Potent nonpeptide endothelin antagonists: Synthesis and structure-activity relationships of pyrazole-5-carboxylic acids

Article abstract of DOI:10.1016/S0960-894X(00)00513-8

We have previously reported the identification of pyrazole-5-carboxylic acids as a new class of endothelin antagonists from low affinity pyrazol-5-ol ligands, which were obtained by random screening assays.¹ We describe herein the synthesis and the structure-activity relationships (SARs) of these pyrazole-5-carboxylic acids with potent ET(A) selective, mixed ET(A)/ET(B) or moderately ET(B) selective antagonist activities. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00513-8

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2575±2578
Potent Nonpeptide Endothelin Antagonists:
Synthesis and Structure±Activity Relationships of
Pyrazole-5-carboxylic Acids
Jidong Zhang,a,* Stanislas Didierlaurent,a Michel Fortin,b
Dominique Lefrancois,a Eric Uridata and Jean Paul Veverta
aMedicinal Chemistry, Hoechst Marion Roussel, 102 route de Noisy, 93235 Romainville Cedex, France
bCentral Research, Hoechst Marion Roussel, 102 route de Noisy, 93235 Romainville Cedex, France
Received 24 May 2000; accepted 11 September 2000
AbstractÐWe have previously reported the identi®cation of pyrazole-5-carboxylic acids as a new class of endothelin antagonists
from low anity pyrazol-5-ol ligands, which were obtained by random screening assays.1 We describe herein the synthesis and the
structure±activity relationships (SARs) of these pyrazole-5-carboxylic acids with potent ETA selective, mixed ETA/ETB or moder-
ately ETB selective antagonist activities. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
A large number of potent, nonpeptide ETA selective
(Ro61-1790, L-754,142, SB217242, BMS-182874,
PD156707 and TBC11251),5 ETB selective (Ro46-8443
and A-308165)6 and ETA/ETB mixed antagonists
(Bosentan, SB209670, L-749329, and LU302872)7 have
been described in the literature. We have previously
reported the preparation of pyrazole-5-carboxylic acids
(i.e. 1) as a new class of endothelin antagonists from low
anity screening hits, pyrazol-5-ols.1 We wish to
describe herein the synthesis and the SAR of these pyr-
azole-5-carboxylic acids, which resulted in potent ETA
selective and mixed ETA/ETB antagonists, but also in
some moderately ETB selective antagonists.
The endothelins (ET-1, 2 and 3) are 21-amino-acid
peptides, with ET-1 being one of the most potent vaso-
constrictors known.2 The endothelins exert their biolo-
gical e€ects by interacting with two speci®c G-protein
coupled membrane receptors (ETA and ETB).3 Elevated
levels of endothelins have been associated with a certain
number of diseases, such as myocardial infarction,
hypertension, heart failure, athero-sclerosis, cerebral
and coronary vasospasm, renal failure and asthma.4
Several studies have shown that the mode of action of
ET-1 as related to the aforementioned pathological
conditions is mediated through the ETA receptor, which
is mainly found in the vascular smooth muscle tissues.
The ETB receptor is expressed on both vascular endo-
thelial and smooth muscle cells, but its role remains
poorly understood. Therefore selective ETA receptor
antagonists may have bene®cial e€ects for patients with
those disease states. In addition, all types of antagonists
(ETA and ETB selective: mixed ETA/ETB) should be
very useful tools for understanding the roles of both
ET receptor subtypes in normal physiological and
pathological conditions.
Chemistry
The structures of the pyrazole-5-carboxylic acids
described in this study are depicted in Tables 1±3. The
synthesis of compound 1 has already been reported.
However, this compound has also been prepared
according to the procedure outlined in Scheme 1, which
was developed for the synthesis of pyrazole acids 7a±q
(Table 1) and 8a±d (Table 3).8 The commercially avail-
able ketones 2 were treated with diethyl oxalate in the
presence of NaOEt in ethanol to give quantitatively the
corresponding a,g-diketoesters 3 according to a litera-
ture method.9 Reactions of a,g-diketoesters 3 with 6-
chloro-piperonyl chloride were performed in the pre-
sence of EtONa and NaI in DMF. The crude
*Corresponding author. Tel.:+33-1-4991-4731; Fax: +33-1-4991-
5087; e-mail: jidong.zhang@aventis.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00513-8

2576
J. Zhang et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2575±2578
compounds 4 were allowed to react directly with
hydrazine monohydrate in ethanol to a€ord the desired
pyrazole esters 5. Regioselective alkylation of 5 with the
requisite alkyl halide or alkyl tosylate in the presence of
NaH in DMF at room temperature gave compounds 6.
Finally, the pyrazole acids 7a±q and 8a±d were obtained
after saponi®cation in good yields.
Pyrazole-5-carboxylic acids 17a±f (Table 2) were
obtained by a new route outlined in Scheme 2. Wittig±
Horner reaction of 6-chloropiperonal 9 with triethyl
phosphonoacetate in DME in the presence of NaH,
a€orded unsaturated ester 10. After reduction of the
double bond with NaBH4/Cu2Cl2 in a mixture of THF/
EtOH,10 the saturated ester derivative 11 was obtained
in modest yield. Reaction of 11 with diethyl oxalate in
toluene in the presence of NaH gave 12, which was
cyclized into the hydroxypyrazole 13 by the reaction
with cyanoethylhydrazine in AcOH. Bromopyrazole 14
was obtained after OH±bromine exchange in pure
POBr3 at 60 ꢀC. The deprotection of the bromopyrazole
14 was realized by a retro-Michael-type reaction on the
cyanoethyl substituent by treatment with NaH in DMF.
The so-obtained ionic pyrazole intermediate was regio-
selectively alkylated in situ with cyclohexylmethyl bro-
mide, which a€orded the desired compound 15. Suzuki
reaction or Stille coupling of 15 with requisite aryl
boronic acids or aryl stannanes gave the corresponding
pyrazoles 16a±f, which after saponi®cation under stan-
dard conditions yielded the desired pyrazole-5-carboxylic
acids 17a±f.
Table 1. In vitro endothelin receptor binding anity (IC50 (nM)) for
compounds 1, 7a±q
IC50 (nM)
a
b
No.
R
ETA
ETB
34
1
18
7a
7b
7c
7d
7e
7f
28
17
34
15
9.7
Results and Discussion
11
We have previously proposed a pharmacophore model
for ET antagonism that contains a central pyrazole-5-
carboxylic acid ¯anked by a piperonyl moiety, a second
benzyl group and an additional hydrophobic substituent
located next to the piperonyl (i.e. 1).1 In order to
increase the ET antagonist potencies, we performed
26
31
3.6
3.4
2.6
Table 2. In vitro endothelin receptor binding anity (IC50 (nM)) for
compounds 7e, 17a±f
4.9
7g
7h
7i
226
131
70
953
810
4.9
IC50 (nM)
7j
2.5
20.5
11
14.8
a
b
No.
Ar
ETA
ETB
7k
7l
3.2
7e
3.6
2.6
2045
17a
17b
17c
17d
17e
17f
4.7
3.4
6.3
7m
7n
7o
7p
7q
6.2
2.5
79
89
44
17.8
2300
178
440
1.3
1.1
1.1
9.8
3.2
1.7
59
3.3
89
4.0
aRat heart ventricles.
bRat cerebellum.
aRat heart ventricles.
bRat cerebellum.

J. Zhang et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2575±2578
2577
further SAR studies on the two later substituents while
the carboxylic acid function and the 6-chloropiperonyl
were kept constant. As in our previous study,1 the inhi-
bition of endothelin binding to ET receptors was mea-
sured using [125I]-labelled ET-1 competition assays.8
Table 1 summarizes the results of this study for the 1-
position of these pyrazoles (Scheme 1). Obviously,
hydrophobic benzylic substituents (compounds 7a±c)
gave balanced ET antagonists, but no signi®cant di€er-
ence in potency has been observed as compared to the
lead compound 1. Interestingly, aliphatic hydrophobic
substituents (compounds 7d±f) were well accepted by
the receptors with one exception for the secondary
cyclohexyl in compound 7g. Introduction of a hetero-
atom like oxygen into these aliphatic substituents resul-
ted in a signi®cant loss in anities for both ET
receptors (compound 7h). The best anities were
obtained with the cyclohexylmethyl group (7e), which
resulted in a 5-fold improved anity for ETA and a 10-
fold improved anity for ETB receptor compared to
compound 1. However, all these compounds showed
mixed ET antagonism, and in our e€ort to search for
ET subtype selective antagonists, additional compounds
(7i±q) containing a second acid functionality were pre-
pared. Amazingly, very good ETA selectivity was
observed following a minor change of the position of
the acid group as demonstrated with the compound 7i
with the carboxylic acid at the 4-position of cyclohex-
ylmethyl. Indeed, this compound showed an ETA/ETB
ratio of 1/165. A carboxylic acid functionality at the 4-
position seems crucial for ETA selectivity, since the cor-
responding aromatic derivative 7l displayed the same
kind of selectivity (1/186). Surprisingly, changing the
position of this second acid function to the 3-position in
7j (cyclohexylmethyl) or in 7m±o (benzyl group) led to
poor ETA selectivity (ETA/ETB ratio varying from 1/6
to 1/14). Finally, ETB selectivity was obtained when this
second acid was introduced to the 2-position of the
cyclohexylmethyl (7k) or the ortho-position of the
benzyl group (7p,q), with 7q being the most ETB selec-
tive antagonist obtained (ETA/ETB: 22/1).
Table 3. In vitro endothelin receptor binding anity (IC50 (nM)) for
compounds 8a±d
With the aim to improve even further the ET receptor
binding anity for pyrazole-5-carboxylic acids, we then
decided to investigate the region occupied by the 3-
phenyl group, which was identi®ed as one of the key
substituents in our previous work.1 Compounds syn-
thesized for this purpose are presented in Table 2
(Scheme 2). Replacement of the phenyl group by a pyr-
idin-2-yl (compound 17a) did not show signi®cant
change in potency, while a pyridin-3-yl (compound 17b)
led to a slight loss in ETA binding. Introduction of a 4-
methoxy substituent on the phenyl ring (compound 17c)
resulted in almost a total loss of the anity for both ET
receptors. Small lipophilic aromatic groups like furanyl
and thienyl (compounds 17d±f) slightly increased ETA
binding anities but had little in¯uence on ETB a-
nities as compared to 7e. Compound 17f was the most
potent mixed ET antagonist with an IC50 of 1.1 nM for
ETA receptor and an IC50 of 1.7 nM for ETB receptor.
IC50 (nM)
a
b
No.
R
ETA
ETB
825
8a
0.81
8b
8c
8d
1.3
9.9
4.6
8.6
28.3
14.9
aRat heart ventricles.
bRat cerebellum.
Scheme 2. Reagents and conditions: (a) EtO2CCH2PO(OEt)2, NaH/
DME, rt, 2.5 h, yield: 90%; (b) NaBH4/Cu2Cl2, THF/EtOH, 0 ꢀC, 5 h,
yield: 57% (c) EtO2CCO2Et, NaOEt/toluene, re¯ux, 3 h, yield: 76%;
(d) NCCH2CH2NHNH2, AcOH, 100 ꢀC, 1 h, yield: 55%; (e) POBr3,
60 ꢀC, 15 min, yield: 85%; (f) (i) NaH, DMF, 0 ꢀC, 1 h; (ii) cyclo-
hexylmethyl bromide, rt to 60 ꢀC, 16 h, yield: 55%; (g) ArSnBu3,
Pd(PPh3)4, toluene, 5h (16a,b, yields from 56 to 67%); ArB(OH)2,
Pd(PPh3)4, K3PO4, DMF, 24 h (16c±f, yields from 55 to 82%); (h)
NaOH (2 N), EtOH, re¯ux, 2 h (yields: from 90 to 100%).
Scheme 1. Reagents and conditions: (a) EtO2CCO2Et, NaOEt/EtOH,
0 ꢀC to rt, 20 h (100%); (b) 6-chloropiperonyl chloride, NaOEt/DMF,
.
NaI, rt, 16 h (crude); (c) H2NNH2 H2O, EtOH, re¯ux, 4 h (yields for
steps b and c: Ar=Ph: 41%; Ar=2-Thienyl: 30%); (d) R0X, NaH/
DMF, rt, 20 h (yields: from 50 to 87%); (e) NaOH (2 N), EtOH,
re¯ux, 2 h (yields: from 75 to 100%).

2578
J. Zhang et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2575±2578
Since the 2-thienyl derivative gave better ET binding
than the corresponding phenyl analogue, it was then
chosen as a new building-block for further selectivity
studies as was done previously with the phenyl series
(compounds 1, 7a±q). Substituents which have shown to
have the most dramatic e€ect on ET binding selectivity
in the former series, were then incorporated into com-
pounds 8a±d (Table 3, Scheme 1). Compound 8a
showed an improved anity for ETA (IC50=0.81 nM)
and unchanged anity for ETB, resulting in a better
selectivity ratio of 1/1020 as compared to 7i. Slightly
better anities for both ETA and ETB receptors were
observed for compound 8b as compared to 7j, and
therefore the selectivity was unchanged. Finally, ETA
binding anities were also slightly increased for 8c,d as
compared to 7p,q, while ETB anities remained
unchanged, which led to diminished ETB selectivities.
(b) Hrenreich, H.; Anderson, R. W.; Fox, C. H.;
Rieckmann, P.; Ho€mann, G. S.; Travis, W. D.; Coligan, J.
E.; Kehrl, J. H.; Fauci, A. S. J. Exp. Chem. 1990, 172, 1741.
(c) Shichiri, M.; Hirata, Y.; Nakajima, T.; Ando, K.; Imai, T.;
Yanagisawa, M.; Masaki, T.; Marumo, F. J. Clin. Invest.
1991, 87, 1867.
4. For reviews, see (a) Naylor, W. Trends Pharmacol. Sci.
1990, 11, 96. (b) Filep, J. G. Life Sci. 1993, 52, 119. (c) Dao,
H. H.; Moreau, P. Expert Opin. Invest. Drugs 1999, 8, 1807.
5. (a) Roux, S.; Breu, V.; Giller, T.; Neidhart, W.; Ramuz, H.;
Coassolo, P.; Clozel, J. P.; Clozel, M. J. Pharmacol. Exp.
Ther. 1997, 283, 1110. (b) Williams, D. L., Jr.; Murphy, K. L.;
Nolan, N. A.; O'Brien, J. A.; Pettibone, D. J.; Kivlighn, S. D.;
Krause, S. M.; Lis, E. V., Jr.; Zingaro, G. J.; Gabel, R. A.;
Clayton, F. C.; Siegel, P. K. S.; Zhang, K.; Naue, J.; Vyas, K.;
Walsh, T. F.; Fitch, K. J.; Chakravarty, P. K.; Greenlee, W.
J.; Clineschmidt, B. V. J. Pharmacol. Exp. Ther. 1995, 275,
1518. (c) Ohlstein, E. H.; Nambi, P.; Largo, A.; Hay, D. W.
P.; Beck, G.; Fong, K.-L.; Eddy, E. P.; Smith, P.; Ellens, H.;
Elliott, J. D. J. Pharmacol. Exp. Ther. 1996, 276, 609. (d)
Stein, P. D.; Hunt, J. T.; Floyd, D. M.; Moreland, S.; Dick-
inson, K. E. J.; Mitchell, C.; Liu, E. C.-K.; Webb, M. L.;
Murugesan, N. J. Med. Chem. 1994, 37, 329. (e) Patt, W. C.;
Reisdorph, B. R.; Repine, J. T.; Doherty, A. M.; Haleen, S. J.;
Walker, D. M.; Welch, K. M.; Flynn, M. A.; Hallak, H.;
Reyner, E. L.; Stewart, B. H. Bioorg. Med. Chem. Lett. 1997,
7, 297. (f) Wu, C.; Chan, M. F.; Stavros, F.; Raju, B.; Okun,
I.; Mong, S.; Keller, K. M.; Brock, T.; Kogan, T. P.; Dixon,
R. A. F. J. Med. Chem. 1997, 40, 1690.
In summary, investigation of SARs of the pyrazole-5-
carboxylic acids resulted in the synthesis of very potent
ET antagonists. Representatives of this family subtype
have been identi®ed: 8aÐpotent and selective ETA anta-
gonist (ETA IC50=0.81 nM; ETA/ETB ratio=1/1020);
8bÐmoderately ETA selective antagonist (ETA
IC50=1.3 nM; ETA/ETB ratio=1/7); 17fÐETA/ETB
balanced antagonist (ETA IC50=1.1 nM; ETB
IC50=1.7 nM), and 7qÐmoderately ETB selective (ETB
IC50=4.0 nM; ETA/ETB ratio=22/1). All these com-
pounds might provide useful tools for the evaluation of
the bene®t of ETA receptor antagonism, and for the
better understanding of the role of the ETB receptor.
6. (a) Breu, V.; Clozel, M.; Burri, K.; Hirth, G.; Neidhart, W.;
Ramuz, H. FEBS 1996, 383, 37. (b) Liu, G.; Kozmina, N. S.;
Winn, M.; von Geldern, T. W.; Chiou, W. J.; Dixon, D. B.;
Nguyen, B.; Marsh, K. C.; Opgenorth, T. J. J. Med. Chem.
1999, 42, 3679.
7. (a) Clozel, M.; Breu, V.; Burri, K.; Cassal, J. M.; Fischli,
W.; Gray, G. A.; Hirth, G.; Loe‚er, B. M.; Mueller, M.;
Neidhart, W.; Ramuz, H. Nature 1993, 365, 759. (b) Elliott, J.
D.; Lago, M. A.; Cousins, R. D.; Gao, A.; Leber, J. D.;
Erhard, K. F.; Nambi, P.; Elshourbagy, N. A.; Kumar, C.;
Lee, J. A.; Bean, J. W.; Debrosse, C. W.; Eggleston, D. S.;
Brooks, D. P.; Feuerstein, E. H. J. Med. Chem. 1994, 37, 1553.
(c) Walsh, T. F.; Fitch, K. J.; Williams, D. L., Jr.; Murphy, K.
L.; Nolan, N. A.; Pettibone, D. J.; Chang, R. S. L.; O'Malley,
S. S.; Clineschmidt, B. V.; Veber, D. F.; Greenlee, W. J.
Bioorg. Med. Chem. Lett. 1995, 5, 1155. (d) Amberg, W.;
Hergenroeder, S.; Hillen, H.; Jansen, R.; Kettschau, G.; Kling,
A.; Klinge, D.; Raschack, M.; Riechers, H.; Unger, L. J. Med.
Chem. 1999, 42, 3026.
Acknowledgement
We would like to thank the Analytical Department
(HMR, Romainville) for performing the spectral analysis.
References and Notes
1. Zhang, J.; Didierlaurent, S.; Fortin, M.; Lefrancois, D.;
Uridat, E.; Vevert, J. P. Bioorg. Med. Chem. Lett. 2000, 10,
1351.
2. (a) Yanagisawa, M.; Kurihara, H.; Kimura, H.; Tomobe,
Y.; Kobayashi, M.; Mitsui, Y.; Goto, K.; Masaki, T. Nature
1988, 332, 411. (b) Doherty, A. M. J. Med. Chem. 1992, 35,
1493.
8. Didierlaurent, S.; Fortin, M.; Zhang, J. PCT WO 9715570,
1997.
9. Cox, A. In Comprehensive Organic Chemistry; Barton, D.,
Ollis, W. D., Sutherland, I. O., Eds.; Pergamon Press: New
York, 1969; Vol. 2, p. 702.
3. (a) Kosaka, T.; Suziki, N.; Matsumoto, H.; Itoh, Y.;
Yasuhara, T.; Onda, H.; Fujino, M. FEBS Lett. 1989, 249, 42.
10. Narisada, M.; Horibe, I.; Watanabe, F.; Takeda, K. J.
Org. Chem. 1989, 54, 5308.