Synthesis and in vitro evaluation of ambrisentan analogues as potential endothelin receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.05.034

A series of novel 2-[(4,6-dimethylpyrimidin-2-yl)oxy]-3,3-diphenyl butyric acid derivatives were synthesized and evaluated for their antagonistic activity for endothelin-1-induced contraction in rabbit aorta. Within this series of compounds, 2-[(4,6-dimet

Full text of DOI:10.1016/j.bmcl.2011.05.034

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3894–3897
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro evaluation of ambrisentan analogues as potential
endothelin receptor antagonists
Jun Xia a, Jianfei Song b, Le Zhen a, Xiuling Zhang a, Xiantao Lei a, Lina Zheng a, Qiujuan Wang b,
Hongbin Sun a,
⇑
a Center for Drug Discovery, College of Pharmacy, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing 210009, China
b Department of Pharmacology, College of Pharmacy, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 2-[(4,6-dimethylpyrimidin-2-yl)oxy]-3,3-diphenyl butyric acid derivatives were synthe-
sized and evaluated for their antagonistic activity for endothelin-1-induced contraction in rabbit aorta.
Within this series of compounds, 2-[(4,6-dimethylpyrimidin-2-yl)oxy]-3-cyano-3,3-diphenylpropionic
acid (4) displays comparable potency with ambrisentan (1), and warrants further investigation.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 3 March 2011
Revised 18 April 2011
Accepted 10 May 2011
Available online 24 May 2011
Keywords:
Endothelin receptor
Ambrisentan analogues
Pulmonary arterial hypertension
SAR analysis
Antagonists
The endothelins consist of three subtypes: endothelin-1, 2 and 3
(ET-1, ET-2, ET-3). The endothelins function by binding to trans-
membrane G-proteincoupled receptors of which two major sub-
types, ETA and ETB, have been identified.1–3 Both receptor
subtypes are found on smooth muscle cells and mediate the vaso-
constrictor and pressor actions of endothelins. Elevated levels of
endothelins have been associated with a number of physiological
and pathological processes such as renal failure,4 heart failure,5
hypertension,6 atherosclerosis,7 acute myocardial infarction,8 pul-
monary arterial hypertension (PAH),9 cerebral vasospasm,10 sub-
arachnoid hemorrhage11 and metastatic prostate cancer.12
Theoretically, selective ETA-receptor antagonists should be more
effective in achieving vasodilation than non-selective ETA/ETB
receptor antagonists, given the role played by ETB receptors in both
vasodilation and ET-1 clearance.13 In animal models of PAH, how-
ever, positive dilatory effects have been observed with both selec-
tive ETA-receptor blockade and non-selective antagonism.14 Hence,
at the present time, there is no specific answer to the question that
which class of antagonists is better.14 In fact, bosentan, which is
the first non-peptidic endothelin receptor antagonist approved
for the treatment of PAH,15 is a mixed ETA/ETB receptor antagonist.
On the other hand, many research have been embarked on the dis-
covery and development of selective ETA receptor antagonists,16–18
of which ambrisentan (1) has been approved for the treatment of
PAH, and is regarded as ‘the best-in-class’ due to its lower hepato-
toxicity and weaker drug interaction.19 It was noticed that modifi-
cations at methoxy group in 1 led to analogues which retained ETA
affinity but exhibited ETB affinity as well.20 In this study, a series of
ambrisentan analogues, 2-[(4,6-dimethylpyrimidin-2-yl)oxy]-3,3-
diphenyl butyric acid derivatives, have been synthesized and
evaluated for their in vitro antagonism of endothelin function in
order to find more potent and safer endothelin receptor antago-
nists.
CO2H
O
N
O
N
1 (Ambrisentan)
We envisioned that the methoxy group in 1 could be replaced
by a hydroxymethyl group, resulting in compound 2, an isomeride
of ambrisentan (1). A series of ether and ester derivatives of 2 were
further designed and synthesized to explore the structure–activity
relationship. On the other hand, compound 4 with a cyano group in
place of the methoxy group in 1 also attracted our attention. More-
over, the effect of introduction of sterically hindered and hydro-
⇑
phobic groups into 1 was studied through the design of
compound 5.
Corresponding author. Tel./fax: +86 25 83271198.
E-mail address: hbsun2000@yahoo.com (H. Sun).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.034

J. Xia et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3894–3897
3895
CN
CO2H N
O
CO2H N
O
CN
OH
HO
RO
NC
c
a
b
BnO
N
N
7
8
6
O
3
CN
OH
2
O
CHO
d
e
CO2H N
O
CO2H N
O
BnO
BnO
OH
NC
N
N
11
10
9
COOH
O
O
N
N
O
N
5
g
f
O
4
HO
N
The synthesis of 2 and its derivatives 14–17 and 20–46 is out-
lined in Scheme 1. Reaction of diphenylacetonitrile 6 with parafor-
2
12
maldehyde gave alcohol 7.21 Benzylation of
7 with benzyl
g, i
g, h
trichloroacetimidate afforded benzyl ether 8 (yield 71% from 6).
Reduction of 8 with DIBAl-H gave aldehyde 9. Treatment of 9 with
NaCN in the presence of NaHSO3 provided cyanohydrine 10 (yield
85% from 8). Unexpectedly, acidic hydrolysis of 10 gave lactone 11
COOBn
COOR
O
N
N
O
HO
as the major product instead of the corresponding a-hydroxy acid
N
RO
N
(yield 72%). Reaction of 11 with 4,6-dimethyl-2-methylsulfonyl-
1,3-pyrimidine in the presence of K2CO3 gave pyrimidine deriva-
tive 12 (yield 73%). Hydrolysis of 12 yielded compound 2 which
could be cyclized again under acidic conditions to give 12, and thus
the acidification during work-up should be very careful, maintain-
ing pH value around 7 (yield 95%). Hydrolysis of 12 and subsequent
complete alkylation of the resulting hydroxyl and carboxylic
groups with reactive alkyl halides (e.g., methyl iodide, ethyl iodide,
allyl bromide and benzyl bromide) in the presence of K2CO3 and
NaOH afforded the corresponding esters 13, which were hydro-
lyzed to give the corresponding carboxylic acids 14–17 (yield
80–88%). When using less reactive alkyl halides such as butyl bro-
mide, the above reactions did not work well. Therefore, we devel-
oped another approach to derivatization of 2. Hydrolysis of 12 and
subsequent selective benzylation of the resulting carboxylic group
with benzyl bromide in the presence of water afforded benzyl ester
18. Alkylation or acylation of 18, followed by debenzylation by
hydrogenolysis with Pd/C provided compounds 20–46 (yield
70–91%).
13
18
g
j, k or l
COOBn
COOH
O
N
N
O
R'O
RO
N
N
19
14-17
m
COOH
O
N
N
R'O
20-46
Scheme 1. Reagents and conditions: (a) HCHO, CaO/THF, H2O, 30 °C; (b) benzyl
trichloroacetimidate, TfOH/cyclohexane, CH2Cl2, rt; (c) Dibal-H, toluene, 0 °C to rt;
(d) NaCN, NaHSO3, THF, H2O, rt; (e) AcOH, 67% H2SO4, 100–110 °C, reflux; (f) 4,6-
dimethyl-2-methylsulfonyl-1,3-pyrimidine, K2CO3, DMF, 90 °C; (g) 10%NaOH,
MeOH, reflux; (h) RX, K2CO3, NaOH, DMF; (i) BnBr, K2CO3, H2O, DMF; (j) R0X,
NaH/THF; (k) (R0CO)2O or R0COCl/Pyr; (l) R0COCl, NaH/THF; (m) H2,10% Pd–C, THF.
The synthesis of 4 is shown in Scheme 2. Reaction of diphenyl-
acetonitrile 6 with ethyl glyoxalate afforded a-hydroxy ester 47 in
87% yield. Reaction of 47 with 4,6-dimethyl-2-methylsulfonyl-1,3-
pyrimidine in the presence of NaH at low temperature provided
pyrimidine derivative 48 in 74% yield. Hydrolysis of 48 produced
carboxylic acid 4 in 99% yield.
The synthesis of 5 is shown in Scheme 3. Reaction of diphenyl-
acetic acid (49) with benzyl bromide in the presence of K2CO3
afforded benzyl ester 50. Alkylation of 50 with benzyl bromide in
the presence of NaH produced compound 51. Reduction of 51 with
LiAlH4 yielded alcohol 52, which was oxidized with PCC to give
aldehyde 53. Treatment of 53 with NaCN in the presence of
NaHSO3 furnished cyanohydrine 54 (yield 81% from 49). Hydrolysis
CO2Et
OH
CO2Et
O
N
N
CN
b
a
NC
NC
6
48
47
of 54 afforded
a-hydroxy acid 55 smoothly, which reacted with
4,6-dimethyl-2-methylsulfonyl-1,3-pyrimidine in the presence of
K2CO3 to yield compound 5 (yield 80% from 54).
COOH
N
c
O
The functional inhibitory potency of the synthesized com-
pounds against the contraction induced by endothelin-1 on rabbit
aortic rings was assessed following the literature procedures22–25
(for details see Supplementary data). The assay results are
summarized in Table 1. As shown in Table 1, some test compounds
(e.g., 2, 4, 15, 16, 23, 30, 43) exhibited potent functional inhibitory
activity against endothelin-1-induced vascular contraction. In this
NC
N
4
Scheme 2. Reagents and conditions: (a) HCOCOOEt, NaH/THF, ꢀ20 °C; (b) 4,6-
dimethyl-2-methylsulfonyl-1,3-pyrimidine, NaH, THF, ꢀ20 °C; (c) 10%NaOH,
MeOH, reflux.

3896
J. Xia et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3894–3897
Table 1 (continued)
COOBn
COOBn
b
Compd
R
Inhibition rate (%)
3.47
COOH
c
a
O
HOOC
33
O
51
50
49
34
8.09
NA
CN
CHO
O
OH
OH
e
d
35
O
O
54
53
52
36
NA
COOH
OH
COOH
N
O
O
37
13.16
f
g
O
N
N
N
55
38
7.82
NA
5
Scheme 3. Reagents and conditions: (a) BnBr, K2CO3, DMF, rt; (b) BnBr, NaH, THF,
0 °C; (c) K2CO3, THF, 0 °C; (d) PCC/CH2Cl2, 0 °C to rt; (e) NaCN, NaHSO3, THF, H2O, rt;
(f) AcOH, 67% H2SO4, 100–110 °C; (g) 4,6-dimethyl-2-methylsulfonyl-1,3-pyrimi-
dine, LiNH2, DMF, 50 °C.
39
40
O
O
NA
Table 1
O
O
Inhibition of endothelin-induced contractions on isolated rabbit aortic rings
41
42
43
44
3.40
15.52
22.02
NA
COOH
O
O
N
N
O
O
O
O
RO
2, 14-17, 20-46
O
Compd
R
Inhibition rate (%)
O
Ambrisentan
Bosentan
2
4
5
—
—
H
—
—
—
43.23
22.54
23.19
42.20
11.77
14.32
NA
20.64
23.44
NA
45
46
13.65
NA
O
Ts
(ET-1: 10ꢀ8 M; test compounds: 10ꢀ6 M).
12
14
15
16
17
18
20
21
22
23
24
25
26
27
Methyl
Ethyl
Allyl
Benzyl
—
n-Propyl
n-Butyl
Acetyl
Propionyl
Butyryl
Valeryl
Caproyl
Capryloyl
O
functional assay, 2, 15, 16, 23, 30, and 43 displayed comparable
potency with bosentan, whereas 4 (42.2% inhibition) was much
more potent than bosentan (22.54% inhibition) and equally potent
with ambrisentan (43.23% inhibition). We further determined the
effects of 4 and ambrisentan on the concentration–contractile re-
sponse curve to ET-1 in isolated rabbit aorta (Figs. 1 and 2). The
results again confirmed that 4 was a very potent antagonist against
endothelin-1-induced vascular contraction.
SAR analysis showed that replacement of the methoxy group in
1 with a hydroxymethyl group retained the activity, albeit result-
ing in a decrease in potency (23.19% inhibition for 2 vs 43.23% inhi-
bition for 1). Alkylation or acylation of the hydroxyl group in 2 led
to mixed results: in some cases the activity remained (e.g., 15, 16,
23, 30, 43), and in other cases a loss in potency was observed (e.g.,
14, 17, 20, 28). The pattern of substitution appeared to be less
crucial, but the size and length seemed to have an impact on the
potency. Moderate substitutions were beneficial to potency (e.g.,
16, 23, 30, 43), while large (e.g., 17, 25, 26, 27, 28, 29, 39, 44) or
small (e.g., 14, 22, 41) substituents led to significant decreases in
potency. Replacement of the methoxy group in 1 with hydrophobic
groups resulted in a decrease in potency (e.g., 5). It was interesting
to note that introduction a cyano group to replace the methoxy
group in 1 resulted in a very potent compound (4).
NA
NA
15.11
9.64
21.68
1.11
5.88
NA
5.61
28
NA
O
29
30
NA
O
21.80
O
N
31
32
NA
NA
O
HOOC

J. Xia et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3894–3897
3897
140%
120%
100%
80%
60%
40%
20%
0%
2, 4, 15, 16, 23, 30, and 43 exhibited potent inhibitory activity
against endothelin-1-induced vascular contraction. Particularly,
2-[(4,6-dimethylpyrimidin-2-yl)oxy]-3-cyano-3,3-diphenylprop-
ionic acid (4) was comparable with ambrisentan (1) in potency.
Since 4 is a racemic compound while ambrisentan is an optically
active enantiomer, optically pure enantiomers of 4 warrants fur-
ther investigation.
Acknowledgments
This work was supported in part by the ‘111 Project’ from the
Ministry of Education of China and the State Administration of For-
eign Expert Affairs of China (No. 111-2-07).
-10 -9.5
-9
-8.5
-8
-7.5
-7
-6.5
-6
Supplementary data
ET-1, Log (M)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.05.034.
Figure 1. Effects of compound 4 and ambrisentan on the concentration–contractile
response curve to ET-1 in isolated rabbit aorta: vehicle (open circle), compound 4:
10-6
M
(open squares), ambrisentan: 10-6
M
(open triangles). The date points
References and notes
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**
**
**
**
**
**
** **
**
**
**
** **
-10 -9.5 -9 -8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5
Log(M)
Figure 2. Effects of ET-1 on the concentration–contractile response cure to
compound
4 (open circle) and ambrisentan (open triangles). The data points
represent the mean percentage of the maximal response s (n = 6), ⁄⁄p <0.01 versus
control (ET-1: 2 ꢁ 10ꢀ8 M).
In summary, a series of novel 2-[(4,6-dimethylpyrimidin-2-
yl)oxy]-3,3-diphenyl butyric acid derivatives were synthesized
and evaluated for their antagonistic activity for endothelin-1-in-
duced contraction in rabbit aorta. Within this series of compounds,
25. Tsuneyuki, T.; Peter, J. B.; Ivana, K.; Magdi, H. Y.; Timothy, D. W.; Maria, G. B.
Eur. J. Pharmacol. 1997, 324, 219.