U.S. patent application number 09/808771 was filed with the patent office on 2001-08-16 for substituted phenyl compounds and derivatives thereof that modulate the activity of endothelin.
Invention is credited to Balaji, Vitukudi Narayanaiyengar, Castillo, Rosario Silvestre, Chan, Ming Fai, Kois, Adam, Raju, Bore Gowda, Wu, Chengde.
Application Number | 20010014694 09/808771 |
Document ID | / |
Family ID | 27078927 |
Filed Date | 2001-08-16 |
United States Patent
Application |
20010014694 |
Kind Code |
A1 |
Chan, Ming Fai ; et
al. |
August 16, 2001 |
Substituted phenyl compounds and derivatives thereof that modulate
the activity of endothelin
Abstract
Methods, compositions, and compounds for modulating the activity
of an endothelin peptide are provided. The methods use compositions
that contain compounds of formula (I): 1 where X and Y are selected
from groups that include O, S, and NH; and Ar.sup.1, Ar.sup.2 and
Ar.sup.3 are independently selected from substituted or
unsubstituted groups that include 5 to 6 membered aryl groups and
heteroaryl groups that contain one or two heteroatom(s). The
methods are effected by contacting endothelin receptors with one or
more of the compounds or with compositions containing one or more
of the compounds prior to, simultaneously with, or subsequent to
contacting the receptors with an endothelin peptide.
Inventors: |
Chan, Ming Fai; (San Diego,
CA) ; Balaji, Vitukudi Narayanaiyengar; (Encinitas,
CA) ; Castillo, Rosario Silvestre; (San Diego,
CA) ; Kois, Adam; (San Diego, CA) ; Raju, Bore
Gowda; (San Diego, CA) ; Wu, Chengde; (San
Diego, CA) |
Correspondence
Address: |
Stephanie Seidman
Heller Ehrman White & McAuliffe LLP
4250 Executive Square, 7th Floor
La Jolla
CA
92037
US
|
Family ID: |
27078927 |
Appl. No.: |
09/808771 |
Filed: |
March 14, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09808771 |
Mar 14, 2001 |
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09327661 |
Jun 8, 1999 |
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09327661 |
Jun 8, 1999 |
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08590139 |
Jan 23, 1996 |
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5977117 |
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08590139 |
Jan 23, 1996 |
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08583871 |
Jan 5, 1996 |
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Current U.S.
Class: |
514/415 ;
514/646; 514/712; 514/717; 564/336; 568/39 |
Current CPC
Class: |
A61K 31/192 20130101;
C07D 317/64 20130101; A61K 31/36 20130101; C07D 405/14 20130101;
A61K 31/506 20130101; A61P 29/00 20180101; A61K 31/505 20130101;
C07D 239/60 20130101; Y10S 206/828 20130101 |
Class at
Publication: |
514/415 ;
514/646; 514/712; 514/717; 564/336; 568/39 |
International
Class: |
A61K 031/135; A61K
031/10; A61K 031/085; A61K 031/405 |
Claims
We claim:
1. A compound that has the formula (I): 14or a pharmaceutically
acceptable salt or ester thereof, wherein: X and Y are
independently selected from O, S, NR.sup.28, --(CH.sub.2).sub.v--,
--NR.sup.28(CH.sub.2).sub.v--, --S--(CH.sub.2).sub.v-- or
--O--(CH.sub.2).sub.v-- where v is 0 to 12, provided that, when
Ar.sup.3 is phenyl, at least one of X and Y is O, S or NR.sup.28;
R.sup.28 is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl,
haloalkyl, alkylaryl, heterocycle, arylalkyl, arylalkoxy, alkoxy,
aryloxy, cycloalkyl, cycloalkenyl and cycloalkynyl; Ar.sup.1 and
Ar.sup.2 are independently selected from among aryl and heteroaryl
groups containing one to three fused rings and from 3 up to about
21 members in the ring(s), in which the heteroaryl groups contain
one to three heteroatoms selected from O, S and N; and Ar.sup.3 is
independently selected from the aryl and heteroaryl groups set
forth for Ar.sup.1 and Ar.sup.2, X, Y, Ar.sup.1, Ar.sup.2 and
Ar.sup.3 are selected with the proviso that the resulting compound
does not have the formula (IV): 15in which R' is lower alkyl, COOH,
C(O)NR.sub.aR.sub.b, where R.sub.a is hydrogen or alkyl containing
1 to 6 carbon atoms in the chain, R.sub.b is alkyl containing 1 to
6 carbon atoms in the chain, OH, methoxy, cyanomethyl, or R.sub.a
and R.sub.b together form --(CH.sub.2).sub.x--, where x is 1 to
6.
2. The compound of claim 1 that has the formula (I): 16or a
pharmaceutically acceptable salt or ester thereof, wherein:
Ar.sup.3 is pyrazinyl, pyridazinyl, pyridyl, oxazolyl, isoxazolyl
or imiazolyl; X and Y are independently selected from O, S,
NR.sup.28, --(CH.sub.2).sub.v--, --NR.sup.28(CH.sub.2).sub.v--,
--S--(CH.sub.2).sub.v-- or --O--(CH.sub.2).sub.v-- where v is 0 to
12; R.sup.28 is selected from hydrogen, alkyl, alkenyl, alkynyl,
aryl, haloalkyl, alkylaryl, heterocyclyl, arylalkyl, arylalkoxy,
alkoxy, aryloxy, cycloalkyl, cycloalkenyl and cycloalkynyl;
Ar.sup.1 and Ar.sup.2 are independently selected from among aryl
and heteroaryl groups containing one or two to three fused rings
and from 3 up to about 21 members in the ring(s), in which the
heteroaryl groups contain one to three heteroatoms selected from O,
S and N.
3. The compound of claim 2, wherein Ar.sup.1 and Ar.sup.2 are each
independently selected from among aryl groups that contain 5 to 6
members in the ring and heteroaryl groups that contain 5 to 6
members in the ring and one or two heteroatom(s).
4. The compound of claim 2, wherein Ar.sup.1 and Ar.sup.2 are
independently selected from naphthyl, phenyl, biphenyl, quinolyl,
thienyl, furyl, isoquinolyl, pyrrolyl, pyridyl, indolyl,
oxadiazolyl, pyrazolyl, isoxazolyl, isothiazolyl, pyrimidyl,
benzo[b]furyl and benzo[b]thienyl.
5. The compound of claim 4, wherein Ar.sup.1 and Ar.sup.2 are
unsubstituted or are substituted with one or more substituents
selected from among alkyl, alkoxy, alkenyl, alkynyl, halo,
pseudohalo, (CH.sub.2).sub.qCOR.sup.16 in which q is 0 to 6,
(alkenyl).sub.rCOR.sup.1- 5 in which alkenyl is a straight or
branched carbon chain containing at least two carbons and one
unsaturated bond so that r, which is the number of carbons in the
chain, is 0 or 2 to 6, (CH.sub.2).sub.tOH in which t is 0 to 6,
(alkenyl).sub.uOH in which alkenyl is a straight or branched carbon
chain containing at least two carbons and one unsaturated bond so
that u is 0 or 2 to 6; R.sup.15 and R.sup.16 are each independently
hydrogen, alkyl, haloalkyl, aryl, aryloxy, heterocyclyl, arylalkyl,
arylalkoxy, cycloalkyl, cycloalkenyl, cycloalkynyl, OH, R.sup.20,
C(O)R.sup.20, CO.sub.2R.sup.20, SH, S(O).sub.nR.sup.20 in which n
is 0-2, HNOH, (CH.sub.2).sub.sR.sup.20 in which s is 1-6,
NR.sup.20R.sup.21, OR.sup.20, R.sup.21NCOR.sup.20 or
R.sup.21NSO.sub.2R.sup.20; R.sup.20 is selected from among
hydrogen, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heterocyclyl,
arylalkyl, cycloalkyl, cycloalkenyl or cycloalkynyl; and R.sup.21
is selected from among hydrogen, alkyl, alkenyl, alkynyl, aryl,
alkylaryl, alkoxy, aryloxy, heterocyclyl, arylalkyl, arylalkoxy,
cycloalkyl, cycloalkenyl and cycloalkynyl.
6. The compound of claim 4, wherein Ar.sup.3 is selected from among
pyrazinyl and pyridyl groups.
7. The compound of claim 2, wherein Ar.sup.3 is selected from among
pyrazinyl and pyridyl groups.
8. The compound of claim 6, wherein Ar.sup.3 is a pyrazinyl
group.
9. The compound of claim 6, wherein Ar.sup.3 is a pyridyl
group.
10. The compound of claim 7, wherein Ar.sup.3 is a pyrazinyl
group.
11. The compound of claim 7, wherein Ar.sup.3 is a pyridyl
group.
12. The compound of claim 2, wherein Ar.sup.1 and Ar.sup.2 are
selected from phenyl, thienyl, furyl, pyrrolyl, pyridyl, pyrazolyl,
isoxazolyl, isothiazolyl and pyrimidyl.
13. The compound of claim 6, wherein Ar.sup.1 and Ar.sup.2 are
selected from phenyl, thienyl, furyl, pyrrolyl, pyridyl, pyrazolyl,
isoxazolyl, isothiazolyl and pyrimidyl.
14. The compound of claim 7, wherein Ar.sup.1 and Ar.sup.2 are
selected from phenyl, thienyl, furyl, pyrrolyl, pyridyl, pyrazolyl,
isoxazolyl, isothiazolyl and pyrimidyl.
15. The compound of claim 2, wherein Ar.sup.3 is substituted with a
carboxyl group or an isostere thereof.
16. The compound of claim 6, wherein at least one of Ar.sup.1 and
Ar.sup.2 is substituted phenyl and at least one of X and Y is O or
S.
17. A pharmaceutical composition, comprising a compound of claim 1
or a pharmaceutically acceptable salt or ester of a compound of
claim 1 in a pharmaceutically acceptable carrier.
18. The pharmaceutical composition of claim 17 that is formulated
for single dosage administration.
19. A method for treating endothelin-mediated disorders, comprising
administering a therapeutically effective amount of a compound of
claim 1 or a pharmaceutically acceptable salt or ester thereof,
wherein the effective amount is sufficient to ameliorate one or
more of the symptoms of the disorder.
20. The method of claim 19, wherein the disorder is selected from
the group consisting of hypertension, cardiovascular disease,
asthma, pulmonary hypertension, inflammatory diseases,
ophthalmologic disease, menstrual disorders, obstetric conditions,
wounds, gastroenteric disease, renal failure,
immunosuppressant-mediated renal vasoconstriction,
erythropoietin-mediated vasoconstriction, endotoxin shock,
anaphylactic shock and hemorrhagic shock.
21. The method of claim 19, wherein the disorder is selected from
the group consisting of asthma and inflammatory diseases.
22. A method for inhibiting the binding of an endothelin peptide to
an endothelin receptor, comprising contacting the receptor with an
endothelin peptide and with one or more compounds of claim 1 or
pharmaceutically acceptable salts or esters thereof, wherein the
contacting is effected prior to, simultaneously with or subsequent
to contacting the receptors with the endothelin peptide.
23. The method of claim 22, wherein the endothelin receptor is an
endothelin.sub.A (ET.sub.A) or endothelin.sub.B (ET.sub.B)
receptor.
24. An article of manufacture, comprising packaging material and a
compound of claim 1 or a pharmaceutically acceptable salt or ester
thereof, wherein the compound is contained within the packaging
material; the compound, or salt or ester thereof, is effective for
antagonizing the effects of endothelin, ameliorating the symptoms
of an endothelin-mediated disorder, or inhibiting the binding of an
endothelin peptide to an endothelin receptor with an IC.sub.50 of
less than about 10 .mu.M, and the packaging material includes a
label that indicates that the compound or salt thereof is used for
antagonizing the effects of endothelin, ameliorating the symptoms
of an endothelin-mediated disorder, or inhibiting the binding of
endothelin to an endothelin receptor with an IC.sub.50 of less than
about 10 .mu.M.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 09/327,661, filed Jun. 8, 1999, entitled "SUBSTITUTED PHENYL
COMPOUNDS AND DERIVATIVES THEREOF THAT MODULATE THE ACTIVITY OF
ENDOTHELIN" to Chan et al.
[0002] U.S. application Ser. No. 09/327,661 is a continuation of
U.S. application Ser. No. 08/590,139, filed Jan. 23, 1996, entitled
"SUBSTITUTED PHENYL COMPOUNDS AND DERIVATIVES THEREOF THAT MODULATE
THE ACTIVITY OF ENDOTHELIN" to Chan et al., now U.S. Pat. No.
5,977,117, which is a continuation-in-part of U.S. application Ser.
No. 08/583,871, filed Jan. 5, 1996, entitled "SUBSTITUTED PHENYL
COMPOUNDS AND DERIVATIVES THEREOF THAT MODULATE THE ACTIVITY OF
ENDOTHELIN" to Chan et al., now abandoned.
FIELD OF THE INVENTION
[0003] The present invention relates to the compounds that modulate
the activity of the endothelin family of peptides. In particular,
the invention relates to substituted phenyl compounds, particularly
substituted benzoic acid compounds and derivatives thereof, and to
the use of such compounds as endothelin agonists and
antagonists.
BACKGROUND OF THE INVENTION
[0004] The vascular endothelium releases a variety of vasoactive
substances, including the endothelium-derived vasoconstrictor
peptide, endothelin (ET) (see, e.g., Vanhoutte et al. (1986) Annual
Rev. Physiol. 48: 307-320; Furchgott and Zawadski (1980) Nature
288: 373-376). Endothelin, which was originally identified in the
culture supernatant of porcine aortic endothelial cells (see,
Yanagisawa et al. (1988) Nature 332: 411-415), is a potent
twenty-one amino acid peptide vasoconstrictor. It is the most
potent vasopressor known and is produced by numerous cell types,
including the cells of the endothelium, trachea, kidney and brain.
Endothelin is synthesized as a two hundred and three amino acid
precursor preproendothelin that contains a signal sequence which is
cleaved by an endogenous protease to produce a thirty-eight (human)
or thirty-nine (porcine) amino acid peptide. This intermediate,
referred to as big endothelin, is processed in vivo to the mature
biologically active form by a putative endothelin-converting enzyme
(ECE) that appears to be a metal-dependent neutral protease (see,
e.g., Kashiwabara et al. (1989) FEBS Lttrs. 247: 337-340). Cleavage
is required for induction of physiological responses (see, e.g.,
von Geldern et al. (1991) Peptide Res. 4: 32-35). In porcine aortic
endothelial cells, the thirty-nine amino acid intermediate, big
endothelin, is hydrolyzed at the Trp.sup.21-Val.sup.22 bond to
generate endothelin-1 and a C-terminal fragment. A similar cleavage
occurs in human cells from a thirty-eight amino acid intermediate.
Three distinct endothelin isopeptides that exhibit potent
vasoconstrictor activity have been identified. They are
endothelin-1, endothelin-2 and endothelin-3.
[0005] The isopeptides are encoded by a family of three genes (see,
e.g., Inoue et al. (1989) Proc. Natl. Acad. Sci. USA 86: 2863-2867;
see, also Saida et al. (1989) J. Biol. Chem. 264: 14613-14616). The
nucleotide sequences of the three human genes are highly conserved
within the region encoding the mature 21 amino acid peptides and
the C-terminal portions of the peptides are identical. Endothelin-2
is (Trp.sup.6,Leu.sup.7) endothelin-1 and endothelin-3 is
(Thr.sup.2,Phe.sup.4,Thr.sup.5,Tyr.sup.6- ,Lys.sup.7,Tyr.sup.14)
endothelin-1. The endothelin peptides exhibit numerous biological
activities in vitro and in vivo. Endothelin provokes a strong and
sustained vasoconstriction in vivo in rats and in isolated vascular
smooth muscle preparations; it also provokes the release of
eicosanoids and endothelium-derived relaxing factor (EDRF) from
perfused vascular beds. Intravenous administration of endothelin-1
and in vitro addition to vascular and other smooth muscle tissues
produce long-lasting pressor effects and contraction, respectively
(see, e.g., Bolger et al. (1991) Can. J. Physiol. Pharmacol. 69:
406-413). In isolated vascular strips, for example, endothelin-1 is
a potent (EC.sub.50=4.times.10.sup.-- 10 M), slow acting, but
persistent, contractile agent. A single dose in vivo elevates blood
pressure in about twenty to thirty minutes. Endothelin-induced
vasoconstriction is not affected by antagonists to known
neurotransmitters or hormonal factors, but is abolished by calcium
channel antagonists. The effect of calcium channel antagonists,
however, is most likely the result of inhibition of calcium influx,
since calcium influx appears to be required for the long-lasting
contractile response to endothelin.
[0006] Endothelin also mediates renin release, stimulates ANP
release and induces a positive inotropic action in guinea pig
atria. In the lung, endothelin-1 acts as a potent
bronchoconstrictor (Maggi et al. (1989) Eur. J. Pharmacol. 160:
179-182). Endothelin increases renal vascular resistance, decreases
renal blood flow, and decreases glomerular filtrate rate. It is a
potent mitogen for glomerular mesangial cells and invokes the
phosphoinositide cascade in such cells (Simonson et al. (1990) J.
Clin. Invest. 85: 790-797). Release of endothelins from cultured
endothelial cells is modulated by a variety of chemical and
physical stimuli and appears to be regulated at the level of
transcription and/or translation. Expression of the gene encoding
endothelin-1 is increased by chemical stimuli, including
adrenaline, thrombin and Ca.sup.2+ ionophore. The production and
release of endothelin from the endothelium is stimulated by
angiotensin II, vasopressin, endotoxin, cyclosporine and other
factors (see, Brooks et al. (1991) Eur. J. Pharm. 194:115-117), and
is inhibited by nitric oxide. Endothelial cells appear to secrete
short-lived endothelium-derived relaxing factors (EDRF), including
nitric oxide or a related substance (Palmer et al. (1987) Nature
327: 524-526), when stimulated by vasoactive agents, such as
acetylcholine and bradykinin. Endothelin-induced vasoconstriction
is also attenuated by atrial natriuretic peptide (ANP).
[0007] There are specific high affinity binding sites (dissociation
constants in the range of 2-6.times.10.sup.-10 M) for the
endothelins in the vascular system and in other tissues, including
the intestine, heart, lungs, kidneys, spleen, adrenal glands and
brain. Binding is not inhibited by catecholamines, vasoactive
peptides, neurotoxins or calcium channel antagonists. Endothelin
binds and interacts with receptor sites that are distinct from
other autonomic receptors and voltage dependent calcium channels.
Competitive binding studies indicate that there are multiple
classes of receptors with different affinities for the endothelin
isopeptides. The sarafotoxins, a group of peptide toxins from the
venom of the snake Atractaspis eingadensis that cause severe
coronary vasospasm in snake bite victims, have structural and
functional homology to endothelin-1 and bind competitively to the
same cardiac membrane receptors (Kloog et al. (1989) Trends
Pharmacol. Sci. 10: 212-214).
[0008] Two distinct endothelin receptors, designated ET.sub.A and
ET.sub.B, have been identified and DNA clones encoding each
receptor have been isolated (Arai et al. (1990) Nature 348:
730-732; Sakurai et al. (1990) Nature 348: 732-735). Based on the
amino acid sequences of the proteins encoded by the cloned DNA, it
appears that each receptor contains seven membrane spanning domains
and exhibits structural similarity to G-protein-coupled membrane
proteins. Messenger RNA encoding both receptors has been detected
in a variety of tissues, including heart, lung, kidney and brain.
The distribution of receptor subtypes is tissue specific (Martin et
al. (1989) Biochem. Biophys. Res. Commun. 162: 130-137). ET.sub.A
receptors appear to be selective for endothelin-1 and are
predominant in cardiovascular tissues. ET.sub.B receptors are
predominant in noncardiovascular tissues, including the central
nervous system and kidney, and interact with the three endothelin
isopeptides (Sakurai et al. (1990) Nature 348: 732-734). In
addition, ET.sub.A receptors occur on vascular smooth muscle, are
linked to vasoconstriction and have been associated with
cardiovascular, renal and central nervous system diseases; whereas
ET.sub.B receptors are located on the vascular endothelium, linked
to vasodilation (Takayanagi et al. (1991) FEBS Lttrs. 282: 103-106)
and have been associated with bronchoconstrictive disorders.
[0009] By virtue of the distribution of receptor types and the
differential affinity of each isopeptide for each receptor type,
the activity of the endothelin isopeptides varies in different
tissues. For example, endothelin-1 inhibits .sup.125I-labelled
endothelin-1 binding in cardiovascular tissues forty to seven
hundred times more potently than endothelin-3. .sup.125I-labelled
endothelin-1 binding in non-cardiovascular tissues, such as kidney,
adrenal gland, and cerebellum, is inhibited to the same extent by
endothelin-1 and endothelin-3, which indicates that ET.sub.A
receptors predominate in cardiovascular tissues and ET.sub.B
receptors predominate in non-cardiovascular tissues.
[0010] Endothelin plasma levels are elevated in certain disease
states (see, e.g., International PCT Application WO 94/27979, and
U.S. Pat. No. 5,382,569, which disclosures are herein incorporated
in their entirety by reference). Endothelin-1 plasma levels in
healthy individuals, as measured by radioimmunoassay (RIA), are
about 0.26-5 pg/ml. Blood levels of endothelin-1 and its precursor,
big endothelin, are elevated in shock, myocardial infarction,
vasospastic angina, kidney failure and a variety of connective
tissue disorders. In patients undergoing hemodialysis or kidney
transplantation or suffering from cardiogenic shock, myocardial
infarction or pulmonary hypertension levels as high as 35 pg/ml
have been observed (see, Stewart et al. (1991) Annals Internal Med.
114: 464-469). Because endothelin is likely to be a local, rather
than a systemic, regulating factor, it is probable that the levels
of endothelin at the endothelium/smooth muscle interface are much
higher than circulating levels.
[0011] Elevated levels of endothelin have also been measured in
patients suffering from ischemic heart disease (Yasuda et al.
(1990) Amer. Heart J. 119:801-806, Ray et al. (1992) Br. Heart J.
67:383-386). Circulating and tissue endothelin immunoreactivity is
increased more than twofold in patients with advanced
atherosclerosis (Lerman et al. (1991) New Engl. J. Med.
325:997-1001). Increased endothelin immunoreactivity has also been
associated with Buerger's disease (Kanno et al. (1990) J. Amer.
Med. Assoc. 264:2868) and Raynaud's phenomenon (Zamora et al.
(1990) Lancet 336 1144-1147). Increased circulating endothelin
levels were observed in patients who underwent percutaneous
transluminal coronary angioplasty (PTCA) (Tahara et al. (1991)
Metab. Clin. Exp. 40:1235-1237; Sanjay et al. (1991) Circulation
84(Suppl. 4):726), and in individuals with pulmonary hypertension
(Miyauchi et al. (1992) Jpn. J. Pharmacol.58:279P; Stewart et al.
(1991) Ann.Internal Medicine 114:464-469). Thus, there is clinical
human data supporting the correlation between increased endothelin
levels and numerous disease states.
[0012] Endothelin Agonists and Antagonists
[0013] Because endothelin is associated with certain disease states
and is implicated in numerous physiological effects, compounds that
can interfere with endothelin-associated activities, such as
endothelin-receptor interaction and vasoconstrictor activity, are
of interest. Compounds that exhibit endothelin antagonistic
activity have been identified. For example, a fermentation product
of Streptomyces misakiensis, designated BE-18257B, has been
identified as an ET.sub.A receptor antagonist. BE-18257B is a
cyclic pentapeptide, cyclo(D-Glu-L-Ala-allo-D-Ile-L-Leu-D-Trp),
which inhibits .sup.125I-labelled endothelin-1 binding in
cardiovascular tissues in a concentration-dependent manner
(IC.sub.50 1.4 .mu.M in aortic smooth muscle, 0.8 .mu.M in
ventricle membranes and 0.5 .mu.M in cultured aortic smooth muscle
cells), but fails to inhibit binding to receptors in tissues in
which ET.sub.B receptors predominate at concentrations up to 100
.mu.M. Cyclic pentapeptides related to BE-18257B, such as
cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123), have been synthesized
and shown to exhibit activity as ET.sub.A receptor antagonists
(see, U.S. Pat. No. 5,114,918 to Ishikawa et al.; see, also, EP A1
0 436 189 to BANYU PHARMACEUTICAL CO., LTD (Oct. 7, 1991)). Studies
that measure the inhibition by these cyclic peptides of
endothelin-1 binding to endothelin-specific receptors indicate that
these cyclic peptides bind preferentially to ET.sub.A receptors.
Other peptidic and non-peptidic ET.sub.A antagonists have been
identified (see, e.g., U.S. Pat. Nos. 5,352,800, 5,334,598,
5,352,659, 5,248,807, 5,240,910, 5,198,548, 5,187,195, 5,082,838).
These include other cyclic peptides, acyltripeptides, hexapeptide
analogs, certain anthraquinone derivatives, indanecarboxylic acids,
certain N-pyrimidylbenzenesulfonamides, certain
benzenesulfonamides, and certain naphthalenesulfonamides (see,
e.g., Nakajima et al. (1991) J. Antibiot. 44:1348-1356; Miyata et
al. (1992) J. Antibiot. 45:74-8; Ishikawa et al. (1992) J.Med.
Chem. 35:2139-2142; U.S. Pat. No. 5,114,918 to Ishikawa et al.; EP
A1 0 569 193; EP A1 0 558 258; EP A1 0 436 189 to BANYU
PHARMACEUTICAL CO., LTD (Oct. 7, 1991); Canadian Patent Application
2,067,288; Canadian Patent Application 2,071,193; U.S. Pat. No.
5,208,243; U.S. Pat. No. 5,270,313; Cody et al. (1993) Med. Chem.
Res. 3:154-162; Miyata et al. (1992) J. Antibiot 45:1041-1046;
Miyata et al. (1992) J. Antibiot 45:1029-1040, Fujimoto et al.
(1992) FEBS Lett. 305:41-44; Oshashi et al. (1002) J. Antibiot
45:1684-1685; EP A1 0 496 452; Clozel et al. (1993) Nature
365:759-761; International Patent Application WO93/08799; Nishikibe
et al. (1993) Life Sci. 52:717-724; and Benigni et al. (1993)
Kidney Int. 44:440-444, U.S. Pat. No. 5,464,853 and others). In
general, these identified compounds have activities in vitro assays
as ET.sub.A antagonists at concentrations on the order of about 50
.mu.M - 100 .mu.M or less. A number of such compounds have also
been shown to possess activity in vivo animal models. Very few, if
any, selective ET.sub.B antagonists have been described.
[0014] Endothelin Agonists and Antagonists as Therapeutic Agents It
has been recognized that compounds that exhibit activity at
IC.sub.50 or EC.sub.50 concentrations on the order of 10.sup.-4M or
lower in standard in vitro assays that assess endothelin antagonist
or agonist activity have pharmacological utility (see, e.g., U.S.
Pat. Nos. 5,352,800, 5,334,598, 5,352,659, 5,248,807, 5,240,910,
5,198,548, 5,187,195, 5,082,838 and 5,464,853). By virtue of this
activity, such compounds are considered to be useful for the
treatment of hypertension such as peripheral circulatory failure,
heart disease such as angina pectoris, cardiomyopathy,
arteriosclerosis, myocardial infarction, pulmonary hypertension,
vasospasm, vascular restenosis, Raynaud's disease, cerebral stroke
such as cerebral arterial spasm, cerebral ischemia, late phase
cerebral spasm after subarachnoid hemorrhage, asthma,
bronchoconstriction, renal failure, particularly post-ischemic
renal failure, cyclosporine nephrotoxicity such as acute renal
failure, colitis, as well as other inflammatory diseases, endotoxic
shock caused by or associated with endothelin, and other diseases
in which endothelin has been implicated.
[0015] Thus, in view of the numerous physiological effects of
endothelin and its association with certain diseases, endothelin is
believed to play a critical role in these pathophysiological
conditions (see, e.g., Saito et al. (1990) Hypertension 15:
734-738; Tomita et al. (1989) N. Engl. J. Med. 321: 1127; Kurihara
et al. (1989) J. Cardiovasc. Pharmacol. 13(Suppl. 5): S13-S17;
Doherty (1992) J. Med. Chem. 35: 1493-1508; Morel et al. (1989)
Eur. J. Pharmacol. 167: 427-428). More detailed knowledge of the
function and structure of the endothelin peptide family should
provide insight in the progression and treatment of such
conditions.
[0016] To aid in gaining further understanding of and to develop
treatments for endothelin-mediated or related disorders, there is a
need to identify compounds that modulate or alter endothelin
activity. Identification of compounds that modulate endothelin
activity, such as compounds that act as specific antagonists or
agonists, may not only aid in elucidating the function of
endothelin, but may yield in therapeutically useful compounds. In
particular, compounds that specifically interfere with the
interaction of endothelin peptides with ET.sub.A, ET.sub.B or other
receptors should be useful in identifying essential characteristics
of endothelin peptides, should aid in the design of therapeutic
agents, and may be useful as disease specific therapeutic
agents.
[0017] Therefore, it is an object herein to provide compounds that
have the ability to modulate the biological activity of one or more
of the endothelin isopeptides. It is another object to provide
compounds that have use as endothelin antagonists. It is also an
object to use compounds that specifically interact with or inhibit
the interaction of endothelin peptides with endothelin receptors as
therapeutic agents for the treatment of endothelin-mediated
diseases and disorders, and/or as reagents for the identification
of endothelin receptor subtypes and for the elucidation of the
physiological and pathophysiological roles of endothelin.
SUMMARY OF THE INVENTION
[0018] Methods, compositions, and compounds for modulating the
activity of an endothelin peptide are provided. In particular the
methods use compositions that contain compounds that modulate the
interaction of an endothelin peptide with ET.sub.A and/or ET.sub.B
receptors. The methods are effected by contacting endothelin
receptors with one or more of the compounds or with compositions
containing one or more of the compounds prior to, simultaneously
with, or subsequent to contacting the receptors with an endothelin
peptide.
[0019] The compounds provided herein that are used in the
compositions and methods have formula (I): 2
[0020] where:
[0021] X and Y are independently selected from O, S, NR.sup.28,
--(CH.sub.2).sub.v--, --NR.sup.28(CH.sub.2).sub.v--,
--S--(CH.sub.2).sub.v-- or --O--(CH.sub.2).sub.v-- where v is 0 to
12, preferably 0 to 6, more preferably 0 to 3, provided that, when
Ar.sup.3 is phenyl, at least one of X and Y is O, S or
NR.sup.28;
[0022] R.sup.28 is selected from hydrogen, alkyl, alkenyl, alkynyl,
aryl, haloalkyl, alkylaryl, heterocyclyl, arylalkyl, arylalkoxy,
alkoxy, aryloxy, cycloalkyl, cycloalkenyl and cycloalkynyl, and is
preferably hydrogen, lower alkyl, lower alkoxy and lower
haloalkyl;
[0023] Ar.sup.1 and Ar.sup.2 are independently selected from among
aryl and heteroaryl groups containing one or more, preferably one
ring or two to three fused rings and from 3 up to about 21 members
in the ring(s), in which the heteroaryl groups contain one or two
to three heteroatoms selected from among O, S and N. In particular
Ar.sup.1 and Ar.sup.2 are independently selected from substituted
or unsubstituted groups that include, but are not limited to, the
following: naphthyl, phenyl, biphenyl, quinolyl, thienyl, furyl,
isoquinolyl, pyrrolyl, pyridyl, indolyl, oxadiazolyl, pyrazolyl,
isoxazolyl, isothiazolyl, pyrimidyl, benzo[b]furan, benzo[b]thienyl
and other such aryl and heteroaryl groups. Preferred among these
groups are 5 to 6 membered aryl groups and heteroaryl groups that
contain one or two heteroatom(s).
[0024] Ar.sup.1 and Ar.sup.2 are unsubstituted or are substituted
with one or more substituents, preferably selected from among
alkyl, alkoxy, alkenyl, alkynyl, halo, pseudohalo,
(CH.sub.2).sub.qCOR.sup.16 in which q is 0 to 6, preferably 0 to 3,
more preferably 0 or 1, (alkenyl).sub.rCOR.sup.15 in which alkenyl
is a straight or branched carbon chain containing at least two
carbons and one unsaturated bond so that r is 0 or 2 to 6,
preferably 0, 2 or 3, tetrazolyl, (CH.sub.2).sub.tOH in which t is
0 to 6, preferably 0 to 3, more preferably 0 or 1,
(alkenyl).sub.uOH in which alkenyl is a straight or branched carbon
chain containing at least two carbons and one unsaturated bond so
that u is 0 or 2 to 6, preferably 0, 2 or 3;
[0025] R.sup.15 and R.sup.16 are independently hydrogen, alkyl,
haloalkyl, aryl, aryloxy, heterocyclyl, arylalkyl, arylalkoxy,
cycloalkyl, cycloalkenyl, cycloalkynyl, OH, R.sup.20, C(O)R.sup.20,
CO.sub.2R.sup.2 , SH, S(O).sub.nR.sup.20 in which n is 0-2, HNOH,
(CH.sub.2).sub.sH, (CH.sub.2).sub.sR.sup.20 in which s is 1-6,
NR.sup.20R.sup.21, OR.sup.20, R.sup.21NCOR.sup.20 and
R.sup.21NSO.sub.2R.sup.20;
[0026] R.sup.20 is selected from among hydrogen, alkyl, alkenyl,
alkynyl, aryl, alkylaryl, heterocyclyl, arylalkyl, cycloalkyl,
cycloalkenyl or cycloalkynyl, where R.sup.20 is preferably alkyl or
aryl; and
[0027] R.sup.21 is selected from among hydrogen, alkyl, alkenyl,
alkynyl, aryl, alkylaryl, alkoxy, aryloxy, heterocyclyl, arylalkyl,
arylalkoxy, cycloalkyl, cycloalkenyl, cycloalkynyl.
[0028] Ar.sup.3 is independently selected from aryl and heteroaryl
groups set forth for Ar.sup.1 and Ar.sup.2. Ar.sup.3 is
particularly selected from among single ring aromatic groups that
contain from 3 to 7, preferably 4 to 6, more preferably 5 or 6
members. In particular, Ar.sup.3 is aryl or is a heterocycle
containing one or two heteroatoms, preferably nitrogen. Thus,
Ar.sup.3 is preferably selected from among phenyl, pyrimidyl,
pyrazinyl, pyridazinyl, pyridyl, oxazolyl, isoxazolyl and
imidazolyl groups. Ar.sup.3 is more preferably phenyl, pyridyl,
pyrimidyl or pyrazinyl and is preferably substituted with an acidic
group, particularly a carboxyl group or an isostere thereof.
[0029] It is noted that the compounds for use in the compositions
do not have the formula (IV): 3
[0030] in which R' is lower alkyl, COOH, C(O)NR.sub.aR.sub.b, where
R.sub.a is hydrogen or C.sub.1-6-alkyl, R.sub.b is C.sub.1-6-alkyl,
OH, methoxy, cyanomethyl, or R.sub.a and R.sub.b together form
--(CH.sub.2).sub.x--, where x is 1 to 6. In the preferred compounds
R' is also (C.sub.1-6-alkyl)COOH. Compounds of formula IV, however,
may be used in the methods.
[0031] More preferred among the compounds that are used in the
methods are those of formula (I) that have formula (II): 4
[0032] where:
[0033] R.sup.1 is hydrogen or --(CH.sub.2).sub.n--A in which n is 0
to 6, preferably 0 to 3, and A is an acidic group, A is preferably
a group such as tetrazolyl or C(O)OR.sup.4 in which R.sup.4 is
hydrogen, lower alkyl or haloalkyl;
[0034] j, o and p are independently 0 or 1, and j is preferably
0;
[0035] R.sup.2, R.sup.3 and R.sup.22 are each independently
selected from alkyl, alkenyl, halo, haloalkyl, alkoxy, -S-alkyl,
-NR.sup.29-alkyl, aryl or heteroaryl, which are preferably single
rings that contain 4 to 7, more preferably 5 or 6, members in the
ring;
[0036] R.sup.29 is selected from hydrogen, alkyl, alkenyl, alkynyl,
aryl, haloalkyl, alkylaryl, heterocyclyl, arylalkyl, arylalkoxy,
alkoxy, arylalkoxy, cycloalkyl, cycloalkenyl and cycloalkynyl, and
is preferably hydrogen, lower alkyl, lower alkoxy and lower
haloalkyl; and
[0037] X, Y, Ar.sup.1 and Ar.sup.2 are as defined above.
[0038] Preferred compounds for use in the compositions have formula
(I) and (II), but with the proviso that the compounds do not have
formula (IV). In particular, when Ar.sup.3 is phenyl, X is O or S,
Y is O or S, and Ar.sup.1 and Ar.sup.2 are unsubstituted or
substituted with halogen or lower alkyl, then R.sup.1 is not COOH
or C(O)NR.sub.aR.sub.b, where R.sub.a is hydrogen or
C.sub.1-6-alkyl, R.sub.b is C.sub.1-6-alkyl, OH, methoxy,
cyanomethyl, or R.sub.a and R.sub.b together form
--(CH.sub.2).sub.x--, where x is 1 to 6.
[0039] In preferred compounds, R.sup.1 is selected from hydrogen
and --(CH.sub.2).sub.n--A in which n is 0 to 6,
--(CH.sub.2).sub.q(CO.sub.2R.- sup.4), --(CH.sub.2).sub.q(OH), CN,
--C(R.sup.7).dbd.NOR.sup.8, NO.sub.2, --(CH.sub.2).sub.qR.sup.9,
--C.ident.CR.sup.10, --CR.sup.11.dbd.C(R.sup.1- 2)(R.sup.13),
tetrazolyl, CONR.sup.27R.sup.26, --(CH.sub.2).sub.qC(.dbd.O)-
CH.sub.2C(.dbd.O)CO.sub.2H, --CO(R.sup.14), alkylthio,
alkylsulfinyl, alkylsulfonyl, carbamoyl, thiocarbamoyl, or a
nitrogen-containing ring, where:
[0040] A is selected from among CO.sub.2R.sup.4, carboxylic acid,
carboxamide, alkylthioic acid, alkyldithoic acid, alkylimidic acid,
sulfinic acid, sulfonic acid, phosphonic, sulfinimidic acid,
sulfonimidic acid, sulfonamide, alkylhydroxamic acid, hydrazide,
amide, hydroxyl, hydrogen,
[0041] q is 0 to 12, preferably 0 to 6;
[0042] R.sup.4 is hydrogen, lower alkyl or haloalkyl;
[0043] R.sup.7 is selected from hydrogen, alkyl or haloalkyl;
[0044] R.sup.8 is hydrogen, arylalkyl or -(lower
alkyl)CO.sub.2R.sup.17;
[0045] R.sup.9 is --CN, --CO.sub.2R.sup.19, --CH.sub.2OH, or
carbamoyl;
[0046] R.sup.10 is --CO.sub.2H or carboxyphenyl;
[0047] R.sup.11 is hydrogen, alkyl or arylalkyl;
[0048] R.sup.12 and R.sup.13 are independently hydrogen,
--CO.sub.2R.sup.18, --CN, aryl, lower alkyl, heteroaryl lower alkyl
or --NHC(O)aryl, provided that one of R.sup.12 and R.sup.13 is
--CO.sub.2H;
[0049] R.sup.14 is hydrogen, alkyl, -(lower alkyl)carboxy,
arylalkenyl, heteroarylalkenyl or --CO.sub.2H;
[0050] R.sup.17, R.sup.18 and R.sup.19 are independently selected
from hydrogen, alkyl or haloalkyl;
[0051] R.sup.26 and R.sup.27 are each independently selected from
hydrogen, alkyl, alkenyl, alkynyl, aryl, haloalkyl, alkylaryl,
heterocyclyl, arylalkyl, arylalkoxy, alkoxy, aryloxy, cycloalkyl,
cycloalkenyl and cycloalkynyl;
[0052] j, o and p are each independently 0 or 1;
[0053] R.sup.2, R.sup.3 and R.sup.22 are each independently
selected from alkyl, alkenyl, halo, haloalkyl, alkoxy, -S-alkyl,
-NR.sup.29-alkyl, aryl or heteroaryl; and
[0054] R.sup.29 is selected from hydrogen, alkyl, alkenyl, alkynyl,
aryl, haloalkyl, alkylaryl, heterocyclyl, arylalkyl, arylalkoxy,
alkoxy, arylalkoxy, cycloalkyl, cycloalkenyl and cycloalkynyl.
[0055] In preferred embodiments, the compounds of formula (I) for
use in the methods and compositions have formula (III): 5
[0056] where Ar.sup.1, Ar.sup.2, R.sup.1, R.sup.2, X, Y and o are
as defined above and Ar.sup.3 is pyrimidyl or phenyl.
[0057] In more preferred embodiments, at least one of Ar.sup.1 and
Ar.sup.2 is substituted phenyl and at least one of X and Y is O or
S. Ar.sup.3 is preferably phenyl or pyrimidyl, more preferably
phenyl. Preferred compounds for use in the methods and compositions
are carboxcylic acid derivatives R.sup.1 is
(CH.sub.2).sub.qCO.sub.2R.sup.4, as defined above) and
pharmaceutically acceptable esters and salts thereof, but with the
proviso that the compounds do not have formula (IV). In particular,
when X and Y are O, and Ar.sup.1 and Ar.sup.2 are unsubstituted or
substituted with halogen or lower alkyl, R.sup.1 is not COOH or
C(O)NR.sub.aR.sub.b, where R.sub.a is hydrogen or C.sub.1-6-alkyl,
R.sub.b is C.sub.1-6-alkyl, OH, methoxy, cyanomethyl, or R.sub.a
and R.sub.b together form --(CH.sub.2).sub.x--, where x is 1 to 6.
In preferred embodiments, alkyl, alkenyl and alkynyl groups
preferably contain six or fewer carbon atoms, such as 1 to 3 or
fewer.
[0058] In the above compounds, the alkyl, alkynyl and alkenyl
portions of each listed substituent are straight or branched
chains. Preferably these groups are lower alkyl, alkynyl and
alkenyl groups having from about 1 (or 2 for alkynyl and alkenyl)
up to about 12 carbons; in more preferred embodiments they have
from 1 (or 2) to 6 carbons. The aryl and heterocyclyl groups are
single or fused rings, having from 3 to 21, generally, 3 to 7, more
often 4 to 7 members, with preferably 5 or 6 members in the rings,
and are preferably single or double fused rings and more preferably
single rings with 4 to 7 members in the ring.
[0059] In all instances, the ring size and carbon chain length are
selected up to a size such that the resulting molecule
competitively inhibits binding of an endothelin peptide, preferably
ET-1, to an endothelin receptor, preferably ET.sub.A or ET.sub.B,
preferably such that the binding of an endothelin peptide to the
endothelin receptor is inhibited by 50%, compared to binding in the
absence of the compound, at a concentration (IC.sub.50) of less
than about 100 .mu.M, when measured as described herein [see
EXAMPLES].
[0060] Of the compounds described herein, of particular interest
are those that inhibit an endothelin-mediated activity by about 50%
at concentrations of less than about 50 .mu.M. More preferred are
those that inhibit an endothelin-mediated activity by about 50% at
concentrations of less than about 10 .mu.M and more preferably at
concentrations of less than about 1 .mu.M. The preferred IC.sub.50
concentrations are set forth with reference to the in vitro assays
exemplified herein. It is understood that these IC.sub.50
concentrations vary from assay to assay. For example, it is noted
that, as described below, the IC.sub.50 concentration determined in
the in vitro assays is a non-linear function of incubation
temperature. The preferred values recited herein refer to the
assays that are performed at 24.degree. C. It is noted however,
that, when the assays are performed at 24.degree. C., somewhat
higher IC.sub.50 concentrations are observed than when they are
performed at 4.degree. C. Accordingly, when the assay is performed
at 24.degree. C., the preferred IC.sub.50 concentrations are about
10-fold higher than when the assay is performed at 4.degree. C.
[0061] Also among the most preferred compounds, for use in the
methods provided herein, are those that are ET.sub.A or ET.sub.B
selective. A compound is considered selective for a particular
endothelin receptor subtype if it inhibits endothelin binding to
the receptor at an IC.sub.50 concentration that is at least 10-fold
lower than its IC.sub.50 concentration for other endothelin
receptor subtypes. In particular, compounds that interact with
ET.sub.A receptors with an IC.sub.50 of less than about 10 .mu.M,
preferably less than 1 .mu.M, but with ET.sub.B receptors with an
IC.sub.50 of greater than about 10 .mu.M or compounds that interact
with ET.sub.B receptors with an IC.sub.50 of less than about 10
.mu.M, preferably less than 1 .mu.M, but with ET.sub.A receptors
with an IC.sub.50 of greater than about 10 .mu.M are preferred.
[0062] Pharmaceutical compositions formulated for administration by
an appropriate route and by appropriate means, containing effective
concentrations of one or more of the compounds provided herein or
pharmaceutically acceptable salts or esters thereof, that deliver
amounts effective for the treatment of hypertension, stroke,
asthma, shock, ocular hypertension, glaucoma, renal failure,
inadequate retinal perfusion or other conditions that are in some
manner mediated by an endothelin peptide or that involve
vasoconstriction or whose symptoms can be ameliorated by
administration of an endothelin antagonist or agonist, are also
provided. Particularly preferred compositions are those that
deliver amounts effective for the treatment of hypertension or
renal failure. The effective amounts and concentrations are
effective for ameliorating any of the symptoms of any of the
disorders.
[0063] Methods for treatment of endothelin-mediated disorders,
including but not limited to, hypertension, asthma, shock, ocular
hypertension, glaucoma, inadequate retinal perfusion and other
conditions that are in some manner mediated by an endothelin
peptide, or for treatment of disorders that involve
vasoconstriction or that are ameliorated by administration of an
endothelin antagonist or agonist are provided.
[0064] In particular, methods of treating endothelin-mediated
disorders by administering effective amounts of the compounds,
prodrugs or other suitable derivatives of the compounds are
provided. Such disorders include but are not limited to
hypertension, cardiovascular diseases, cardiac diseases including
myocardial infarction, pulmonary hypertension,
erythropoietin-mediated hypertension, respiratory diseases and
inflammatory diseases, including asthma, bronchoconstriction,
ophthalmologic diseases, gastroenteric diseases, renal failure,
endotoxin shock, menstrual disorders, obstetric conditions, wounds,
anaphylactic shock, hemorrhagic shock, and other diseases in which
endothelin mediated physiological responses are implicated.
Treatment involves, for instance, administration of effective
amounts of one or more of the compounds provided herein in
pharmaceutically acceptable carriers as provided herein. Preferred
methods of treatment are methods for treatment of hypertension and
renal failure.
[0065] More preferred methods of treatment are those in which the
compositions contain at least one compound that inhibits the
interaction of endothelin-1 with ET.sub.A or ET.sub.B receptors at
an IC.sub.50 of less than about 10 M, preferably less than about 5
.mu.M and more preferably less than about 1 .mu.M. Other preferred
methods are those in which the compositions contain one or more
compounds that is (are) ET.sub.A selective or one or more compounds
that is (are) ET.sub.B selective. Methods in which the compounds
are ET.sub.A selective are for treatment of disorders, such as
hypertension; and methods in which the compounds are ET.sub.B
selective are for treatment of disorders, such as asthma, that
require bronchodilation.
[0066] In practicing the methods, effective amounts of compositions
containing therapeutically effective concentrations of the
compounds formulated for oral, intravenous, local and topical
application for the treatment of hypertension, cardiovascular
diseases, cardiac diseases, including myocardial infarction,
respiratory diseases, including asthma, inflammatory diseases,
ophthalmologic diseases, gastroenteric diseases, renal failure,
immunosuppressant-mediated renal vasoconstriction,
erythropoietin-mediated vasoconstriction, endotoxin shock,
anaphylactic shock, hemorrhagic shock, pulmonary hypertension, and
other diseases in which endothelin mediated physiological responses
are implicated are administered to an individual exhibiting the
symptoms of one or more of these disorders. The amounts are
effective to ameliorate or eliminate one or more symptoms of the
disorders.
[0067] Methods for the identification and isolation of endothelin
receptor subtypes are also provided. In particular, methods for
detecting, distinguishing and isolating endothelin receptors using
the disclosed compounds are provided.
[0068] In addition, methods for identifying compounds that are
suitable for use in treating particular diseases based on their
preferential affinity for a particular endothelin receptor subtype
are provided.
[0069] Also provided are methods for elucidating the physiological
and/or pathophysiological roles of endothelin using the compounds
disclosed herein.
[0070] Articles of manufacture containing packaging material, a
compound provided herein, which is effective for ameliorating the
symptoms of an endothelin-mediated disorder, antagonizing the
effects of endothelin or inhibiting binding of an endothelin
peptide to an ET receptor with an IC.sub.50 of less than about 50
.mu.M, within the packaging material, and a label that indicates
that the compound or salt thereof is used for antagonizing the
effects of endothelin, treating an endothelin-mediated disorder, or
inhibiting the binding of an endothelin peptide to an ET receptor
are provided.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0071] Definitions
[0072] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which this invention belongs. All patents
and publications referred to herein are incorporated by
reference.
[0073] As used herein, endothelin (ET) peptides include peptides
that have substantially the amino acid sequence of endothelin-1,
endothelin-2 or endothelin-3 and that act as potent endogenous
vasoconstrictor peptides.
[0074] As used herein, an endothelin-mediated condition is a
condition that is caused by abnormal endothelin activity or one in
which compounds that inhibit endothelin activity have therapeutic
use. Such diseases include, but are not limited to hypertension,
cardiovascular disease, asthma, inflammatory diseases,
ophthalmologic disease, menstrual disorders, obstetric conditions,
gastroenteric disease, renal failure, pulmonary hypertension,
endotoxin shock, anaphylactic shock, or hemorrhagic shock.
Endothelin-mediated conditions also include conditions that result
from therapy with agents, such as erythropoietin and
immunosuppressants, that elevate endothelin levels.
[0075] As used herein an effective amount of a compound for
treating a particular disease is an amount that is sufficient to
ameliorate, or in some manner reduce the symptoms associated with
the disease. Such amount may be administered as a single dosage or
may be administered according to a regimen, whereby it is
effective. The amount may cure the disease but, typically, is
administered in order to ameliorate the symptoms of the disease.
Typically, repeated administration is required to achieve the
desired amelioration of symptoms.
[0076] As used herein, an endothelin agonist is a compound that
potentiates or exhibits a biological activity associated with or
possessed by an endothelin peptide.
[0077] As used herein, an endothelin antagonist is a compound, such
as a drug or an antibody, that inhibits endothelin-stimulated
vasoconstriction and contraction and other endothelin-mediated
physiological responses. The antagonist may act by interfering with
the interaction of the endothelin with an endothelin-specific
receptor or by interfering with the physiological response to or
bioactivity of an endothelin isopeptide, such as vasoconstriction.
Thus, as used herein, an endothelin antagonist interferes with
endothelin-stimulated vasoconstriction or other response or
interferes with the interaction of an endothelin with an
endothelin-specific receptor, such as ET.sub.A receptors, as
assessed by assays known to those of skill in the art.
[0078] The effectiveness of potential agonists and antagonists can
be assessed using methods known to those of skill in the art. For
example, endothelin agonist activity can be identified by its
ability to stimulate vasoconstriction of isolated rat thoracic
aorta or portal vein ring segments (Borges et al. (1989) "Tissue
selectivity of endothelin" Eur. J. Pharmacol. 165: 223-230).
Endothelin antagonist activity can be assessed by the ability to
interfere with endothelin-induced vasoconstriction. Exemplary
assays are set forth in the EXAMPLES. As noted above, the preferred
IC.sub.50 concentration ranges are set forth with reference to
assays in which the test compound is incubated with the ET
receptor-bearing cells at 24.degree. C. (assays in which the
incubation step is performed at 4.degree. C. may also be performed,
and in general yield lower IC.sub.50 concentrations). It is
understood that for purposes of comparison, these IC.sub.50 values
determined at 24.degree. C. are somewhat higher than the
concentrations determined at 4.degree. C.
[0079] As used herein, the biological activity or bioactivity of
endothelin includes any activity induced, potentiated or influenced
by endothelin in vivo. It also includes the ability to bind to
particular receptors and to induce a functional response, such as
vasoconstriction. It may be assessed by in vivo assays or by in
vitro assays, such as those exemplified herein. The relevant
activities include, but are not limited to, vasoconstriction,
vasorelaxation and bronchodilation. For example, ET.sub.B receptors
appear to be expressed in vascular endothelial cells and may
mediate vasodilation and other such responses; whereas ET.sub.A
receptors, which are endothelin-1-specific, occur on smooth muscle
and are linked to vasoconstriction Any assay known to those of
skill in the art to measure or detect such activity may be used to
assess such activity (see, e.g., Spokes et al. (1989) J.
Cardiovasc. Pharmacol. 13(Suppl. 5):S191-S192; Spinella et al.
(1991) Proc. Natl. Acad. Sci. USA 88: 7443-7446; Cardell et al.
(1991) Neurochem. Int. 18:571-574); and the Examples herein).
[0080] As used herein, the IC.sub.50 refers to an amount,
concentration or dosage of a particular test compound that achieves
a 50% inhibition of a maximal response, such as binding of
endothelin to tissue receptors, in an assay that measures such
response.
[0081] As used herein, EC.sub.50 refers to a dosage, concentration
or amount of a particular test compound that elicits a
dose-dependent response at 50% of maximal expression of a
particular response that is induced, provoked or potentiated by the
particular test compound.
[0082] As used herein, a compound that is ET.sub.A selective are
those that exhibit an IC.sub.50 that is at least about 3-fold,
preferably 5- to 10-fold or more, lower with respect to ET.sub.A
receptors than ET.sub.B receptors.
[0083] As used herein, a compound that is ET.sub.B selective are
those that exhibit an IC.sub.50 that is at least about 3-fold,
preferably 5- to 10-fold or more, lower with respect to ET.sub.B
receptors than ET.sub.A receptors.
[0084] As used herein, pharmaceutically acceptable salts, esters or
other derivatives of the compounds include any salts, esters or
derivatives that may be readily prepared by those of skill in this
art using known methods for such derivatization and that produce
compounds that may be administered to animals or humans without
substantial toxic effects and that either are pharmaceutically
active or are prodrugs. For example, hydroxy groups can be
esterified or etherified.
[0085] As used herein, treatment means any manner in which the
symptoms of a conditions, disorder or disease are ameliorated or
otherwise beneficially altered. Treatment also encompasses any
pharmaceutical use of the compositions herein, such as use as
contraceptive agents.
[0086] As used herein, amelioration of the symptoms of a particular
disorder by administration of a particular pharmaceutical
composition refers to any lessening, whether permanent or
temporary, lasting or transient that can be attributed to or
associated with administration of the composition.
[0087] As used herein, substantially pure means sufficiently
homogeneous to appear free of readily detectable impurities as
determined by standard methods of analysis, such as thin layer
chromatography (TLC), gel electrophoresis and high performance
liquid chromatography (HPLC), used by those of skill in the art to
assess such purity, or sufficiently pure such that further
purification would not detectably alter the physical and chemical
properties, such as enzymatic and biological activities, of the
substance. Methods for purification of the compounds to produce
substantially chemically pure compounds are known to those of skill
in the art. A substantially chemically pure compound may, however,
be a mixture of stereoisomers. In such instances, further
purification might increase the specific activity of the
compound.
[0088] As used herein, biological activity refers to the in vivo
activities of a compound or physiological responses that result
upon in vivo administration of a compound, composition or other
mixture. Biological activity, thus, encompasses therapeutic effects
and pharmaceutical activity of such compounds, compositions and
mixtures.
[0089] As used herein, a prodrug is a compound that, upon in vivo
administration, is metabolized or otherwise converted to the
biologically, pharmaceutically or therapeutically active form of
the compound. To produce a prodrug, the pharmaceutically active
compound is modified such that the active compound will be
regenerated by metabolic processes. The prodrug may be designed to
alter the metabolic stability or the transport characteristics of a
drug, to mask side effects or toxicity, to improve the flavor of a
drug or to alter other characteristics or properties of a drug. By
virtue of knowledge of pharmacodynamic processes and drug
metabolism in vivo, those of skill in this art, once a
pharmaceutically active compound is known, can design prodrugs of
the compound (see, e.g., Nogrady (1985) Medicinal Chemistry A
Biochemical Approach, Oxford University Press, New York, pages
388-392). For example, succinyl-sulfathiazole is a prodrug of
4-amino-N-(2-thiazoyl)benzenesulfonamide (sulfathiazole) that
exhibits altered transport characteristics.
[0090] As used herein, a replacement analog of a group refers to a
second group in which one or more atoms in the first group are
replaced with homologous atoms or a similar behaving atom or group
of atoms such as, replacement of O with S or N.
[0091] As used herein, alkyl means an aliphatic hydrocarbon group
that is a straight or branched chain preferably having about 1 to
12 carbon atoms in the chain. Preferred alkyl groups are lower
alkyl groups which are alkyls containing 1 to about 6 carbon atoms
in the chain. Branched means that one or more lower alkyl groups
such as methyl, ethyl or propyl are attached to a linear alkyl
chain. The alkyl group may be unsubstituted or independently
substituted by one or more groups, such as, but not limited to:
halo, carboxy, formyl, sulfo, sulfino, carbamoyl, amino and imino.
Exemplary alkyl groups include methyl, ethyl, propyl, methanoic
acid, ethanoic acid, propanoic acid, ethanesulfinic acid and ethane
sulfonic acid.
[0092] As used herein the term lower describes alkyl, alkenyl and
alkynyl groups containing about 6 carbon atoms or fewer. It is also
used to describe aryl groups or heteroaryl groups that contain 6 or
atoms in the ring.
[0093] As used herein, alkenyl means an aliphatic hydrocarbon group
containing a carbon-carbon double bond and which may be straight or
branched chained having from about 2 to about 10 carbon atoms in
the chain. Preferred alkenyl groups have 2 to about 4 carbon atoms
in the chain. Branched means that one or more lower alkyl or lower
alkenyl groups are attached to a linear alkenyl chain. The alkenyl
group may be unsubstituted or independently substituted by one or
more groups, such as halo, carboxy, formyl, sulfo, sulfino,
carbamoyl, amino and imino. Exemplary alkenyl groups include
ethenyl, propenyl, carboxyethenyl, carboxypropenyl, sulfinoethenyl
and sulfonoethenyl.
[0094] As used herein, alkynyl means an aliphatic hydrocarbon group
containing a carbon-carbon triple bond and which may be straight or
branched having about 2 to 10 carbon atoms in the chain. Branched
means that one or more lower alkyl, alkenyl or alkynyl groups are
attached to a linear alkynyl chain. An exemplary alkynyl group is
ethynyl.
[0095] As used herein, aryl means an aromatic monocyclic or
multicyclic hydrocarbon ring system containing about 6 to about 10
carbon atoms, which may be unsubstituted or independently
substituted with one or more substituents as set forth herein.
Exemplary aryl groups are phenoxy, (methylenedioxy)phenoxy,
thiophenoxy, alkylphenyl and alkenylphenyl.
[0096] As used herein, cycloalkyl refers to saturated cyclic carbon
chains; cycloalkyenyl and cycloalkynyl refer to cyclic carbon
chains that include at least one unsaturated double or triple bond,
respectively. The cyclic portions of the carbon chains may include
one ring or two or more fused rings.
[0097] As used herein, cycloalkenyl means a non-aromatic monocyclic
or multicyclic ring system containing a carbon-carbon double bond
and having about 3 to about 10 carbon atoms. Exemplary monocyclic
cycloalkenyl rings include cyclopentenyl or cyclohexenyl; preferred
is cyclohexenyl. An exemplary multicyclic cycloalkenyl ring is
norbornylenyl. The cycloalkenyl group may be independently
substituted by one or more halo or alkyl groups.
[0098] As used herein, alkoxy means an alkyl-O group in which the
alkyl group is as previously described. Exemplary alkoxy groups
include methoxy, ethoxy and propoxy.
[0099] As used herein, aryloxy means an aryl-O group in which the
aryl group is as previously described. Exemplary aryloxy groups
include phenoxy and naphthoxy. "Replacement analogs of aryloxy
groups" means an aryloxy group, such as phenoxy, where the oxygen
atom is replaced by a functionally similar atom such as sulfur or
nitrogen; for example, thiophenoxy.
[0100] As used herein, alkylenedioxy means an -O-alkyl-O- group in
which the alkyl group is as previously described. A replacement
analog of alkylenedioxy means an alkylenedioxy in which one or both
of the oxygen atoms is replaced by a similar behaving atom or group
of atoms such as, S, N, NH, Se. An exemplary replacement
alkylenedioxy group is ethylenebis(sulfandiyl). Alkylenethioxyoxy
is --S-alkyl-O-- and alkylenedithioxy is --S-alkyl-O--.
[0101] As used herein, heteroaryl means an aromatic monocyclic or
fused ring system in which one or more of the carbon atoms in the
ring system is(are) replaced by an element(s) other than carbon,
for example nitrogen, oxygen or sulfur. Similarly to "aryl groups",
the heteroaryl groups may be unsubstituted or substituted by one or
more substituents. Exemplary heteroaryl groups include pyrazinyl,
pyrazolyl, tetrazolyl, furanyl, (2- or 3-)thienyl, (2-,3- or
4-)pyridyl, imidazoyl, pyrimidyl, isoxazolyl, thiazolyl,
isothiazolyl, quinolinyl, indolyl, isoquinolinyl, oxazolyl and
1,2,4-oxadiazolyl. Preferred heteroaryl groups include 5 to
6-membered nitrogen-containing rings, such as pyrmidinyl.
[0102] As used herein, heterocycle means a ring system that
includes one or more heteroatoms selected from S, O or N.
Heterocycles include aliphatic rings and heteroaryl rings.
Preferred cyclic groups contain one or two fused rings and include
from about 3 to about 7 members in each ring.
[0103] As used herein a nitrogen-containing ring means a
heterocycle, preferably containing 3 to 7 members, more preferably
5 or 6 members, in the ring including at least one nitrogen atom
and optionally containing one or more additional heteroatoms
selected from nitrogen, oxygen and sulphur in addition to the
carbon atom(s) present. The ring system may be unsaturated or
partially saturated and may be unsubstituted or substituted, for
example by hydroxymethyl, carbamoyl, thiocarbamoyl, or -(lower
alkyl).sub.kCO.sub.2R", where k is 0 or 1 and R" is hydrogen and
alkyl, carbamoyl, or thiocarbamoyl. Exemplary nitrogen-containing
ring systems include pyrimidyl, tetrazolyl, substituted tetrazolyl,
1,2,4-oxadiazolyl, substituted isoxazolyl, isothiazolyl,
substituted thiazolyl, pyrazolyl, substituted pyrazolyl, pyridyl,
oxazolyl, substituted oxazolyl and dihydrooxazolyl.
[0104] As used herein, arylalkyl means an aryl-alkyl- group.
Preferred aralkyls are aryl lower alkyls. Exemplary aralkyl groups
include benzyl and phenethyl.
[0105] As used herein, aralkenyl means an aryl-alkenyl group.
Preferred aralkenyls are aryl lower alkenyls. An exemplary
aralkenyl group is styryl.
[0106] As used herein, aralkynyl means an aryl-alkynyl group.
Preferred aralkynyls are aryl lower alkynyls. An exemplary
aralkenyl group is phenylethynyl.
[0107] As used herein, heteroaralkyl means a heteroaryl-alkyl
group. Preferred heteroaralkyls are heteroaryl lower alkyls.
Exemplary heteroaralkyl groups include pyrid(2- or 3-)ylmethyl,
pyrid(2- or 3-)ylethyl, thienylethyl, thienylmethyl,
indol-3-ylmethyl or furylmethyl.
[0108] As used herein, heteroaralkenyl means an heteroaryl-alkenyl-
group in which the heteroaryl and alkenyl are as previously
described. Preferred heteroaralkenyls are heteroaryl lower
alkenyls. An exemplary heteroaralkenyl group is
3-(2-pyridyl)prop-2-enyl.
[0109] As used herein, aryloxy means an aryl-O group. Exemplary
aryloxy groups include phenoxy and naphthoxy.
[0110] As used herein, aralkyloxy means an aralkyl-O- group.
Preferred aralkyloxys are aryl lower alkoxys. Exemplary aralkyloxy
groups include benzyloxy, phenylethoxy, phenylpropyloxy, (1- or
2-naphthalene)ethoxy and (o-tolyl)ethoxy; preferred are
1-phenylethoxy and 1-(o-tolyl)ethoxy.
[0111] As used herein, heteroaralkyloxy means an
heteroaryl-alkyl-O- group. Preferred heteroaralkyloxys are
heteroaryl lower alkoxys. Exemplary heteroaralkyloxy groups include
pyrid(2 or 3-)ylethoxy, pyrid(2- or 3-)ylmethoxy, thienylmethoxy
and thienylethoxy.
[0112] As used herein, aklylthio means alkyl-S-. Preferred
alkylthios are lower alkylthios. An exemplary alkylthio group is
methylthio.
[0113] As used herein, alkylsulfinyl means alkyl-SO-. Preferred
alkylsulfinyls are lower alkylsulfinyls. An exemplary alkylsulfinyl
group is methylsulfinyl.
[0114] As used herein, alkylsulfonyl means alkyl-SO.sub.2--. An
exemplary alkylsulfonyl group is methylsulfonyl.
[0115] As used herein, alkoxycarbonyl means an alkyl-O-CO- group.
Exemplary alkoxycarbonyl groups include methoxy- and
ethoxycarbonyl.
[0116] As used herein, carbamoyl means --CONH.sub.2. As with all
groups described herein, these groups may be unsubstituted or
substituted. Substituted carbamoyl includes groups such as
--CONY.sup.2Y.sup.3 in which Y.sup.2 and Y.sup.3 are independently
hydrogen, alkyl, cyano(lower alkyl), aryalkyl, heteroaralkyl,
carboxy(lower alkyl), carboxy(aryl substituted lower alkyl),
carboxy(carboxy substituted lower alkyl), carboxy(hydroxy
substituted lower alkyl), carboxy(heteroaryl substituted lower
alkyl), carbamoyl(lower alkyl), alkoxycarbonyl(lower alkyl) or
alkoxycarbonyl(aryl substituted lower alkyl), provided that only
one of Y.sup.2 and Y.sup.3 may be hydrogen and when one of Y.sup.2
and Y.sup.3 is carboxy(lower alkyl), carboxy(aryl substituted lower
alkyl), carbamoyl(lower alkyl), alkoxycarbonyl(lower alkyl) or
alkoxycarbonyl(aryl substituted lower alkyl) then the other of
Y.sup.2 and Y.sup.3 is hydrogen or alkyl. Preferred for Y.sup.2 and
Y.sup.3 are independently hydrogen, alkyl, cyano(lower alkyl),
aryalkyl, heteroaralkyl, carboxy(lower alkyl), carboxy(aryl
substituted lower alkyl) and carbamoyl(lower alkyl).
[0117] As used herein, thiocarbamoyl means --CSNH.sub.2.
Substituted thiocarbamoyl includes groups such as
--CSNY.sup.2Y.sup.3 in which Y.sup.2 and Y.sup.3 are as defined
above.
[0118] As used herein, alkoxycarbonyl(lower alkyl) means
alkoxy-CO-lower alkyl.
[0119] As used herein, carboxy(aryl substituted lower alkyl) means
a lower alkyl group substituted by an aryl moiety and a carboxy
moiety, where the alkyl and aryl moieties are as defined
herein.
[0120] As used herein, alkoxycarbonyl(aryl substituted lower alkyl)
means a lower alkyl group substituted by an aryl moiety and an
alkoxy moiety, where the alkyl, aryl and alkoxy moieties are as
defined herein.
[0121] As used herein, aryl lower alkylthio means aryl-lower
alkyl-S-.
[0122] As used herein, heteroaryl lower alkylthio means
heteroaryl-lower alkyl-S-.
[0123] As used herein, acid isostere means a group that is
significantly ionized at physiological pH. Examples of suitable
acid isosteres include sulfo, phosphono, alkylsulfonylcarbamoyl,
tetrazolyl, arylsulfonylcarbamoyl or
heteroarylsulfonylcarbamoyl.
[0124] As used herein, halo or halide refers to the halogen atoms;
F, Cl, Br and I.
[0125] As used herein, pseudohalides are compounds that behave
substantially similar to halides. Such compounds can be used in the
same manner and treated in the same manner as halides (X.sup.-, in
which X is a halogen, such as Cl or Br). Pseudohalides include, but
are not limited to cyanide, cyanate, thiocyanate, selenocyanate and
azide.
[0126] As used herein, haloalkyl refers to a lower alkyl radical in
which one or more of the hydrogen atoms are replaced by halogen
including, but not limited to, chloromethyl, trifluoromethyl,
1-chloro-2-fluoroethyl and the like.
[0127] As used herein, aminocarbonyl refers to --C(O)NH.sub.2.
[0128] As used herein, "carboxamide" refers to groups of formula
R.sub.pCONH.sub.2 in which R is selected from alkyl or aryl,
preferably lower alkyl or lower aryl and p is 0 or 1.
[0129] As used herein, alkylthioic acid refers to groups of the
formula RC(S)OH and RC(O)SH, where R is alkyl, preferably lower
alkyl.
[0130] As used herein, alkylimidic acid refers to groups of the
formula RC(NH)OH, where R is alkyl, preferably lower alkyl.
[0131] As used herein, alkyldithoic acid refers to groups of the
formula RC(S)SH, where R is alkyl, preferably lower alkyl.
[0132] As used herein, alkylhydroxamic acid refers to groups of the
formula R(O)NHOH, where R is alkyl, preferably lower alkyl.
[0133] As used herein, an isostere refers to atoms or groups of
atoms that are of similar size to the atom or group of atoms that
is to be replaced by the isostere and that is selected such that
the compound containing the replacement atom or group of atoms
retains, to a substantial degree, the pharmaceutical activity (i.e.
modulation of the activity of an endothelin peptide) of the
original compound. See, et al. Nelson et al., at pp. 227, 271 and
285, respectively, in Burger's Medicinal Chemistry, Part 1, the
Basis of Medicinal Chemistry, 4th Edition, M. E. Wolff, ed. (John
Wiley & Sons, NY).
[0134] As used herein, the abbreviations for any protective groups,
amino acids and other compounds, are, unless indicated otherwise,
in accord with their common usage, recognized abbreviations, or the
IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972)
Biochem. 11:942-944).
[0135] A. Compounds
[0136] The compounds that are used in the compositions and methods
have formula (I): 6
[0137] where:
[0138] X and Y are independently selected from O, S, NR.sup.28,
--(CH.sub.2).sub.v--, --NR.sup.28(CH.sub.2).sub.v--,
--S--(CH.sub.2).sub.v-- or --O--(CH.sub.2).sub.v-- where v is 0 to
12, preferably 0 to 6, more preferably 0 to 3, provided that, when
Ar.sup.3 is phenyl, then at least one of X and Y is O, S or
NR.sup.28;
[0139] R.sup.28 is selected from hydrogen, alkyl, alkenyl, alkynyl,
aryl, haloalkyl, alkylaryl, heterocyclyl, arylalkyl, arylalkoxy,
alkoxy, aryloxy, cycloalkyl, cycloalkenyl and cycloalkynyl, and is
preferably hydrogen, lower alkyl, lower alkoxy and lower haloalkyl;
and
[0140] Ar.sup.1 and Ar.sup.2 are independently selected from among
aryl and heteroaryl groups containing one or more, preferably one
or two to three fused rings and from 3 up to about 21 members in
the ring(s), in which the heteroaryl groups contain one to three
heteroatoms selected from among O, S and N. In particular Ar.sup.1
and Ar.sup.2 are independently selected from substituted or
unsubstituted groups that include, but are not limited to, the
following: naphthyl, phenyl, biphenyl, quinolyl, thienyl, furyl,
isoquinolyl, pyrrolyl, pyridyl, indolyl, oxadiazolyl, pyrazolyl,
isoxazolyl, isothiazolyl, pyrimidyl, benzo[b]furan, benzo[b]thienyl
and other such aryl and heteroaryl groups. Preferred among these
groups are 5 to 6 membered aryl groups and heteroaryl groups that
contain one or two heteroatom(s).
[0141] Ar.sup.1 and Ar.sup.2 are unsubstituted or are substituted
with one or more substituents, preferably selected from among
alkyl, alkoxy, alkenyl, alkynyl, halo, pseudohalo,
(CH.sub.2).sub.qCOR.sup.16 in which q is 0 to 6, preferably 0 to 3,
more preferably 0 or 1, (alkenyl).sub.rCOR.sup.15 in which alkenyl
is a straight or branched carbon chain containing at least two
carbons and one unsaturated bond so that r is 0 or 2 to 6,
preferably 0, 2 or 3, tetrazolyl, (CH.sub.2).sub.tOH in which t is
0 to 6, preferably 0 to 3, more preferably 0 or 1,
(alkenyl).sub.uOH in which alkenyl is a straight or branched carbon
chain containing at least two carbons and one unsaturated bond so
that u is 0 or 2 to 6, preferably 0, 2 or 3;
[0142] R.sup.15 and R.sup.16 are independently hydrogen, alkyl,
haloalkyl, aryl, aryloxy, heterocyclyl, arylalkyl, arylalkoxy,
cycloalkyl, cycloalkenyl, cycloalkynyl, OH, R.sup.20, C(O)R.sup.20,
CO.sub.2R.sup.20, SH, S(O).sub.nR.sup.20 in which n is 0-2, HNOH,
(CH.sub.2).sub.sH, (CH.sub.2).sub.sR.sup.20 in which s is 1-6,
NR.sup.20R.sup.21, OR.sup.20, R.sup.21NCOR.sup.20 and
R.sup.21NSO.sub.2R.sup.20;
[0143] R.sup.20 is selected from among hydrogen, alkyl, alkenyl,
alkynyl, aryl, alkylaryl, heterocyclyl, arylalkyl, cycloalkyl,
cycloalkenyl or cycloalkynyl, where R.sup.20 is preferably alkyl or
aryl; and
[0144] R.sup.21 is selected from among hydrogen, alkyl, alkenyl,
alkynyl, aryl, alkylaryl, alkoxy, aryloxy, heterocyclyl, arylalkyl,
arylalkoxy, cycloalkyl, cycloalkenyl, cycloalkynyl.
[0145] Ar.sup.3 is independently selected from the aryl and
heteroaryl groups set forth for Ar.sup.1 and Ar.sup.2. Ar.sup.3 is
particularly selected from among single ring aromatic groups that
contain from 3 to 7, preferably 4 to 6, more preferably 5 or 6
members. In particular, Ar.sup.3 is aryl or is a heterocycle
containing one or two heteroatoms, preferably nitrogen. Thus,
Ar.sup.3 is preferably selected from among phenyl, pyrimidyl,
pyrazinyl, pyridazinyl, pyridyl, oxazolyl, isoxazolyl and
imidazolyl groups. Ar.sup.3 is more preferably phenyl, pyridinyl,
pyrimidyl or pyrazinyl and is preferably substituted with an acidic
group or isostere thereof, particularly a carboxyl group.
[0146] It is noted that the compounds for use in the compositions
are preferably do not have the formula (IV): 7
[0147] in which R' is lower alkyl, COOH, C(O)NR.sub.aR.sub.b, where
R.sub.a is hydrogen or C.sub.1-6-alkyl, R.sub.b is C.sub.1-6-alkyl,
OH, methoxy, cyanomethyl, or R.sub.a and R.sub.b together form
--(CH.sub.2).sub.x--, where x is 1 to 6.
[0148] In the above compounds, the alkyl, alkynyl and alkenyl
portions of each listed substituent are straight or branched
chains. Preferably these groups are lower alkyl, alkynyl and
alkenyl groups having from about 1 up to about 12 carbons; in more
preferred embodiments they have from 1 to 6 carbons. The aryl and
heterocyclyl groups are single or fused rings, have from 3 to 21,
generally, 3 to 7, more often 4 to 6 members, with preferably 5 to
7 members in the rings, and may be single or fused rings.
[0149] In all instances, the ring size and carbon chain length are
selected up to a size such that the resulting molecule binds to an
endothelin receptor and retains activity as an endothelin
antagonist or agonist; for example, such that the binding of an
endothelin peptide to the endothelin receptor is inhibited by 50%,
compared to binding in the absence of the compound, at a
concentration (IC.sub.50) of less than about 50 .mu.M.
[0150] More preferred among the compounds that are used in the
methods are those of formula (I) that have formula (II): 8
[0151] where:
[0152] R.sup.1 is selected from hydrogen and --(CH.sub.2).sub.n--A
in which n is 0 to 6, preferably 0 to 3, more preferably 0 or 1,
and A is an acidic group or and isostere thereof, such as
CO.sub.2R.sup.4, carboxylic acid, carboxamide, alkylthioic acid,
alkyldithoic acid, alkylimidic acid, tetrazolyl, sulfinic acid,
sulfonic acid, phosphonic acid, tetrazolyl, sulfinimidic acid,
sulfonimidic acid, sulfonamide, alkylhydroxamic acid, hydrazide,
amide, hydroxyl, and CONR.sup.27R.sup.26, and is more preferably a
group such as C(O)OR.sup.4 in which R.sup.4 is hydrogen, lower
alkyl or lower haloalkyl;
[0153] R.sup.26 and R.sup.27 are each independently selected from
hydrogen, alkyl, alkenyl, alkynyl, aryl, haloalkyl, alkylaryl,
heterocyclyl, arylalkyl, arylalkoxy, alkoxy, aryloxy, cycloalkyl,
cycloalkenyl and cycloalkynyl, and is preferably hydrogen, lower
alkyl, lower alkoxy and lower haloalkyl, and is preferably hydrogen
or lower alkyl;
[0154] o, j and p are independently 0 or 1, and j is preferably
0;
[0155] R.sup.2, R.sup.3, and R.sup.22 are independently selected
from alkyl, alkenyl, halo, haloalkyl, alkoxy, -S-alkyl,
-NR.sup.29-alkyl, aryl or heteroaryl, which are preferably single
rings that contain 4 to 7, more preferably 5 or 6, members in the
ring;
[0156] X and Y are independently selected from O, S, NR.sup.28,
--(CH.sub.2).sub.v--, --NR.sup.28(CH.sub.2).sub.v--
--S--(CH.sub.2).sub.v-- or --O--(CH.sub.2).sub.v--, where v is 0 to
12, preferably 0 to 6, more preferably 0 to 3, and X and Y are
preferably O, S, lower alkyl, preferably --CH.sub.2--,
--S--CH.sub.2--, or lower alkoxy, preferably --O--CH.sub.2--, with
the proviso that, when Ar.sup.3 is phenyl (i.e., when the compounds
of formula II have formula II (a)) at least one of X and Y is O, S
or NR.sup.28;
[0157] R.sup.28 and R.sup.29 are each independently selected from
hydrogen, alkyl, alkenyl, alkynyl, aryl, haloalkyl, alkylaryl,
heterocyclyl, arylalkyl, arylalkoxy, alkoxy, aryloxy, cycloalkyl,
cycloalkenyl and cycloalkynyl, and is preferably hydrogen, lower
alkyl, lower alkoxy and lower haloalkyl; and
[0158] Ar.sup.1 and Ar.sup.2 are as defined above.
[0159] In certain embodiments R.sup.1 is hydrogen,
--(CH.sub.2).sub.q(CO.s- ub.2R.sup.4), --(CH.sub.2).sub.q(OH), CN,
--C(R.sup.7).dbd.NOR.sup.8, NO.sub.2, --(CH.sub.2).sub.qR.sup.9,
--C.ident.CR.sup.10, --CR.sup.11.dbd.C(R.sup.12)(R.sup.13),
--(CH.sub.2).sub.qC(.dbd.O)CH.sub.- 2C(.dbd.O)CO.sub.2H preferably
--C(.dbd.O)CH.sub.2C(.dbd.O)CO.sub.2H, --CO(R.sup.14),
CONR.sup.27R.sup.26, tetrazolyl, alkylthio, alkylsulfinyl,
alkylsulfonyl, carbamoyl, thiocarbamoyl, or a nitrogen-containing
ring with, preferably from 5 to 7 members in the ring, each of
which groups may be unsubstituted or substituted with one more
substituents that are preferably alkyl, halo or haloalkyl in which
the alkyl groups are preferably lower alkyl, where:
[0160] q is 0 to 6, preferably 0 to 3;
[0161] R.sup.4 is hydrogen, lower alkyl or haloalkyl;
[0162] R.sup.7 is selected from hydrogen, alkyl or haloalkyl;
[0163] R.sup.8 is hydrogen, arylalkyl or -(lower
alkyl)CO.sub.2R.sup.17;
[0164] R.sup.9 is --CN, --CO.sub.2R.sup.19, --CH.sub.2OH, or
carbamoyl;
[0165] R.sup.10 is hydrogen, --CO.sub.2H or carboxyphenyl;
[0166] R.sup.11 is hydrogen, alkyl or arylalkyl;
[0167] R.sup.12 and R.sup.13 are independently hydrogen,
--CO.sub.2R.sup.18, --CN, aryl, lower alkyl, heteroaryl, lower
alkyl or --NHC(O)aryl, provided that one of R.sup.12 and R.sup.13
is --CO.sub.2H;
[0168] R.sup.14 is hydrogen, alkyl, -(lower alkyl)carboxy,
arylalkenyl, heteroarylalkenyl or --CO.sub.2H;
[0169] R.sup.17, R.sup.18 and R.sup.19 are independently selected
from hydrogen, alkyl or haloalkyl;
[0170] R.sup.26 and R.sup.27 are each independently selected from
hydrogen, alkyl, alkenyl, alkynyl, aryl, haloalkyl, alkylaryl,
heterocyclyl, arylalkyl, arylalkoxy, alkoxy, aryloxy, cycloalkyl,
cycloalkenyl and cycloalkynyl, and is preferably hydrogen, lower
alkyl, lower alkoxy and lower haloalkyl, and is preferably hydrogen
or lower alkyl.
[0171] In more preferred embodiments, the compounds of formula (II)
for use in the methods and compositions have formula (III): 9
[0172] where Ar.sup.1, Ar.sup.2, R.sup.2, X, Y and o are as defined
above, j and p are 0, and Ar.sup.3 is pyrimidyl or phenyl. The
compounds are preferably selected with the above-noted proviso.
Compounds in which j and/or p are 1 are also contemplated.
[0173] In more preferred embodiments, at least one of Ar.sup.1 and
Ar.sup.2 is substituted phenyl and at least one of X and Y is O or
S. Preferred compounds for use in the methods and compositions are
aromatic carboxylic acid derivatives (R.sup.1 is (lower
alkyl).sub.qCO.sub.2R.sup.- 4), particularly those of formula (III)
and pharmaceutically acceptable esters and salts thereof, but with
the proviso that the compounds do not have formula (IV). In
particular, when X and Y are O, and Ar.sup.1 and Ar.sup.2 are
unsubstituted or substituted with halogen or lower alkyl, R.sup.1
is not COOH or C(O)NR.sub.aR.sub.b, where R.sub.a is hydrogen or
C.sub.1-6-alkyl, R.sub.b is C.sub.1-6-alkyl, OH, methoxy,
cyanomethyl, or R.sub.a and R.sub.b together form
--(CH.sub.2).sub.x--, where x is 1 to 6.
[0174] Of particular interest are compounds of formula (II),
preferably of formula (III) in which:
[0175] R.sup.1 is carboxylic acid, lower alkyl carboxylic acid,
sulfonic acid, phosphonic acid, tetrazolyl or other such group, as
defined above. In particular, R.sup.1 is selected from among
(CH.sub.2).sub.qCO.sub.2R.s- up.4 in which q is 0 to 6, preferably
0 to 3, preferably 0 or 1, tetrazolyl, hydrogen,
--(CH.sub.2).sub.q(CO.sub.2R.sup.4), --(CH.sub.2).sub.q(OH), CN,
--C(R.sup.7).dbd.NOR.sup.8, NO.sub.2, --(CH.sub.2).sub.qR.sup.9,
--C.ident.CR.sup.10, CONR.sup.27R.sup.26--CR.s-
up.11.dbd.C(R.sup.12)(R.sup.13),
--(CH.sub.2).sub.qC(.dbd.O)CH.sub.2C(.dbd- .O)CO.sub.2H,
--CO(R.sup.14), alkylthio, alkylsulfinyl, alkylsulfonyl, carbamoyl,
thiocarbamoyl, or a nitrogen-containing ring with, preferably from
5 to 7 members in the ring, each of which groups may be
unsubstituted or substituted with one more substituents that are
preferably alkyl, halo or haloalkyl in which the alkyl groups are
preferably lower alkyl;
[0176] o is 0 or 1;
[0177] R.sup.2 is selected from alkyl, alkenyl, halo, haloalkyl, or
aryl or heteroaryl, which are preferably single rings that contain
4 to 7, more preferably 5 or 6, members in the ring;
[0178] X and Y are independently selected from O, S, NR.sup.28,
(CH.sub.2).sub.v, --NR.sup.28(CH.sub.2).sub.v--,
--O--(CH.sub.2).sub.v--, --S--(CH.sub.2).sub.v--, where v is 0 to
12, preferably 0 to 6, more preferably 0 to 3, and X and Y are
preferably O, S, lower alkyl, preferably --CH.sub.2--, or lower
alkoxy, preferably --O--CH.sub.2--, provided that when Ar.sup.3 is
phenyl at least one of X and Y is O, S or NR.sup.28;
[0179] R.sup.4 is hydrogen, lower alkyl or haloalkyl,
[0180] R.sup.5 is hydrogen or lower alkyl;
[0181] R.sup.8 is hydrogen, arylalkyl or -(lower
alkyl)CO.sub.2R.sup.17,
[0182] R.sup.9 is --CN, --CO.sub.2R.sup.19, --CH.sub.2OH, or
carbamoyl;
[0183] R.sup.10 is hydrogen, --CO.sub.2H or carboxyphenyl;
[0184] R.sup.11 is hydrogen, alkyl or arylalkyl;
[0185] R.sup.12 and R.sup.13 are independently hydrogen,
--CO.sub.2R.sup.18, --CN, aryl, lower alkyl, heteroaryl, lower
alkyl or --NHC(O)aryl, provided that one of R.sup.12 and R.sup.13
is --CO.sub.2H;
[0186] R.sup.14 is hydrogen, alkyl, -(lower alkyl)carboxy,
arylalkenyl, heteroarylalkenyl or --CO.sub.2H;
[0187] R.sup.7, R.sup.17, R.sup.18 and R.sup.19 are independently
selected from hydrogen, alkyl or haloalkyl;
[0188] R.sup.26 and R.sup.27 are each independently selected from
hydrogen, alkyl, alkenyl, alkynyl, aryl, haloalkyl, alkylaryl,
heterocyclyl, arylalkyl, arylalkoxy, alkoxy, aryloxy, cycloalkyl,
cycloalkenyl and cycloalkynyl, and is preferably hydrogen, lower
alkyl, lower alkoxy and lower haloalkyl, and is preferably hydrogen
or lower alkyl;
[0189] R.sup.28 is selected from hydrogen, alkyl, alkenyl, alkynyl,
aryl, haloalkyl, alkylaryl, heterocyclyl, arylalkyl, arylalkoxy,
alkoxy, aryloxy, cycloalkyl, cycloalkenyl and cycloalkynyl, and is
preferably hydrogen, lower alkyl, lower alkoxy and lower haloalkyl;
and
[0190] Ar.sup.1 and Ar.sup.2 are selected as described above and
are preferably pyrimidyl or phenyl, most preferably phenyl, and
most preferably substituted phenyl, which is substituted as
described below.
[0191] In preferred embodiments, the compounds of formula (II) have
formula (III), as set forth above, in which where Ar.sup.1,
Ar.sup.2, R.sup.2, R.sup.3, X, Y, and o are as defined above and
Ar.sup.3 is pyrimidyl or phenyl. Again preferred compounds do not
have formula (IV). Compounds for use in the methods may have
formula (IV).
[0192] Of the preferred compounds those in which R.sup.1 is
selected from among (CH.sub.2).sub.qCO.sub.2R.sup.4 in which q is 0
to 6, preferably 0 to 3, more preferably 0 or 1, tetrazolyl,
--CH.dbd.CH--Z, --C(R.sup.4).dbd.C(R.sup.4)--Z, --C.ident.CZ,
--O--(CH.sub.2).sub.qZ, --CO.sub.2H, --S--(CH.sub.2).sub.qZ, and
--(CH.sub.2).sub.qC(O)Z, in which q is 0 to 6, more preferably 0 to
3 and Z is carboxylic acid, carboxamide, alkylthioic acid,
alkyldithoic acid, alkylimidic acid, sulfinic acid, sulfonic acid,
phosphonic acid, sulfinimidic acid, sulfonimidic acid, sulfonamide,
alkylhydroxamic acid, hydrazide, amide, hydroxyl, hydrogen, alkyl,
or alkenyl, tetrazolyl, preferably COOH or tetrazolyl, and R.sup.4
is as defined above. R.sup.1 is more preferably
(CH.sub.2).sub.qCO.sub.2R.sup.4 or tetrazolyl, and most preferably
(CH.sub.2).sub.qCO.sub.2R.sup.4 in which q is 0 to 3.
[0193] In more preferred embodiments, at least one of Ar.sup.1 and
Ar.sup.2 is substituted phenyl and at least one of X and Y is O.
Preferred compounds are aromatic carboxylic acid derivatives
(R.sup.1 is (lower alkyl).sub.qCO.sub.2R.sup.4) and
pharmaceutically acceptable esters and salts thereof of formula
(III) but that do not have formula (IV). In preferred embodiments,
alkyl, alkenyl and alkynyl groups preferably contain six or fewer
carbon atoms, more preferably 3 or fewer.
[0194] In particular embodiments disclosed herein, the compounds
herein, and derivatives thereof, have formula (V): 10
[0195] more preferably formula (VI): 11
[0196] where:
[0197] R.sup.1 is as defined above, is preferably
(CH.sub.2).sub.qCO.sub.2- R.sup.4, where q is preferably 0 to 3,
where R.sup.4 is hydrogen or lower alkyl, preferably containing 1
to 3 carbon atoms, and R.sup.1 is most preferably C(O)OH;
[0198] X and Y are independently selected from O, S, CH.sub.2 or
NR.sup.28 in which R.sup.28 is selected from hydrogen, alkyl,
alkenyl, alkynyl, aryl, haloalkyl, alkylaryl, heterocyclyl,
arylalkyl, arylalkoxy, alkoxy, aryloxy, cycloalkyl, cycloalkenyl
and cycloalkynyl, and is preferably hydrogen, lower alkyl, lower
alkoxy and lower haloalkyl; preferably, Y and Z are, independently,
O or S, and most preferably O.
[0199] R.sup.32, R.sup.33, R.sup.34, R.sup.35, R.sup.36 and
R.sup.37 are each independently selected from (i), (ii) or (iii) as
follows:
[0200] (i) R.sup.32, R.sup.33, R.sup.34, R.sup.35, R.sup.36 and
R.sup.37 are each independently selected from among H, NHR.sup.38,
CONR.sup.38, NO.sub.2, halide, pseudohalide, alkyl, alkenyl,
alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino,
alkylthio, alkoxy, haloalkyl, alkylsulfinyl, alkylsulfonyl,
alkoxycarbonyl, alkylcarbonyl, alkenylthio, alkenylamino,
alkenyloxy, alkenyl sulfinyl, alkenylsulfonyl, aminocarbonyl,
carboxy, carboxyalkyl, carboxyalkenyl, and formyl; or
[0201] (ii) at least two of R.sup.32, R.sup.33 and R.sup.34 or
R.sup.35, R.sup.36 and R.sup.37 are substituting adjacent carbons
on the ring and together form alkylenedioxy, alkylenethioxyoxy or
alkylenedithioxy (i.e. --O--(CH.sub.2).sub.n--O--,
--S--(CH.sub.2).sub.n--O--, --S--(CH.sub.2).sub.n--S--, where n is
1 to 4, preferably 1 or 2,) which is unsubstituted or substituted
by replacing one or more hydrogens with halide, lower alkyl, lower
alkoxy or lower haloalkyl, and the others of R.sup.32, R.sup.33,
R.sup.34, R.sup.35, R.sup.36 and R.sup.37 are selected as in (i);
or
[0202] (iii) at least two of R.sup.32, R.sup.33 and R.sup.34 are
substituting adjacent carbons on the ring and together form
alkylenedioxy, alkylenethioxyoxy or alkylenedithioxy (i.e.
--O--(CH.sub.2).sub.n--O--, --S--(CH.sub.2).sub.n--O--,
--S--(CH.sub.2).sub.n--S--, where n is 1 to 4, preferably 1 or 2,)
which is unsubstituted or substituted by replacing one or more
hydrogens with halide, lower alkyl, lower alkoxy or lower
haloalkyl, and at least two of R.sup.35, R.sup.36 and R.sup.37 are
substituting adjacent carbons on the ring and together form
alkylenedioxy, alkylenethioxyoxy or alkylenedithioxy (i.e.
--O--(CH.sub.2).sub.n--O--, --S--(CH.sub.2).sub.n--O--,
--S--(CH.sub.2).sub.n--S--, where n is 1 to 4, preferably 1 or 2,)
which is unsubstituted or substituted by replacing one ore more
hydrogens with halide, lower alkyl, lower alkoxy or lower
haloalkyl, and the others of R.sup.32, R.sup.33, R.sup.34,
R.sup.35, R.sup.36 and R.sup.37 are selected as in (i); and
[0203] R.sup.38 is selected from hydrogen, alkyl, alkenyl, alkynyl,
aryl, haloalkyl, alkylaryl, heterocyclyl, arylalkyl, arylalkoxy,
alkoxy, aryloxy, cycloalkyl, cycloalkenyl and cycloalkynyl, and is
preferably hydrogen, lower alkyl, lower alkoxy and lower
haloalkyl.
[0204] Preferably, at least one of R.sup.32, R.sup.33, R.sup.34,
R.sup.35, R.sup.36 and R.sup.37 on each ring is H and the others
are selected from among (i), (ii) or (iii) as follows:
[0205] (i) alkoxy, halo, alkylcarbonyl, formyl, and alkyl, in which
the alkyl portions or groups contain from 1 to 3 carbons, provided
that at least one of R.sup.32, R.sup.33, R.sup.34, R.sup.35,
R.sup.36 and R.sup.37 on each ring is H;
[0206] (ii) at least two of R.sup.32, R.sup.33, R.sup.34 are
substituting adjacent carbons and together form alkylenedioxy and
the other is H, and R.sup.35, R.sup.36 and R.sup.37 are selected as
set forth in (i); or
[0207] (iii) at least two of R.sup.32, R.sup.33, R.sup.34 are
substituting adjacent carbons and together form alkylenedioxy, and
at least two of R.sup.35, R.sup.36 and R.sup.37 are substituting
adjacent carbons and together form alkylenedioxy, and the others of
R.sup.32, R.sup.33, R.sup.34, R.sup.35, R.sup.36 and R.sup.37 are
H.
[0208] Compounds that are also contemplated herein include
compounds of formula (VII): 12
[0209] and formula (VIII): 13
[0210] The substituents are selected with the preferences as
described above for the compounds of formulae (V) and (VI).
[0211] In the most active compounds provided herein, as evidenced
by in vitro binding assays at least one of the rings is substituted
with alkylenedioxy and the other ring is substituted with
alkylenedioxy, preferably methylenedioxy, lower alkyl, lower
alkoxy, carboxylower alkyl or carboxy-lower alkenyl.
[0212] Preferred compounds for use in the compositions have formula
(V) and (VI), but with the proviso that the compounds do not have
formula (IV). In particular, when and Ar.sup.3 is phenyl, X and Y
are O, and Ar.sup.1 and Ar.sup.2 are otherwise unsubstituted or
substituted with halogen or lower alkyl, R.sup.1 is not COOH or
C(O)NR.sub.aR.sub.b, where R.sub.a is hydrogen or C.sub.1-6-alkyl,
R.sub.b is C.sub.1-6-alkyl, OH, methoxy, cyanomethyl, or R.sub.a
and R.sub.b together form --(CH.sub.2).sub.x--, where x is 1 to
6.
[0213] In the presently preferred compounds X and Y are O, R.sup.1
is COOH, and one of Ar.sup.1 and Ar.sup.2 is substituted with
methylenedioxy and the other is substituted with one or more
substituents selected from methylenedioxy, methoxy, carboxy,
carboxylower alkyl, carboxylower alkenyl, and lower alkyl, in which
alkenyl groups preferably contain 2 or 3 carbons and the alkyl
groups 1 to 3 carbons.
[0214] Compounds provided herein include, but are not limited to:
2-(4-methoxyphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic acid,
2-[3,4-(methylenedioxy)phenoxy]-6-(4-methylphenoxy)benzoic acid,
2-(4-fluorphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic acid,
2-(3,4-dimethoxyphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid, 2,6-bis-(3,4-methylenedioxyphenoxy)benzoic acid,
2,6-bis-(4-methoxyphenox- y)benzoic acid,
2-(2-bromo-4-methylphenoxy)-6-[3,4-(methylenedioxy)phenoxy-
]benzoic acid, 2,6-bis-(4-methylphenoxy)benzoic acid,
2-[3,4-(methylenedioxy) phenoxy]-6-(3-methylphenoxy)benzoic acid,
2-(4-methoxythiophenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid,
2-[(3-carboxymethyl)phenoxy]-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid,
2-[(4-carboxymethyl)phenoxy]-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid,
2-[(4-carboxyethyl)phenoxy]-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid,
2-(3,4-methylenedioxy)phenoxy-6-[2-propyl-4,5-(methylenedioxy)phenoxy]ben-
zoic acid,
2-[3,4-(methylenedioxy)phenoxy]-6-[2-(1-propenyl)-3,4-(methylen-
edioxy)phenoxy]benzoic acid,
2-[3,4-(methylenedioxy)phenoxy]-6-(3-methoxyp- henoxy)benzoic acid,
2-[(3-carboxyethyl)phenoxy]-6-[3,4-(methylenedioxy)ph-
enoxy]benzoic acid,
2-(3,4-methylenedioxy)phenoxy-6-[2-carboxyethyl-4,5-(m-
ethylenedioxy)phenoxy]benzoic acid,
2-(3,4-methylenedioxy)phenoxy-6-[2-car-
boxy-trans-ethenyl-4,5-(methylenedioxy)phenoxy]benzoic acid,
4,6-bis[2-carboxy-3,4-(methylenedioxy)phenoxy]-2-(methylthio)pyrimidine,
4-[2-carboxy-3,4-(methylenedioxy)phenoxy]-6-[3,4-(methylenedioxy)phenoxy]-
-2-(methylthio)-pyrimidine,
4,6-diphenoxy-2-(methylthio)-pyrimidine-5-carb- oxylic acid,
2,6-bis-[3,4-(methylenedioxy)phenoxy]phenyl acetic acid, ethyl
2-[3,4-(methylenedioxy)phenoxy]-6-[2-(carboxyethyl)-4-methoxyphenox-
y]benzoate,
2-[3,4-(methylenedioxy)phenoxy]-6-[(2-carboxyethyl)-4-methoxyp-
henoxy]benzoic acid. Preferred compounds for use in the methods
include the preceding compounds. Compounds for use in the methods
also include compounds of formula IV, including
2,6-diphenoxybenzoic acid and 2,6-diphenoxyphenylacetic acid.
[0215] More active among these compounds are
2-(4-methoxyphenoxy)-6-[3,4-(- methylenedioxy)phenoxy]benzoic acid,
2-[3,4-(methylenedioxy)phenoxy]-6-(4-- methylphenoxy)benzoic acid,
2,6-bis-(3,4-methylenedioxyphenoxy)benzoic acid,
2-(2-bromo-4-methylphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid, 2-[3,4-(methylenedioxy)phenoxy]-6-(3-methylphenoxy)benzoic
acid,
2-(4-methoxythiophenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid,
2-(3,4-methylenedioxy)phenoxy-6-[2-propyl-4,5-(methylenedioxy)phenoxy]ben-
zoic acid,
2-[3,4-(methylenedioxy)phenoxy]-6-[2-(1-propenyl)-3,4-(methylen-
edioxy)phenoxy]benzoic acid,
2-[3,4-(methylenedioxy)phenoxy]-6-(3-methoxyp- henoxy)benzoic acid,
2-(3,4-methylenedioxy)phenoxy-6-[2-carboxyethyl-4,5-(-
methylenedioxy)phenoxy]benzoic acid,
2-(3,4-methylenedioxy)phenoxy-6-[2-ca-
rboxy-trans-ethenyl-4,5-(methylenedioxy)phenoxy]benzoic acid, ethyl
2-[3,4-(methylenedioxy)phenoxy]-6-[2-(carboxyethyl)-4-methoxyphenoxy]benz-
oate and
2-[3,4-(methylenedioxy)phenoxy]-6-[(2-carboxyethyl)-4-methoxyphen-
oxy]benzoic acid.
[0216] Preferred among the compounds provided herein are those that
have an IC.sub.50 for ET.sub.A and/or ET.sub.B receptors less than
10 .mu.M, more preferably less than 1 .mu.M, even more preferably
less than 0.5 .mu.M, in the assays exemplified herein, when
measured at 24.degree. C. (less than 0.1 .mu.M when measured as
4.degree. C.) as described in the Examples When measured at
24.degree. C., the most preferred compounds herein have IC.sub.50
concentrations for inhibiting binding of endothelin-1 to the
ET.sub.A and/or ET.sub.B receptor of about 0.5 .mu.M or lower.
[0217] The preferred compounds include, but are not limited to:
2-(4-methoxyphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic acid,
2-[3,4-(methylenedioxy)phenoxy]-6-(4-methylphenoxy)benzoic acid,
2,6-bis-(3,4-methylenedioxyphenoxy)benzoic acid,
2-(2-bromo-4-methylpheno-
xy)-6-[3,4-(methylenedioxy)phenoxy]benzoic acid,
2-(4-methoxythiophenoxy)-- 6-[3,4-(methylenedioxy)phenoxy]benzoic
acid, 2-[3,4-(methylenedioxy)phenox-
y]-6-[2-(1-propenyl)-3,4-(methylenedioxy)phenoxy]benzoic acid,
2-(3,4-methylenedioxy)phenoxy-6-[2-carboxyethyl-4,5-(methylenedioxy)pheno-
xy]benzoic acid,
2-(3,4-methylenedioxy)phenoxy-6-[2-carboxy-trans-ethenyl--
4,5-(methylenedioxy)phenoxy]benzoic acid, ethyl
2-[3,4-(methylenedioxy)phe-
noxy]-6-[2-(carboxyethyl)-4-methoxyphenoxy]-benzoate and
2-[3,4-(methylenedioxy)phenoxy]-6-[(2-carboxyethyl)-4-methoxyphenoxy]benz-
oic acid.
[0218] Also preferred among the compounds herein are compounds that
are ET.sub.A or ET.sub.B selective (i.e., interact with greater
affinity (preferably at least 3-fold greater, more preferably
5-fold or more) with one receptor sub-type than with the other
receptor sub-type). Included among ET.sub.A selective compounds are
2-[3,4-(methylenedioxy)phenoxy]-6--
[3-(carboxymethyl)phenoxy]benzoic acid,
2-[3,4-(methylenedioxy)phenoxy]-6--
[4-(carboxymethyl)phenoxy]benzoic acid,
4,6-bis-[2-carboxy-3,4-(methylened-
ioxy)phenoxy]-2-(methylthio)pyrimidine, and
4-[2-carboxy-3,4-(methylenedio-
xy)phenoxy]-6-[3,4-(methylenedioxy)phenoxy]-2-(methylthio)-pyrimidine.
Included among ET.sub.B selective compounds are
2,6-bis-(4-methylphenoxy)- benzoic acid;
2-(3,4-methylenedioxy)phenoxy-6-[2-carboxyethyl-4,5-(methyle-
nedioxy)phenoxy]benzoic acid;
2-[3,4-(methylenedioxy)phenoxy]-6-[2-(1-prop-
enyl)-3,4-(methylenedioxy)phenoxy]benzoic acid;
2-[(3-carboxymethyl)phenox-
y]-6-[3,4-(methylenedioxy)phenoxy]benzoic acid;
2-[3,4-(methylenedioxy)phe-
noxy]-6-[2-(1-propenyl)-3,4-(methylenedioxy)phenoxy]benzoic acid,
and 2,6-bis-(4-methoxyphenoxy)benzoic acid.
[0219] B. Preparation of the Compounds
[0220] The preparation of some of the above compounds are described
in detail in the Examples. Any such compound may be synthesized
according to a method discussed in general below and set forth in
detail in the Examples by selecting appropriate starting materials
and readily available reagents as exemplified.
[0221] In general, most of the syntheses involve the reaction of an
alkyl dihalobenzoate with the sodium salt of a substituted phenol
in DMSO (dimethyl sulfoxide), and then reaction of the product with
a second sodium phenoxide. The alkyl dihalobenzoates and sodium
phenoxides either can be obtained commercially or synthesized
according to methods described in the Examples or using other
methods available to those of skill in this art.
[0222] Prodrugs and other derivatives of the compounds suitable for
administration to humans may also be designed and prepared by
methods known to those of skill in the art (see, e.g., Nogrady
(1985) Medicinal Chemistry A Biochemical Approach, Oxford
University Press, New York, pages 388-392).
[0223] Compounds described herein have been synthesized and tested
for activity in in vitro assays recognized as indicative of
activity as endothelin antagonists. Nuclear magnetic resonance
spectroscopic (NMR), mass spectrometric, infrared spectroscopic and
high performance liquid chromatographic analyses indicated that the
synthesized compounds have structures consistent with those
expected for such compounds and are generally at least about 98%
pure. All tested compounds have exhibited activity in the assay
described in the EXAMPLES.
[0224] C. Evaluation of the Bioactivity of the Compounds
[0225] Standard physiological, pharmacological and biochemical
procedures are available for testing the compounds to identify
those that possess any of the biological activities of an
endothelin peptide or the ability to interfere with or inhibit
endothelin peptides. Compounds that exhibit in vitro activities,
such as the ability to bind to endothelin receptors or to compete
with one or more of the endothelin peptides for binding to
endothelin receptors can be used in the methods for isolation of
endothelin receptors and the methods for distinguishing the
specificities of endothelin receptors and the methods for
elucidating the physiological and/or pathophysiological roles of
endothelin, and are candidates for use in the methods of treating
endothelin-mediated disorders.
[0226] Thus, other preferred compounds of formulas I and II, in
addition to those specifically identified herein, that are
endothelin antagonists or agonists may be identified using such
screening assays.
[0227] 1. Identifying Compounds That Modulate the Activity of an
Endothelin Peptide
[0228] The compounds are tested for the ability to modulate the
activity of endothelin-1. Numerous assays are known to those of
skill in the art for evaluating the ability of compounds to
modulate the activity of endothelin (see, e.g., U.S. Pat. No.
5,114,918 to Ishikawa et al.; EP A1 0 436 189 to BANYU
PHARMACEUTICAL CO., LTD. (Oct. 7, 1991); Borges et al. (1989) Eur.
J. Pharm. 165: 223-230; Filep et al. (1991) Biochem. Biophys. Res.
Commun. 177: 171-176). in vitro studies may be corroborated with in
vivo studies (see, e.g., U.S. Pat. No. 5,114,918 to Ishikawa et
al.; EP A1 0 436 189 to BANYU PHARMACEUTICAL CO., LTD. (Oct. 7,
1991)) and pharmaceutical activity thereby evaluated. Such assays
are described in the Examples herein and include the ability to
compete for binding to ET.sub.A and ET.sub.B receptors present on
membranes isolated from cell lines that have been genetically
engineered to express either ET.sub.A or ET.sub.B receptors on
their cell surfaces.
[0229] The properties of a potential antagonist may be assessed as
a function of its ability to inhibit an endothelin induced activity
in vitro using a particular tissue, such as rat portal vein and
aorta as well as rat uterus, trachea and vas deferens (see e.g.,
Borges, R., Von Grafenstein, H. and Knight, D. E., "Tissue
selectivity of endothelin," Eur. J. Pharmacol 165:223-230, (1989)).
The ability to act as an endothelin antagonist in vivo can be
tested in hypertensive rats, ddy mice or other recognized animal
models (see, Kaltenbronn et al. (1990) J. Med. Chem. 33:838-845,
see, also, U.S. Pat. No. 5,114,918 to Ishikawa et al.; and EP A1 0
436 189 to BANYU PHARMACEUTICAL CO., LTD (Oct. 7, 1991); see, also
Bolger et al. (1983) J. Pharmacol. Exp. Ther. 225291-309). Using
the results of such animal studies, pharmaceutical effectiveness
may be evaluated and pharmaceutically effective dosages determined.
A potential agonist may also be evaluated using in vitro and in
vivo assays known to those of skill in the art.
[0230] Endothelin activity can be identified by the ability of a
test compound to stimulate constriction of isolated rat thoracic
aorta (Borges et al. (1989) "Tissue selectivity of endothelin" Eur.
J. Pharmacol. 165: 223-230). To perform the assay, the endothelium
is abraded and ring segments mounted under tension in a tissue bath
and treated with endothelin in the presence of the test compound.
Changes in endothelin induced tension are recorded. Dose response
curves may be generated and used to provide information regarding
the relative inhibitory potency of the test compound. Other
tissues, including heart, skeletal muscle, kidney, uterus, trachea
and vas deferens, may be used for evaluating the effects of a
particular test compound on tissue contraction.
[0231] Endothelin isotype specific antagonists may be identified by
the ability of a test compound to interfere with endothelin binding
to different tissues or cells expressing different
endothelin-receptor subtypes, or to interfere with the biological
effects of endothelin or an endothelin isotype (Takayanagi et al.
(1991) Reg. Pep. 32: 23-37, Panek et al. (1992) Biochem. Biophys.
Res. Commun. 183: 566-571). For example, ET.sub.B receptors are
expressed in vascular endothelial cells, possibly mediating the
release of prostacyclin and endothelium-derived relaxing factor (De
Nucci et al. (1988) Proc. Natl. Acad. Sci. USA 85:9797). ET.sub.A
receptors are not detected in cultured endothelial cells, which
express ET.sub.B receptors.
[0232] The binding of compounds or inhibition of binding of
endothelin to ET.sub.B receptors can be assessed by measuring the
inhibition of endothelin-1-mediated release of prostacyclin, as
measured by its major stable metabolite, 6-keto PGF.sub.1.alpha.,
from cultured bovine aortic endothelial cells (see, e.g., Filep et
al. (1991) Biochem. and Biophys Res. Commun. 177: 171-176). Thus,
the relative affinity of the compounds for different endothelin
receptors may be evaluated by determining the inhibitory dose
response curves using tissues that differ in receptor subtype.
[0233] Using such assays, the relative affinities of the compounds
for ET.sub.A receptors and ET.sub.B receptors have been and can be
assessed. Those that possess the desired properties, such as
specific inhibition of binding of endothelin-1, are selected. The
selected compounds that exhibit desirable activities may be
therapeutically useful and are tested for such uses using the
above-described assays from which in vivo effectiveness may be
evaluated (see, e.g., U.S. Pat. No. 5,248,807; U.S. Pat. No.
5,240,910; U.S. Pat. No. 5,198,548; U.S. Pat. No. 5,187,195; U.S.
Pat. No. 5,082,838; U.S. Pat. No. 5,230,999; published Canadian
Application Nos. 2,067,288 and 2071193; published Great Britain
Application No. 2,259,450; Published International PCT Application
No. WO 93/08799; Benigi et al. (1993) Kidney International
44:440-444; and Nirei et al. (1993) Life Sciences 52:1869-1874).
Compounds that exhibit in vitro activities that correlate with in
vivo effectiveness will then be formulated in suitable
pharmaceutical compositions and used as therapeutics.
[0234] The compounds also may be used in methods for identifying
and isolating endothelin-specific receptors and aiding in the
design of compounds that are more potent endothelin antagonists or
agonists or that are more specific for a particular endothelin
receptor.
[0235] 2. Isolation of Endothelin Receptors
[0236] A method for identifying endothelin receptors is provided.
In practicing this method, one or more of the compounds is linked
to a support and used in methods of affinity purification of
receptors. By selecting compounds with particular specificities,
distinct subclasses of ET receptors may be identified.
[0237] One or more of the compounds may be linked to an appropriate
resin, such as Affi-gel, covalently or by other linkage, by methods
known to those of skill in the art for linking endothelin to such
resins (see, Schvartz et al. (1990) Endocrinology 126: 3218-3222).
The linked compounds can be those that are specific for ET.sub.A or
ET.sub.B receptors or other subclass of receptors.
[0238] The resin is pre-equilibrated with a suitable buffer
generally at a physiological pH (7 to 8). A composition containing
solubilized receptors from a selected tissue are mixed with the
resin to which the compound is linked and the receptors are
selectively eluted. The receptors can be identified by testing them
for binding to an endothelin isopeptide or analog or by other
methods by which proteins are identified and characterized.
Preparation of the receptors, the resin and the elution method may
be performed by modification of standard protocols known to those
of skill in the art (see, e.g., Schvartz et al. (1990)
Endocrinology 126: 3218-3222).
[0239] Other methods for distinguishing receptor type based on
differential affinity to any of the compounds herein are provided.
Any of the assays described herein for measuring the affinity of
selected compounds for endothelin receptors may also be used to
distinguish receptor subtypes based on affinity for particular
compounds provided herein. In particular, an unknown receptor may
be identified as an ET.sub.A or ET.sub.B receptor by measuring the
binding affinity of the unknown receptor for a compound provided
herein that has a known affinity for one receptor over the other.
Such preferential interaction is useful for determining the
particular disease that may be treated with a compound prepared as
described herein. For example, compounds with high affinity for
ET.sub.A receptors and little or no affinity for ET.sub.B receptors
are candidates for use as hypertensive agents; whereas, compounds
that preferentially interact with ET.sub.B receptors are candidates
for use as anti-asthma agents.
[0240] D. Formulation and Administration of the Compositions
[0241] Effective concentrations of one or more of the compounds of
formula I, II or III pharmaceutically acceptable salts, esters or
other derivatives thereof are mixed with a suitable pharmaceutical
carrier or vehicle. In instances in which the compounds exhibit
insufficient solubility, methods for solubilizing compounds may be
used. Such methods are known to those of skill in this art, and
include, but are not limited to, using cosolvents, such as
dimethylsulfoxide (DMSO), using surfactants, such as tween, or
dissolution in aqueous sodium bicarbonate. Derivatives of the
compounds, such as salts of the compounds or prodrugs of the
compounds may also be used in formulating effective pharmaceutical
compositions.
[0242] The concentrations or the compounds are effective for
delivery of an amount, upon administration, that ameliorates the
symptoms of the endothelin-mediated disease. Typically, the
compositions are formulated for single dosage administration.
[0243] Upon mixing or addition of the compound(s), the resulting
mixture may be a solution, suspension, emulsion or the like. The
form of the resulting mixture depends upon a number of factors,
including the intended mode of administration and the solubility of
the compound in the selected carrier or vehicle. The effective
concentration is sufficient for ameliorating the symptoms of the
disease, disorder or condition treated and may be empirically
determined.
[0244] Pharmaceutical carriers or vehicles suitable for
administration of the compounds provided herein include any such
carriers known to those skilled in the art to be suitable for the
particular mode of administration. In addition, the compounds may
be formulated as the sole pharmaceutically active ingredient in the
composition or may be combined with other active ingredients.
[0245] The active compounds can be administered by any appropriate
route, for example, orally, parenterally, intravenously,
intradermally, subcutaneously, or topically, in liquid, semi-liquid
or solid form and are formulated in a manner suitable for each
route of administration. Preferred modes of administration include
oral and parenteral modes of administration.
[0246] The active compound is included in the pharmaceutically
acceptable carrier in an amount sufficient to exert a
therapeutically useful effect in the absence of undesirable side
effects on the patient treated. The therapeutically effective
concentration may be determined empirically by testing the
compounds in known in vitro and in vivo systems (see, e.g., U.S.
Pat. No. 5,114,918 to Ishikawa et al.; EP A1 0 436 189 to BANYU
PHARMACEUTICAL CO., LTD (Oct. 7, 1991); Borges et al. (1989) Eur.
J. Pharm. 165: 223-230;: Filep et al. (1991) Biochem. Biophys. Res.
Commun. 177: 171-176) and then extrapolated therefrom for dosages
for humans.
[0247] The concentration of active compound in the drug composition
will depend on absorption, inactivation and excretion rates of the
active compound, the dosage schedule, and amount administered as
well as other factors known to those of skill in the art. For
example, the amount that is delivered is sufficient to treat the
symptoms of hypertension. The effective amounts for treating
endothelin-mediated disorders are expected to be higher than the
amount of the compound that would be administered for treating
bacterial infections.
[0248] Typically a therapeutically effective dosage should produce
a serum concentration of active ingredient of from about 0.1 ng/ml
to about 50-100 .mu.g/ml. The pharmaceutical compositions typically
should provide a dosage of from about 0.01 mg to about 2000 mg of
compound per kilogram of body weight per day. The active ingredient
may be administered at once, or may be divided into a number of
smaller doses to be administered at intervals of time. It is
understood that the precise dosage and duration of treatment is a
function of the disease being treated and may be determined
empirically using known testing protocols or by extrapolation from
in vivo or in vitro test data. It is to be noted that
concentrations and dosage values may also vary with the severity of
the condition to be alleviated. It is to be further understood that
for any particular subject, specific dosage regimens should be
adjusted over time according to the individual need and the
professional judgment of the person administering or supervising
the administration of the compositions, and that the concentration
ranges set forth herein are exemplary only and are not intended to
limit the scope or practice of the claimed compositions.
[0249] If oral administration is desired, the compound should be
provided in a composition that protects it from the acidic
environment of the stomach. For example, the composition can be
formulated in an enteric coating that maintains its integrity in
the stomach and releases the active compound in the intestine. The
composition may also be formulated in combination with an antacid
or other such ingredient.
[0250] Oral compositions will generally include an inert diluent or
an edible carrier and may be compressed into tablets or enclosed in
gelatin capsules. For the purpose of oral therapeutic
administration, the active compound or compounds can be
incorporated with excipients and used in the form of tablets,
capsules or troches. Pharmaceutically compatible binding agents and
adjuvant materials can be included as part of the composition.
[0251] The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder, such as microcrystalline cellulose, gum
tragacanth and gelatin; an excipient such as starch and lactose, a
disintegrating agent such as, but not limited to, alginic acid and
corn starch; a lubricant such as, but not limited to, magnesium
stearate; a glidant, such as, but not limited to, colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; and a
flavoring agent such as peppermint, methyl salicylate, and fruit
flavoring.
[0252] When the dosage unit form is a capsule, it can contain, in
addition to material of the above type, a liquid carrier such as a
fatty oil. In addition, dosage unit forms can contain various other
materials which modify the physical form of the dosage unit, for
example, coatings of sugar and other enteric agents. The compounds
can also be administered as a component of an elixir, suspension,
syrup, wafer, chewing gum or the like. A syrup may contain, in
addition to the active compounds, sucrose as a sweetening agent and
certain preservatives, dyes and colorings and flavors.
[0253] The active materials can also be mixed with other active
materials which do not impair the desired action, or with materials
that supplement the desired action, such as antacids, H2 blockers,
and diuretics. For example, if the compound is used for treating
asthma or hypertension, it may be used with other bronchodilators
and antihypertensive agents, respectively.
[0254] Solutions or suspensions used for parenteral, intradermal,
subcutaneous, or topical application can include any of the
following components: a sterile diluent, such as water for
injection, saline solution, fixed oil, polyethylene glycol,
glycerine, propylene glycol or other synthetic solvent;
antimicrobial agents, such as benzyl alcohol and methyl parabens;
antioxidants, such as ascorbic acid and sodium bisulfite; chelating
agents, such as ethylenediaminetetraacetic acid (EDTA); buffers,
such as acetates, citrates and phosphates; and agents for the
adjustment of tonicity such as sodium chloride or dextrose.
Parenteral preparations can be enclosed in ampules, disposable
syringes or multiple dose vials made of glass, plastic or other
suitable material.
[0255] If administered intravenously, suitable carriers include
physiological saline or phosphate buffered saline (PBS), and
solutions containing thickening and solubilizing agents, such as
glucose, polyethylene glycol, and polypropylene glycol and mixtures
thereof. Liposomal suspensions, including tissue-targeted
liposomes, may also be suitable as pharmaceutically acceptable
carriers. These may be prepared according to methods known to those
skilled in the art. For example, liposome formulations may be
prepared as described in U.S. Pat. No. 4,522,811.
[0256] The active compounds may be prepared with carriers that
protect the compound against rapid elimination from the body, such
as time release formulations or coatings. Such carriers include
controlled release formulations, such as, but not limited to,
implants and microencapsulated delivery systems, and biodegradable,
biocompatible polymers, such as collagen, ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid
and others. Methods for preparation of such formulations are known
to those skilled in the art.
[0257] The compounds may be formulated for local or topical
application, such as for topical application to the skin and mucous
membranes, such as in the eye, in the form of gels, creams, and
lotions and for application to the eye or for intracisternal or
intraspinal application. Such solutions, particularly those
intended for ophthalmic use, may be formulated as 0.01%-10%
isotonic solutions, pH about 5-7, with appropriate salts. The
compounds may be formulated as aeorsols for topical application,
such as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126,
4,414,209, and 4,364,923, which describe aerosols for delivery of a
steroid useful for treatment inflammatory diseases, particularly
asthma).
[0258] Finally, the compounds may be packaged as articles of
manufacture containing packaging material, a compound provided
herein, which is effective for antagonizing the effects of
endothelin, ameliorating the symptoms of an endothelin-mediated
disorder, or inhibiting binding of an endothelin peptide to an ET
receptor with an IC.sub.50 of less than about 10 .mu.M, more
preferably less than 1 .mu.M, within the packaging material, and a
label that indicates that the compound or salt thereof is used for
antagonizing the effects of endothelin, treating
endothelin-mediated disorders or inhibiting the binding of an
endothelin peptide to an ET receptor.
[0259] The following examples are included for illustrative
purposes only and are not intended to limit the scope of the
invention.
EXAMPLE 1
2-(4-Methoxyphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid
[0260] A. Sodium 3,4-(methylenedioxy)phenoxide
[0261] Sesamol (415 mg, 3.0 mmol) was added, as a solution in THF
(2 ml), to a suspension of mineral oil-free NaH under a water
cooling bath. The mixture was stirred at 25.degree. C. for 20 min.
yielding a clear solution. The solvent was evaporated at 40.degree.
C. under reduced pressure and the resulting solid sodium
3,4-(methylenedioxy)phenoxide was dried in vacuo at 25.degree. C.
for 30 min before use.
[0262] B. 2,6-Difluorobenzoyl chloride
[0263] A solution of 2,6-difluorobenzoic acid (1.58 g, 10.0 mmol)
and SOCl.sub.2 (1.1 ml, 15 mmol) were refluxed at 80.degree. C. for
1 hr. Two drops of DMF were added and reflux continued for 15 min.
to dissolve any remaining solid. Excess SOCl.sub.2 was evaporated
in vacuo to give 2,6-difluorobenzoyl chloride as a light yellow
oil.
[0264] C. Ethyl 2,6-difluorobenzoate
[0265] 2,6-Difluorobenzoyl chloride was diluted with
CH.sub.2Cl.sub.2 (5 ml) and cooled to 0.degree. C. A mixture of
EtOH (1.2 ml, 20 mmol) and pyridine (1.6 ml, 20 mmol) was added,
dropwise, over 5 minutes to the reaction mixture. The ice bath was
removed after 5 minutes and the reaction mixture stirred at
25.degree. C. for 45 min. The solvent and volatiles were evaporated
under reduced pressure and the residue was partitioned between 1N
HCl and EtOAc (10 ml each). The aqueous phase was separated and
extracted with 2.times.7 ml EtOAc. The combined organic extracts
was washed with NaHCO.sub.3 (10 ml), brine (7 ml), dried over
MgSO.sub.4 and concentrated to give 1.562 g ethyl
2,6-difluorobenzoate as an almost colorless oil. (84% yield from
steps B and C).
[0266] D. Ethyl 2-fluoro-6-
[3,4-(methylene)dioxyphenoxy]benzoate
[0267] Ethyl 2,6-difluorobenzoate (558 mg, 3.0 mmol) was added to
dry sodium 3,4-methylenedioxyphenoxide (3.0 mmol, prepared as
described in part A) in DMSO followed by addition of dry DMSO (2
ml). The reaction mixture was warmed to 50.degree. C. forming a
solution which turned from brown to green upon further heating.
After 90 min., the reaction was cooled to 25.degree. C., diluted
with water (10 ml) and extracted with EtOAc (3.times.10 ml). The
organic extract was washed with water (2.times.7 ml) and brine (7
ml), dried (MgSO.sub.4) and concentrated to give a light yellow
oil. The product was purified by flash chromatography on SiO.sub.2
(5-20% EtOAc/hexane) yielding 532 mg of ethyl
2-fluoro-6-[3,4-(methylenedioxy)phenoxy]benzoate as a colorless oil
(58% yield).
[0268] E. Ethyl
2-(4-methoxyphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoa- te
[0269] Sodium 4-methoxyphenoxide was prepared as described in part
A using 4-methoxyphenol (156 mg, 1.25 mmol) and was dried in vacuo.
A solution of ethyl
2-fluoro-6-[3,4-(methylenedioxy)phenoxy]benzoate (304 mg, 1.0 mmol)
in DMSO was added to the sodium 4-methoxyphenoxide at 25.degree. C.
and the mixture was stirred until all the solid had dissolved
(approximately 20 min.). Stirring was continued at 25.degree. C.
for an additional 24 hr. The reaction mixture was then warmed to
60.degree. C. and stirred for 2 hr., then stirred for 4 hr. at
80.degree. C. At this time all of the ethyl
2-fluoro-6-[3,4-(methylenedioxy)phenoxy]benzoate had been consumed,
as judged by thin layer chromatography (TLC). The reaction was
cooled to 25.degree. C., diluted with water (10 ml) and extracted
with EtOAc (3.times.10 ml). The organic extract was washed with
water (2.times.7 ml) and brine (7 ml), dried (MgSO.sub.4) and
concentrated to give a light yellow oil. The crude product was
purified by flash chromatography (SiO.sub.2, 50%
CH.sub.2Cl.sub.2/hexane-CH.sub.2Cl.sub.2) giving 195 mg of ethyl
2-(4-methoxyphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoate. (47%
yield, m.p. 84.5-85.degree. C.).
[0270] F.
2-(4-methoxyphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid
[0271] NaOH/EtOH (0.5 M) was added to ethyl
2-(4-methoxyphenoxy)-6-[3,4-(m- ethylenedioxy)phenoxy]benzoate (175
mg, 0.429 mmol) at 25.degree. C. The mixture (solid and solution)
was heated under reflux for 20 hr. until TLC indicated the absence
of starting material and the formation of a polar material. The
solvent was evaporated and the residue was diluted with H.sub.2O (7
ml, pH=7.5) and extracted with EtOAc (2.times.7 ml) to give 132 mg
2-(4-methoxyphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic acid as
a yellow foam which was recrystallized from MeOH/H.sub.2O (twice)
to give 102 mg of colorless needles, m.p. 164-165.degree. C.
[0272] The aqueous solution from above was cooled and acidified to
pH 1. It was then extracted with EtOAc (3.times.7 ml) to give a
further 40 mg of
2-(4-methoxyphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic acid as
a white solid, m.p. 163-165.degree. C. This resulted in a total
yield of 142 mg (87%).
EXAMPLE 2
2-[3,4-(methylenedioxy)phenoxy]-6-(4-methylphenoxy)benzoic acid
[0273] 2-[3,4-(methylenedioxy)phenoxy]-6-(4-methylphenoxy)benzoic
acid was prepared as described in Example 1 using sodium
4-methylphenoxide instead of 4-methoxyphenoxide in step E. The
crude product was recrystallized from MeOH/H.sub.2O giving
2-[3,4-(methylenedioxy)phenoxy]-6-(4-methylphen- oxy)benzoic acid
as a white solid. (33% yield, m.p. 180-182.degree. C.).
EXAMPLE 3
2-(4-fluorphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic acid
[0274] 2-(4-fluorphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid was prepared as described in Example 1 using sodium
4-fluorophenoxide instead of sodium 4-methoxyphenoxide in step E.
The crude product was recrystallized from MeOH/H.sub.2O giving
2-(4-fluorophenoxy)-6-[3,4-(meth- ylenedioxy)phenoxy]benzoic acid
as a white solid. (37% yield, m.p. 164-166.degree. C.).
EXAMPLE 4
2-(3,4-dimethoxyphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid
[0275]
2-(3,4-dimethoxyphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid was prepared as described in Example 1 using sodium
3,4-dimethoxyphenoxide instead of sodium 4-methoxyphenoxide in step
E. The crude product was recrystallized from EtOAc/H.sub.2O giving
2-(3,4-dimethoxyphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic as
a tan solid. (35% yield, m.p. 98-100.degree. C.).
EXAMPLE 5
2,6-bis-[3,4-(methylenedioxy)phenoxy]benzoic acid
[0276] A. Ethyl 2,6-bis-[3,4-(methylenedioxy)phenoxy]benzoate
[0277] Ethyl 2,6-bis-[3,4-(methylenedioxy)phenoxy]benzoate was
prepared as described in Example 1D using four equivalents of
sodium 3,4-methylenedioxyphenoxide (Example 1A) per one equivalent
of ethyl 2,6-difluorobenzoate (Example 1C).
[0278] B. 2,6-bis-[3,4-(methylenedioxy)phenoxy]benzoic acid
[0279] 2,6-bis-(3,4-methylenedioxyphenoxy)benzoic acid was prepared
as described in Example 1F with the exception that KOH/EtOH was
used in place of NaOH/EtOH and the reflux reaction was carried out
for 1-12 hours rather than 20 hours. Recrystallization from
MeOH/H.sub.2O gave 2,6-bis-(3,4-methylenedioxyphenoxy)benzoic acid
as a white solid in 40% overall yield, m.p. 117-178.degree. C.
EXAMPLE 6
2,6-bis-(4-methoxyphenoxy)benzoic acid
[0280] 2,6-bis-(4-methoxyphenoxy)benzoic acid was prepared as
described in Example 5, using four equivalents sodium
3-methoxyphenoxide. Recrystallization from MeOH/H.sub.2O gave
2,6-bis-(3-methoxyphenoxy)benzo- ic acid as a white solid in 52%
overall yield, m.p. 222-224.degree. C.
EXAMPLE 7
2-(2-Bromo-4-methylphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid
[0281]
2-(2-Bromo-4-methylphenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid was prepared as described in Example 1, using sodium
2-bromo-4-methylphenoxide instead of 4-methyoxyphenoxide in Step E.
Recrystallization from MeOH/H.sub.2O gave
2-(2-bromo-4-methylphenoxy)-6-[-
3,4-(methylenedioxy)phenoxy]benzoic acid as a white solid in 11%
overall yield, m.p. 191-192.degree. C.
EXAMPLE 8
2,6-bis-(4-methylphenoxy)benzoic acid
[0282] 2,6-bis-(4-methylphenoxy)benzoic acid was prepared as
described in Example 5 using four equivalents of sodium
4-methylphenoxide. Recrystallization from MeOH/H.sub.2O gave
2,6-bis-(4-methylphenoxy)benzoi- c acid as a white solid, in 48%
overall yield, m.p. 205-206.degree. C.
EXAMPLE 9
2,6-diphenoxybenzoic acid
[0283] 2,6-diphenoxybenzoic acid was prepared as described in U.S.
Pat. No. 4,191,554, resulting in a 52% yield from
1,3-diphenoxybenzene, m.p 150-153.degree. C.
EXAMPLE 10
2,6-diphenoxyphenylacetic acid
[0284] nBuLi (2.64 ml, 2.38 M in hexane, 6.29 mmol) was added to a
solution of 1,3-diphenoxybenzene (1.5 g, 5.72 mmol) in THF (50 ml)
at 0.degree. C. After the addition was complete, the ice-bath was
removed and the mixture was stirred at room temperature for 2
hours. The reaction mixture was then poured onto freshly crushed
dry-ice (5 g) and the resulting solution was stirred until it
warmed to room temperature. The THF was removed by evaporation and
the residue was partitioned between 1 N NaOH and Et.sub.2O. The
aqueous layer was acidified with concentrated HCl to pH 1 with
cooling and extracted with EtOAc. The organic layer was dried
(MgSO.sub.4) and the solid was filtered. The filtrate was
concentrated to give a yellow solid (1.6 g). SOCl.sub.2 (20 ml) was
added to this yellow solid and the resulting red mixture was
refluxed for 30 minutes. The mixture was then allowed to cool to
room temperature. Volatiles were evaporated on a rotovap. An excess
amount of ethereal CH.sub.2N.sub.2 was added to the residue and the
resulting mixture was stirred at RT for 12 hours. The volatiles
were again evaporated on a rotovap and the residue was suspended in
a 1:1 mixture of THF and water. A catalytic amount of Ag.sub.2O was
added. The mixture was stirred at RT overnight. The resulting grey
solid was filtered off. The filtrate was concentrated and the
aqueous residue was acidified with concentrated HCl to pH 1 and
extracted with EtOAc. The organic layer was dried (MgSO4), the
solid was filtered and the filtrate was concentrated. A portion of
the residue was purified via HPLC to give
2,6-diphenoxyphenyl-acetic acid (20 mg) as a yellowish powder, m.p.
32-38.degree. C.
EXAMPLE 11
2-[3,4-(methylenedioxy)phenoxy]-6-(3-methylphenoxy)benzoic acid
[0285] 2-[3,4-(methylenedioxy)phenoxy]-6-(3-methylphenoxy)benzoic
acid was prepared as described in Example 1 using sodium
3-methylphenoxide instead of sodium 4-methoxyphenoxide in step E.
The product was a light brown solid, m.p. 169-172.degree. C.
EXAMPLE 12
2-(4-methoxythiophenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid
[0286]
2-(4-methoxythiophenoxy)-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid was prepared as described in Example 1 using
4-methoxybenzenethiol (7 eq.) in step E. The final product was a
colorless oil which solidified on standing; 10% yield, m.p.
165-166.degree. C.
EXAMPLE 13
2-[(3-Carboxymethyl)phenoxy]-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid
[0287] A. Ethyl
2-{[3-(carboethoxy)methyl]phenoxy}-6-[3,4-(methylenedioxy)-
phenoxy]benzoate
[0288] Ethyl
2-{[3-(carboethoxy)methyl]phenoxy}-6-[3,4-(methylenedioxy)phe-
noxy]benzoate was prepared in the same manner as described in
Example 1E using 2-fluoro-6-[3,4-(methylenedioxy)phenoxy]benzoate
and sodium 3-(carboethoxy)methyl]phenoxide in DMSO at 100.degree.
C. for 48 hours (28% yield, oil).
[0289] B.
2-[(3-Carboxymethyl)phenoxy]-6-[3,4-(methylenedioxy)phenoxy]benz-
oic acid
[0290]
2-[(3-Carboxymethyl)phenoxy]-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid was prepared in the same manner as described in Example 1F
using ethyl
2-{[3-(carboethoxy)methyl]phenoxy}-6-[3,4-(methylenedioxy)phenoxy]b-
enzoate by basic hydrolysis at refluxing temperature for 4 hours
(83% yield, m.p. 142-145.degree. C.).
EXAMPLE 14
2-[(4-Carboxymethyl)phenoxy]-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid
[0291] A. Ethyl
2-{[4-(carboethoxy)methyl]phenoxy}-6-[3,4-(methylenedioxy)-
phenoxy]benzoate
[0292] Ethyl
2-{[4-(carboethoxy)methyl]phenoxy}-6-[3,4-(methylenedioxy)phe-
noxy]benzoate was prepared in the same manner as described in
Example 1E using 2-fluoro-6-[3,4-(methylenedioxy)phenoxy]benzoate
and sodium 4-(carboethoxy)methylphenoxide in DMSO at 100.degree. C.
for 48 hours (23% yield, oil).
[0293] B.
2-[(4-Carboxymethyl)phenoxy]-6-[3,4-(methylenedioxy)phenoxy]benz-
oic acid
[0294]
2-[(4-Carboxymethyl)phenoxy]-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid was prepared in the same manner as described in Example 1F
using ethyl
2-{[4-(carboethoxy)methyl]phenoxy}-6-[3,4-(methylenedioxy)phenoxy]b-
enzoate by basic hydrolysis at refluxing temperature for 4 hours
(76% yield, m.p. 204-206.degree. C.).
EXAMPLE 15
2-[(4-Carboxyethyl)phenoxy]-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid
[0295] A. Ethyl
2-{[4-(carboethoxy)ethyl]phenoxy}-6-[3,4-(methylenedioxy)p-
henoxy]benzoate
[0296] Ethyl
2-{[4-(carboethoxy)ethyl]phenoxy}-6-[3,4-(methylenedioxy)phen-
oxy]benzoate was prepared in the same manner as described in
Example 1E using 2-fluoro-6-[3,4-(methylenedioxy)phenoxy]benzoate
and sodium 4-(carboethoxy)ethylphenoxide in DMSO at 100.degree. C.
and stirred for 48 hours (28% yield, oil).
[0297] B.
2-[(4-Carboxyethyl)phenoxy]-6-[3,4-(methylenedioxy)phenoxy]benzo-
ic acid
[0298]
2-[(4-Carboxyethyl)phenoxy]-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid was prepared in the same manner as described in Example 1F
using ethyl
2-{[4-(carboethoxy)ethyl]phenoxy}-6-[3,4-(methylenedioxy)phenoxy]be-
nzoate by basic hydrolysis at refluxing temperature for 4 hours
(40% yield, m.p. 138-140.degree. C.).
EXAMPLE 16
2-[(3-Carboxyethyl)phenoxy]-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid
[0299] A. Ethyl
2-{[3-(carboethoxy)ethyl]phenoxy}-6-[3,4-(methylenedioxy)p-
henoxy]benzoate
[0300] Ethyl
2-{[3-(carboethoxy)ethyl]phenoxy}-6-[3,4-(methylenedioxy)phen-
oxy]benzoate was prepared in the same manner as described in
Example 1E using 2-fluoro-6-[3,4-(methylenedioxy)phenoxy]benzoate
(Example 1D) and sodium 3-(carboethoxy)ethylphenoxide in DMSO at
100.degree. C. and stirring for 48 hours (29% yield, oil).
[0301] B.
2-[(3-Carboxyethyl)phenoxy]-6-[3,4-(methylenedioxy)phenoxy]benzo-
ic acid
[0302]
2-[(3-Carboxyethyl)phenoxy]-6-[3,4-(methylenedioxy)phenoxy]benzoic
acid was prepared in the same manner as described in Example 1F
using ethyl
2-{[3-(carboethoxy)ethyl]phenoxy}-6-[3,4-(methylenedioxy)phenoxy]be-
nzoate by basic hydrolysis at refluxing temperature for 4 hours
(73% yield, m.p. 100-114.degree. C.).
EXAMPLE 17
2-[3,4-(methylenedioxy)phenoxy]-6-[2-(1-propenyl)-3,4-(methylenedioxy)phen-
oxy]benzoic acid
[0303] A. Allyl 3,4-(methylenedioxy)phenyl ether
[0304] Allyl bromide (1.75 g., 14 mmol) was added to a solution of
sesamol (2.0 g, 14 mmol) that had been dissolved in 50 ml of dry
acetone, followed by addition of powdered potassium carbonate (2.4
g, 17 mmol) and the resulting cloudy solution was refluxed for 18
h. The solution was cooled and the solvent removed in vacuo. The
remaining suspension was extracted into ether and the organic layer
was washed with water (1.times.25 ml), brine solution (1.times.25
ml), and dried over MgSO.sub.4. Concentration of solvent yielded
2.4 g (94%) of a pale yellow oil which was used in the next step
with no further purification.
[0305] B. 2-allyl-4,5-(methylenedioxy)phenol
[0306] A solution of allyl 3,4-(methylenedioxy)phenyl ether (2.4 g,
13 mmol), was dissolved in 30 ml of 1,2-dichlorobenzene and the
solution was refluxed for 18 h. The solvent was removed under high
vacuum in a water bath set at 60.degree. C. The remaining oil was
extracted into ether, washed with brine and dried over MgSO.sub.4.
The solvent was removed under vacuum to give a yellow oil which was
further purified by flash column chromatography. Elution with 10%
ethyl acetate/hexanes gave 2.2 g (92%) of the pure compound as a
pale yellow oil.
[0307] C. Ethyl
2-fluoro-6-[2-(1-propenyl)-4,5-(methylenedioxy)phenoxy]ben-
zoate
[0308] A solution of 2-allyl-4,5-(methylenedioxy)phenoxy (2.2 g.,
12 mmol) in dry THF under nitrogen was added to mineral oil free
sodium hydride (480 mg, 12 mmol) at 0.degree. C. Bubbles of H.sub.2
gas persisted for about 10 minutes. The solution was allowed to
come to room temperature, and stirring was continued for an
additional 10 min. resulting in a clear pale yellow solution. The
THF was removed under vacuum and the sodium salt of
2-allyl-4,5(methylenedioxy)phenol was dried under high vacuum for
10 minutes. The salt was then dissolved in 15 ml. of dry DMSO and
treated with ethyl 2,6-difluoro benzoate (2.5 g, 12 mmol)
previously dissolved in dry DMSO (10 ml). The resulting solution,
which turned dark brown almost immediately, was heated at
50.degree. C. overnight (18 h.) Water (30 ml) followed by ethyl
acetate (50 ml) was added to the reaction mixture upon cooling and
the layers were separated. The aqueous layer was extracted with an
additional 50 ml of EtOAC. The combined organic fractions was then
washed with brine and dried over MgSO.sub.4. Evaporation of
solvents yielded an oil which was purified by flash column
chromatography. Elution with 2% ethyl acetate-hexanes gave 2.41 g
(54%) of a yellow oil. .sup.1H NMR analysis (CDCl.sub.3) of this
oil showed that the desired terminal methylene unit was not present
as the olefin migrated to the more stable conjugated position upon
exposure to heat and base.
[0309] D. Ethyl
2-(3,4-methylenedioxy)phenoxy-6-[2-(1-propenyl)-4,5-(methy-
lenedioxy)phenoxy]benzoate
[0310] Sodium hydride (308 mg, 7.69 mmol) was added to a solution
of sesamol (967 mg, 6.99 mmol) in dry THF (5 ml), and the solution
was stirred at room temperature for 20 minutes. The THF was then
removed under pressure and the remaining sodium salt of sesamol was
placed under vacuum for 10 minutes. Dry DMSO (8 ml) followed
immediately by ethyl-2-fluoro-6-[2-(1-propenyl)-3,4
(methylenedioxy)phenoxy]benzoate (2.41 g, 6.99 mmol) in dry DMSO
were added. The resulting dark brown mixture was heated at
80.degree. C. for 18 h. Water (30 ml) followed by ethyl acetate (50
ml) was added to the reaction mixture upon cooling and the layers
were separated. The aqueous layer was extracted with an additional
50 ml of EtOAC. The combined organic fractions was then washed with
brine and dried over MgSO.sub.4. Evaporation of solvents yielded an
oil which was purified by flash column chromatography using
5-10%-ethyl acetate-hexanes giving 1.4 g (43%) of the unsaturated
ester as a yellow oil.
[0311] E.
2-(3,4-methylenedioxy)phenoxy-6-[2-(1-propenyl)-4,5-(methylenedi-
oxy)phenoxy]benzoic acid
[0312] KOH (140 mg, 2.8 mmol), which had been dissolved in ethanol,
was added to an ethanolic solution of ethyl
2-(3,4-methylenedioxy)phenoxy-6-[- 2-(1-propenyl)-4,5
methylenedioxy)phenoxy]benzoate (200 mg, 0.43 mmol), and the
solution, which turned into a white suspension, was refluxed for 12
hours. The ethanol was removed under vacuum and the remaining solid
was dissolved in water (20 ml) and extracted with ethyl acetate
(1.times.5 ml). The aqueous fraction was then acidified to pH 2.0
with 6N HCl and extracted into ethyl acetate. The organic layer was
washed with brine and dried over MgSO.sub.4. After evaporation of
solvent
2-[3,4-(methylenedioxy)phenoxy]-6-[2-(1-propenyl)-3,4-(methylenedioxy)phe-
noxy]benzoic acid remained as a yellow-brown oil (136 mg, 72%
yield). The oil, shown to be 96% pure by analytical HPLC, was
further purified by preparative HPLC to give the colorless, pure
acid (m.p. 175-177.degree. C.).
EXAMPLE 18
2-(3,4-methylenedioxy)phenoxy-6-[2-propyl-4,5-(methylenedioxy)phenoxy]benz-
oic acid
[0313] A. Ethyl
2-(3,4-methylenedioxy)phenoxy-6-(2-propyl-4,5-(methylenedi-
oxy)phenoxy benzoate
[0314] Palladium on activated carbon (50 mg of 10%) was added to a
solution of ethyl
2-(3,4-methylenedioxy)phenoxy-6-[2-(1-propenyl)-4,5(met-
hylenedioxy)phenoxy]benzoate (250 mg, 0.54 mmol) (Example 17D) in
ethanol (10 ml). The solution was hydrogenated at room temperature
at 50 psi for 12 hours. Filtration of the catalyst through celite
and evaporation of solvent left a clear oil (251 mg, quantitative)
that was used in the next step without further purification.
[0315] B.
2-(3,4-methylenedioxy)phenoxy-6-[2-propyl-4,5-(methylenedioxy)ph-
enoxy]benzoic acid
[0316]
2-(3,4-methylenedioxy)phenoxy-6-[2-propyl-4,5-(methylenedioxy)pheno-
xy]benzoic acid was prepared in the same manner as described in
Example 17E from ethyl
2-(3,4-methylenedioxy)phenoxy-6-[2-propyl-4,5-(methylenedi-
oxy)phenoxy]benzoate (251 mg, 0.5 mmol) in 46% yield. A portion of
the crude product was further purified by preparative HPLC to give
the pure acid as a white powder (m.p. 168.degree. C.).
EXAMPLE 19
2-(3,4-methylenedioxy)phenoxy-6-[2-carboxyethyl-4,5-(methylenedioxy)phenox-
y]benzoic acid
[0317] A.
Ethyl-2-(3,4-methylenedioxy)phenoxy-6-[2-formyl-4,5-(methylenedi-
oxy)phenoxy]benzoate
[0318] Sodium periodate (810 mg, 3.78 mmol) was added to a solution
of ethyl
2-(3,4-methylenedioxy)phenoxy-6-[2-(1-propenyl)-4,5-(methylenedioxy-
)phenoxy]benzoate (876 mg, 1.89 mmol; Example 17D) in dioxane (20
ml) and water (10 ml) followed immediately by addition of osmium
tetroxide (41 mg). The brown solution was stirred at room
temperature for 2 hours, and the reaction was judged complete by
TLC (20% ethyl acetate-hexanes). The reaction was quenched by
addition of 5 ml of NaHSO.sub.3. The solvents were removed in vacuo
giving a dark brownish oil. The oil was extracted into ethyl
acetate and then washed with water (1.times.20 ml), brine
(1.times.20 ml) and dried over MgSO.sub.4. Evaporation of solvents
left a dark oil which was purified by flash column chromatography
using 20% ethyl acetate-hexanes, yielding 380 mg (45%) of a light
brown oil.
[0319] B. Ethyl
2-(3,4-methylenedioxy)phenoxy-6-[2-(tert-butoxycarbonyl-tr-
ans-ethenyl)-4,5-(methylenedioxy)phenoxy]benzoate
[0320] NaH in one lot (25 mg, 0.574 mmol) was added to a solution
of t-butyl diethylphosphonoacetate (157 mg, 0.626 mmol) in dry THF.
The resulting solution was stirred at 0.degree. C. for 5 minutes,
then at room temperature for an additional 10 minutes. Ethyl
2-(3,4-methylenedioxy)phenoxy-6-[2-formyl-4,5-(methylenedioxy)phenoxy]ben-
zoate (235 mg, 0.522 mmol) that had been dissolved in THF was added
dropwise via syringe. After the reaction mixture was stirred at
room temperature for 2 hours, it was judged complete by TLC (20%
ethyl acetate-hexanes). The solvent was removed in vacuo leaving a
brownish residue which was extracted into ethyl acetate. The
organic fraction was washed with brine (1.times.20 ml) and dried
over MgSO.sub.4. Evaporation of solvents left an oil that was
purified by flash column chromatography. Elution with 10% ethyl
acetate-hexanes gave 240 mg (84% yield) of the pure unsaturated
ester as a clear white semisolid.
[0321] C. Ethyl
2-(3,4-methylenedioxy)phenoxy-6-[2-(tert-butoxycarbonyleth-
yl)-4,5-(methylenedioxy)phenoxy]benzoate
[0322] Ethyl
2-(3,4-methylenedioxy)phenoxy-6-[2-(tert-butoxycarbonylethyl)-
-4,5-(methylenedioxy)phenoxy]benzoate was prepared in the same
manner as described in Example 18A from ethyl
2-(3,4-methylenedioxy)phenoxy-6-[2-(t-
ert-butoxycarbonyl-trans-ethenyl)-4,5-(methylenedioxy)phenoxy]benzoate
(140 mg, 0.255 mmol). After hydrogenation at 50 psi for 12 hours,
filtration of the Pd/C catalyst and evaporation of solvents gave
112 mg (80% yield) of the saturated ester as a clear viscous oil
that was taken to the next step without further purification.
[0323] D.
2-(3,4-methylenedioxy)phenoxy-6-[2-carboxyethyl-4,5-(methylenedi-
oxy)phenoxy]benzoic acid
[0324]
2-(3,4-methylenedioxy)phenoxy-6-[2-carboxyethyl-4,5-(methylenedioxy-
)phenoxy]benzoic acid was prepared in the same manner as described
in Example 17E from ethyl
2-(3,4-methylenedioxy)phenoxy-6-[2-(tert-butoxycar-
bonyl-trans-ethenyl)-4,5-(methylenedioxy)phenoxy]benzoate (100 mg,
0.180 mmol). Purification of the crude product was achieved by
preparative HPLC to give 58 mg (69% yield) of the pure diacid as a
light brown semisolid.
EXAMPLE 20
2-(3,4-methylenedioxy)phenoxy-6-[2-carboxy-trans-ethenyl-4,5-(methylenedio-
xy)phenoxy]benzoic acid
[0325]
2-(3,4-methylenedioxy)phenoxy-6-[2-carboxy-trans-ethenyl-4,5-(methy-
lenedioxy)phenoxy]benzoic acid was prepared in the same manner as
described in Example 17E by hydrolysis of ethyl
2-(3,4-methylenedioxy)phe-
noxy-6-[2-(tert-butoxycarbonyl-trans-ethenyl)-4,5-(methylenedioxy)phenoxy]-
benzoate (100 mg, 0.21 mmol; Example 19B). Purification of the
crude product was achieved by preparative HPLC to give 49 mg (58%
yield) of the pure diacid as a tan colored powder (m.p.
242-254.degree. C.).
EXAMPLE 21
2,6-bis-[3,4-(methylenedioxy)phenoxy]phenyl acetic acid
[0326] A. 2,6-bis-[3,4-(methylenedioxy)phenoxy]benzyl chloride
[0327] A solution of 2,6-bis-[3,4-(methylenedioxy)phenoxy]benzoic
acid (454 mg, 1.15 mmol; Example 5) was added to a suspension of
lithium aluminum hydride (88 mg, 2.30 mmol) in THF (12 ml) and the
solution was refluxed for 12 hours. The THF was then removed by
evaporation and the remaining residue was partitioned between ethyl
acetate (30 ml) and 1N HCl (20 ml). The organic fraction was
collected, washed with brine (1.times.15 ml) and dried over
MgSO.sub.4. Evaporation of the solvents yielded 550 mg of a brown
residue, which was taken to the next step without further
purification.
[0328] The crude alcohol from above (550 mg) was dissolved in
CHCl.sub.3 along with two drops of dimethyl formamide. To this was
then added thionyl chloride (430 .mu.L, 5.75 mmol) and the
resulting pale yellow solution was refluxed for 1.5 hours. After
cooling, the solution was washed with brine and the organic layer
was dried over MgSO.sub.4. Evaporation of solvents left a brownish
solid which was purified by column chromatography using 5% ethyl
acetate-hexanes to give 435 mg of the pure benzyl chloride as a
brown solid (94% yield for both steps).
[0329] B. 2,6-bis-[3,4-(methylenedioxy)phenoxy]benzyl cyanide
[0330] Sodium cyanide (85 mg, 1.6 mmol) was added to a solution of
2,6-bis-[(3,4-(methylenedioxy)phenoxy]-benzyl chloride (435 mg, 1.1
mmol) in DMF (3 ml) at room temperature. The resulting dark brown
solution was stirred at room temperature for 18 hours. The DMF was
removed under high vacuum and the remaining brown residue was
extracted into ethyl acetate and washed with brine (1.times.5 ml).
After drying over MgSO.sub.4, the solvent was removed by
evaporation and the remaining solid was purified by column
chromatography using 10% ethyl acetate-hexanes which yielded 217 mg
(51% yield) of the pure benzyl cyanide as a white powder.
[0331] C. Preparation of
2,6-bis-[3,4-(methylenedioxy)phenoxy]phenylacetic acid
[0332] Potassium hydroxide (304 mg, 5.4 mmol) was added to a
solution of 2,6-bis-[3,4-(methylenedioxy)phenoxy]benzyl cyanide
(211 mg, 0.54 mmol) in ethanol (.about.5 ml) that had been
dissolved in water (2 ml). The resulting suspension was then
refluxed for 15 hours giving a pale yellow clear solution. The
solvents were removed in vacuo and the remaining residue was
dissolved in water. The aqueous solution was extracted with ethyl
acetate (1.times.5 ml) to remove any unreacted starting material,
and then the pH was adjusted to .about.2.0 using concentrated HCl.
The aqueous solution was then extracted into ethyl acetate and the
organic phase was washed with brine (1.times.5 ml) and dried
(MgSO.sub.4). Evaporation of solvents left a pale brown residue
which was crystallized from MeOH/H.sub.2O to give 186 mg (81%
yield) of the pure acid as a light tan colored solid (mp.
176-177.degree. C.).
EXAMPLE 22
Ethyl
2-[3,4-(methylenedioxy)phenoxy]-6-[2-(carboxyethyl)-4-methoxyphenoxy-
]benzoate
[0333] A. Allyl(4-methoxyphenyl)ether
[0334] Allyl(4-methoxyphenyl)ether was prepared in the same manner
as described in Example 17A, but using 4-methoxyphenol (10 g, 84
mmol) and allyl bromide (9.7 g, 84 mmol), in 98% yield as a yellow
oil.
[0335] B. 2-Allyl-4-methoxyphenol
[0336] 2-Allyl-4-methoxyphenol was prepared in the same manner as
described in Example 17B, but using allyl(4-methoxyphenyl) ether
(13.0 g, 79 mmol). Column chromatography using 10% ethyl
acetate-hexanes resulted in 11.6 g (89% yield) of the pure phenol
as a pale yellow oil.
[0337] C. Ethyl
2-fluoro-6-[2-(1-propenyl)-4-methoxyphenoxy]benzoate
[0338] Ethyl 2-fluoro-6-[2-(1-propenyl)-4-methoxyphenoxy]benzoate
was prepared in the same manner as described in Example 17C, but
using 2-allyl-4-methoxyphenol (1.73 g, 10.5 mmol) and ethyl
2,6-difluorobenzoate (1.78 g, 9.6 mmol). Column chromatography
using 2% ethyl acetate-hexanes resulted in 1.48 g (43% yield) of
the pure benzoate as a pale yellow oil.
[0339] D. Ethyl
2-[3,4-(methylenedioxy)phenoxy]-6-[2-(1-propenyl)-4-methox-
yphenoxy]benzoate
[0340] Ethyl
2-[3,4-(methylenedioxy)phenoxy]-6-[2-(1-propenyl)-4-methoxyph-
enoxy]benzoate was prepared in the same manner as described in
Example 17D from ethyl
2-fluoro-6-[2-(1-propenyl)-4-methoxyphenoxy]benzoate (500 mg, 1.51
mmol) and sesamol (256 mg, 1.82 mmol). Column chromatography using
15% ethyl acetate/hexanes resulted in 246 mg (37% yield) of the
pure benzoate as a yellow oil.
[0341] E. Ethyl
2-[3,4-(methylenedioxy)phenoxy]-6-(2-formyl-4-methoxypheno-
xy)benzoate
[0342] Ethyl
2-[3,4-(methylenedioxy)phenoxy]-6-(2-formyl-4-methoxyphenoxy)-
benzoate was prepared in the same manner as described in Example
19A from ethyl
2-[3,4-(methylenedioxy)phenoxy]-6-[2-(1-propenyl)-4-methoxyphenoxy]-
benzoate (246 mg, 0.55 mmol). Column chromatography using 10% ethyl
acetate-hexanes resulted in 87 mg (36% yield) of the pure aldehyde
as a pale yellow oil.
[0343] F. Ethyl
2-[3,4-(methylenedioxy)phenoxy]-6-{2-[(methoxycarbonyl)-vi-
nyl]-4-methoxyphenoxy}benzoate
[0344] Methyl(triphenylphosphoranylidene)acetate (80 mg, 0.24 mmol)
was added to a solution of ethyl
2-[3,4-(methylenedioxy)phenoxy]-6-(2-formyl--
4-methoxyphenoxy)benzoate (87 mg, 0.2 mmol) that had been dissolved
in THF (5 ml). The resulting solution was refluxed for 16 hours.
The reaction mixture was then cooled and the solvent removed in
vacuo leaving an oily residue that was chromatographed directly
using 15% ethyl acetate-hexanes to give 80 mg (81% yield) of the
pure diester as a yellow oil.
[0345] G. Ethyl
2-[3,4-(methylenedioxy)phenoxy]-6-[2-(carboxyethyl)-4-meth-
oxyphenoxy]benzoate
[0346] Ethyl
2-[3,4-(methylenedioxy)phenoxy]-6-[2-(carboxyethyl)-4-methoxy-
phenoxy]benzoate was prepared in the same manner as described in
Example 18A by hydrogenation of ethyl
2-[3,4-(methylenedioxy)phenoxy]-6-{2-[2-(me-
thoxycarbonyl)vinyl]-4-methoxyphenoxy}benzoate (80 mg, 0.16 mmol).
Concentration of solvents gave 57 mg (71% yield) of the crude
benzoate which was taken to the next step with no further
purification.
EXAMPLE 23
2-[3,4-(methylenedioxy)phenoxy]-6-[(2-carboxyethyl)-4-methoxyphenoxy]benzo-
ic acid
[0347]
2-[3,4-(Methylenedioxy)phenoxy]-6-[(2-carboxyethyl)-4-methoxyphenox-
y]benzoic acid was prepared by hydrolysis of ethyl
2-[3,4-(methylenedioxy)-
phenoxy]-6-[2-(carboxyethyl)-4-methoxyphenoxy]benzoate as described
in Example 17E. Initial treatment of the diester with KOH provided
a 1:1 mixture of the desired diacid and preferential cleavage of
the methyl ester to give an ethyl
2-[3,4-(methylenedioxy)phexoy]-6-[2-(carboxyethyl)-
-4-methoxyphenoxy]benzoate A fraction of this was further purified
by preparative HPLC. The remaining portion of the crude material
was rehydrolyzed with excess KOH to give the diacid as a white
semisolid.
EXAMPLE 24
4,6-Bis[2-carboxy-3,4-(methylenedioxy)phenoxy]-2-(methylthio)pyrimidine
and
4-[2-carboxy-3,4-(methylenedioxy)phenoxy]-6-[3,4-(methylenedioxy)phen-
oxy]-2-(methylthio)-pyrimidine
[0348] A.
4,6-Bis[3,4-(methylenedioxyphenoxy]-2-(methylthio)pyrimidine
[0349] Sesamol [3,4-(methylenedioxy)phenol, 564 mg, 4.0 mmol] was
added as a solution in THF (1.0 ml) to a suspension of mineral oil
free sodium hydride (115 mg, 4.8 mmol) in THF (0.5 ml) under water
cooling. The mixture was stirred at 25.degree. C. for 20 min. The
solvent was evaporated at 40.degree. C. under reduced pressure to
leave a solid.
[0350] A solution of 4,6-dichloro-2-(methylthio)pyrimidine (398 mg,
2.0 mmol) in DMSO (0.6 ml) was added to a solution of the solid
prepared above in DMSO (1.0 ml). The brownish-yellow solution was
heated at 70.degree. C. for 1 hour and at 100.degree. C. for
another hour. The solvent was evaporated, the solid filtered,
washed with water several times and dried. The crude product was
purified by flash column chromatography on silica gel using 5-10%
ethyl acetate in hexane as solvent to give
4-chloro-6-[3,4-(methylenedioxy)phenoxy]-2-(methylthio)py- rimidine
(196 mg, 33% yield) and 4,6-bis[3,4-(methylenedioxy)phenoxy]-2-(m-
ethylthio)pyrimidine (510 mg, 64% yield).
[0351] B.
4,6-Bis[2-carboxy-3,4-(methylenedioxy)phenoxy]-2-(methylthio)-py-
rimidine and
4-[2-carboxy-3,4-(methylenedioxy)phenoxy]-6-[3,4-(methylenedi-
oxy)phenoxy]-2-(methylthio)-pyrimidine
[0352] n-BuLi (1.0 M, 1.5 mmol) was added to a stirred solution of
4,6-bis-[3,4-(methylenedioxy)phenoxy]-2-(methylthio)pyrimidine (509
mg, 1.25 mmol) in THF (6 ml) at -78.degree. C. After 2 hours of
stirring, the solution was warmed up to 0.degree. C. and excess dry
ice (5 pieces) was added in small pieces over 20 minutes. After the
mixture has warmed to room temperature for 30 minutes, the reaction
mixture was acidified with dilute HCl and extracted with EtOAc. The
organic extract was dried over MgSO.sub.4, filtered and evaporated
in vacuo. The crude products were separated by HPLC to give
4,6-bis-[2-carboxy-3,4-(methylenedioxy)-phenoxy-
]-2-(methylthio)pyrimidine as an off-white solid, m.p. 190.degree.
C. (64 mg, 10.6% yield) and
4-[2-carboxy-3,4-(methylenedioxy)-phenoxy]-6-[3,4-(m-
ethylenedioxy)-phenoxy]-2-(methylthio)pyrimidine as an off-white
solid, m.p. 180-181.degree. C. (344 mg, 62% yield).
EXAMPLE 25
4,6-Diphenoxy-2-(methylthio)-pyrimidine-5-carboxylic acid
[0353] A. 4,6-Diphenoxy-2-(methylthio)pyrimidine
[0354] Phenol (188 mg, 2.0 mmol) was added as a solution in THF
(0.5 ml) to a suspension of mineral oil free sodium hydride (52 mg,
2.4 mmol) in THF (0.5 ml) under water cooling. The mixture was
stirred at 25.degree. C. for 20 minutes. The solvent was evaporated
at 80.degree. C. under reduced pressure to give an off white
solid.
[0355] A solution of 4,6-dichloro-2-(methylthio)pyrimidine (299 mg,
1.5 mmol) in DMSO (0.3 ml) was added to a solution of the solid
prepared above in DMSO (1.0 ml). The resulting pink solution was
heated at 70.degree. C. for 1 hour and at 100.degree. C. for
another hour. The solvent was evaporated, the solid filtered,
washed with water several times and dried. The crude product was
purified by flash column chromatography on silica gel using 5-10%
ethyl acetate in hexanes as eluent to give
4-chloro-6-phenoxy-2-(methylthio) pyrimidine (99.7 mg, 26% yield)
and 4,6-diphenoxy-2-(methylthio)pyrimidine (311 mg, 70% yield).
[0356] B. 4,6-Diphenoxy-2-(methylthio)-pyrimidine-5-carboxylic
acid
[0357] n-BuLi (1.0 M, 1.2 mmol) was added to a stirred solution of
4,6-diphenoxy-2-(methylthio)pyrimidine (298 mg, 1.0 mmol) in THF (2
ml) at -78.degree. C. The reaction mixture was stirred for 2 hours
and warmed up to 0.degree. C. Dry ice (5 pieces) was added in small
pieces over 10 minutes and the reaction was warmed to room
temperature for 30 minutes. Dilute HCl was added and the mixture
was extracted with EtOAc. The combined organic extract was dried
over MgSO.sub.4, filtered and evaporated in vacuo to give a yellow
solid that was recrystallized from ethyl acetate/hexanes to give a
yellow solid (243 mg, 71% yield), m.p. 97-98.degree. C.
EXAMPLE 26
2-[3,4-(methylenedioxy)phenoxy]-6-(3-methoxyphenoxy)benzoic
acid
[0358] A.
Ethyl-2-fluoro-6[(3,4-methylenedioxy)phenoxy]benzene-carboxylate
[0359] Sesamol (3,4-methylenedioxyphenol; 423 mg, 3.0 mmol) was
added as a solution in THF (1.0 ml) to a suspension of mineral oil
free-sodium hydride (72 mg, 3.0 mmol) in THF (0.5 ml) under water
cooling. The mixture was stirred at 25.degree. C. for 20 minutes.
The solvent was evaporated at 40.degree. C. under reduced
pressure.
[0360] A solution of ethyl-2,6-difluorobenzene-carboxylate (EXAMPLE
1C; 373 mg, 2.0 mmol) in DMSO (0.5 ml) was added to a solution of
the solid prepared above in DMSO (1.0 ml). The brownish solution
was heated at 80.degree. C. for 2 hours. The solvent was
evaporated, the solid filtered, washed with water several times and
dried. The crude product was purified by flash column
chromatography on silica gel using 3-15% ethyl acetate in hexane to
give 371 mg of the product (61% yield) as a light yellow oil and
unreacted sesamol.
[0361] B.
Ethyl-2-(3-methoxy)phenoxy-6-[(3,4-methylenedioxy)phenoxy]benzen-
e-carboxylate
[0362] 3-Methoxy phenol (0.172 ml, 1.5 mmol) was added as a
solution in THF (0.5 ml) to a suspension of mineral oil free sodium
hydride (36 mg, 1.5 mmol) in THF (0.5 ml) under water cooling. The
mixture was stirred at 25.degree. C. for 20 minutes. The solvent
was evaporated under reduced pressure.
[0363] A solution of
ethyl-2-fluoro-6-[(3,4-methylenedioxy)phenoxy]benzene- -carboxylate
(305.0 mg, 1 mmol) in DMSO (0.5 ml) was added to a solution of the
solid prepared above in DMSO (1 ml). The solution was heated at
80.degree. C. overnight. CU(I)I (catalytic amount) was added and
the solution was heated to 100.degree. C. for 48 hours. The solvent
was evaporated. The residue was diluted with water, acidified to
pH.apprxeq.3% in HCl and extracted with ethyl acetate. The organic
extract was washed with water and brine, dried over MgSO.sub.4 and
concentrated to give a brown oil. The crude product was purified by
flash column chromatography on silica gel using 3-15% ethyl acetate
in hexane to yield 236 mg (58% yield) of brown oil.
[0364] C.
2-[3,4-(methylenedioxy)phenoxy]-6-(3-methoxyphenoxy)benzoic
acid
[0365] KOH (371 mg, 5.7 mmol) that was dissolved in methanol was
added to a solution of
ethyl-2-(3-methoxyphenoxy)-6-[3,4-(methylenedioxy)phenoxy]b-
enzoate (200 mg, 0.57 mmol) in methanol (5 ml). The mixture was
heated under reflux for approximately 20 hours at which time TLL
had indicated the formation of a polar material. The solvent was
evaporated and the remaining residue was diluted with water (7 ml,
pH.apprxeq.7.5) and extracted with EtOAc (2.times.10 ml). The
aqueous fraction was then acidified to pH.about.2.5 using
concentrated HCl and then extracted with ethyl acetate (2.times.10
ml). Evaporation of solvents gave an off-white solid which was
recrystallized from methanol/H.sub.2O to give 146 mg (67%) of a
white solid, mp 146-148.degree. C.
EXAMPLE 27
Assays for Identifying Compounds That Exhibit Endothelin
Antagonistic and/or Agonist Activity
[0366] Compounds that are potential endothelin antagonists are
identified by testing their ability to compete with
.sup.125I-labeled ET-1 for binding to human ET.sub.A receptors or
ET.sub.B receptors present on isolated cell membranes. The
effectiveness of the test compound as an antagonist or agonist of
the biological tissue response of endothelin can also be assessed
by measuring the effect on endothelin induced contraction of
isolated rat thoracic aortic rings. The ability of the compounds to
act as antagonists or agonists for ET.sub.B receptors can be assess
by testing the ability of the compounds are to inhibit endothelin-1
induced prostacyclin release from cultured bovine aortic
endothelial cells.
[0367] A. Endothelin Binding Inhibition--Binding Test #1:
Inhibition of Binding to ET.sub.A Receptors
[0368] TE 671 cells (ATCC Accession No. HTB 139) express ET.sub.A
receptors. These cells were grown to confluence in T-175 flasks.
Cells from multiple flasks were collected by scraping, pooled and
centrifuged for 10 min at 190.times.g. The cells were resuspended
in phosphate buffered saline (PBS) containing 10 mM EDTA using a
Tenbroeck homogenizer. The suspension was centrifuged at 4.degree.
C. at 57,800.times.g for 15 min, the pellet was resuspended in 5 ml
of buffer A (5 mM HEPES buffer, pH 7.4 containing aprotinin (100
KIU/ml)) and then frozen and thawed once. 5 ml of Buffer B (5 mM
HEPES Buffer, pH 7.4 containing 10 mM MnCl.sub.2 and 0.001%
deoxyribonuclease Type 1) was added, the suspension mixed by
inversion and then incubated at 37.degree. C. for 30 minutes. The
mixture was centrifuged at 57,800.times.g as described above, the
pellet washed twice with buffer A and then resuspended in buffer C
(30 mM HEPES buffer, pH 7.4 containing aprotinin (100 KIU/ml) to
give a final protein concentration of 2 mg/ml and stored at
-70.degree. C. until use.
[0369] The membrane suspension was diluted with binding buffer (30
mM HEPES buffer, pH 7.4 containing 150 mM NaCl, 5 mM MgCl.sub.2,
0.5% Bacitracin) to a concentration of 8 .mu.g/50 .mu.l.
.sup.25I-endothelin-1 (3,000 cpm, 50 ml) was added to 50 .mu.l of
either: (A) endothelin-1 (for non specific binding) to give a final
concentration 80 nM); (B) binding buffer (for total binding); or
(C) a test compound (final concentration 1 nM to 100 .mu.M). The
membrane suspension (50 .mu.l), containing up to 8 .mu.g of
membrane protein, was added to each of (A), (B), or (C). Mixtures
were shaken, and incubated at 4.degree. C. for 16-18 hours, and
then centrifuged at 4.degree. C. for 25 min at 2,500.times.g.
Alternatively, the incubation was conducted at 24.degree. C. When
incubated at 24.degree. C., the IC.sub.50 concentrations are 2- to
10-fold higher than when the incubation is conducted at 4.degree.
C. This, must be kept in mind when comparing IC.sub.50
concentrations among compounds provided herein.
[0370] The supernatant, containing unbound radioactivity, was
decanted and the pellet counted on a Genesys multiwell gamma
counter. The degree of inhibition of binding (D) was calculated
according to the following equation: 1 % D = 100 - ( C ) - ( A ) (
B ) - ( A ) .times. 100
[0371] Each test was generally performed in triplicate.
[0372] B. Endothelin Binding Inhibition--Binding Test #2:
Inhibition of Binding to ET.sub.B Receptors
[0373] COS7 cells were transfected with DNA encoding the ET.sub.B
receptor, The resulting cells, which express the human ET.sub.B
receptor, were grown to confluence in T-150 flasks. Membrane was
prepared as described above. The binding assay was performed as
described above using the membrane preparation diluted with binding
buffer to a concentration of 1 .mu.g/50 .mu.l.
[0374] Briefly, the COS7 cells, described above, that had been
transfected with DNA encoding the ET.sub.B receptor and express the
human ET.sub.B receptor on their surfaces were grown to confluence
in T-175 flasks. Cells from multiple flasks were collected by
scraping, pooled and centrifuged for 10 min at 190.times.g. The
cells were resuspended in phosphate buffered saline (PBS)
containing 10 mM EDTA using a Tenbroeck homogenizer. The suspension
was centrifuged at 4.degree. C. 57,800.times.g for 15 min, the
pellet was resuspended in 5 ml of buffer A (5 mM HEPES buffer, pH
7.4 containing aprotinin (100 KIU/ml)) and then frozen and thawed
once. Five ml of Buffer B (5 mM HEPES Buffer, pH 7.4 containing 10
mM MnCl.sub.2 and 0.001% deoxyribonuclease Type 1) was added, the
suspension mixed by inversion and then incubated at 37.degree. C.
for 30 minutes. The mixture was centrifuged at 57,800.times.g as
described above, the pellet washed twice with buffer A and then
resuspended in buffer C (30 mM HEPES buffer, pH 7.4 containing
aprotinin (100 KIU/ml) to give a final protein concentration of 2
mg/ml.
[0375] The binding assay was performed as described above using the
membrane preparation diluted to give 1 .mu.g/50 .mu.l of binding
buffer.
[0376] C. Test for Activity Against Endothelin-induced Contraction
of Isolated Rat Thoracic Aortic Rings
[0377] The effectiveness of the test compound as an antagonist or
agonist of the biological tissue response of endothelin also is
assessed by measuring the effect on endothelin induced contraction
of isolated rat thoracic aortic rings (see, e.g., Borges et al.
(1989) Eur. J. Pharmacol. 165:223-230) or by measuring the ability
to contract the tissue when added alone.
[0378] Compounds to be tested are prepared as 100 .mu.M stocks. If
necessary to effect dissolution, the compounds are first dissolved
in a minimum amount of DMSO and diluted with 150 mM NaCl. Because
DMSO can cause relaxation of the aortic ring, control solutions
containing varying concentrations of DMSO were tested.
[0379] The thoracic portion of the adult rat aorta is excised, the
endothelium abraded by gentle rubbing and then cut into 3 mm ring
segments. Segments are suspended under a 2 g preload in a 10 ml
organ bath filled with Krebs'-Henseleit solution saturated with a
gas mixture of 95% O.sub.2 and 5% CO.sub.2 (118 mM NaCl, 4.7 mM
KCl, 1.2 mM MgSO.sub.4, 1.2 mM KH.sub.2PO.sub.4, 25 mM NaHCO.sub.3,
2.5 mM CaCl.sub.2, 10 mM D-glucose).
[0380] There is a correlation between activity as an antagonist of
endothelin-induced thoracic aortic ring contraction and activity as
an inhibitor of binding of endothelin to endothelin receptors. The
pA.sub.2 is a linear function of the log of the IC.sub.50.
[0381] D. Assay for Identifying Compounds That Have Agonist and/or
Antagonistic Activity Against ET.sub.B Receptors
[0382] 1. Stimulation of Prostacyclin Release
[0383] Since endothelin-1 stimulates the release of prostacyclin
from cultured bovine aortic endothelial cells, the compounds that
have agonist or antagonist activity are identified by their ability
to inhibit endothelin-1 induced prostacyclin release from such
endothelial cells by measuring 6-keto PGF.sub.1.alpha.
substantially as described by (Filep et al. (1991) Biochem.
Biophys. Res. Commun. 177 171-176. Bovine aortic cells are obtained
from collagenase-treated bovine aorta, seeded into culture plates,
grown in Medium 199 supplemented with heat inactivated 15% fetal
calf serum, and L-glutamine (2 mM), penicillin, streptomycin and
fungizone, and subcultured at least four timies. The cells are then
seeded in six-well plates in the same medium. Eight hours before
the assay, after the cells reach confluence, the medium is
replaced. The cells are then incubated with a) medium alone, b)
medium containing endothelin-1 (10 nM), c) test compound alone, and
d) test compound+endothelin-1 (10 nM).
[0384] After a 15 min incubation, the medium is removed from each
well and the concentrations of 6-keto PGF.sub.1.alpha. are measured
by a direct immunoassay. Prostacyclin production is calculated as
the difference between the amount of 6-keto PGF.sub.1.alpha.
released by the cells challenged with the endothelin-1 minus the
amount released by identically treated unchallenged cells.
Compounds that stimulate 6-keto PGF.sub.1.alpha. release possess
agonist activity and those which inhibit endothelin-1 6-keto
PGF.sub.1.alpha. release possess antagonist activity.
[0385] 2. Inhibition of Sarafotoxin 6c Induced Contraction
[0386] Sarafotoxin 6c is a specific ET.sub.B antagonist that
contracts rat fundal stomach strips. The effectiveness of test
compounds to inhibit this sarafotoxin 6c-induced contraction of rat
fundal stomach strips is used as a measure ET.sub.B antagonist
activity. Two isolated rat fundal stomach strips are suspended
under a 1 g load in a 10 ml organ bath filled with Krebs'-Henseleit
solution containing 10 .mu.M cyclo(D-Asp-Pro-D-Val-Leu-D-Trp)
(BQ-123; see, U.S. Pat. No. 5,114,918 to Ishikawa et al.), 5 .mu.M
indomethacin, and saturated with a gas mixture of 95% O.sub.2/5%
CO.sub.2. Changes in tension are measured isometrically and
recorded using a Grass Polygraph coupled to a force transducer.
Sarafotoxin 6c is added cumulatively to one strip while the second
strip is preincubated for 15 min with a test compound prior to
addition of cumulative doses of sarafotoxin 6c. The effects of the
test compounds on the concentration-response curve for sarafotoxin
6c are examined.
[0387] E. Deoxycorticosterone Acetate (DOCA)-salt Hypertensive Rat
Model for Assessing in vivo Activity of Selected Compounds
[0388] Selected compounds disclosed herein are tested for activity
in the deoxycorticosterone acetate (DOCA)-salt hypertensive rat
model. To perform these tests, silastic MDX4-4210 elastomer
implants containing 47 mg (DOCA) are prepared according to the
method of Ornmsbee et al. ((1973) the J. Pharm. Sci. 62:255-257).
Briefly, DOCA is incorporated into silicon rubber implants for
sustained release. To prepare the implants the DOCA is incorporated
into unpolymerized silicone rubber, catalyst is added and the
mixture is cast in a hemicylindrical shape.
[0389] Sprague Dawley rats (7-8 weeks old) are unilaterally
nephrectomized under ketamine anesthesia and a DOCA-implant is
placed on the left lateral dorsal abdomen of the animal. The rats
are allowed to recover for about three weeks. During recovery they
are permitted free access to normal rat chow and 0.9% NaCl drinking
solution in place of drinking water. The rats develop hypertension
within 3 weeks.
[0390] All animals are used in the tests between 21 and 30 days
post surgery. The mean arterial blood pressure in these animals
ranges from about 165-200 mm Hg.
[0391] On the day of experimentation, catheters are inserted under
brevital anesthesia into the right femoral artery for measurement
of blood pressure, and into the right femoral vein for
administration of a selected compound. The animals are placed in a
restrainer and allowed to recover for a minimum of 60 min or until
a steady mean arterial blood pressure is recorded. At that time,
the selected compound or control vehicle is administered either
intravenously, as a 60 minute infusion, or orally by oral gavage.
Blood pressure was recorded continuously for a further 10 hrs.
[0392] F. Effect of Intravenous Administration on ET-1-induced
Pressor Responses in Conscious, Autonomically Blocked Rats; a Model
for Assessing in vivo Activity of Selected Compounds
[0393] Male Sprague Dawley rats (250-450 g) are anesthetized
(Brevital 50 mg/kg, IP) and cannulae were placed in the femoral
artery to measure mean arterial pressure (MAP) and in the femoral
vein for intravenous drug administration. Animals are placed in a
restrainer and allowed to regain consciousness. Thirty minutes
later autonomic blockade is administered (atropine methyl nitrate,
3 mg/kg, IV, followed by propranalol, 2 mg/kg, IV). An hour later
animals receive a bolus injection of vehicle (0.5 ml) followed
thirty minutes later by intravenous bolus administration of ET-1
(Control, 1 .mu.g/kg). Following recovery from this challenge,
test-compounds are administered by intravenous bolus administration
(0.5 ml) and then re-challenged with ET-1 thirty minutes later.
Results are expressed as the percent inhibition of the ET-1-induced
pressor response after administration of the test compound compared
to the pressor response induced by the control ET-1 challenge. In
some cases a third ET-1 challenge is administered ninety minutes
after administration of the test compound.
[0394] G. Results
[0395] The IC.sub.50 for each of the compounds of the preceding
Examples for ET.sub.A and ET.sub.B receptors has been measured.
Almost all of the compounds have an IC.sub.50 of less than 10 .mu.M
and many have an IC.sub.50 less than about 1 .mu.M for either or
both of the ET.sub.A and ET.sub.B receptors. A number of the
compounds have an IC.sub.50 for one receptor sub-type that is
substantially less (at least 10-fold or more) than for the other
receptor sub-type. Thus, these compounds are selective for either
the ET.sub.A receptor sub-type or the ET.sub.B receptor
sub-type.
[0396] Since modifications will be apparent to those of skill in
this art, it is intended that this invention be limited only by the
scope of the appended claims.
* * * * *