U.S. patent application number 11/517906 was filed with the patent office on 2007-01-04 for sulfonamides as potassium channel blockers.
This patent application is currently assigned to Icagen, Inc.. Invention is credited to Robert N. Atkinson, Grant A. McNaughton-Smith, Aimee D. Reed.
Application Number | 20070004784 11/517906 |
Document ID | / |
Family ID | 31891407 |
Filed Date | 2007-01-04 |
United States Patent
Application |
20070004784 |
Kind Code |
A1 |
McNaughton-Smith; Grant A. ;
et al. |
January 4, 2007 |
Sulfonamides as potassium channel blockers
Abstract
Compounds, compositions and methods are provided which are
useful in the treatment of diseases through the modulation of
potassium ion flux through voltage-dependent potassium channels.
More particularly, the invention provides sulfonamides, and
compositions and methods utilizing sulfonamides that are useful in
the treatment of diseases by blocking potassium channels associated
with the onset or recurrence of the indicated conditions. Exemplary
diseases treatable with the compounds, compositions and methods of
the invention include sickle cell disease and glaucoma.
Inventors: |
McNaughton-Smith; Grant A.;
(Morrisville, NC) ; Reed; Aimee D.; (Willoughby,
OH) ; Atkinson; Robert N.; (Raleigh, NC) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Icagen, Inc.
Durham
NC
|
Family ID: |
31891407 |
Appl. No.: |
11/517906 |
Filed: |
September 7, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10641686 |
Aug 14, 2003 |
7119112 |
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11517906 |
Sep 7, 2006 |
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10376878 |
Feb 28, 2003 |
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10641686 |
Aug 14, 2003 |
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60403898 |
Aug 15, 2002 |
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60360644 |
Feb 28, 2002 |
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Current U.S.
Class: |
514/359 ;
514/365; 514/397; 514/406; 514/422; 548/203; 548/255; 548/311.1;
548/335.5; 548/517 |
Current CPC
Class: |
C07D 307/52 20130101;
A61K 9/0048 20130101; A61P 11/00 20180101; A61P 35/00 20180101;
C07D 333/70 20130101; C07D 271/06 20130101; A61P 29/00 20180101;
C07D 263/32 20130101; C07C 317/44 20130101; A61K 45/06 20130101;
A61P 11/06 20180101; A61P 27/06 20180101; C07C 311/20 20130101;
A61P 7/06 20180101; A61K 31/165 20130101; A61P 9/12 20180101; A61P
43/00 20180101; A61P 27/02 20180101; C07C 2601/16 20170501; C07C
323/56 20130101 |
Class at
Publication: |
514/359 ;
514/406; 548/255; 548/335.5; 514/365; 548/203; 514/397; 514/422;
548/311.1; 548/517 |
International
Class: |
A61K 31/427 20060101
A61K031/427; A61K 31/4192 20060101 A61K031/4192; A61K 31/4178
20060101 A61K031/4178; A61K 31/4025 20060101 A61K031/4025; C07D
417/02 20060101 C07D417/02; C07D 405/02 20060101 C07D405/02 |
Claims
1-8. (canceled)
9. A method of inhibiting potassium flux of a cell, said method
comprising contacting said cell with an effective amount of a
compound having the formula: ##STR18## wherein ring system Z is a
member selected from the group consisting of substituted or
unsubstituted C.sub.5-C.sub.7 carbocycle, substituted or
unsubstituted aryl, substituted and unsubstituted membered
heteroaryl and substituted and unsubstituted 5-7-membered
heterocycloalkyl; A is a member selected from --NHS(O).sub.2--,
--S(O).sub.2NH--, --C(R.sup.4R.sup.5)S(O).sub.n--,
--S(O).sub.nC(R.sup.4R.sup.5)--, --C(R.sup.4R.sup.5)NHS(O).sub.n--,
--S(O).sub.nNHC(R.sup.4R.sup.5)--,
--C(.sup.4R.sup.5)S(O).sub.nNH--, and
--HNS(O).sub.nC(R.sup.4R.sup.5)-- wherein n is selected from the
integers from 0 to 2; and R' is a member selected from the group of
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted
(C.sub.5-C.sub.7)carbocycle, and substituted or unsubstituted
5-7-membered heterocycloalkyl, in an amount effective to inhibit
said flux.
10. The method according to claim 9, wherein said compound has a
structure according to FIG. 1.
11. A method for reducing intraocular pressure in a subject in need
thereof by decreasing potassium ion flow through intermediate
conductance potassium channels in a cell, the method comprising the
step of administering to the subject a compound having the formula:
##STR19## wherein ring system Z is a member selected from the group
consisting of substituted or unsubstituted aryl, substituted or
unsubstituted C.sub.5-C.sub.7 carbocycle, substituted and
unsubstituted heteroaryl and substituted and unsubstituted
5-7-membered heterocycloalkyl; A is a member selected from
--NHS(O).sub.2--, --S(O).sub.2NH--,
--C(R.sup.4R.sup.5)S(O).sub.n--, --S(O).sub.nC(R.sup.4R.sup.5)--,
--C(R.sup.4R.sup.5)NHS(O).sub.n--,
--S(O).sub.nNHC(R.sup.4R.sup.5)--,
--C(R.sup.4R.sup.5)S(O).sub.nNH--, and
--HNS(O).sub.nC(R.sup.4R.sup.5)-- wherein n is selected from the
integers from 0 to 2; and R' is a member selected from the group of
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted
(C.sub.5-C.sub.7)carbocycle, and substituted or unsubstituted
5-7-membered heterocycloalkyl, in an amount sufficient to decrease
potassium ion flow through intermediate conductance, calcium
activated potassium channels, thereby reducing intraocular
pressure.
12. The method according to claim 11, wherein said compound has a
structure according to FIG. 1.
13. The method of claim 11, wherein the subject has glaucoma
characterized by increased intraocular pressure.
14. The method of claim 11 wherein the method prevents glaucoma
characterized by increased intraocular pressure.
15. The method of claim 14, wherein additionally one or more agents
selected from the group consisting of miotics, beta blockers,
alpha-2 agonists, carbonic anhydrase inhibitors, beta adrenergic
blockers, prostaglandins and docosanoid are administered to said
subject.
16. A method of preventing or retarding dehydration of erythrocytes
comprising contacting said erythrocyte with a compound having the
formula: ##STR20## wherein ring system Z is a member selected from
the group consisting of substituted or unsubstituted aryl,
substituted and unsubstituted heteroaryl and substituted and
unsubstituted 5-7-membered heterocycloalkyl; A is a member selected
from --NHS(O).sub.2--, --S(O).sub.2NH--,
--C(R.sup.4R.sup.5)S(O).sub.n--, -S(O).sub.nC(R.sup.4R.sup.5)--,
--C(R.sup.4R.sup.5)NHS(O).sub.n--,
--S(O).sub.nNHC(R.sup.4R.sup.5)--,
--C(.sup.4R.sup.5)S(O).sub.nNH--, and
--HNS(O).sub.nC(R.sup.4R.sup.5)-- wherein n is selected from the
integers from 0 to 2; and R.sup.1 is a member selected from the
group of substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
(C.sub.5-C.sub.7)carbocycle, and substituted or unsubstituted
5-7-membered heterocycloalkyl, in an amount effective to prevent or
retard said dehydration.
17. The method according to claim 16, wherein said compound has a
structure according to FIG. 1.
18. A method for treating or preventing sickle cell disease
comprising administering to a subject suffering sickle cell disease
a therapeutically effective amount of a compound having the
formula: ##STR21## wherein ring system Z is a member selected from
the group consisting of substituted or unsubstituted aryl,
substituted or unsubstituted C.sub.5-C.sub.7 carbocycle,
substituted and unsubstituted heteroaryl and substituted and
unsubstituted 5-7-membered heterocycloalkyl; A is a member selected
from --NHS(O).sub.2--, --S(O).sub.2NH--,
--C(R.sup.4R.sup.5)S(O).sub.n--, --S(O).sub.nC(R.sup.4R.sup.5)--,
--C(R.sup.4R.sup.5)NHS(O).sub.n--,
--S(O).sub.nNHC(R.sup.4R.sup.5)--,
--C(R.sup.4R.sup.5)S(O).sub.nNH--, and
--HNS(O).sub.nC(.sup.4R.sup.5)-- wherein n is selected from the
integers from 0 to 2; and R.sup.1 is a member selected from the
group of substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
(C.sub.5-C.sub.7)carbocycle, and substituted or unsubstituted
5-7-membered heterocycloalkyl.
19. The method according to claim 18, wherein said compound has a
structure according to FIG. 1.
20. A method of treating or preventing a disease state which is a
member selected from inflammation and abnormal cell proliferation
in a subject, said method comprising administering to said subject
a therapeutically effective amount of a compound having the
formula: ##STR22## wherein ring system Z is a member selected from
the group consisting of substituted or unsubstituted aryl,
substituted or unsubstituted C.sub.5-C.sub.7 carbocycle,
substituted and unsubstituted heteroaryl and substituted and
unsubstituted 5-7-membered heterocycloalkyl; A is a member selected
from --NHS(O).sub.2--, --S(O).sub.2NH--,
--C(R.sup.4R.sup.5)S(O).sub.n--, --S(O).sub.nC(.sup.4R.sup.5)--,
--C(R.sup.4R.sup.5)NHS(O).sub.n--,
--S(O).sub.nNHC(.sup.4R.sup.5)--,
--C(R.sup.4R.sup.5)S(O).sub.nNH--, and
--HNS(O).sub.nC(R.sup.4R.sup.5)-- wherein n is selected from the
integers from 0 to 2; and R.sup.1 is a member selected from the
group of substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
(C.sub.5-C.sub.7)carbocycle, and substituted or unsubstituted
(C.sub.5-C.sub.7)heterocycloalkyl.
21. The method according to claim 20, wherein said inflammation is
respiratory inflammation.
22. The method according to claim 21, wherein said respiratory
inflammation is a member selected from acute respiratory distress
syndrome, chronic obstructive pulmonary disease, and asthma.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the use of sulfonamides as
potassium channel blockers and to the treatment of diseases
modulated by potassium channels. Additionally, this invention
relates to sulfonamide compounds that are useful as potassium
channel blockers.
BACKGROUND OF THE INVENTION
[0002] Ion channels are cellular proteins that regulate the flow of
ions, including calcium, potassium, sodium and chloride into and
out of cells. These channels are present in all human cells and
affect such physiological processes as nerve transmission, muscle
contraction, cellular secretion, regulation of heartbeat, dilation
of arteries, release of insulin, and regulation of renal
electrolyte transport. Among the ion channels, potassium channels
are the most ubiquitous and diverse, being found in a variety of
animal cells such as nervous, muscular, glandular, immune,
reproductive, and epithelial tissue. These channels allow the flow
of potassium in and/or out of the cell under certain conditions.
For example, the outward flow of potassium ions upon opening of
these channels makes the interior of the cell more negative,
counteracting depolarizing voltages applied to the cell. These
channels are regulated, e.g., by calcium sensitivity,
voltage-gating, second messengers, extracellular ligands, and
ATP-sensitivity.
[0003] Potassium channels are made by alpha subunits that fall into
at least 8 families, based on predicted structural and functional
similarities (Wei et al., Neuropharmacology 35(7): 805-829 (1997)).
Three of these families (Kv, eag-related, and KQT) share a common
motif of six transmembrane domains and are primarily gated by
voltage. Two other families/also contain this motif but are gated
by cyclic nucleotides (CNG) and calcium (small conductance and
intermediate conductance potassium channels), respectively. The
small conductance and intermediate conductance, calcium activated
potassium channels comprise a family of calcium activated potassium
channels gated solely by calcium, with a unit conductance of 2-20
and 20-85 pS, respectively. Macroscopic and unitary intermediate
conductance, calcium activated potassium channel currents show
inward rectification (see, e.g., Ishii et al., Proc. Natl. Acad.
Sci USA 94: 11651-11656 (1997). The three other families of
potassium channel alpha subunits have distinct patterns of
transmembrane domains. Slo family potassium channels, or BK
channels have seven transmembrane domains (Meera et al., Proc.
Natl. Acad. Sci. U.S.A. 94(25): 14066-71 (1997)) and are gated by
both voltage and calcium or pH (Schreiber et al., J. Biol. Chem.
273: 3509-16 (1998)). Another family, the inward rectifier
potassium channels (Kir), belongs to a structural family containing
two transmembrane domains, and an eighth functionally diverse
family (TP, or "two-pore") contains two tandem repeats of this
inward rectifier motif.
[0004] Potassium channels are typically formed by four alpha
subunits, and can be homomeric (made of identical alpha subunits)
or heteromeric (made of two or more distinct types of alpha
subunits). In addition, potassium channels made from Kv, KQT and
Slo or BK subunits have often been found to contain additional,
structurally distinct auxiliary, or beta, subunits. These subunits
do not form potassium channels themselves, but instead they act as
auxiliary subunits to modify the functional properties of channels
formed by alpha subunits. For example, the Kv beta subunits are
cytoplasmic and are known to increase the surface expression of Kv
channels and/or modify inactivation kinetics of the channel
(Heinemann et al., J. Physiol. 493: 625-633 (1996); Shi et al.,
Neuron 16(4): 843-852 (1996)). In another example, the KQT family
beta subunit, minK, primarily changes activation kinetics
(Sanguinetti et al., Nature 384: 80-83 (1996)).
[0005] The intermediate conductance, calcium activated potassium
channel is also called SK4, KCa4, IKCa, SMK, and Gardos.
Intermediate conductance, calcium activated potassium channels have
been previously described in the literature by their
electrophysiology. For example, the Gardos channel, a well-known
intermediate conductance, calcium activated potassium channel, is
opened by submicromolar concentrations of internal calcium and has
a rectifying unit conductance, ranging from 50 pS at -120 mV to 13
pS at 120 mV (symmetrical 120 mM K+; Christopherson, J. Membrane
Biol. 119: 75-83 (1991)). Intermediate conductance, calcium
activated potassium channels are blocked by charybdotoxin (CTX) but
not the structurally related peptide iberiotoxin (IBX), both of
which block BK channels (Brugnara et al., J. Membr. Biol. 147:
71-82 (1995)). Intermediate conductance, calcium activated
potassium channels are also blocked by maurotoxin. Apamin, a potent
blocker of certain native (Vincent et al., J. Biochem. 14: 2521
(1975); Blatz & Magleby, Nature 323: 718-720 (1986)) and cloned
SK channels does not block intermediate conductance, calcium
activated potassium channels (de-Allie et al., Br. J. Pharm. 117:
479-487 (1996)). The Gardos channel is also blocked by some
imidazole compounds, such as clotrimazole, but not ketoconazole
(Brugnara et al, J. Clin. Invest., 92: 520-526 (1993)).
Intermediate conductance, calcium activated potassium channels can
therefore be distinguished from the other calcium activated
potassium channels by their biophysical and pharmacological
profiles. Intermediate conductance, calcium activated potassium
channels from different tissues have been reported to possess a
wide range of unit conductance values.
[0006] Human intermediate conductance, calcium activated potassium
channels have been cloned and characterized (see, e.g., Ishii et
al., Proc. Natl. Acad. Sci. USA 94: 11651-11656 (1997); Genbank
Accession No. AF0225150; Joiner et al., Proc. Natl. Acad. Sci. USA
94: 11013-11018 (1997); Genbank Accession No. AF000972; Lodsdon et
al., J. Biol. Chem. 272: 32723-32726 (1997); Genbank Accession No.
AF022797; and Jensen et al., Am. J. Physiol. 275: C848-856 (1998);
see also WO 98/11139; WO 99/03882; WO 99/25347; and WO 00/12711).
Non-human intermediate conductance, calcium activated potassium
channels have also been cloned, e.g., from mouse and rats (see,
e.g., Vandorpe et al., J. Biol. Chem. 273: 21542-21553 (1998);
Genbank Accession No. NM.sub.--032397; Warth et al., Pflugers Arch.
438: 437-444 (1999); Genbank Accession No. AJ133438; and Neylon et
al., Circ. Res. (online)85: E33-E43 (1999); Genbank Accession No.
AF190458). The gene for the intermediate conductance, calcium
activated potassium channels is named KCNN4 and it is located on
chromosome 19q13.2 (Ghanshani et al., Genomics 51: 160-161
(1998)).
[0007] The intermediate conductance, calcium activated potassium
channel is implicated in the regulation of mammalian cell
proliferation (see, for example, Wulff et al., Proc. Nat. Acad.
Sci. USA 97: 8151-8156 (2000)) and the dehydration and sickling of
erythrocytes in sickle cell disease. Sickle cell disease has been
recognized within West Africa for several centuries. Sickle cell
anemia and the existence of sickle hemoglobin (Hb S) was the first
genetic disease to be understood at the molecular level. It is
recognized today as the morphological and clinical result of a
glycine to valine substitution at the No. 6 position of the
beta-globin chain (Ingram, Nature 178: 792-794 (1956)). The origin
of the amino acid change and of the disease state is the
consequence of a single nucleotide substitution (Marotta et al., J.
Biol. Chem. 252: 5040-5053 (1977)).
[0008] Normal erythrocytes are comprised of approximately 70%
water. Water crosses a normal erythrocyte membrane in milliseconds.
Loss of cell water causes an exponential increase in cytoplasmic
viscosity as the mean cell hemoglobin concentration (MCHC) rises
above about 32 g/dl. Since cytoplasmic viscosity is a major
determinate of erythrocyte deformability and sickling, the
dehydration of the erythrocyte has substantial Theological and
pathological consequences. Regulation of erythrocyte dehydration is
recognized as an important therapeutic approach for treating sickle
cell disease. Since cell water follows any osmotic change in
intracellular ion concentration, maintaining the red cell's
potassium concentration is of particular importance (Stuart et al.,
Brit J. Haematol. 69: 1-4 (1988)).
[0009] An approach towards therapeutically treating dehydrated
sickle cells involves altering erythrocyte potassium flux by
targeting a calcium-dependent potassium channel. This calcium
activated potassium channel is also referred to as the Gardos
channel (Brugnara et al, J. Clin. Invest. 92: 520-526 (1993)).
Recently, a cloned human intermediate conductance, calcium
activated potassium channel, was shown to be substantially similar
to the Gardos channel in terms of both its biophysical and
pharmacological properties (Ishii et al., Proc. Natl. Acad. Sci.
USA 94: 11651-11656 (1997)).
[0010] In vitro studies have shown that clotrimazole, an
imidazole-containing antimycotic agent, blocks Ca.sup.2+-activated
K.sup.+ flux and cell dehydration in sickle erythrocytes (Brugnara
et al., J. Clin. Invest. 92: 520-526 (1993)). Studies in a
transgenic mouse model for sickle cell disease, SAD-1 mouse (Trudel
et al., EMBO J. 11: 3157-3165 (1991)), show that oral
administration of clotrimazole leads to inhibition of the red cell
Gardos channel, increased red cell K.sup.+ content, a decreased
mean corpuscular hemoglobin concentration (MCHC) and decreased cell
density (De Franceschi et al., J. Clin. Invest. 93: 1670-1676
(1994)). Moreover, therapy with oral clotrimazole induces
inhibition of the Gardos channel and reduces erythrocyte
dehydration in patients with sickle cell disease (Brugnara et al.,
J. Clin. Invest. 97: 1227-1234 (1996)). Other antimycotic agents,
which inhibit the Gardos channel in vitro, include miconazole,
econazole, butoconazole, oxiconazole and sulconazole (U.S. Pat. No.
5,273,992 to Brugnara et al.). All of these compounds contain an
imidazole-like ring. i.e., a heteroaryl ring containing two or more
nitrogens.
[0011] Although of demonstrable efficacy, the imidazole-based
Gardos channel inhibitors that have been explored to date are
hampered by several shortcomings including a well-documented
potential for hepatotoxicity. This toxicity is exacerbated by the
inhibitors' low potencies, non-specific interactions with potassium
channels other than the Gardos channel and low bioavailabilities,
each of which motivate for the administration of higher and more
frequent dosages of the inhibitors.
[0012] Glaucoma is a disease characterized by increased intraocular
pressure. Increased intraocular pressure is associated with many
diseases including, but not limited to, primary open-angle
glaucoma, normal tension glaucoma, angle-closure glaucoma, acute
glaucoma, pigmentary glaucoma, neovascular glaucoma, or trauma
related glaucoma, Sturge-Weber syndrome, uveitis, and exfoliation
syndrome.
[0013] Currently, there are a variety of drugs available that
employ different mechanisms to lower intraocular pressure, e.g.,
timolol, betaxolol, levobunolol, acetazolamide, methazolamide,
dichlorphenamide, dorzolamide, brinzolamide, latanoprost,
brimonidine, and rescula (see, e.g., U.S. Pat. No. 6,172,054, U.S.
Pat. No. 6,172,109, and U.S. Pat. No. 5,652,236). Miotics, beta
blockers, alpha-2 agonists, carbonic anhydrase inhibitors, beta
adrenergic blockers, prostaglandins and docosanoid are all
currently used alone or in combination to treat glaucoma. Miotics
and prostaglandins are believed to lower intraocular pressure by
increasing drainage of the intraocular fluid, while beta blockers,
alpha-2 agonists and carbonic anhydrase are believed to lower
intraocular pressure by decreasing production of intraocular fluid
thereby reducing the flow of fluid into the eye. All are
characterized by side effects ranging from red eye and blurring of
vision to decreased blood pressure and breathing difficulties.
[0014] In view of the above-described shortcomings of currently
known methods of treating diseases in which the intermediate
conductance, calcium activated potassium channel is implicated, a
substantial advance in the treatment of diseases related to
potassium flux is expected from the discovery of new intermediate
conductance, calcium activated potassium channel inhibitors. The
present invention provides a new genus of such ion channel
inhibitors based on a sulfonamide-containing scaffold.
SUMMARY OF THE INVENTION
[0015] The present invention provides compounds capable of
inhibiting the intermediate conductance, calcium activated
potassium channel thus providing a novel approach towards the
treatment and/or prevention of diseases in which said channel is
implicated, as described below. Compounds capable of inhibiting the
intermediate conductance, calcium activated potassium channel are
highly desirable, and are an object of the present invention.
[0016] Thus, in one aspect, the present invention provides
compounds according to Formula I: ##STR1## in which the ring system
Z is selected from substituted or unsubstituted aryl, substituted
or unsubstituted heteroaryl, substituted or unsubstituted
carbocycle, or substituted or unsubstituted heterocycloalkyl. The
symbol A represents --NHS(O).sub.2--, --S(O).sub.2NH--,
--C(R.sup.4R.sup.5)S(O).sub.n--, --S(O).sub.nC(R.sup.4R.sup.5)--,
--C(R.sup.4R.sup.5)NHS(O).sub.n--,
--S(O).sub.nNHC(R.sup.4R.sup.5)--,
--C(R.sup.4R.sup.5)S(O).sub.nNH--, or
--HNS(O).sub.nC(R.sup.4R.sup.5)--. The symbol R.sup.1 represents a
substituted or unsubstituted aryl group, a substituted or
unsubstituted heteroaryl group, a substituted or unsubstituted
carbocycle, or substituted or unsubstituted heterocycloalkyl. The
symbol R.sup.2 represents COOR.sup.3, substituted or unsubstituted
2-furan, substituted or unsubstituted 2-thiazole or ##STR2##
[0017] The symbol R.sup.3 represents a substituted or unsubstituted
C.sub.1-C.sub.4 alkyl group, e.g, methyl, ethyl, or --CF.sub.3. X
represents --N.dbd.N--, --N.dbd.C(R.sup.4)--, --C(R.sup.4).dbd.N--,
--C(R.sup.4R.sup.5)--C(R.sup.4R.sup.5)-- or
--C(R.sup.4).dbd.C(R.sup.5)--, in which R.sup.4 and R.sup.5
independently represent hydrogen, halogen, substituted and
unsubstituted lower alkyl, --OR.sup.6 or --CF.sub.3. The symbol Y
represents O, NR.sup.11 or S, in which R.sup.11 is --H, lower alkyl
or --CF.sub.3. The symbol R.sup.6 represents a member selected from
hydrogen, substituted or unsubstituted lower alkyl or
--CF.sub.3.
[0018] In another aspect, the present invention provides
pharmaceutical compositions comprising a pharmaceutically
acceptable excipient and a compound of Formula I.
[0019] Controlling diseases (e.g., sickle cell disease, glaucoma,
rheumatoid arthritis, uveitis, diseases characterized by abnormal
cell proliferation, among others ) via altering cellular ionic
fluxes of cells affected by a disease is a powerful therapeutic
approach. Moreover, basic understanding of the role of cellular
ionic fluxes in both disease processes and normal physiology
promises to provide new therapeutic modalities, regimens and
agents. Compounds that alter cellular ion fluxes, particularly
those that inhibit potassium flux, are highly desirable as both
drugs and as probes for elucidating the basic mechanisms underlying
these ion fluxes. Similarly, methods utilizing these compounds in
basic research and in therapeutic applications are valuable tools
in the arsenal of both the researcher and clinician. Therefore such
compounds and methods are also an object of the present
invention.
[0020] Thus, in a third aspect, the present invention provides a
method of inhibiting potassium flux of a cell. The method comprises
contacting a cell with an amount of a compound according to Formula
I, effective to inhibit the potassium flux.
[0021] An important therapeutic pathway for treatment of sickle
cell disease is preventing or retarding the dehydration of
erythrocytes by manipulating the cellular ion fluxes of
erythrocytes. Thus, in another aspect, the invention provides a
method for reducing erythrocyte dehydration. The method comprises
contacting an erythrocyte with an amount of a compound according to
Formula I, which is effective to reduce erythrocyte
dehydration.
[0022] In a fifth aspect, the invention provides a method of
treating or preventing sickle cell disease. The method comprises
administering to a subject suffering sickle cell disease a
therapeutically effective amount of a compound having a structure
according to Formula I.
[0023] In a sixth aspect, the present invention provides a method
for reducing intraocular pressure. The method includes delivering
to an eye, an amount of a compound according to Formula I
sufficient to lower said intraocular pressure.
[0024] In a seventh aspect, the invention provides a method of
treating or preventing glaucoma. The method comprises delivering to
a subject suffering from or at risk of developing glaucoma a
therapeutically effective amount of a compound according to Formula
I.
[0025] In another aspect, the invention is also directed to methods
of treating or preventing mammalian cell proliferation. Thus, in
another aspect, the invention provides methods of inhibiting
mammalian cell proliferation as an approach towards the treatment
or prevention of diseases characterized by unwanted or abnormal
cell proliferation. In its broadest sense, these methods involve
only a single step--the administration of an effective amount of at
least one pharmacologically active compound according to the
invention to a mammalian cell in situ. In exemplary embodiment, the
compounds may act cytostatically, cytotoxically, or by a
combination of both mechanisms to inhibit cell proliferation.
Mammalian cells treatable in this manner include, e.g., vascular
smooth muscle cells, fibroblasts, endothelial cells, various
pre-cancer cells and various cancer cells. In a preferred
embodiment, cell proliferation is inhibited in a subject suffering
from a disorder that is characterized by unwanted or abnormal cell
proliferation. Such diseases are described more fully below.
[0026] In an exemplary method of the invention, an effective amount
of at least one compound according to the invention, or a
pharmaceutical composition thereof, is administered to a patient
suffering from a disorder that is characterized by abnormal cell
proliferation. While not intending to be bound by any particular
theory, it is believed that administration of an appropriate amount
of a compound according to the invention to a subject inhibits cell
proliferation by altering the ionic fluxes associated with early
mitogenic signals. Such alteration of ionic fluxes is thought to be
due to the ability of the compounds of the invention to inhibit
potassium channels of cells. The method can be used
prophylactically to prevent unwanted or abnormal cell
proliferation, or may be used therapeutically to reduce or arrest
proliferation of abnormally proliferating cells. The compound, or a
pharmaceutical formulation thereof, can be applied locally to
proliferating cells to arrest or inhibit proliferation at a desired
time, or may be administered to a subject systemically to arrest or
inhibit cell proliferation.
[0027] Diseases which are characterized by abnormal cell
proliferation that can be treated or prevented by means of the
present invention include, but are not limited to, blood vessel
proliferative disorders, fibrotic disorders, atherosclerotic
disorders and various cancers. Blood vessel proliferation disorders
generally refer to angiogenic and vasculogenic disorders generally
resulting in abnormal proliferation of blood vessels. The formation
and spreading of blood vessels, or vasculogenesis and angiogenesis,
respectively, play important roles in a variety of physiological
processes such as embryonic development, corpus luteum formation,
wound healing and organ regeneration. They also play a pivotal role
in cancer development. Other examples of blood vessel proliferative
disorders include arthritis, where new capillary blood vessels
invade the joint and destroy cartilage and ocular diseases such as
diabetic retinopathy, where new capillaries in the retina invade
the vitreous, bleed and cause blindness and neovascular
glaucoma.
[0028] Another example of abnormal neovascularization is that
associated with solid tumors. It is now established that
unrestricted growth of tumors is dependent upon angiogenesis and
that induction of angiogenesis by liberation of angiogenic factors
can be an important step in carcinogenesis. For example, basic
fibroblast growth factor (bFGF) is liberated by several cancer
cells and plays a crucial role in cancer angiogenesis. The
demonstration that certain animal tumors regress when angiogenesis
is inhibited has provided the most compelling evidence for the role
of angiogenesis in tumor growth. Other cancers that are associated
with neovascularization include hemangioendotheliomas, hemangiomas
and Kaposi's sarcoma.
[0029] Proliferation of endothelial and vascular smooth muscle
cells is the main feature of neovascularization. The invention is
useful in inhibiting such proliferation, and therefore in
inhibiting or arresting altogether the progression of the
angiogenic condition which depends in whole or in part upon such
neovascularization. The invention is particularly useful when the
condition has an additional element of endothelial or vascular
smooth muscle cell proliferation that is not necessarily associated
with neovascularization. For example, psoriasis may additionally
involve endothelial cell proliferation that is independent of the
endothelial cell proliferation associated with neovascularization.
Likewise, a solid tumor which requires neovascularization for
continued growth may also be a tumor of endothelial or vascular
smooth muscle cells. In this case, growth of the tumor cells
themselves, as well as the neovascularization, is inhibited by the
compounds described herein.
[0030] The invention is also useful for the treatment of fibrotic
disorders such as fibrosis and other medical complications of
fibrosis which result in whole or in part from the proliferation of
fibroblasts. Medical conditions involving fibrosis (other than
atherosclerosis, discussed below) include undesirable tissue
adhesion resulting from surgery or injury.
[0031] Other cell proliferative disorders which can be treated by
means of the invention include arteriosclerotic conditions.
Arteriosclerosis is a term used to describe a thickening and
hardening of the arterial wall. An arteriosclerotic condition as
used herein means classical atherosclerosis, accelerated
atherosclerosis, atherosclerotic lesions and any other
arteriosclerotic conditions characterized by undesirable
endothelial and/or vascular smooth muscle cell proliferation,
including vascular complications of diabetes.
[0032] Proliferation of vascular smooth muscle cells is a main
pathological feature in classical atherosclerosis. It is believed
that liberation of growth factors from endothelial cells stimulates
the proliferation of subintimal smooth muscle which, in turn,
reduces the caliber and finally obstructs the artery. The invention
is useful in inhibiting such proliferation, and therefore in
delaying the onset of, inhibiting the progression of, or even
halting the progression of such proliferation and the associated
atherosclerotic condition.
[0033] Proliferation of vascular smooth muscle cells produces
accelerated atherosclerosis, which is the main reason for failure
of heart transplants that are not rejected. This proliferation is
also believed to be mediated by growth factors, and can ultimately
result in obstruction of the coronary arteries. The invention is
useful in inhibiting such obstruction and reducing the risk of, or
even preventing, such failures.
[0034] Vascular injury can also result in endothelial and vascular
smooth muscle cell proliferation. The injury can be caused by any
number of traumatic events or interventions, including vascular
surgery and balloon angioplasty. Restenosis is the main
complication of successful balloon angioplasty of the coronary
arteries. It is believed to be caused by the release of growth
factors as a result of mechanical injury to the endothelial cells
lining the coronary arteries. Thus, by inhibiting unwanted
endothelial and smooth muscle cell proliferation, the compounds
described herein can be used to delay, or even avoid, the onset of
restenosis.
[0035] Other atherosclerotic conditions which can be treated or
prevented by means of the present invention include diseases of the
arterial walls that involve proliferation of endothelial and/or
vascular smooth muscle cells, such as complications of diabetes,
diabetic glomerulosclerosis and diabetic retinopathy.
[0036] The compounds described herein are also useful in treating
or preventing various types of cancers. Cancers which can be
treated by means of the present invention include, but are not
limited to, biliary tract cancer; brain cancer, including
glioblastomas and medulloblastomas; breast cancer; cervical cancer;
choriocarcinoma; colon cancer; endometrial cancer; esophageal
cancer; gastric cancer; hematological neoplasms, including acute
and chronic lymphocytic and myelogenous leukemia, multiple myeloma,
AIDS associated leukemias and adult T-cell leukemia lymphoma;
intraepithelial neoplasms, including Bowen's disease and Paget's
disease; liver cancer; lung cancer; lymphomas, including Hodgkin's
disease and lymphocytic lymphomas; neuroblastomas; oral cancer,
including squamous cell carcinoma; ovarian cancer, including those
arising from epithelial cells, stromal cells, germ cells and
mesenchymal cells; pancreas cancer; prostate cancer; rectal cancer;
sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma,
fibrosarcoma and osteosarcoma; skin cancer, including melanoma,
Kaposi's sarcoma, basocellular cancer and squamous cell cancer;
testicular cancer, including germinal tumors (seminoma,
non-seminoma (teratomas, choriocarcinomas)), stromal tumors and
germ cell tumors; thyroid cancer, including thyroid adenocarcinoma
and medullar carcinoma; and renal cancer including adenocarcinoma
and Wilms tumor.
[0037] The compounds of the invention are useful with hormone
dependent and also with nonhormone dependent cancers. They also are
useful with prostate and nonprostate cancers and with breast and
nonbreast cancers. They further are useful with multidrug resistant
strains of cancer.
[0038] In addition to the particular disorders enumerated above,
the invention is also useful in treating or preventing
dermatological diseases including keloids, psoriasis, dermatitis,
hypertrophic scars, seborrheic dermatosis, papilloma virus
infection (e.g., producing verruca vulgaris, verruca plantaris,
verruca plan, condylomata, etc.), eczema and epithelial
precancerous lesions such as actinic keratosis. Other inflammatory
disease states may also benefit from the methods described herein
including arthritis, chronic obstructive pulnonary disease (COPD),
acute respiratory distress syndrome (ARDS), asthma and other
respiratory ailments mediated by the inflammatory process;
atherosclerosis; keratoconjunctivitis; uveitis; inflammatory bowel
disease; proliferative glomerulonephritis; lupus erythematosus (and
other auto-immune diseases); scleroderma; temporal arthritis;
thromboangiitis obliterans; mucocutaneous lymph node syndrome; and
other pathologies mediated by growth factors including uterine
leiomyomas; multiple sclerosis; shock, sepsis; ischemia; and
reperfusion injury.
[0039] These and other objects and advantages of the present
invention will be apparent from the detailed description and
examples that follow. All publications, patents and patent
applications are incorporated herein by reference in their
entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 displays structures of representative compounds of
the invention.
DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED
EMBODIMENTS
Abbreviations and Definitions:
[0041] The abbreviations used herein have their conventional
meaning within the chemical and biological arts. For example: ;
Et.sub.3N, triethylamine; MeOH, methanol; and DMSO,
dimethylsulfoxide; MCHC, mean corpuscular hemoglobin concentration;
SAD-1, a transgenic mouse model of sickle cell disease as described
by Trudel et.al., EMBO J., 10 (11): 3157-3165 (1991).
[0042] "Blocking" and "inhibiting," are used interchangeably herein
to refer to the partial or full blockade of an intermediate
conductance, calcium activated potassium channel by one or more
compound(s) of the invention.
[0043] Where substituent groups are specified by their conventional
chemical formulae, written from left to right, they equally
encompass the chemically identical substituents which would result
from writing the structure from right to left, e.g., --CH.sub.2O--
is intended to also recite --OCH.sub.2--.
[0044] The term "alkyl," by itself or as part of another
substituent, means, unless otherwise stated, a straight or branched
chain, or cyclic hydrocarbon radical, or combination thereof, which
may be fully saturated, mono- or polyunsaturated and can include
di- and multivalent radicals, having the number of carbon atoms
designated (i.e. C.sub.1-C.sub.10 means one to ten carbons).
Examples of saturated hydrocarbon radicals include, but are not
limited to, groups such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,
(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for
example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An
unsaturated alkyl group is one having one or more double bonds or
triple bonds. Examples of unsaturated alkyl groups include, but are
not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,
2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-
and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The
term "alkyl," unless otherwise noted, is also meant to include
those derivatives of alkyl defined in more detail below, such as
"heteroalkyl." Alkyl groups which are limited to hydrocarbon groups
are termed "homoalkyl".
[0045] The term "alkylene" by itself or as part of another
substituent means a divalent radical derived from an alkane, as
exemplified, but not limited, by
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, and further includes those
groups described below as "heteroalkylene." Typically, an alkyl (or
alkylene) group will have from 1 to 24 carbon atoms, with those
groups having 10 or fewer carbon atoms being preferred in the
present invention. A "lower alkyl" or "lower alkylene" is a shorter
chain alkyl or alkylene group, generally having eight or fewer
carbon atoms.
[0046] The terms "alkoxy," "alkylamino" and "alkylthio" (or
thioalkoxy) are used in their conventional sense, and refer to
those alkyl groups attached to the remainder of he molecule via an
oxygen atom, an amino group, or a sulfur atom, respectively.
[0047] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or cyclic hydrocarbon radical, or combinations
thereof, consisting of the stated number of carbon atoms and at
least one heteroatom selected from the group consisting of O, N, Si
and S, and wherein the nitrogen and sulfur atoms may optionally be
oxidized and the nitrogen heteroatom may optionally be quaternized.
The heteroatom(s) O, N and S and Si may be placed at any interior
position of the heteroalkyl group or at the position at which the
alkyl group is attached to the remainder of the molecule. Examples
include, but are not limited to, --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3, and
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3 and
--CH.sub.2--O--Si(CH.sub.3).sub.3. Similarly, the term
"heteroalkylene" by itself or as part of another substituent means
a divalent radical derived from heteroalkyl, as exemplified, but
not limited by, --CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For
heteroalkylene groups, heteroatoms can also occupy either or both
of the chain termini (e.g., alkyleneoxy, alkylenedioxy,
alkyleneamino, alkylenediamino, and the like). Still further, for
alkylene and heteroalkylene linking groups, no orientation of the
linking group is implied by the direction in which the formula of
the linking group is written. For example, the formula
--C(O).sub.2R'-- represents both --C(O).sub.2R'-- and
--R'C(O).sub.2--.
[0048] The terms "cycloalkyl" and "heterocycloalkyl", by themselves
or in combination with other terms, represent, unless otherwise
stated, cyclic versions of "alkyl" and "heteroalkyl", respectively.
Additionally, for heterocycloalkyl, a heteroatom can occupy the
position at which the heterocycle is attached to the remainder of
the molecule. Examples of cycloalkyl include, but are not limited
to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl,
cycloheptyl, and the like. Examples of heterocycloalkyl include,
but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),
1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,
3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,
tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,
2-piperazinyl, and the like.
[0049] The terms "halo" or "halogen," by themselves or as part of
another substituent, mean, unless otherwise stated, a fluorine,
chlorine, bromine, or iodine atom. Additionally, terms such as
"haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl.
For example, the term "halo(C.sub.1-C.sub.4)alkyl" is mean to
include, but not be limited to, trifluoromethyl,
2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the
like.
[0050] The term "aryl" means, unless otherwise stated, a
polyunsaturated, aromatic, hydrocarbon substituent which can be a
single ring or multiple rings (preferably from 1 to 3 rings) which
are fused together or linked covalently. The term "heteroaryl"
refers to aryl groups (or rings) that contain from one to four
heteroatoms selected from N, O, and S, wherein the nitrogen and
sulfur atoms are optionally oxidized, and the nitrogen atom(s) are
optionally quaternized. A heteroaryl group can be attached to the
remainder of the molecule through a heteroatom. Non-limiting
examples of aryl and heteroaryl groups include phenyl, 1-naphthyl,
2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,
3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,
4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl,
4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,
2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl,
4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
Substituents for each of the above noted aryl and heteroaryl ring
systems are selected from the group of acceptable substituents
described below.
[0051] For brevity, the term "aryl" when used in combination with
other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both
aryl and heteroaryl rings as defined above. Thus, the term
"arylalkyl" is meant to include those radicals in which an aryl
group is attached to an alkyl group (e.g., benzyl, phenethyl,
pyridylmethyl and the like) including those alkyl groups in which a
carbon atom (e.g., a methylene group) has been replaced by, for
example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,
3-(1-naphthyloxy)propyl, and the like).
[0052] Each of the above terms (e.g., "alkyl," "heteroalkyl,"
"aryl" and "heteroaryl") are meant to include both substituted and
unsubstituted forms of the indicated radical. Preferred
substituents for each type of radical are provided below.
[0053] Substituents for the alkyl and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one
or more of a variety of groups selected from, but not limited to:
--OR', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'', --SR', -halogen,
--SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R', --CONR'R'',
--OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O).sub.2R', --NR--C(NR'R''R''').dbd.NR'''',
--NR--C(NR'R'').dbd.NR''', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --CN and --NO.sub.2 in a number
ranging from zero to (2m'+1), where m' is the total number of
carbon atoms in such radical. R', R'', R''' and R'''' each
preferably independently refer to hydrogen, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g.,
aryl substituted with 1-3 halogens, substituted or unsubstituted
alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a
compound of the invention includes more than one R group, for
example, each of the R groups is independently selected as are each
R', R'', R''' and R'''' groups when more than one of these groups
is present. When R' and R'' are attached to the same nitrogen atom,
they can be combined with the nitrogen atom to form a 5-, 6-, or
7-membered ring. For example, --NR'R'' is meant to include, but not
be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above
discussion of substituents, one of skill in the art will understand
that the term "alkyl" is meant to include groups including carbon
atoms bound to groups other than hydrogen groups, such as haloalkyl
(e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like).
[0054] Similar to the substituents described for the alkyl radical,
substituents for the aryl and heteroaryl groups are varied and are
selected from, for example: halogen, --OR', .dbd.O, .dbd.NR',
.dbd.N--OR', --NR'R'', --SR', -halogen, --SiR'R''R''', --OC(O)R',
--C(O)R', --CO.sub.2R', --CONR'R'', --OC(O)NR'R'', --NR''C(O)R',
--NR'--C(O)NR''R''', --NR''C(O).sub.2R',
--NR--C(NR'R''R''').dbd.NR'''', --NR--C(NR'R'').dbd.NR''',
--S(O)R', --S(O).sub.2R', --S(O).sub.2NR'R'', --NRSO.sub.2R', --CN
and --NO.sub.2, --R', --N.sub.3, --CH(Ph).sub.2,
fluoro(C.sub.1-C.sub.4)alkoxy, and fluoro(C.sub.1-C.sub.4)alkyl, in
a number ranging from zero to the total number of open valences on
the aromatic ring system; and where R', R'', R''' and R'''' are
preferably independently selected from hydrogen,
(C.sub.1-C.sub.8)alkyl and heteroalkyl, unsubstituted aryl and
heteroaryl, (unsubstituted aryl)-(C.sub.1-C.sub.4)alkyl, and
(unsubstituted aryl)oxy-(C.sub.1-C.sub.4)alkyl. When a compound of
the invention includes more than one R group, for example, each of
the R groups is independently selected as are each R', R'', R'''
and R'''' groups when more than one of these groups is present.
[0055] Two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally be replaced with a substituent of
the formula -T-C(O)--(CRR').sub.q--U--, wherein T and U are
independently --NR--, --O--, --CRR'-- or a single bond, and q is an
integer of from 0 to 3. Alternatively, two of the substituents on
adjacent atoms of the aryl or heteroaryl ring may optionally be
replaced with a substituent of the formula
-A-(CH.sub.2).sub.r--B--, wherein A and B are independently
--CRR'--, --O--, --NR--, --S--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2NR'-- or a single bond, and r is an integer of from 1
to 4. One of the single bonds of the new ring so formed may
optionally be replaced with a double bond. Alternatively, two of
the substituents on adjacent atoms of the aryl or heteroaryl ring
may optionally be replaced with a substituent of the formula
--(CRR').sub.s--X--(CR''R''').sub.d--, where s and d are
independently integers of from 0 to 3, and X is --O--, --NR'--,
--S--, --S(O)--, --S(O).sub.2--, or --S(O).sub.2NR'--. The
substituents R, R', R'' and R''' are preferably independently
selected from hydrogen or substituted or unsubstituted
(C.sub.1-C.sub.6)alkyl.
[0056] As used herein, the term "heteroatom" is meant to include
oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).
[0057] The term "pharmaceutically acceptable salts" is meant to
include salts of the active compounds which are prepared with
relatively nontoxic acids or bases, depending on the particular
substituents found on the compounds described herein. When
compounds of the present invention contain relatively acidic
functionalities, base addition salts can be obtained by contacting
the neutral form of such compounds with a sufficient amount of the
desired base, either neat or in a suitable inert solvent. Examples
of pharmaceutically acceptable base addition salts include sodium,
potassium, calcium, ammonium, organic amino, or magnesium salt, or
a similar salt. When compounds of the present invention contain
relatively basic functionalities, acid addition salts can be
obtained by contacting the neutral form of such compounds with a
sufficient amount of the desired acid, either neat or in a suitable
inert solvent. Examples of pharmaceutically acceptable acid
addition salts include those derived from inorganic acids like
hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,
phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from relatively nontoxic organic acids
like acetic, propionic, isobutyric, maleic, malonic, benzoic,
succinic, suberic, fumaric, lactic, mandelic, phthalic,
benzenesulfonic, p-tolylsulfonic, citric, tartaric,
methanesulfonic, and the like. Also included are salts of amino
acids such as arginate and the like, and salts of organic acids
like glucuronic or galactunoric acids and the like (see, for
example, Berge et al., "Pharmaceutical Salts", Journal of
Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds
of the present invention contain both basic and acidic
functionalities that allow the compounds to be converted into
either base or acid addition salts.
[0058] The neutral forms of the compounds are preferably
regenerated by contacting the salt with a base or acid and
isolating the parent compound in the conventional manner. The
parent form of the compound differs from the various salt forms in
certain physical properties, such as solubility in polar
solvents.
[0059] In addition to salt forms, the present invention provides
compounds, which are in a prodrug form. Prodrugs of the compounds
described herein are those compounds that readily undergo chemical
changes under physiological conditions to provide the compounds of
the present invention. Additionally, prodrugs can be converted to
the compounds of the present invention by chemical or biochemical
methods in an ex vivo environment. For example, prodrugs can be
slowly converted to the compounds of the present invention when
placed in a transdermal patch reservoir with a suitable enzyme or
chemical reagent.
[0060] Certain compounds of the present invention can exist in
unsolvated forms as well as solvated forms, including hydrated
forms. In general, the solvated forms are equivalent to unsolvated
forms and are encompassed within the scope of the present
invention. Certain compounds of the present invention may exist in
multiple crystalline or amorphous forms. In general, all physical
forms are equivalent for the uses contemplated by the present
invention and are intended to be within the scope of the present
invention.
[0061] Certain compounds of the present invention possess
asymmetric carbon atoms (optical centers) or double bonds; the
racemates, diastereomers, geometric isomers and individual isomers
are encompassed within the scope of the present invention.
[0062] The compounds of the present invention may also contain
unnatural proportions of atomic isotopes at one or more of the
atoms that constitute such compounds. For example, the compounds
may be radiolabeled with radioactive isotopes, such as for example
tritium (.sup.3H), iodine-125 (.sup.125I) or carbon-14 (.sup.14C).
All isotopic variations of the compounds of the present invention,
whether radioactive or not, are intended to be encompassed within
the scope of the present invention.
[0063] The term "glaucoma" refers to an optic neuropathy or
degenerative state usually associated with elevation of intraocular
pressure. See, Shields, TEXTBOOK OF GLAUCOMA (4.sup.th Ed.), 1997,
Lippincott, Williams and Wilkins, which is incorporated herein by
reference. The mechanism by which elevated eye pressure injures the
optic nerve is not well understood. It is known that axons entering
the inferotemporal and superotemporal aspects of the optic disc are
damaged. As fibers of the disc are destroyed, the neural rim of the
optic disc shrinks and the physiologic cup within the optic disc
enlarges. A term known as pathologic "cupping" refers to this
shrinking and enlarging process. Although it is possible to measure
the cup-to-disc ratio, it is not a useful diagnostic tool because
it varies widely in the population. However, it can be used to
measure the progression of the disease by a series of measurements
in a time period.
[0064] Glaucoma is not a single disease but a group of conditions
with various causes. In most cases, these conditions produce
increased pressure within the eye.
[0065] Ultimately glaucoma can lead to optic nerve damage and the
loss of visual function. It is not unusual for persons who exhibit
gradual development of intraocular pressure to exhibit no symptoms
until the end-stage of the disease is reached.
[0066] The term "open angle glaucoma"--refers to a chronic type of
glaucoma. Occurring in approximately 1% of Americans, open-angle
glaucoma is the most common type of glaucoma. Open-angle glaucoma
is characterized by a very gradual, painless rise of pressure
within the eye. Subjects with open-angle glaucoma exhibit no
outward manifestations of disease until irreversible vision
impairment.
[0067] "Normal tension glaucoma" commonly referred to as low
tension glaucoma is a form of open angle glaucoma that accounts for
about 1/3 of open-angle glaucoma cases in the United States.
[0068] "Angle closure glaucoma" is a glaucoma most prevalent in
people who are far-sighted. In angle closure glaucoma, the anterior
chamber of the eye is smaller than average hampering the ability of
the aqueous humor to pass between the iris and the lens on its way
to the anterior chamber, causing fluid pressure to build up between
the iris.
[0069] "Acute glaucoma" is caused by a sudden increase in
intraocular pressure. This intense rise in pressure is accompanied
by severe pain. In acute glaucoma, there are outward manifestations
of the disease including red eye, cornea swelling and clouding
over.
[0070] The term "pigmentary glaucoma" refers to a hereditary
condition which develops more frequently in men than in woman and
begins in the twenties or thirties. pigmentary glaucoma affects
persons of near-sightedness. Myopic eyes have a concave-shaped iris
creating an unusually wide angle. The wideness of the angle causes
the pigment layer of the eye to rub on the lens when the pupil
constricts and dilates during normal focusing. The rubbing action
ruptures the cells of the iris pigment epithelium, thereby
releasing pigment particles into the aqueous humor and trabecular
meshwork. If pigment plugs the pores of the trabecular meshwork,
drainage is inhibited.
[0071] The term "exfoliation syndrome" refers to a type of glaucoma
most common in persons of European descent. Exfoliation syndrome is
characterized by a whitish material that builds on the lens of the
eye. Movement of the iris causes this material to be rubbed off the
lens along with some pigment from the iris. Both the pigment and
the whitish exfoliation material clog the meshwork, inhibiting
drainage of the aqueous humor.
[0072] The term "trauma related Glaucoma" refers to a type of
glaucoma caused by an external force acting upon the eye, i.e.,
chemical burn, blow to the eye. Trauma related glaucoma occurs when
this external force causes a mechanical disruption or physical
change with in the eye's drainage system.
[0073] "Congenital glaucoma" occurs in about 1 in 10,000 births. It
may appear up until age 4. Primary congenital glaucoma is due to
abnormal development of the trabecular meshwork. Congenital
glaucoma can be hereditary as well as non-hereditary. In congenital
glaucoma, the eye enlarges or the cornea becomes hazy. The
stretching of the cornea causes breaks to occur in the inner
lining. The breaks allow aqueous humor to enter the cornea causing
it to swell. As the cornea continues to stretch, more aqueous humor
is let in and there is an increase in edema and haze in the
cornea.
[0074] The term "Sturge-Weber Syndrome" refers to a rare syndrome
characterized by a facial birthmark port wine in color. The
birthmark is associated with an abnormal blood vessels on the
surface of the brain. These vascular malformations may affect the
eyelids, sclera, conjunctiva, and iris. One third of patients with
Sturge-Weber syndrome suffer from increased intraocular pressure.
This increased pressure leads to glaucoma. A vascular malformation
of the sclera causes elevated pressure in the veins. This elevated
pressure in the veins drains the eye thereby causing the
intraocular pressure to rise and resulting in damage to the
drainage system of the eye.
[0075] The term "uveitis" refers to a disease characterized by
inflammation of the choroid, ciliary body and iris. In anterior
uveitis, a decrease in aqueous humor formation may cause
dangerously low levels of pressure within the eye. In other forms
of uveitis, i.e., posterior uveitis, the intraocular pressure is
elevated. The elevation may be caused by active inflammation,
insufficient anti-inflammatory therapy, excessive corticosteroid
use or insufficient glaucoma therapy. If the inflammation is
chronic and not properly controlled, it can lead to trabecular cell
death.
[0076] The term "chronic elevation" refers to increased pressure
caused by a condition that is reoccurring and not treatable.
[0077] The term "acute elevation" refers to a sudden increase in
intraocular eye pressure. The sudden rise can occur within hours
and causes pain within the eye and may even cause nausea and
vomiting
[0078] The term "gradual elevation" refers to a slow increase of
pressure within the eye. There are no symptoms associated with the
increased rise.
[0079] An "ophthalmically acceptable carrier" is a carrier that has
substantially no long term or permanent detrimental effect on the
eye to which it is administered.
Introduction
[0080] As discussed above, the blockade of the intermediate
conductance, calcium activated potassium channel is a powerful
therapeutic approach for the treatment of disease states in which
said channel plays a therapeutically relevant role as a drug
target. Representative diseases that may be treated by inhibition
of the intermediate conductance, calcium activated potassium
channel include, but are not limited to sickle cell disease,
inflammation and glaucoma.
[0081] The present invention is illustrated by reference to the use
of the compounds of the invention in treating sickle cell disease
and glaucoma. The focus on the two selected diseases is for clarity
of illustration only and is not intended to define or otherwise
limit the scope of the present invention.
[0082] The prevention of sickle cell dehydration via inhibition of
the intermediate conductance, calcium activated potassium channel
(i.e., the Gardos channel) is useful in the treatment and/or
prevention of sickle cell disease. Moreover, physiological studies
show that intermediate conductance, calcium activated potassium
channels play a role in secretion of Cl.sup.- and water from
epithelial tissue. Given that the intraocular pressure of the eye
is maintained, in part, by secretion of aqueous humor, the
inhibition of aqueous humor secretion by an antagonist of the
intermediate conductance, calcium activated potassium channel
reduces intraocular pressure. For example, in rabbits, topical
application of a compound of the invention was demonstrated to
result in a dose-dependent, long duration reduction in intraocular
pressure. Thus, the blockade of intermediate conductance, calcium
activated potassium channels in the eye is of benefit for the
treatment of glaucoma.
[0083] The present invention provides sulfonamide compounds,
compositions containing these compounds, and methods for using
these compounds and compositions to decrease ion flux in
intermediate conductance, calcium activated potassium channels.
Inhibition of said channel reduces mammalian cell proliferation,
intraocular pressure, erythrocyte dehydration, sickle cell
dehydration, and delays the occurrence of acute sickle cell
episodes. Thus, the present invention also provides methods of
using the compounds of the invention to treat and prevent diseases
in which inhibition of ion flux through intermediate conductance,
calcium activated potassium channels may prove beneficial.
Description of the Embodiments
I. Modulators of Intermediate Conductance Calcium Activated
Potassium Channels
[0084] In a first aspect, the present invention provides compounds
according to Formula I: ##STR3## in which the ring system Z is
selected from substituted or unsubstituted aryl, substituted or
unsubstituted carbocycle, substituted or unsubstituted heteroaryl,
or substituted or unsubstituted heterocycloalkyl. The symbol A
represents --NHS(O).sub.2--, --S(O).sub.2NH--,
--C(R.sup.4R.sup.5)S(O).sub.n--, --S(O).sub.nC(R.sup.4R.sup.5)--,
--C(R.sup.4R.sup.5)NHS(O).sub.n--,
--S(O).sub.nNHC(R.sup.4R.sup.5)--,
--C(R.sup.4R.sup.5)S(O).sub.nNH--, or
--HNS(O).sub.nC(R.sup.4R.sup.5)--. The symbol R.sup.1 represents a
substituted or unsubstituted aryl group, a substituted or
unsubstituted heteroaryl group, a substituted or unsubstituted
carbocycle, or substituted or unsubstituted heterocycloalkyl. The
symbol R.sup.2 represents COOR.sup.3, substituted or unsubstituted
2-furan, substituted or unsubstituted 2-thiazole or ##STR4##
[0085] The symbol R.sup.3 represents a substituted or unsubstituted
C.sub.1-C.sub.4 alkyl group, e.g, methyl, ethyl, or --CF.sub.3. X
represents --N.dbd.N--, --N.dbd.C(R.sup.4)--, --C(R.sup.4).dbd.N--,
--C(R.sup.4R.sup.5)--C(R.sup.4R.sup.5)-- or
--C(R.sup.4).dbd.C(R.sup.5)--, in which R.sup.4 and R.sup.5
independently represent hydrogen, halogen, substituted and
unsubstituted lower alkyl, --OR.sup.6 or --CF.sub.3. The symbol Y
represents O, NR.sup.11 or S, in which R.sup.11 is --H, lower alkyl
or --CF.sub.3. The symbol R.sup.6 represents a member selected from
hydrogen, or substituted or unsubstituted lower alkyl.
[0086] In an exemplary embodiment, the invention provides compounds
as described above in which Z is selected from substituted or
unsubstituted phenyl and substituted or unsubstituted thiophene. In
another exemplary embodiment, compounds of the invention include a
group for R.sup.2 that is selected from substituted or
unsubstituted 2-furan, and ##STR5## in which X is
--N.dbd.C(R.sup.4)--, --C(R.sup.4).dbd.N--,
--C(R.sup.4R.sup.5)--C(R.sup.4R.sup.5)-- or
--C(R.sup.4).dbd.C(R.sup.5)--; and Y is O or S.
[0087] In yet another exemplary embodiment, R.sup.1 is a selected
from ##STR6## in which the symbols R.sup.7, R.sup.8 and R.sup.9
independently represent H, halogen, lower alkyl, OR.sup.10,
--OCF.sub.3, CF.sub.3, and NO.sub.2. The symbol R.sup.10 represents
H, lower alkyl, or substituted lower alkyl.
[0088] In a still further exemplary embodiment, the invention
provides compounds in which the symbol R.sup.1 represents the
group: ##STR7## wherein R.sup.13 is a member selected from halogen,
substituted or unsubstituted C.sub.1-C.sub.4 alkyl, CF.sub.3 and
OCF.sub.3.
[0089] Representative compounds of the invention according to
Formula I are set forth in FIG. 1.
[0090] Also within the scope of the present invention are compounds
of the invention that are poly- or multi-valent species, including,
for example, species such as dimers, trimers, tetramers and higher
homologs of the compounds of the invention or reactive analogues
thereof. The poly- and multi-valent species can be assembled from a
single species or more than one species of the invention. For
example, a dimeric construct can be "homo-dimeric" or
"heterodimeric." Moreover, poly- and multi-valent constructs in
which a compound of the invention or a reactive analogue thereof,
is attached to an oligomeric or polymeric framework (e.g.,
polylysine, dextran, hydroxyethyl starch and the like) are within
the scope of the present invention. The framework is preferably
polyfunctional (i.e. having an array of reactive sites for
attaching compounds of the invention). Moreover, the framework can
be derivatized with a single species of the invention or more than
one species of the invention.
[0091] Moreover, the present invention includes compounds within
the motif set forth in Formula I, which are functionalized to
afford compounds having a water-solubility that is enhanced
relative to analogous compounds that are not similarly
functionalized. Methods of enhancing the water-solubility of
organic compounds are known in the art. Such methods include, but
are not limited to, functionalizing an organic nucleus with a
permanently charged moiety, e.g., quaternary ammonium, or a group
that is charged at a physiologically relevant pH, e.g. carboxylic
acid, amine. Other methods include, appending to the organic
nucleus hydroxyl- or amine-containing groups, e.g. alcohols,
polyols, polyethers, and the like. Representative examples include,
but are not limited to, polylysine, polyethyleneimine,
poly(ethyleneglycol) and poly(propyleneglycol). Suitable
functionalization chemistries and strategies for these compounds
are known in the art. See, for example, Dunn, R. L., et al., Eds.
POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series
Vol. 469, American Chemical Society, Washington, D.C. 1991.
Preparation of Potassium Channel Blockers
[0092] Compounds of the present invention can be prepared using
readily available starting materials or known intermediates. For
example, furan derivatized bis-aryl sulfonamides are readily
prepared the method of Scheme A: ##STR8##
[0093] In Scheme A, and each of the succeeding schemes, each of the
reaction components can bear one or more substituents ("R groups")
other than a locus of reaction. The symbols R', R'', R''', etc.
generally represent substituents for aryl or heteroaryl groups as
described in the definitions section herein.
[0094] In scheme A, the iodo aniline substrate a is coupled with
the furan moiety via a Pd mediated reaction with a boronic acid
derivative to afford compound b. The resulting adduct is reacted
with an activated sulfonic acid derivative to produce adduct c.
##STR9##
[0095] Scheme B sets out an exemplary route to
oxadiazolyl-containing compounds of the invention. Thus, amidine d
is acylated with a benzoyl chloride species, affording compound e.
Compound e is cyclized to compound f. The nitro group of compound f
is reduced and the resulting amine is converted to the correspond
sulfonamide h. ##STR10##
[0096] Scheme C sets forth a representative route to
oxazole-containing compounds of the invention. Acyl halide i is
converted to oxazole j by the action of triazole in sulfalone. The
nitro group of j is reduced, affording the corresponding amine k,
which is converted to a sulfone l by the action of an activated
sulfonic acid derivative. ##STR11##
[0097] Scheme D provides an exemplary route to bis-aryl
sulfonamides of the invention. Benzyl halide m is reacted with an
appropriate thiol n, forming sulfide o, which is subsequently
oxidized to sulfonamide p.
[0098] Methods for preparing dimers, trimers and higher homologs of
small organic molecules, such as those of the present invention, as
well as methods of functionalizing a polyfunctional framework
molecule are well known to those of skill in the art. For example,
an aromatic amine of the invention is converted to the
corresponding isothiocyanate by the action of thiophosgene. The
resulting isothiocyanate is coupled to an amine of the invention,
thereby forming either a homo- or heterodimeric species.
Alternatively, the isothiocyanate is coupled with an
amine-containing backbone, such as polylysine, thereby forming a
conjugate between a polyvalent framework and a compound of the
invention. If it is desired to prepare a hetereofuntionalized
polyvalent species, the polylysine is underlabeled with the first
isothiocyanate and subsequently labeled with one or more different
isothiocyanates. Alternatively, a mixture of isothiocyanates is
added to the backbone. Purification proceeds by, for example, size
exclusion chromatography, dialysis, nanofiltration and the
like.
Compound Stability
[0099] Compounds of the present invention useful as intermediate
conductance, calcium activated potassium channel inhibitors,
preferably exhibit both acceptable bioavailability and stability in
vivo. The stability of the compounds of the invention in various
biological milieus can be assayed by methods known in the art. In
one embodiment, the stability of the compounds is assayed in an in
vitro preparation. In a preferred embodiment, the in vitro
preparation is a liver microsome preparation. The results of such
in vitro assays provide data relevant to the in vivo stability of
the compounds of the invention. Other in vitro assays useful in
assaying the stability of the compounds of the invention are known
in the art.
[0100] In addition to in vitro methods, in vivo methods such as
pharmacokinetic studies can be performed in a range of animal
models. One or more compounds of the invention can be administered
to an animal, preferably a rat, at different dosages and/or by
different routes (e.g., i.v., i.p., p.o). Blood, urine and/or feces
samples can be collected at serial time points and the samples
assayed for the presence and/or concentration of the compound(s) of
the invention and/or the metabolites of the compound(s).
[0101] Any appropriate quantity can be utilized to compare data
from different compounds. Exemplary quantities include, half-life,
bioavailability, amount of compound remaining intact after a
predetermined time period and the like. In a preferred embodiment,
the amount of compound remaining intact after a predetermined time
period is utilized. As used herein, "intact" refers to compound
that has not been metabolized or other wise degraded into a species
different from the original compound.
[0102] Any technique that allows the detection and, preferably, the
quantitation of the compound(s) and/or metabolites is appropriate
for use in assaying the compounds of the invention. These methods
include, but are not limited to, spectrometric methods (e.g., NMR
(e.g., .sup.19F NMR), MS, IR, UV/vis), chromatographic methods
(e.g., LC, GC, HPLC) and hybrid methods utilizing both
spectrometric and chromatographic methods (e.g., GC/MS, LC/MS,
LC/MS/MS). Further, the methods can utilize detectable labels such
as compounds of the invention that are labeled with radioisotopes
(e.g., .sup.3H, .sup.15N, .sup.14C) or fluorescent labels (e.g.,
fluorescein, rhodamine). Other methods for assaying the in vivo
persistence of small organic molecules, particularly those
applicable to bioactive molecules, will be apparent to those of
skill in the art.
Compound Activity
[0103] To develop pharmaceutically useful intermediate conductance,
calcium activated potassium channel inhibitors, candidate compounds
must demonstrate acceptable activity towards the target channel.
The activity of the compounds of the invention towards these ion
channels, such as the Gardos channel, can be assayed utilizing
methods known in the art.
[0104] Compounds that decrease ion flow through intermediate
conductance, calcium activated potassium channels are tested using
biologically active channels, either recombinant or naturally
occurring. Intermediate conductance, calcium activated potassium
channels, preferably human channels, can be found in native cells,
isolated in vitro, co-expressed or expressed in a cell, or
expressed in membrane derived from a cell. Modulation by a compound
of the invention is tested using standard in vitro or in vivo
assays such as those well known in the art or as otherwise
described herein. Compounds that decrease the flux of ions will
cause a detectable decrease in the ion current density by
decreasing the probability of the channel being open, by increasing
the probability of it being closed, by decreasing conductance
through the channel, and by hampering the passage of ions.
[0105] Decreased flux of potassium may be assessed by determining
changes in polarization (i.e., electrical potential) of a cell
which expresses, for example, the intermediate conductance, calcium
activated potassium channel known as the Gardos channel. One method
of determining changes in cellular polarization is the
voltage-clamp technique e.g., the "cell attached" mode, the "inside
out" mode, and the "whole cell" mode (see, e.g., Ackerman et al.,
New Engl. J. Med. 336:1575-1595 (1997)). Other known assays include
radiolabeled rubidium flux assays and fluorescence assays using
voltage-sensitive dyes. See, e.g., Vestergarrd-Bogind et al., J.
Membrane Biol., 88:67-75 (1988); Danel et al., J Pharmacol. Meth.,
25:185-193 (1991); Holevinsky et al., J. Membrane Biology,
137:59-70 (1994). Assays for compounds capable of inhibiting or
increasing potassium flux through the intermediate conductance,
calcium activated potassium channel protein can be performed by
application of the compounds to a bath solution in contact with and
comprising cells having said channel. See, e.g., Blatz et al.,
Nature, 323:718-720 (1986); Park, J. Physiol., 481:555-570 (1994).
Generally the compounds to be tested are present in the range from
1 pM to 100 mM. Changes in function of the channels can be measured
in the electrical currents or ionic flux, or by the consequences of
changes in currents and flux.
[0106] The effects of the test compounds upon the function of the
channels can be measured by changes in the electrical currents or
ionic flux or by the consequences of changes in currents and flux.
Changes in electrical current or ion flux are measured either by
increases or decreases in flux of cations such as potassium or
rubidium ions. The cations can be measured in a variety of standard
ways. They can be measured directly by concentration changes of the
ions or indirectly by membrane potential or by radiolabeling of the
ions. Consequences of the test compound on ion flux can be quite
varied. Accordingly, any suitable physiological parameter can be
used to assess the influence of a test compound on the channels of
this invention. Changes in channel function can be measured by
ligand displacement such as CTX release. When the functional
consequences are determined using intact cells or animals, one can
also measure a variety of effects such as transmitter release
(e.g., dopamine), hormone release.(e.g., insulin), transcriptional
changes to both known and uncharacterized genetic markers (e.g.,
northern blots), cell volume changes (e.g., in red blood cells),
immune-responses (e.g., T cell activation), changes in cell
metabolism such as cell growth or pH changes.
[0107] For compounds of interest in the modulation of sickle cell
disease, the inhibition by test compounds of an erythrocyte Gardos
channel can be assayed using human red blood cells. The degree of
inhibition can be measured using a detectable material such as
.sup.86Rb. In an exemplary assay, utilizing .sup.86Rb, Gardos
channel inhibition can be assayed by exposing red blood cells to
.sup.86Rb and a test compound and measuring the amount of .sup.86Rb
taken up by the cells. Numerous variations on this assay will be
apparent to those of skill in the art.
[0108] The potency of the compounds of the invention can be assayed
using erythrocytes by a method such as that disclosed by Brugnara
et al., J. Clin. Invest., 92: 520-526 (1993); and Brugnara et al.,
J. Biol. Chem., 268(12): 8760-8768 (1993). Utilizing the methods
described in these references, both the percent inhibition of the
Gardos channel and the IC.sub.50 of the compounds of the invention
can be assayed. Briefly, erythrocytes are exposed to a test
compound and a .sup.86Rb-containing medium. The initial rate of
.sup.86Rb transport can be calculated from a parameter such as the
linear least square slope of .sup.86Rb uptake by the cell(s).
Inhibitory constants can be calculated by standard methods using
computer-assisted nonlinear curve fitting.
[0109] When used to modulate intraocular pressure, the activity of
a compound of the invention towards an intermediate conductance,
calcium activated potassium channel can be assessed using a variety
of in vitro and in vivo assays. In one embodiment, the in vivo
assays conducted in mammals and disclosed herein, e.g., the rabbit
assay in the examples section, are used to identify intermediate
conductance, calcium activated potassium channel blockers for
treatment of increased intraocular pressure. In another embodiment,
the in vitro assays described herein are used, e.g., radiolabeled
rubidium flux. Such assays are used to test for inhibitors of
intermediate conductance, calcium activated potassium channels and
for the identification of compounds that reduce intraocular
pressure in a subject. Assays for modulatory compounds include,
e.g., measuring current; measuring membrane potential; measure ion
flux; e.g., potassium or rubidium; measuring potassium
concentration; measuring second messengers and transcription
levels; using potassium-dependent yeast growth assays; measuring
intraocular pressure, e.g., by administering a compound able to
decrease ion flow through intermediate conductance, calcium
activated potassium channels to a subject and measuring changes in
intraocular pressure.
[0110] Other methods for assaying the activity of ion channels and
the activity of agents that affect the ion channels are known in
the art. The selection of an appropriate assay methods is well
within the capabilities of those of skill in the art who. See, for
example, Hille, B., IONIC CHANNELS OF EXCITABLE MEMBRANES, Sinaner
Associates, Inc. Sunderland, Mass. (1992).
[0111] Activities for selected compounds of the invention were
determined using the assay set forth in Example 4, and are
presented in Table 1. TABLE-US-00001 TABLE 1 Relative potencies, in
the intermediate conductance, calcium activated potassium channel
blocker assay, for a collection of compounds. Compound ID# Activity
1 + 4 ++ 11 +++ 13 +++ 16 ++ 31 ++ 33 +++ 40 +++ 41 +++ 42 ++ 48 +
50 ++ 74 ++ 76 +++ 77 +++ 82 ++ 83 ++ 95 ++ 96 + + Represents 10
.mu.M < IC.sub.50 < 2 .mu.M; ++ represents 2 .mu.M <
IC.sub.50 < 0.5 .mu.M; +++ represents IC.sub.50 < 0.5
.mu.M.
II. Pharmaceutical Compositions of Potassium Channel Blockers
[0112] In another aspect, the present invention provides
pharmaceutical compositions comprising a pharmaceutically
acceptable excipient and a compound of the invention.
Formulation of the Compounds (Compositions)
[0113] The compounds of the present invention can be prepared and
administered in a wide variety of oral, parenteral and topical
dosage forms. Thus, the compounds of the present invention can be
administered by injection, that is, intravenously, intramuscularly,
intracutaneously, subcutaneously, intraduodenally, or
intraperitoneally. Also, the compounds described herein can be
administered by inhalation, for example, intranasally.
Additionally, the compounds of the present invention can be
administered transdermally. Accordingly, the present invention also
provides pharmaceutical compositions comprising a pharmaceutically
acceptable carrier or excipient and one or more compounds of the
invention.
[0114] For preparing pharmaceutical compositions from the compounds
of the present invention, pharmaceutically acceptable carriers can
be either solid or liquid. Solid form preparations include powders,
tablets, pills, capsules, cachets, suppositories, and dispersible
granules. A solid carrier can be one or more substances, which may
also act as diluents, flavoring agents, binders, preservatives,
tablet disintegrating agents, or an encapsulating material.
[0115] In powders, the carrier is a finely divided solid, which is
in a mixture with the finely divided active component. In tablets,
the active component is mixed with the carrier having the necessary
binding properties in suitable proportions and compacted in the
shape and size desired.
[0116] The powders and tablets preferably contain from 5% or 10% to
70% of the active compound. Suitable carriers are magnesium
carbonate, magnesium stearate, talc, sugar, lactose, pectin,
dextrin, starch, gelatin, tragacanth, methylcellulose, sodium
carboxymethylcellulose, a low melting wax, cocoa butter, and the
like. The term "preparation" is intended to include the formulation
of the active compound with encapsulating material as a carrier
providing a capsule in which the active component with or without
other carriers, is surrounded by a carrier, which is thus in
association with it. Similarly, cachets and lozenges are included.
Tablets, powders, capsules, pills, cachets, and lozenges can be
used as solid dosage forms suitable for oral administration.
[0117] For preparing suppositories, a low melting wax, such as a
mixture of fatty acid glycerides or cocoa butter, is first melted
and the active component is dispersed homogeneously therein, as by
stirring. The molten homogeneous mixture is then poured into
convenient sized molds, allowed to cool, and thereby to
solidify.
[0118] Liquid form preparations include solutions, suspensions, and
emulsions, for example, water or water/propylene glycol solutions.
For parenteral injection, liquid preparations can be formulated in
solution in aqueous polyethylene glycol solution.
[0119] Aqueous solutions suitable for oral use can be prepared by
dissolving the active component in water and adding suitable
colorants, flavors, stabilizers, and thickening agents as desired.
Aqueous suspensions suitable for oral use can be made by dispersing
the finely divided active component in water with viscous material,
such as natural or synthetic gums, resins, methylcellulose, sodium
carboxymethylcellulose, and other well-known suspending agents.
[0120] Also included are solid form preparations, which are
intended to be converted, shortly before use, to liquid form
preparations for oral administration. Such liquid forms include
solutions, suspensions, and emulsions. These preparations may
contain, in addition to the active component, colorants, flavors,
stabilizers, buffers, artificial and natural sweeteners,
dispersants, thickeners, solubilizing agents, and the like.
[0121] The pharmaceutical preparation is preferably in unit dosage
form. In such form the preparation is subdivided into unit doses
containing appropriate quantities of the active component. The unit
dosage form can be a packaged preparation, the package containing
discrete quantities of preparation, such as packeted tablets,
capsules, and powders in vials or ampoules. Also, the unit dosage
form can be a capsule, tablet, cachet, or lozenge itself, or it can
be the appropriate number of any of these in packaged form.
[0122] The quantity of active component in a unit dose preparation
may be varied or adjusted from 0.1 mg to 10000 mg, more typically
1.0 mg to 1000 mg, most typically 10 mg to 500 mg, according to the
particular application and the potency of the active component. The
composition can, if desired, also contain other compatible
therapeutic agents.
[0123] Any method of administering drugs directly to a mammalian
eye may be employed to administer, in accordance with the present
invention, the compound or compounds to the eye to be treated. The
primary effect on the mammal resulting from the direct
administration of the compound or compounds to the mammal's eye is
a reduction in intraocular pressure. More preferably, one or more
intermediate conductance, calcium activated potassium channel
blockers and/or additional compounds known to reduce intraocular
pressure are applied topically to the eye or are injected directly
into the eye. Particularly useful results are obtained when the
compound or compounds are applied topically to the eye in an
ophthalmic preparation, e.g., as ocular solutions, suspensions,
gels or creams, as examples of topical ophthalmic preparations used
for dose delivery.
[0124] In accordance with the invention the compounds are typically
administered in an ophthalmically acceptable carrier in sufficient
concentration so as to deliver an effective amount of the compound
or compounds to the eye. The compounds are administered in
accordance with the present invention to the eye, typically admixed
with an ophthalmically acceptable carrier, and optionally with
another compound suitable for treatment of glaucoma and/or
reduction of intraocular pressure. Any suitable, e.g.,
conventional, ophthalmically acceptable carrier may be employed
including water (distilled or deionized water), saline and other
aqueous media, with or without solubility enhancers such as any of
the ophthalmically acceptable beta-cyclodextrins. The compounds may
be soluble in the carrier which is employed for their
administration, so that the compounds are administered to the eye
in the form of a solution. Alternatively, a suspension of the
compound or compounds (or salts thereof) in a suitable carrier may
also be employed.
[0125] When forming compositions for topical administration, the
compounds are generally formulated as between about 0.001% to 10%
w/v, more preferably between about 0.1% to 5% w/v. In one
embodiment, the formulation is 1.0% w/v. In one embodiment, the
formulations are solutions in water at a pH preferably between
about 7.0 to 7.6 pH, preferably pH 7.4.+-.0.3. In another aspect of
the invention, the compounds are formulated as suspensions. In a
preferred embodiment, the formulation is in a 1% w/v ophthalmic
suspension: 1.0% compound of formula V, micronized; 0.06% carbomer
(carbopol 1382 ), NF; 1.0% poloxamer 188, NF; 2.5% glycerin, USP;
0.01% benzalkonium chloride, NF; sodium hydroxide, NF, q.s. pH
7.4.+-.0.3; and purified water, USP (the formulation may be
prepared as % w/w for convenience, and higher grades of water, USP,
may be substituted). Various preservatives may be used in an
ophthalmic preparation. Preservatives include, but are not limited
to, benzalkonium chloride, chlorobutanol, thimerosal,
phenylmercuric acetate, and phenylmercuric nitrate. Likewise,
various vehicles may be used in such ophthalmic preparation. These
vehicles include, but are not limited to, polyvinyl alcohol,
povidone, cyclodextrins, hydroxypropyl methyl cellulose,
poloxamers, carboxymethyl cellulose and hydroxyethyl cellulose.
Such preservatives, if utilized, will typically be employed in an
amount between about 0.001 and about 1.0 wt %.
[0126] Tonicity adjusters may be added as needed or convenient.
They include, but are not limited to, salts, particularly sodium
chloride, potassium chloride etc., mannitol and glycerin, or any
other suitable ophthalmically acceptable tonicity adjuster. Such
agents, if utilized, will typically be employed in an amount
between about 0.1 and about 10.0 wt %.
[0127] Various buffers and means for adjusting pH may be used so
long as the resulting preparation is ophthalmically acceptable.
Accordingly, buffers include but are not limited to, acetate
buffers, titrate buffers, phosphate buffers, and borate buffers.
Acids or bases may be used to adjust the pH of these formulations
as needed.
[0128] In a similar vein, ophthalmically acceptable antioxidants
include, but are not limited to, sodium metabisulfite, sodium
thiosulfate, acetylcysteine, butylated hydroxyanisole, and
butylated hydroxytoluene.
[0129] Some compounds may have limited solubility in water and
therefore may require a surfactant or other appropriate co-solvent
in the composition. Such co-solvents include: Polysorbate 20, 60
and 80; Pluronic F-68, F-84 and P-103; cyclodextrin; polyoxyl 35
castor oil; or other agents known to those skilled in the art. Such
co-solvents are typically employed at a level between about 0.01%
and about 2% by weight.
[0130] Viscosity greater than that of simple aqueous solutions may
be desirable to increase ocular absorption of the compound, to
decrease variability in dispensing the formulations, to decrease
physical separation of components of a suspension or emulsion of
formulation and/or otherwise to improve the ophthalmic formulation.
Such viscosity building agents include, for example, polyvinyl
alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy propyl
methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose,
hydroxy propyl cellulose, chondroitin sulfate and salts thereof,
hyaluronic acid and salts thereof, combinations of the foregoing,
and other agents known to those skilled in the art. Such agents are
typically employed at a level between about 0.01% and about 2% by
weight. Determination of acceptable amounts of any of the above
adjuvants is readily ascertained by one skilled in the art.
[0131] The ophthalmic solution (ocular drops) may be administered
to the mammalian eye as often as necessary to maintain an
acceptable level of intraocular pressure in the eye. In other
words, the ophthalmic solution (or other formulation) is
administered to the mammalian eye as often as necessary to maintain
the beneficial effect of the active ingredient in the eye. Those
skilled in the art will recognize that the frequency of
administration depends on the precise nature of the active
ingredient and its concentration in the ophthalmic formulation.
Within these guidelines it is contemplated that the ophthalmic
formulation of the present invention will be administered to the
mammalian eye once daily. The formulations may be administered to
the mammalian eye anywhere from about 1-4.times. daily, or as
otherwise deemed appropriate by the attending physician. The
formulations may also be administered in combination with one or
more other pharmaceutical compositions known to reduce intraocular
pressure in a subject or otherwise have a beneficial effect in a
subject, including miotics (e.g., pilocarpine, carbachol, and
acetylcholinesterase inhibitors); sympathomimetics (e.g.,
epinephrine and dipivalylepinephrine); beta-blockers (e.g.,
betaxolol, levobunolol and timolol); alpha-2 agonists (e.g.,
para-amino clonidine); carbonic anhydrase inhibitors (e.g.,
acetazolamide, methazolamide and ethoxzolamide); and prostaglandins
and their analogs and derivatives (e.g., latanaprost).
[0132] The compositions of the present invention may additionally
include components to provide sustained release and/or comfort.
Such components include high molecular weight, anionic mucomimetic
polymers, gelling polysaccharides and finely-divided drug carrier
substrates. These components are discussed in greater detail in
U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The
entire contents of these patents are incorporated herein by
reference.
[0133] As will likewise be appreciated by those skilled in the art,
the compositions may be formulated in various dosage forms suitable
for topical ophthalmic delivery, as described above, including
solutions, suspensions, emulsions, gels, and erodible solid ocular
inserts. The compositions are preferably aqueous suspensions or
solutions. Further, such formulated compositions may also include
one or more additional active ingredients in a single vial for
delivery to the patient. That is to say, in addition to one or more
potassium channel inhibitors present in a single formulation, the
present invention additionally contemplates the presence of one or
more of the following therewith: miotics (e.g., pilocarpine,
carbachol, and acetylcholinesterase inhibitors); sympathomimetics
(e.g., epinephrine and dipivalylepinephrine); beta-blockers (e.g.,
betaxolol, levobunolol and timolol); alpha-2 agonists (e.g.,
para-amino clonidine); carbonic anhydrase inhibitors (e.g.,
acetazolamide, methazolamide and ethoxzolamide); and prostaglandins
and their analogs and derivatives (e.g., latanaprost) in a single
formulation for administration. One skilled in the art will
recognize due care will need to be given in selecting such agents
for co-administration from a single formulation with due regard for
chemical stability and compatibility with other agents (whether
active therapeutic agents or excipients) in the composition made
available to the patient.
Effective Dosages
[0134] Pharmaceutical compositions provided by the present
invention include compositions wherein the active ingredient is
contained in a therapeutically effective amount, i.e., in an amount
effective to achieve its intended purpose. The actual amount
effective for a particular application will depend, inter alia, on
the condition being treated. For example, when administered in
methods to reduce sickle cell dehydration and/or delay the
occurrence of erythrocyte sickling or distortion in situ, such
compositions will contain an amount of active ingredient effective
to achieve this result. Similarly, when the pharmaceutical
composition is used to treat or prevent glaucoma, a therapeutically
effective amount will reduce intraocular pressure below a
predetermined pressure threshold. Determination of a
therapeutically effective amount of a compound of the invention is
well within the capabilities of those skilled in the art,
especially in light of the detailed disclosure herein.
[0135] For any compound described herein, the therapeutically
effective amount can be initially determined from cell culture
assays. Target concentrations will be those concentrations of
active compound(s) that are capable of inducing inhibition of the
intermediate conductance, calcium activated potassium channel. In
preferred embodiments, said channel activity is at least 25%
inhibited. Concentrations of active compound(s) that are capable of
inducing at least about 50%, 75%, or even 90% or higher inhibition
of the ion channel potassium flux are presently preferred. The
percentage of inhibition of the intermediate conductance, calcium
activated potassium channel in the patient can be monitored to
assess the efficacy of the drug concentration achieved, and the
dosage can be adjusted upwards or downwards by the medical
practitioner to achieve the desired percentage of inhibition.
[0136] As is well known in the art, therapeutically effective
amounts for use in humans can also be determined from animal
models. For example, a dose for humans can be formulated to achieve
a concentration that has been found to be effective in animals. A
particularly useful animal model for sickle cell disease is the
SAD-1 mouse model (Trudel et al., EMBO J. 11: 31573165 (1991)). The
dosage in humans can be adjusted by monitoring Gardos channel
inhibition and adjusting the dosage upwards or downwards, as
described above.
[0137] A therapeutically effective dose can also be determined from
human data for compounds which are known to exhibit similar
pharmacological activities, such as clotrimazole and other
antimycotic agents (see, e.g., Brugnara et al., JPET 273:266272
(1995)); Benzaquen et al., Nature Medicine 1: 534-540 (1995);
Brugnara et al., J. Clin. Invest. 97(5): 1227-1234 (1996)). The
applied dose can be adjusted based on the relative bioavailability
and potency of the administered compound as compared with
clotrimazole.
[0138] Adjusting the dose to achieve maximal efficacy in humans
based on the methods described above and other methods as are
well-known in the art is well within the capabilities of the
ordinarily skilled artisan.
[0139] In the case of local administration, the systemic
circulating concentration of administered compound will generally
not be of particular importance. In such instances, the compound is
administered so as to achieve a concentration at the local area
effective to achieve the intended result.
[0140] By way of example, when a compound of the invention is used
in the prophylaxis and/or treatment of sickle cell disease,
including both chronic sickle cell episodes and acute sickle cell
crisis, a circulating concentration of administered compound of
about 0.001 .mu.M to 20 .mu.M is considered to be effective, with
about 0.01 .mu.M to 5 .mu.M being preferred.
[0141] Patient doses for oral administration of the compounds
described herein, which is the preferred mode of administration for
prophylaxis and for treatment of chronic sickle cell episodes,
typically range from about 0.01 mg/day to about 100 mg/day, more
typically from about 0.1 mg/day to about 10 mg/day, and most
typically from about 0.50 mg/day to about 5 mg/day. Stated in terms
of patient body weight, typical dosages range from about 0.0001 to
about 0.150 mg/kg/day, more typically from about 0.001 to about
0.015 mg/kg/day, and most typically from about 0.01 to about 0.10
mg/kg/day.
[0142] Dosages may be varied depending upon the requirements of the
patient and the compound being employed. The dose administered to a
patient, in the context of the present invention should be
sufficient to effect a beneficial therapeutic response in the
patient over time. The size of the dose also will be determined by
the existence, nature, and extent of any adverse side-effects.
Determination of the proper dosage for a particular situation is
within the skill of the practitioner. Generally, treatment is
initiated with smaller dosages which are less than the optimum dose
of the compound. Thereafter, the dosage is increased by small
increments until the optimum effect under circumstances is reached.
In one embodiment of the invention, the dosage range is 0.001% to
10% w/v. In another embodiment, the dosage range is 0.1% to 5% w/v.
In another embodiment, the dosage range is 10-1000 .mu.g per eye.
In another embodiment, the dosage range is 75-150 .mu.g per
eye.
[0143] For other modes of administration, dosage amount and
interval can be adjusted individually to provide levels of the
administered compound effective for the particular clinical
indication being treated. For example, if acute sickle crises are
the most dominant clinical manifestation, in one embodiment, a
compound according to the invention can be administered in
relatively high concentrations multiple times per day.
Alternatively, if the patient exhibits only periodic sickle cell
crises on an infrequent, periodic or irregular basis, in one
embodiment, it may be more desirable to administer a compound of
the invention at minimal effective concentrations and to use a less
frequent administration regimen. This will provide a therapeutic
regimen that is commensurate with the severity of the individual's
sickle cell disease.
[0144] Utilizing the teachings provided herein, an effective
prophylactic or therapeutic treatment regimen can be planned which
does not cause substantial toxicity and yet is entirely effective
to treat the clinical symptoms demonstrated by the particular
patient. This planning should involve the careful choice of active
compound by considering factors such as compound potency, relative
bioavailability, patient body weight, presence and severity of
adverse side effects, preferred mode of administration and the
toxicity profile of the selected agent.
Compound Toxicity
[0145] The ratio between toxicity and therapeutic effect for a
particular compound is its therapeutic index and can be expressed
as the ratio between LD.sub.50 (the amount of compound lethal in
50% of the population) and ED.sub.50 (the amount of compound
effective in 50% of the population). Compounds that exhibit high
therapeutic indices are preferred. Therapeutic index data obtained
from cell culture assays and/or animal studies can be used in
formulating a range of dosages for use in humans. The dosage of
such compounds preferably lies within a range of plasma
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized. See,
e.g. Fingl et al., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS,
Ch. 1, p. 1, 1975. The exact formulation, route of administration
and dosage can be chosen by the individual physician in view of the
patient's condition and the particular method in which the compound
is used.
III. Methods for Decreasing Ion Flow in Intermediate Conductance,
Calcium Activated Potassium Channels
[0146] In addition to the compounds and pharmaceutical formulations
discussed in detail above, the present invention provides a number
of methods in which the compounds of the invention find use. The
methods include, but are not-limited to, those that are used in a
laboratory setting to probe the basic mechanisms of intermediate
conductance, calcium activated potassium channels and
channel-active compounds, e.g., pharmacokinetics, drug activity,
disease origin and progression and the like.
[0147] Thus, in another aspect, the invention provides a method of
inhibiting potassium flux of a cell. The method comprises,
contacting a cell with an effective amount of a compound of the
invention.
[0148] This aspect of the invention has a wide range of uses, but
it is preferred as a modality for the study of the basic mechanisms
underlying potassium flux and the mechanism of activity of agents
that modulate this flux. Further, the compounds of the invention
can be utilized as tools in the discovery of new agents that
modulate potassium flux. For example, the compounds of the
invention can be utilized in assays, such as competitive assays, to
test the efficacy of putative inhibitors of potassium flux. These
methods of the invention can be performed both in vitro and in
vivo. Assays according to the present invention can be carried out
by, for example, modifying art-recognized methods to allow the
incorporation of the compounds of the invention into them. Such
modification is well within the skill of those of skill in the
art.
[0149] The methods provided in this aspect of the invention are
also useful for the diagnosis of conditions that can be treated by
modulating ion flux through intermediate conductance, calcium
activated potassium channels, or for determining if a patient will
be responsive to therapeutic agents, which act by blocking
potassium channels. In particular, a patient's cell sample can be
obtained and contacted with a compound of the invention and the ion
flux can be measured relative to a cell's ion flux in the absence
of a compound of the invention. A decrease in ion flux will
typically indicate that the patient will be responsive to a
therapeutic regimen of ion channel openers.
IV. Methods for Treating Conditions Mediated by Intermediate
Conductance, Calcium Activated Potassium Channels
[0150] In another preferred embodiment, this method is used to
treat or prevent a condition that can be positively affected by
modulating potassium flux. In a presently preferred embodiment, the
condition is sickle cell disease or glaucoma and inflammation. For
example, in sickle cell disease, the invention provides a method
for reducing erythrocyte dehydration. This method comprises,
contacting an erythrocyte with an effective amount of a compound of
the invention. This aspect of the invention can be used for a range
of purposes including, for example, study of the mechanism of
erythrocyte dehydration, investigation of compounds that inhibit or
reverse erythrocyte dehydration and the treatment or prevention of
conditions associated with erythrocyte dehydration.
[0151] In another aspect, the invention provides a method of
treating or preventing sickle cell disease. The method comprises
administering to a subject suffering sickle cell disease a
therapeutically effective amount of one or more compounds of the
invention with or without one or more other agents useful in
ameliorating the effects of the disease. This aspect of the
invention can be utilized to prevent the onset of acute sickle cell
events or to ameliorate the effects of these events. Further, the
method can be used to treat and/or prevent chronic sickle cell
disease. The method can make use of the compounds of the invention
per se or, preferably, the pharmaceutical formulations of the
invention. The relevant modes of administration, choice of dosage
levels and frequency of dosing are discussed above.
[0152] The following examples are provided by way of illustration
only and not by way of limitation. Those of skill in the art will
readily recognize a variety of non-critical parameters that can be
changed or modified to yield similar results.
EXAMPLES
[0153] Examples 1-3 illustrate methods for the synthesis and
characterization of compounds of the invention. The compounds of
the invention were isolated in substantially pure form utilizing
the methods detailed in these Examples.
[0154] Example 4 illustrates the use of a rubidium flux assay to
determine the activity of the compounds of the invention.
[0155] In the examples below, unless otherwise stated, temperatures
are given in degrees Celsius (.degree. C.); operations were carried
out at room or ambient temperature (typically a range of from about
18-25.degree. C.; evaporation of solvent was carried out using a
rotary evaporator under reduced pressure (typically, 4.5-30 mmHg)
with a bath temperature of up to 60.degree. C.; the course of
reactions was typically followed by TLC and reaction times are
provided for illustration only; melting points are uncorrected;
products exhibited satisfactory .sup.1H-NMR and/or microanalytical
data; yields are provided for illustration only; and the following
conventional abbreviations are also used: rt (room temperature
.about.25.degree. C.), mp (melting point), L (liter(s)), mL
(milliliters), mmol (millimoles), g (grams), mg (milligrams), min
(minutes), and h (hours).
Example 1
1.1
2-(3-trifluoromethyl-benzenesulfonylamino)-cyclohex-1-enecarboxylic
acid methyl ester (31)
[0156] 3-Trifluoromethylsulfonyl chloride (544 .mu.L, 3.4 mmol) was
added drop wise to a stirring solution of
2-amino-cyclohex-1-enecarboxylic acid methyl ester (530 mg, 3.4
mmol) in pyridine (4 mL). After 18 h at rt the solvent was removed
under reduced pressure. Column chromatography (hexane/ethyl
acetate; 3:1) gave the desired product as a colorless oil which
solidified upon standing (524 mg, 42%); .sup.1H NMR .delta.
(CDCl.sub.3) 11.70 (s, 1H), 8.14 (s, 1H), 8.06 (d, J=7.8 Hz, 1H),
7.82 (d, J=7.7 Hz, 1H), 7.68 (t, J=7.8 Hz, 1H), 3.77 (s, 3H),
2.43-2.40 (m, 2H), 2.25-2.22 (m, 2H), 1.56-1.50 (m, 4H); MS (EI)
found (M+1) 364.4.
1.2 N-(2-furan-2-yl-phenyl)-3-trifluoromethyl-benzenesulfonamide
(76)
[0157] ##STR12##
[0158] 2-Iodoaniline (200 mg, 0.913 mmol) and furan-2-boronic acid
(107 mg, 0.959 mmol) were combined in 2 mL of ethanol and stirred
for 30 minutes. Palladium acetate (6 mg, 0.03 mmol),
triphenylphosphine (22 mg, 0.82 mmol) and a 2N aqueous solution of
sodium carbonate (0.57 mL, 1.1 mmol) were added to the reaction
mixture successively. The reaction was heated to 75.degree. C. and
stirred for 5 h. It was then cooled to 55.degree. C. and stirred
for another 14 h. After cooling the reaction mixture to room
temperature, water (5 mL) was added and the mixture was extracted
with ethyl acetate (2.times.10 mL). The organic layers were
combined and dried (Na.sub.2SO.sub.4). Column chromatography
(hexanes/ethyl acetate; 9:1) gave the desired product (A) as a
white solid (73 mg, 50%): .sup.1H NMR .delta. (CDCl.sub.3) 7.50 (d,
J=1.0 Hz, 1H), 7.48 (dd, J=1.4, 7.7 Hz, 1H), 7.12 (dt, J=1.4, 7.7
Hz, 1H), 6.80 (t, J=7.1 Hz, 1H), 6.75 (d, J=8.0 Hz, 1H), 6.59 (d,
J=3.3 Hz, 1H), 6.51 (dd, J=1.9, 3.3 Hz, 1H).
[0159] A solution of 3-trifluoromethylsulfonyl chloride (42 mg,
0.17 mmol) and pyridine (38 .mu.L, 0.47 mmol) in acetonitrile (2
mL) was added to a solution 2-furan-2-yl-phenylamine (A) (25 mg,
0.16 mmol) in acetonitrile (0.5 mL). After 18 h a saturated aqueous
solution of sodium bicarbonate (5 mL) was added and the mixture was
extracted with ethyl acetate (2.times.10 mL). The organic layers
were combined and dried (Na.sub.2SO.sub.4). Column chromatography
(hexanes/ethyl acetate; 9:1) gave the desired product (76) as a
white solid (40 mg, 70%). .sup.1H NMR .delta. (CDCl.sub.3) 7.89 (s,
1H), 7.62 (m, 4H), 7.35 (m, 4H), 7.24 (m, 1H), 6.35 (dd, J=1.8, 3.4
Hz, 1H), 6.21 (d, J=3.4 Hz, 1H).
1.3
N-[2-(3-methyl-[1,2,4]oxadiazol-5-yl)-phenyl]-3-trifluoromethyl-benzen-
esulfonamide (82)
[0160] ##STR13##
[0161] Acetonitrile (0.10 mL, 2.0 mmol) and hydroxylamine (0.49 mL,
8.0 mmol) were combined in ethanol (10 mL). The mixture was heated
to 60.degree. C. for 3 h. After cooling to room temperature, all
solvents were removed to yield the crude amidine (B) as a white
solid. The crude amidine (B) and diisopropylethylamine (0.70 mL,
4.0 mmol) were combined in dichloromethane (10 mL) and stirred for
30 min. A solution of 2-nitrobenzoyl chloride (0.32 mL, 2.4 mmol)
in dichloromethane (2 mL) was slowly added to the reaction mixture.
After 18 h, water (5 mL) was added to the mixture and the organics
were extracted with dichloromethane (2.times.5 mL), combined and
dried (Na.sub.2SO.sub.4). Recrystallization from ethyl acetate and
hexanes gave (C) as a yellow solid as a mixture of E- and Z-isomers
(440 mg, 98%): .sup.1H NMR .delta. (CDCl.sub.3) 7.93 (dd, J=1.3,
7.8 Hz, 1H), 7.83 (dd, J=1.5, 7.6 Hz, 1H), 7.74-7.62 (m, 2H), 4.78
(broad s, 2H), 2.01 (s, 3H).
[0162] A 1.0 M solution of tetrabutylammonium fluoride in
tetrahydrofuran (0.66 mL, 0.66 mmol) was added to a solution of
compound (C) (440 mg, 1.97 mmol) in tetrahydrofuran (5 mL). The
reaction mixture was stirred for 18 h after which water (5 mL) was
added. The organics were extracted with ethyl acetate (2.times.5
mL), combined and dried (Na.sub.2SO.sub.4). Column chromatography
(hexanes/ethyl acetate; 4:1) gave (D) as an off-white solid (386
mg, 95%): .sup.1H NMR .delta. (CDCl.sub.3) 7.99 (m, 1H), 7.93 (m,
1H), 7.77 (m, 2H), 2.50 (s, 3H).
[0163] Compound (D) (100 mg, 0.487 mmol) and glacial acetic acid
(0.12 mL, 2.1 mmol) were dissolved in 1.5 mL of water and 3.0 mL of
ethanol and then heated at reflux. The reaction vessel was removed
from the bath to allow it to cool slightly and iron (109 mg, 1.95
mmol) was added in portions. The reaction mixture was again heated
at reflux for 20 min after which the reaction was cooled to room
temperature and basified with 30% aqueous ammonium hydroxide
solution to a pH.about.9. The mixture was filtered through Celite
and the ethanol was removed by rotary evaporation. The residue was
extracted with ethyl acetate (2.times.5 mL) and dried
(Na.sub.2SO.sub.4). Column chromatography (hexanes/ethyl acetate;
4:1) gave (E) as a beige solid (39 mg, 46%). .sup.1H NMR .delta.
(CDCl.sub.3) 7.91 (d, J=7.1 Hz, 1H), 7.30 (dd, J=1.4, 8.0 Hz, 1H),
6.76 (m, 2H), 2.46 (s, 3H).
[0164] Compound (82) was prepared as described in the procedure of
(76) using compound (E) (30 mg, 0.17 mmol) as the aniline.
Purification was accomplished by column chromatography using 5:1
hexanes:ethyl acetate as the eluent. This yielded the desired
product (33 mg, 50%) as an off white solid.
1.4
N-[4,5-dimethoxy-2-(3-methyl-[1,2,4]oxadiazol-5-yl)-phenyl]-3-trifluor-
omethyl-benzenesulfonamide (41)
[0165] ##STR14##
[0166] Compound (F) was prepared as described in the procedure of
compound (C). The 3,4-dimethoxy-5-nitrobenzoyl chloride was
prepared by the reaction of 3,4-dimethoxy-5-nitrobenzoic acid (585
mg, 2.57 mmol) with oxalyl chloride (0.36 mL, 4.1 mmol) in
dichloromethane using N,N-dimethylformamide as a catalyst. The
resulting mixture was stirred at room temperature for 1 h, the
volatile liquids were removed and the resulting acid chloride was
used without further purification.
[0167] Purification of (F) was accomplished by column
chromatography using 1:4 hexanes:ethyl acetate followed by 100%
ethyl acetate. This yielded (F) as a mixture of E- and Z-isomers
(397 mg, 73%): .sup.1H NMR .delta. (CDCl.sub.3) for the E-isomer
7.70 (s, 1H), 7.08 (s, 1H), 4.83 (broad s, 2H), 4.11 (s, 6H), 2.92
(s, 3H) and for the Z-isomer 7.46 (s, 1H), 7.16 (s, 1H), 4.82
(broad s, 2H), 3.98 (s, 6H), 2.00 (s, 3H).
[0168] Compound G was prepared as described in the procedure of
compound (D). Purification was accomplished using 1:1 hexanes:ethyl
acetate as the eluent. This gave the desired product as a yellow
solid (281 mg, 76%): .sup.1H NMR .delta. (CDCl.sub.3) 7.58 (s, 1H),
7.18 (s, 1H), 4.02 (s, 3H), 4.00 (s, 3H), 2.49 (s, 3H).
1.5
N-[2-(3-methoxy-[1,2,4]oxadiazol-5-yl)-phenyl]-3-trifluoromethyl-benze-
nesulfonamide (83)
[0169] ##STR15##
[0170] O-Methylisourea hydrochloride (I) (553 mg, 5.00 mmol) was
added to cooled (0.degree. C.) 2N aqueous sodium hydroxide (7.0 mL,
14 mmol). 2-Nitrobenzoyl chloride (0.66 mL, 5.0 mmol) was slowly
added to the reaction mixture. After 1.5 h the organics were
extracted with ethyl acetate (2.times.10 mL), combined and dried
(Na.sub.2SO.sub.4). Removal of the solvent by rotary evaporation
gave crude (J) (776 mg, 70%) as a white solid. .sup.1H NMR .delta.
(CDCl.sub.3) 9.03 (broad s, 1H), 7.94 (dd, J=1.6, 7.5 Hz, 1H),
7.74-7.50 (m, 3H), 5.73 (broad s, 1H), 3.83 (s, 3H).
[0171] Compound (J) (250 mg, 1.12 mmol) was suspended in ether (3
mL) and cooled to 0.degree. C. A solution of tert-butyl
hypochlorite (0.14 mL, 1.2 mmol) in ether (0.5 mL) was then slowly
added. The mixture was stirred for 30 minutes. A 2N aqueous sodium
hydroxide solution was added and the reaction mixture was warmed to
room temperature and was stirred for 1 hour. The ether was removed
by rotary evaporation and methanol (2 mL) was added. The solution
was then warmed to 60.degree. C. for 5 hours and then cooled to
room temperature. The resulting solution was extracted with ethyl
acetate (2.times.5 mL). The organic layers were combined and dried
(Na.sub.2SO.sub.4). Purification was accomplished by column
chromatography using 8:1 and then 1:1 hexanes:ethyl acetate as the
eluent. Compound (K) was obtained as a white solid (89 mg, 36%).
.sup.1H NMR .delta. (CDCl.sub.3) 8.00 (m, 1H), 7.90 (m, 1H), 7.76
(m, 2H), 4.12 (s, 3H).
[0172] Compound (K) (89 mg, 0.402 mmol) was dissolved in dioxane (1
mL). In a separate vessel, sodium sulfide (242 mg, 1.01 mmol) was
dissolve in water (1 mL). Both solutions were heated at 80.degree.
C. and the aqueous solution was added to the dioxane solution.
After 20 minutes the reaction was cooled to room temperature. The
organics were extracted with EtOAc (3.times.10 mL), combined and
dried (Na.sub.2SO.sub.4). Purification of the resulting residue by
column chromatography (hexanes:ethyl acetate; 8:1) gave compound
(L) as a white solid (52 mg, 67%). .sup.1H NMR .delta. (CDCl.sub.3)
7.88 (dd, J=1.6, 8.4 Hz, 1H), 7.31 (dt, J=1.6, 7.8 Hz, 1H), 6.75
(m, 2H), 4.11 (s, 3H); MS (EI) found (M+1) 192.1.
[0173] A 0.3 mL solution of 3-(trifluoromethyl)sulfonyl chloride
(37 mg, 0.15 mmol) in acetonitrile (1 mL) was added to a solution
of (L) (26 mg, 0.14 mmol) and pyridine (33 .mu.L, 0.41 mmol) in
acetonitrile (0.5 mL) and the resulting solution was stirred for 15
h. The mixture was diluted with ethyl acetate (5 mL), washed with 1
N hydrochloric acid and dried (Na.sub.2SO.sub.4). Purification by
column chromatography (hexanes:ethyl acetate; 10:1) yielded the
desired product (83) as a white solid (12 mg, 22%). .sup.1H NMR
.delta. (CDCl.sub.3) 10.36 (s, 1H), 8.11 (s, 1H), 7.96 (dd, J=1.6,
7.0 Hz, 1H), 7.80 (d, J=8.3 Hz, 1H), 7.75 (d, J=7.3 Hz, 1H), 7.54
(m, 2H), 7.22 (t, J=8.2 Hz, 1H), 4.13 (s, 3H); MS (EI) found (M+1)
399.9.
Example 2
2.1 General Procedure for the Preparation of Oxazole Functionalized
Bisaryl Sulfonamides
[0174] As shown in Scheme 5, a solution of acid chloride (M) (1 eq)
in DCM was added slowly to a stirring solution of 1,2,3-triazole (1
eq) and Hunigs base (1.2 eq) in DCM at rt. After TLC had shown
conversion was complete the solution was washed with water (5 mL/1
mmol) and the organic layer dried (Na.sub.2SO.sub.4). The solvent
was removed under reduced pressure. The crude material was
dissolved in sulfalone (5 ml/1 mmol) and heated to 180-200.degree.
C. for 2-10 h. The solution was diluted with water (15 ml/1 mmol)
and the product (N) was extracted with diethyl ether (2.times.5
mL/1 mmol). The organic layers were combined and dried
(Na.sub.2SO.sub.4). Selective reduction of the nitro group over
other substituents could be accomplished using one or more of the
methods highlighted in scheme 5. Coupling of anilines (O) with
arylsulfonyl chlorides was achieved using pyridine in acetonitrile
at either ambient or elevated temperatures. Oxazole substituted
bisaryl sulfonamides (P) were isolated in yields of about 20-60%.
##STR16##
2.2
N-(4-Methyl-2-oxazol-2-yl-phenyl)-3-trifluoromethyl-benzenesulfonamide
(77)
[0175] .sup.1H NMR .delta. (d.sub.6-DMSO) 11.17 (s, 1H), 8.24 (s,
1H), 7.93 (t, J=7.3 Hz, 2H), 7.86 (s, 1H), 7.71-7.67 (m, 2H),
7.52-7.48 (m, 2H), 7.33-7.30 (m, 1H), 2.28 (s, 3H); MS (EI) found
(M+1) 383.4.
2.3 N-(2-Oxazol-2-yl-phenyl)-3-trifluoromethyl-benzenesulfonamide
(74)
[0176] .sup.1H NMR .delta. (d.sub.6-DMSO) 11.57 (s, 1H), 8.08 (s,
1H), 7.94 (d, J=7.7 Hz, 1H), 7.87 (d, J=7.7 Hz, 1H), 7.75 (d, J=8.0
Hz, 1H), 7.70-7.67 (m, 2H), 7.48 (t, J=7.8 Hz, 1H), 7.39 (t, J=7.7
Hz, 1H), 7.28 (s, 1H), 7.14 (t, J=7.6 Hz, 1H); MS (EI) found (M+1)
369.4.
2.4
N-(4-Methoxy-2-oxazol-2-yl-phenyl)-3-trifluoromethyl-benzenesulfonamid-
e (40)
[0177] .sup.1H NMR .delta. (CDCl.sub.3) 10.93 (s, 1H), 7.96 (s,
1H), 7.80 (d, J=7.8 Hz, 1H), 7.72 (d, J=9.1 Hz, 1H), 7.65-7.63 (m,
2H), 7.41 (t, J=7.8 Hz, 1H), 7.32 (d, J=3.0 Hz, 1H), 7.24 (s, 1H),
6.97 (dd, J=3.0 and 9.0 Hz, 1H), 3.81 (s, 3H); MS (EI) found (M+1)
399.4.
Example 3
3.1 General Procedure for the Preparation of Aryl
Benzalsulfones
[0178] ##STR17##
[0179] Thioethers of type (S) were prepared by reacting thiophenols
with functionalized benzyl bromides (prepared either from toluoyl
derivatives by bromination or by functionalization of commercially
available benzyl bromides) using K.sub.2CO.sub.3 in DMSO at room
temperature. Oxidation of the thioethers (S) using mCPBA afforded
the corresponding sulfones (T) in high yield.
3.2 2-(3-Trifluoromethyl-phenylsulfanylmethyl)-benzoic acid methyl
ester (96)
[0180] .sup.1H NMR .delta. (CDCl.sub.3) 7.96 (dd, J=1.6 and 7.7 Hz,
1H), 7.50 (s, 1H), 7.44-7.21 (m, 5H), 7.19 (d, J=6.2 Hz, 1H), 4.56
(s, 2H), 3.89 (s, 3H); MS (EI) found (M+1) 327.4.
3.3 2-(3-Trifluoromethyl-benzenesulfonylmethyl)-benzoic acid methyl
ester (95)
[0181] .sup.1H NMR .delta. (CDCl.sub.3) 7.88 (dd, J=1.0 and 7.5 Hz,
1H), 7.83 (d, J=7.2 Hz, 1H), 7.80 (s, 2H), 7.58 (t, J=7.8 Hz, 1H),
7.50 (dd, J=1.2 and 7.5 Hz, 1H), 7.43 (dt, J=1.2 and 7.5 Hz, 1H),
7.36 (d, J=7.5 Hz, 1H), 5.11 (s, 2H), 3.71 (s, 3H); MS (EI) found
(M+1) 359.6.
Example 4
[0182] Example 4 describes a bioassay for measuring the inhibition
of a calcium activated potassium channel, the Gardos channel, in
red blood cells by the compounds of the invention.
4.1 Materials and Methods
[0183] Heparinized whole blood was washed three times with Modified
Flux Buffer (MFB: 140 mM NaCl; 5 mM KCl; 10 mM Tris; 0.1 mM EGTA;
pH=7.4). Red blood cells ("RBCs") at an approximate 10% hematocrit.
Washed cells were then incubated for 3 hours with .sup.86Rb (5
.mu.Ci/mL). After this incubation period, the RBCs were washed
three times with cold MFB. Washed .sup.86Rb loaded RBCs were then
incubated with a test compound of the invention for 10 minutes.
.sup.86Rb flux was then initiated by the addition of 10 .mu.L/mL of
a MFB solution containing 10 mM CaCl.sub.2 and 100 .mu.M A23187, a
calcium ionophore. This yielded a final concentration of 100 .mu.M
CaCl.sub.2 and 10 .mu.M A23187 n the incubation medium. Cells were
incubated for 10 minutes, spun down and the supernatant was
removed. Samples were counted in a Wallace Microbeta liquid
scintillation counter by Cerenkov emission. Total RBC .sup.86Rb
content was determined by lysing the RBCs with water and then
precipitating protein using a 50:50 mixture of ethanol:chloroform.
After a 20 minute microfuge spin, the aqueous and organic layers
separated and the aqueous layer was removed and counted. Efflux is
expressed as a percentage of the initial cell content of
.sup.86Rb.
4.2 Results
[0184] The above-described bioassay demonstrated that the compounds
of the invention are excellent inhibitors of the Gardos channel.
Results for the inhibition studies are displayed in Table 1,
supra.
[0185] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
* * * * *