, J = 1.5 Hz and 9.0 Hz, ArH),

666
J. Cai et al. / Bioorg. Med. Chem. 23 (2015) 657–667
4.1.39. 4-(Benzo[d][1,3]dioxol-4-ylmethoxy)-2-(3-carboxy-1-
phenylpropoxy)benzoic acid (6l)
2H, –OCH2Ar), 6.44 (d, 1H, J = 2.1 Hz, ArH), 6.48 (dd, 1H,
J = 2.1 Hz and 8.9 Hz, ArH), 7.19–7.31 (m, 5H, ArH), 7.37 (d, 1H,
J = 8.6 Hz, ArH), 7.63 (d, 1H, J = 8.7 Hz, ArH), 7.96 (s, 1H, ArH),
8.01 (d, 1H, J = 8.9 Hz, ArH); 13C NMR (75 MHz, CDCl3) d: 30.3,
32.7, 69.8, 78.5, 92.7, 103.3, 108.0, 116.3, 120.6, 122.1, 125.3,
126.7, 127.4, 128.1, 129.8, 133.3, 134.4, 137.6, 154.5, 154.7,
158.1, 163.6, 170.3; MS (ESI(À)) m/e: 444 (MÀÀ1), 282 (100). Anal.
Calcd for C24H19N3O4S: C, 64.72; H, 4.27; N, 9.44. Found: C, 64.87;
H, 4.47; N, 9.66.
White acicular crystal, yield: 79%. Mp: 137–139 °C. IR (cmÀ1):
3083, 2896, 1693, 1608, 1567, 1501, 1448, 1254, 1186, 1039,
1024, 930, 770; 1H NMR (300 MHz, CDCl3) d: 2.46–2.68 (m, 4H,
–CHCH2CH2–), 3.81 (s, 3H, –COCH3), 4.86 (s, 2H, –OCH2Ar), 5.43–
5.47 (m, 1H, –OCH(Ar)CH2–), 5.98 (s, 2H, –OCH2O–), 6.46 (d, 1H,
J = 2.2 Hz, ArH), 6.59 (dd, 1H, J = 2.2 Hz and 8.8 Hz, ArH), 6.78–
7.39 (m, 8H, ArH), 8.05 (d, 1H, J = 8.8 Hz, ArH); 13C NMR
(75 MHz, CDCl3) d: 30.4, 32.5, 70.3, 78.8, 101.1, 102.6, 108.0,
108.2, 108.6, 110.3, 121.1, 126.3, 128.6, 129.1, 129.8, 133.4,
134.7, 147.6, 148.1, 157.8, 164.0, 165.7, 170.6; MS (ESI(À)) m/e:
449 (MÀÀ1), 287 (100). Anal. Calcd for C25H22O8: C, 66.67; H,
4.89. Found: C, 66.21; H, 5.18.
4.1.44. 4-(Benzyloxy)-2-(3-cyano-1-phenylpropoxy)benzoic acid
(6q)
Colorless acicular crystal, yield: 37%. Mp: 142–144 °C. IR
(cmÀ1): 3029, 2934, 2252, 1692, 1674, 1608, 1571, 1498, 1453,
1276, 1190, 1153, 1099, 1047, 1026, 827, 732, 701; 1H NMR
(300 MHz, CDCl3) d: 2.23–2.82 (m, 4H, –C, –CHCH2CH2–), 4.95 (s,
2H, –OCH2Ar), 5.37–5.41 (m, 1H, –OCH(Ar)CH2–), 6.39 (d, 1H,
J = 2.2 Hz, ArH), 6.61 (dd, 1H, J = 2.1 Hz and 8.9 Hz, ArH), 7.30–
7.40 (m, 10H, ArH), 8.04 (d, 1H, J = 8.8 Hz, ArH); 13C NMR
(75 MHz, CDCl3) d: 15.1, 32.9, 69.6, 78.8, 101.3, 102.8, 108.2,
111.6, 119.5, 126.3, 127.2, 127.5, 128.8, 128.9, 129.3, 130.5,
133.6, 133.9, 156.7, 163.8, 165.8; MS (ESI(À)) m/e: 386 (MÀÀ1).
Anal. Calcd for C24H21NO4: C, 74.42; H, 5.43; N, 3.62. Found: C,
74.55; H, 5.40; N, 4.05.
4.1.40. 4-(5-(Benzyloxy)-2-cyanophenoxy)-4-phenylbutanoic
acid (6m)
Light yellow crystal, yield: 77%. Mp: 130–132 °C. IR (cmÀ1):
3062, 3032, 2929, 2220, 1705, 1603, 1575, 1505, 1431, 1397,
1328, 1298, 1268, 1216, 1186, 1114, 1026, 835, 736, 700; 1H
NMR (300 MHz, CDCl3) d: 2.11–2.59 (m, 4H, –CHCH2CH2–), 5.05
(s, 2H, –OCH2Ar), 5.15–5.19 (m, 1H, –OCH(Ar)CH2–), 6.41 (dd, 1H,
J = 2.2 Hz and 8.6 Hz, ArH), 6.47 (d, 1H, J = 2.1 Hz, ArH), 7.25–7.38
(m, 11H, ArH); 13C NMR (75 MHz, CDCl3) d: 30.3, 32.8, 69.5, 78.6,
95.7, 101.3, 102.8, 108.5, 116.6, 126.1, 127.0, 127.3, 128.5, 128.8,
129.2, 132.5, 133.3, 133.8, 157.6, 163.5, 170.8; MS (SCI) m/e: 388
(M++1). Anal. Calcd for C24H21NO4: C, 74.40; H, 5.46. Found: C,
74.22; H, 5.68.
4.1.45. 4-(Benzo[d][1,3]dioxol-4-ylmethoxy)-2-(3-cyano-1-
phenylpropoxy)benzoic acid (6r)
Colorless acicular crystal, yield: 32%. Mp: 162–164 °C. IR
(cmÀ1): 2889, 2244, 1681, 1663, 1605, 1573, 1494, 1449, 1254,
1188, 1099, 1038, 1014, 990, 813; 1H NMR (300 MHz, CDCl3) d:
2.17–2.93 (m, 4H, –CHCH2CH2–), 4.80 (s, 2H, –OCH2Ar), 5.27–
5.31 (m, 1H, –OCH(Ar)CH2–), 5.97 (s, 2H, –OCH2O–), 6.28 (d, 1H,
J = 2.1 Hz, ArH), 6.51 (dd, 1H, J = 2.2 Hz and 8.7 Hz, ArH), 6.72–
7.37 (m, 8H, ArH), 7.85 (d, 1H, J = 8.6 Hz, ArH); 13C NMR
(75 MHz, CDCl3) d: 15.1, 32.8, 70.4, 78.6, 101.3, 102.3, 108.1,
108.2, 108.5, 110.4, 119.5, 121.2, 126.5, 128.3, 129.2, 129.8,
133.5, 134.4, 147.6, 148.3, 157.9, 164.1, 165.8; MS (ESI(À)) m/e:
430 (MÀÀ1) (100). Anal. Calcd for C25H21NO6: C, 69.61; H, 4.87;
N, 3.25. Found: C, 69.75; H, 5.03; N, 3.42.
4.1.41. 4-(5-(Benzo[d][1,3]dioxol-4-ylmethoxy)-2-cyano-
phenoxy)-4-phenylbutanoic acid (6n)
Light yellow oil, yield: 81%. IR (cmÀ1): 3062, 3027, 2928, 2222,
1710, 1605, 1573, 1502, 1445, 1250, 1178, 1120, 1038, 929, 808,
755, 702; 1H NMR (300 MHz, CDCl3) d: 2.18–2.64 (m, 4H, –CHCH2
CH2–), 4.95 (s, 2H, –OCH2Ar), 5.14–5.18 (m, 1H, –OCH(Ar)CH2–),
5.95 (s, 2H, –OCH2O–), 6.44 (dd, 1H, J = 2.2 Hz and 8.6 Hz, ArH),
6.78 (d, 1H, J = 2.3 Hz, ArH), 6.85 (d, 1H, J = 1.1 Hz, ArH), 7.27–
7.35 (m, 8H, ArH); 13C NMR (75 MHz, CDCl3) d: 30.2, 32.5, 70.8,
79.2, 94.3, 101.3, 102.6, 108.1, 108.3, 108.6, 116.5, 121.2, 126.2,
128.6, 129.0, 132.6, 133.5, 134.9, 147.5, 148.3, 157.4, 163.6,
170.1; MS (ESI(À)) m/e: 430 (MÀÀ1), 268 (100). Anal. Calcd for
4.2. Biological evaluation
C25H21NO6: C, 69.60; H, 4.91. Found: C, 69.23; H, 5.12.
SD rats (200–220 g) were used through the studies. They were
housed at room temperature of 25 2 °C and humidity of
60 5%. Animals were free access to feed and water ad libitum dur-
ing the experimental period. The animal experiments were carried
out according to the Regulation of Experimental Animal Adminis-
tration (Committee for the purpose of Control and Supervision of
Experimentation on Animals guideline) issued by State Committee
of Science and Technology of PR China on 14 November, 1988 and
Institutional Animal Ethics Committee approved all the procedures
for the investigating experimental pain in conscious animals.
4.1.42. 4-(5-(Benzo[c][1,2,5]oxadiazol-4-ylmethoxy)-2-cyano-
phenoxy)-4-phenylbutanoic acid (6o)
Light yellow acicular crystal, yield: 90%. Mp: 132–134 °C. IR
(cmÀ1): 3451, 3087, 2936, 2223, 1708, 1610, 1576, 1503, 1454,
1431, 1300, 1255, 1201, 1124, 1044, 837, 750, 702; 1H NMR
(300 MHz, CDCl3) d: 2.17–2.28 (m, –2H, CHCH2CH2–), 2.45–2.60
(m, 2H, –CHCH2CH2–), 5.10 (s, 2H, –OCH2Ar), 5.20–5.24 (m, 1H,
–OCH(Ar)CH2–), 6.46–6.49 (m, 2H, ArH), 7.21–7.33 (m, 5H, ArH),
7.37 (d, 1H, J = 9.3 Hz, ArH), 7.43 (d, 1H, J = 9.3 Hz, ArH), 7.83 (s,
1H, ArH), 7.86 (d, 1H, J = 8.9 Hz, ArH); 13C NMR (75 MHz, CDCl3)
d: 30.4, 32.8, 69.9, 78.5, 92.5, 103.4, 108.1, 112.2, 116.5, 117.0,
125.2, 126.7, 127.2, 128.3, 130.0, 133.2, 134.3, 137.4, 147.3,
147.5, 158.3, 163.5, 170.5; MS (ESI(À)) m/e: 428 (MÀÀ1) (100).
Anal. Calcd for C24H19N3O5: C, 67.13; H, 4.43; N, 9.79. Found: C,
67.12; H, 4.60; N, 9.45.
4.2.1. Inhibition of ET-1 induced contraction of rat thoracic
aortic ring
SD rats were sacrificed by decapitation and the thoracic aorta
was quickly removed and placed in a Krebs–Henseleit 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 ten-
sion change. The ring was stretched by a resting force of 1 g and
4.1.43. 4-(5-(Benzo[c][1,2,5]thiadiazol-4-ylmethoxy)-2-cyano-
phenoxy)-4-phenylbutanoic acid (6p)
Light yellow crystal, yield: 83%. Mp: 120–122 °C. IR (cmÀ1):
3459, 3028, 2933, 2222, 1707, 1608, 1575, 1502, 1454, 1430,
1307, 1256, 1189, 1123, 1039, 1018, 815, 702; 1H NMR
(300 MHz, CDCl3) d: 2.13–2.23 (m, –2H, CHCH2CH2–), 2.44–2.59
(m, 2H, –CHCH2CH2–), 5.16–5.20 (m, 1H, –OCH(Ar)CH2–), 5.23 (s,

J. Cai et al. / Bioorg. Med. Chem. 23 (2015) 657–667
667
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
catheters connected with a pressure transducer were inserted into
the right jugular vein and then the vena cava and the right ventri-
cle for measuring the CVP and RVSP, respectively. Signals were
recorded by hemodynamic analyzer. After hemodynamic measure-
ments, rats were sacrificed, the hearts removed, washed with 0.9%
NaCl, and carefully divided into the right and left ventricle (includ-
ing septum). The weight index of the right ventricle was evaluated
as two ratios: RV/BW and RV/(LV + S).
tissues were contracted with phenylephrine (1
lM) followed by
challenge with acetylcholine (1 M). A negative relaxant response
l
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
experiments. 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 pretreat-
ment period. Contractile responses were expressed as a percentage
of the response.34
Acknowledgments
This work is supported by the Fundamental Research Funds for
the Central Universities (2242014R30019), Technology Supporting
Program of Jiangsu Province (BE2012657) and National Basic
Research Program of China (No. 2011CB933503).
4.2.2. 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
References and notes
150
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L [125I] ET-1. The
l
L. [125I] ET-1 was added to reach the concentration in seven
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SD Rats were randomly divided into control, untreated hypoxic
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in CMC and the control group given vehicle only.
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