U.S. patent application number 13/043895 was filed with the patent office on 2012-02-09 for modulators of atp-binding cassette transporters.
Invention is credited to Peter D.J. Grootenhuis, Sara S. Hadida-Ruah, Matthew Hamilton, Mark Miller, Ashvani Singh.
Application Number | 20120035179 13/043895 |
Document ID | / |
Family ID | 34435000 |
Filed Date | 2012-02-09 |
United States Patent
Application |
20120035179 |
Kind Code |
A1 |
Hadida-Ruah; Sara S. ; et
al. |
February 9, 2012 |
MODULATORS OF ATP-BINDING CASSETTE TRANSPORTERS
Abstract
The present invention provides compounds of Formula I useful as
modulators of ABC transporter activity, ##STR00001## or a
pharmaceutically acceptable salt thereof, wherein R.sup.B, n, B,
R.sup.C, R.sup.D, R.sup.E, A, and Z are described generally and in
classes and subclasses below. The present invention also provides
pharmaceutical compositions, methods and kits associated with
Formula I, useful for as modulators, and for the treatments of
disease and disease conditions associated with ABC transporter
proteins.
Inventors: |
Hadida-Ruah; Sara S.; (La
Jolla, CA) ; Miller; Mark; (San Diego, CA) ;
Singh; Ashvani; (San Diego, CA) ; Hamilton;
Matthew; (Hackettstown, NJ) ; Grootenhuis; Peter
D.J.; (San Diego, CA) |
Family ID: |
34435000 |
Appl. No.: |
13/043895 |
Filed: |
March 9, 2011 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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12541196 |
Aug 14, 2009 |
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13043895 |
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10961485 |
Oct 8, 2004 |
7598412 |
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12541196 |
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60509642 |
Oct 8, 2003 |
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Current U.S.
Class: |
514/248 ; 435/23;
435/29; 435/375; 435/7.21; 514/307; 514/346; 514/406; 514/452;
514/459; 514/469; 514/471; 514/604; 514/617 |
Current CPC
Class: |
A61K 31/495 20130101;
A61P 19/00 20180101; C07D 215/54 20130101; C07D 231/56 20130101;
A61P 27/04 20180101; A61P 35/00 20180101; A61K 45/06 20130101; C07D
213/82 20130101; A61P 7/00 20180101; A61P 19/08 20180101; A61P 1/12
20180101; C07C 233/22 20130101; A61K 31/335 20130101; A61P 5/00
20180101; C07C 235/48 20130101; A61P 25/00 20180101; C07C 311/17
20130101; C07D 307/85 20130101; A61P 5/16 20180101; C07C 235/46
20130101; C07D 307/87 20130101; A61P 21/02 20180101; C07D 217/26
20130101; C07D 407/12 20130101; A61K 31/38 20130101; C07C 2601/08
20170501; C07C 311/16 20130101; A61P 11/00 20180101; A61K 31/435
20130101; A61P 25/14 20180101; C07D 231/14 20130101; A61P 25/16
20180101; C07C 235/34 20130101; C07D 209/42 20130101; C07C 233/73
20130101; A61K 31/41 20130101; A61P 1/18 20180101; A61P 43/00
20180101; A61P 13/12 20180101; A61P 13/02 20180101; C07D 237/28
20130101; C07D 333/32 20130101; A61K 31/40 20130101; A61P 7/02
20180101; C07D 261/20 20130101; C07D 307/68 20130101; A61P 21/00
20180101; A61K 31/00 20130101; A61P 3/10 20180101; A61P 7/10
20180101; A61P 17/00 20180101; A61P 5/18 20180101; C07C 233/60
20130101; C07C 233/65 20130101; A61P 25/28 20180101; A61P 27/02
20180101; A61P 3/06 20180101; C07D 333/70 20130101; C07C 323/60
20130101 |
Class at
Publication: |
514/248 ;
514/469; 514/617; 514/604; 514/471; 514/346; 514/452; 514/459;
514/406; 514/307; 435/375; 435/29; 435/23; 435/7.21 |
International
Class: |
A61K 31/502 20060101
A61K031/502; A61K 31/166 20060101 A61K031/166; A61K 31/18 20060101
A61K031/18; A61K 31/341 20060101 A61K031/341; A61K 31/4412 20060101
A61K031/4412; A61K 31/357 20060101 A61K031/357; A61K 31/351
20060101 A61K031/351; A61K 31/415 20060101 A61K031/415; A61K 31/472
20060101 A61K031/472; C12N 5/071 20100101 C12N005/071; C12Q 1/02
20060101 C12Q001/02; C12Q 1/37 20060101 C12Q001/37; G01N 33/53
20060101 G01N033/53; A61P 11/00 20060101 A61P011/00; A61P 27/04
20060101 A61P027/04; A61P 3/06 20060101 A61P003/06; A61P 7/10
20060101 A61P007/10; A61P 1/12 20060101 A61P001/12; A61P 3/10
20060101 A61P003/10; A61P 5/18 20060101 A61P005/18; A61P 19/08
20060101 A61P019/08; A61P 35/00 20060101 A61P035/00; A61P 5/16
20060101 A61P005/16; A61P 25/28 20060101 A61P025/28; A61P 25/16
20060101 A61P025/16; A61K 31/343 20060101 A61K031/343 |
Claims
1-44. (canceled)
45. A method of modulating ABC transporter activity comprising the
step of contacting said transporter with a compound of formula I or
a pharmaceutically acceptable salt thereof: ##STR00135## or a
pharmaceutically acceptable salt thereof, wherein: A is C(O), or
SO.sub.2; R.sup.C and R.sup.D are independently selected from H,
(C1-C4)alkyl, and aryl, or may be taken together to form a
(C3-C8)cycloalkyl or heterocyclic; R.sup.E is H, (C1-C4)alkyl
optionally substituted with a substituent selected from CN,
NO.sub.2, CF.sub.3, OCF.sub.3, OH, SR.sup.6, S(O)R.sup.6,
SO.sub.2R.sup.6, COOH, COOR.sup.6, OR.sup.6 or phenyl optionally
substituted with R.sup.Z; B is aryl or heterocyclic; Z is
##STR00136## wherein, L is (C1-C6)alkylidene,
--O--((C1-C6)alkylidene), ((C1-6)alkylidene)--O--, or a bond,
wherein up to two carbon atoms in said alkylidene in L are
independently replaced with O, S, or N; W is aryl, heterocyclic, or
(C5-C7)cycloalkyl; m and n are independently 0 to 5; and R.sup.B
and R.sup.Z are independently selected from R.sup.1, R.sup.2,
R.sup.3, R.sup.4, or R.sup.5, wherein: R.sup.1 is oxo, R.sup.6 or
((C1-C4)aliphatic).sub.n-Y; n is 0 or 1; Y is halo, CN, NO.sub.2,
CF.sub.3, OCF.sub.3, OH, SR.sup.6, S(O)R.sup.6, SO.sub.2R.sup.6,
NH.sub.2, NHR.sup.6, N(R.sup.6).sub.2, NR.sup.6R.sup.8,
N(R.sup.8).sub.2, COOH, COOR.sup.6 or OR.sup.6; or two R.sup.1 on
adjacent ring atoms, taken together, form 1,2-methylenedioxy or
1,2-ethylenedioxy; R.sup.2 is aliphatic, wherein each R.sup.2
optionally comprises up to 2 substituents independently selected
from R.sup.1, R.sup.4, or R.sup.5; R.sup.3 is a cycloaliphatic,
aryl, heterocyclic, or heteroaryl ring optionally comprising up to
3 substituents, independently selected from R.sup.1, R.sup.2,
R.sup.4 or R.sup.5; R.sup.4 is OR.sup.5, OR.sup.6, OC(O)R.sup.6,
OC(O)R.sup.5, OC(O)OR.sup.6, OC(O)OR.sup.5, OC(O)N(R.sup.6).sub.2,
OC(O)N(R.sup.5).sub.2, OC(O)N(R.sup.6R.sup.5), SR.sup.6, SR.sup.5,
S(O)R.sup.6, S(O)R.sup.5, SO.sub.2R.sup.6, SO.sub.2R.sup.5,
SO.sub.2N(R.sup.6).sub.2, SO.sub.2N(R.sup.5).sub.2,
SO.sub.2NR.sup.5R.sup.6, SO.sub.3R.sup.6, SO.sub.3R.sup.5,
C(O)R.sup.5, C(O)OR.sup.5, C(O)R.sup.6, C(O)OR.sup.6,
C(O)N(R.sup.6).sub.2, C(O)N(R.sup.5).sub.2, C(O)N(R.sup.5R.sup.6),
C(O)N(OR.sup.6)R.sup.6, C(O)N(OR.sup.5)R.sup.6,
C(O)N(OR.sup.6)R.sup.5, C(O)N(OR.sup.5)R.sup.5,
C(NOR.sup.6)R.sup.6, C(NOR.sup.6)R.sup.5, C(NOR.sup.5)R.sup.6,
C(NOR.sub.5)R.sup.5, N(R.sup.6).sub.2, N(R.sup.5).sub.2,
N(R.sup.5R.sup.6), NR.sup.5C(O)R.sup.5, NR.sup.6C(O)R.sup.6,
NR.sup.5C(O)R.sup.6, NR.sup.6C(O)R.sup.5, NR.sup.6C(O)OR.sup.6,
NR.sup.5C(O)OR.sup.6, NR.sup.6C(O)OR.sup.5, NR.sup.5C(O)OR.sup.5,
NR.sup.6C(O)N(R.sup.6).sub.2, NR.sup.6C(O)NR.sup.5R.sup.6,
NR.sup.6C(O)N(R.sup.5).sub.2, NR.sup.5C(O)N(R.sup.6).sub.2,
NR.sup.5C(O)NR.sup.5R.sup.6, NR.sup.5C(O)N(R.sup.5).sub.2,
NR.sup.6SO.sub.2R.sup.6, NR.sup.6SO.sub.2R.sup.5,
NR.sup.5SO.sub.2R.sup.5, NR.sup.5SO.sub.2R.sup.6,
NR.sup.6SO.sub.2N(R.sup.6).sub.2, NR.sup.5SO.sub.2N(R.sup.6).sub.2,
NR.sup.6SO.sub.2NR.sup.5R.sup.6, NR.sup.6SO.sub.2N(R.sup.5).sub.2,
NR.sup.5SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2N(R.sup.5).sub.2,
N(OR.sup.6)R.sup.6, N(OR.sup.6)R.sup.5, N(OR.sup.5)R.sup.5, or
N(OR.sup.5)R.sup.6; R.sup.5 is a cycloaliphatic, aryl,
heterocyclic, or heteroaryl ring, optionally comprising up to 3
R.sup.1 substituents; R.sup.6 is H or aliphatic, wherein R.sup.6
optionally comprises a R.sup.7 substituent; R.sup.7 is a
cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, and each
R.sup.7 optionally comprises up to 2 substituents independently
chosen from H, (C.sub.1-C.sub.6)-straight or branched alkyl,
(C.sub.2-C.sub.6) straight or branched alkenyl or alkynyl,
1,2-methylenedioxy, 1,2-ethylenedioxy, or (CH.sub.2).sub.n-Q; Q is
selected from halo, CN, NO.sub.2, CF.sub.3, OCF.sub.3, OH,
S-aliphatic, S(O)-aliphatic, SO.sub.2-aliphatic, NH.sub.2,
NH(aliphatic), N(aliphatic).sub.2, N(aliphatic)R.sup.8, NHR.sup.8,
N(R.sup.8).sub.2, COOH, C(O)O-(aliphatic), or O-aliphatic; and
R.sup.8 is an amino protecting group provided that when L is a
bond, R.sup.E is hydrogen and A is C(O), then the following
compound is excluded: TABLE-US-00005 ring W together with R.sup.Z
R.sup.C & R.sup.D together and m ring B with R.sup.B & n
cyclopentyl benzofuran-2-yl 3,4-dimethoxyphenyl
46. (canceled)
47. The method according to claim 45, wherein said disease is
selected from cystic fibrosis, hereditary emphysema, hereditary
hemochromatosis, coagulation-cibrinolysis deficiencies, such as
protein C deficiency, Type 1 hereditary angioedema, lipid
processing deficiencies, such as familial hypercholesterolemia,
Type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage
diseases, such as I-cell disease/Pseudo-Hurler, secretory diarrhea
or polycystic kidney disease, mucopolysaccharidoses,
Sandhof/Tay-Sachs, Crigler-Najjar type II,
polyendocrinopathy/Hyperinsulemia, Diabetes mellitus, Laron
dwarfism, myleoperoxidase deficiency, primary hypoparathyroidism,
melanoma, glycanosis CDG type 1, hereditary emphysema, congenital
hyperthyroidism, osteogenesis imperfecta, hereditary
hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),
neurophyseal DI, Neprogenic DI, Charcot-Marie Tooth syndrome,
Perlizaeus-Merzbacher disease, neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, Amyotrophic lateral
sclerosis, progressive supranuclear plasy, Pick's disease, several
polyglutamine neurological disorders such as Huntington,
Spinocerebullar ataxia type I, spinal and bulbar muscular atrophy,
Dentatorubal pallidoluysian, and Myotonic dystrophy, as well as
Spongiform encephalopathies, such as Hereditary Creutzfeldt-Jakob
disease (due to Prion protein processing defect), Fabry disease,
Straussler-Scheinker syndrome, COPD, dry eye disease, or Sjogren's
disease.
48. The method according to claim 45, wherein said disease is
cystic fibrosis.
49. A method of modulating activity of an anion channel in vitro or
in vivo, comprising the step of contacting said channel with a
compound according to claim 45.
50. The method according to claim 49, wherein said anion channel is
a chloride channel or a bicarbonate channel.
51. The method according to claim 50, wherein said anion channel is
a chloride channel.
52. A method of treating an anion channel mediated disease in a
mammal, comprising the step of administering to said mammal a
composition comprising a compound according to claim 45.
53. The method according to claim 52, wherein said disease is
cystic fibrosis.
54. (canceled)
55. A kit for use in measuring the activity of a ABC transporter or
a fragment thereof in a biological sample in vitro or in vivo,
comprising: (i) a composition comprising a compound according to
claim 45; and (ii) instructions for: a) contacting the composition
with the biological sample; b) measuring activity of said ABC
transporter or a fragment thereof.
56. The kit according to claim 55, wherein said ABC transporter is
CFTR.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 12/541,196 filed Aug. 14, 2009 and entitled
"MODULATORS OF ATP-BINDING CASSETTE TRANSPORTERS," which is a
division of U.S. patent application Ser. No. 10/961,485 filed Oct.
8, 2004 and entitled "MODULATORS OF ATP-BINDING CASSETTE
TRANSPORTERS," which claims the benefit under 35 U.S.C. .sctn.119
of U.S. Provisional Application No. 60/509,642 filed Oct. 8, 2003
the entire contents of each of the above applications being
incorporated herein by reference.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to modulators of ATP-Binding
Cassette ("ABC") transporters or fragments thereof, including CF
Transmembrane Regulator ("CFTR"), compositions thereof, and methods
therewith. The present invention also relates to methods of
treating ABC transporter mediated diseases using such
modulators.
BACKGROUND OF THE INVENTION
[0003] ABC transporters are a group of membrane transporter
proteins that play a major role in the transport and protection of
cells against a wide variety of pharmacological agents, potentially
toxic drugs, and xenobiotics. ABC transporters are homologous
membrane proteins that bind and use cellular adenosine triphosphate
(ATP) for their specific activities. Some of these transporters
were discovered as multidrug resistance proteins (like the MDR1-P
glycoprotein, or the multidrug resistance protein, MRP1), defending
malignant cancer cells against chemotherapeutic agents. Up until
the present time, 48 Human ABC Transporters have been identified,
and these have been arranged into 7 families based on their
sequence identity and function.
[0004] ABC transporters play a variety of important physiological
roles within the body, as well as providing a defense against
harmful compounds from the environment. Moreover they represent
important potential drug targets both in their own right, as well
as, because in many cases therapeutic drugs are also transported
out of the target cell by these molecules.
[0005] One of the members of the ABC transporter family, namely,
CFTR, is believed be the chloride channel responsible for
cAMP-mediated chloride secretion in epithelial cells, and to play a
key role in the secretion of chloride and maintenance of normal
electrolyte transport throughout the body. CFTR is a protein of
approximately 1480 amino acids made up of two repeated elements,
each comprising six transmembrane segments and a nucleotide-binding
domain. The two repeats are separated by a large, polar, regulatory
(R)-domain containing multiple potential phosphorylation sites.
[0006] The gene associated with CFTR has been identified and
sequenced (See Gregory, R. J. et al. (1990) Nature 347:382-386;
Rich, D. P. et al. (1990) Nature 347:358-362), (Riordan, J. R. et
al. (1989) Science 245:1066-1073). A defect in this gene leads to
cystic fibrosis (hereinafter "CF"), the most common fatal genetic
disease in humans affecting approximately one in every 2,500
infants born in the United States. Within the general United States
population, up to 10 million people carry a single copy of the
defective gene without apparent ill effects. In contrast,
individuals with two copies of the CF associated gene suffer from
the chronic effects of CF, including chronic lung destruction and
death.
[0007] In patients with CF, expression of the CF associated gene in
airway cells, leads to reduced cellular apical chloride conductance
causing an imbalance in ion and fluid transport. It is widely
believed that this leads to the abnormal mucus secretion in
pancreatic ductules and in the airways that ultimately results in
the pulmonary infections and epithelial cell damage typically
associated with disease progression in CF. In addition to
respiratory problems, CF patients typically suffer from
gastrointestinal problems, and pancreatic insufficiency. Males are
almost uniformly infertile and fertility is decreased in females.
In contrast to the severe effects of two copies of the CF
associated gene, individuals with a single copy of the CF
associated gene exhibit increased resistance to cholera and to
dehydration resulting from diarrhea--perhaps explaining the
relatively high frequency of the CF gene within the population.
[0008] Sequence analysis of the CFTR gene of CF chromosomes has
revealed a variety of disease causing mutations (Cutting, G. R. et
al. (1990) Nature 346:366-369; Dean, M. et al. (1990) Cell
61:863:870; and Kerem, B-S. et al. (1989) Science 245:1073-1080;
Kerem, B-S et al. (1990) Proc. Natl. Acad. Sci. USA 87:8447-8451).
At present, more than 1000 mutations in the CF gene have been
identified (http://www.genet.sickkids.on.ca/cftr/), but population
studies have indicated that the most common CF mutation, a deletion
of the 3 nucleotides that encode phenylalanine at position 508 of
the CFTR amino'acid sequence, is associated with approximately 70%
of the cases of cystic fibrosis. The mutated CFTR protein is
referred to as .DELTA.F508.
[0009] It is believed that the deletion of residue 508 in
.DELTA.F508-CFTR prevents the nascent protein from folding
correctly, resulting in the inability of this mutant protein to
exit the endoplasmic reticulum (hereinafter "ER"), and traffic to
the plasma membrane. As a result, insufficient amounts of the
mature protein are present at the plasma membrane and chloride
transport within epithelial tissues is significantly reduced
(Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Hence, the cellular
phenomenon of defective ER processing of other proteins like CFTR,
by the ER machinery, has been shown to be the underlying basis for
a wide range of isolated and inherited diseases. The two ways that
the ER machinery can malfunction is either by loss of coupling to
ER export of the proteins leading to degradation, or by the ER
accumulation of these defective/misfolded proteins [Aridor M, et
al., Nature Med., 5(7), pp 745-751 (1999); Shastry, B. S., et al.,
Neurochem. International, 43, pp 1-7 (2003); Rutishauser, J., et
al., Swiss Med Wkly, 132, pp 211-222 (2002); Morello, J P et al.,
TIPS, 21, pp. 466-469 (2000); Bross P., et al., Human Mut., 14, pp.
186-198 (1999)]. Studies have shown, however, that
.DELTA.F508-CFTR, when presented at the plasma membrane is
functional as a cAMP-responsive Cl.sup.- channel (Dalemans et al.
(1991), Nature Lond. 354: 526-528; Denning et al., supra.; Pasyk
and Foskett (1995), J. Cell. Biochem. 270: 12347-50).
[0010] Although CFTR transports a variety of molecules in addition
to anions, this role of transporting anions represents an important
element in the overall cellular machinery for transporting ions and
water across the epithelium. The other elements include the
epithelial Na.sup.+ channel, ENaC, Na.sup.+/2Cl.sup.-/K.sup.+
co-transporter, Na.sup.+--K.sup.+-ATPase pump and the basolateral
membrane K.sup.+ channels, that are responsible for the uptake of
chloride into the cell.
[0011] These elements work together to achieve directional
transport across the epithelium via their selective expression and
localization within the cell. Chloride absorption takes place by
the coordinated activity of: (i) ENaC and CFTR present on the
apical membrane; and (ii) the Na.sup.+-K.sup.+-ATPase pump and Cl--
channels expressed on the basolateral surface of the cell.
Secondary active transport of chloride from the luminal side leads
to the accumulation of intracellular chloride, which can then
passively leave the cell via Cl.sup.- channels, resulting in a
vectorial transport. Arrangement of Na.sup.+/2Cl.sup.-/K.sup.+
co-transporter, Na.sup.+-K.sup.+-ATPase pump and the basolateral
membrane K.sup.+ channels on the basolateral surface and CFTR on
the luminal side coordinate the secretion of chloride via CFTR on
the luminal side. Because water is probably never actively
transported itself, its flow across epithelia depends on tiny
transepithelial osmotic gradients generated by the bulk flow of
sodium and chloride.
[0012] In addition to CF, modulation of CFTR activity may be
beneficial for other diseases not directly caused by mutations in
CFTR, such as secretory diseases and other protein folding diseases
mediated by CFTR. These include, but are not limited to, chronic
obstructive pulmonary disease (hereinafter "COPD"), dry eye
disease, and Sjogren's Syndrome.
[0013] COPD is characterized by airflow limitation that is
progressive and not fully reversible. The airflow limitation is due
to mucus hypersecretion, emphysema, and bronchiolitis. Activators
of mutant or wild-type CFTR offer a potential treatment of mucus
hypersecretion and impaired mucociliary clearance that is common in
COPD. Specifically, increasing anion secretion across CFTR may
facilitate fluid transport into the airway surface liquid to
hydrate the mucus and optimized periciliary fluid viscosity. This
would lead to enhanced mucociliary clearance and to a reduction in
the symptoms associated with COPD. Dry eye disease is characterized
by a decrease in tear aqueous production and abnormal tear film
lipid, protein and mucin profiles. There are many causes of dry
eye, some of which include age, Lasik eye surgery, arthritis,
medications, chemical/thermal burns, allergies, and diseases, such
as CF and Sjogrens's syndrome. Increasing anion secretion via CFTR
would enhance fluid transport from the corneal endothelial cells
and secretory glands surrounding the eye to increase corneal
hydration. This would help to alleviate the symptoms associated
with dry eye disease. Sjogrens's syndrome is an autoimmune disease
in which the immune system attacks moisture-producing glands
throughout the body, including the eye, mouth, skin, respiratory
tissue, liver, vagina, and gut. Symptoms include dry eye, mouth,
and vagina, as well as lung disease. The disease is also associated
with rheumatoid arthritis, systemic lupus, systemic sclerosis, and
polymypositis/dermatomyositis. Defective protein trafficking is
believed to cause the disease for which treatment options are
limited. Modulators of CFTR activity may hydrate the various organs
afflicted by the disease and help to elevate the associated
symptoms.
[0014] As discussed above, it is believed that the deletion of
residue 508 in .DELTA.F508-CFTR prevents the nascent protein from
folding correctly, resulting in the inability of this mutant
protein to exit the ER, and traffic to the plasma membrane. As a
result, insufficient amounts of the mature protein are present at
the plasma membrane and chloride transport within epithelial
tissues is significantly reduced. In fact, this cellular phenomenon
of defective ER processing of ABC transporters by the ER machinery,
has been shown to be the underlying basis not only for CF disease,
but for a wide range of other isolated and inherited diseases. The
two ways that the ER machinery can malfunction is either by loss of
coupling to ER export of the proteins leading to degradation, or by
the ER accumulation of these defective/misfolded proteins [Aridor
M, et al., Nature Med., 5(7), pp 745-751 (1999); Shastry, B. S., et
al., Neurochem. International, 43, pp 1-7 (2003); Rutishauser, J.,
et al., Swiss Med Wkly, 132, pp 211-222 (2002); Morello, J P et
al., TIPS, 21, pp. 466-469 (2000); Bross P., et al., Human Mut.,
14, pp. 186-198 (1999)].
[0015] The diseases associated with the first class of ER
malfunction are CF (due to misfolded .DELTA.F508-CFTR), hereditary
emphysema (due to .alpha.1-antitrypsin; non Piz variants),
hereditary hemochromatosis, coagulation-fibrinolysis deficiencies,
such as protein C deficiency, Type 1 hereditary angioedema, lipid
processing deficiencies, such as familial hypercholesterolemia,
Type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage
diseases, such as I-cell disease/pseudo-Hurler,
mucopolysaccharidoses (due to lysosomal processing enzymes),
Sandhof/Tay-Sachs (due to .beta.-hexosaminidase), Crigler-Najjar
type II (due to UDP-glucuronyl-sialyc-transferase),
polyendocrinopathy/hyperinsulemia, Diabetes mellitus (due to
insulin receptor), Laron dwarfism (due to growth hormone receptor),
myleoperoxidase deficiency, primary hypoparathyroidism (due to
preproparathyroid hormone), melanoma (due to tyrosinase). The
diseases associated with the latter class of ER malfunction are
glycanosis CDG type 1, hereditary emphysema (due to
.beta.1-antitrypsin (PiZ variant), congenital hyperthyroidism,
osteogenesis imperfecta (due to Type I, II, IV procollagen),
hereditary hypofibrinogenemia (due to fibrinogen), ACT deficiency
(due to .alpha.1-antichymotrypsin), Diabetes insipidus (DI),
neurophyseal DI (due to Vasopressin hormone/V2-receptor),
neprogenic DI (due to aquaporin II), Charcot-Marie Tooth syndrome
(due to Peripheral myelin protein 22), Perlizaeus-Merzbacher
disease, neurodegenerative diseases such as Alzheimer's disease
(due to .beta.APP and presenilins), Parkinson's disease,
amyotrophic lateral sclerosis, progressive supranuclear plasy,
Pick's disease, several polyglutamine neurological disorders such
as Huntington, spinocerebullar ataxia type I, spinal and bulbar
muscular atrophy, dentatorubal pallidoluysian, and myotonic
dystrophy, as well as Spongiform encephalopathies, such as
hereditary Creutzfeldt-Jakob disease (due to Prion protein
processing defect), Fabry disease (due to lysosomal
.alpha.-galactosidase A) and Straussler-Scheinker syndrome (due to
Prp processing defect).
[0016] In CF, chloride transport mediated by the CFTR is reduced
resulting in the abnormal mucus secretion that characterizes the
disease. By contrast in secretory diarrheas epithelial water
transport is dramatically increased as a result of secretagogue
activated chloride transport. The mechanism involves elevation of
cAMP and stimulation of CFTR.
[0017] Although there are numerous causes of diarrhea, the major
consequences of diarrheal diseases, resulting from excessive
chloride transport are common to all, and include dehydration,
acidosis, death and impaired growth.
[0018] Acute and chronic diarrheas represent a major medical
problem in many areas of the world. Diarrhea is both a significant
factor in malnutrition and the leading cause of death (5,000,000
deaths/year) in children less than five years old.
[0019] Secretory diarrheas are also a dangerous condition in
patients of acquired immunodeficiency syndrome (AIDS) and chronic
inflammatory bowel disease (IBD). Sixteen million travelers to
developing countries from industrialized nations every year develop
diarrhea, with the severity and number of cases of diarrhea varying
depending on the country and area of travel.
[0020] Diarrhea in barn animals and pets such as cows, pigs and
horses, sheep, goats, cats and dogs, also known as scours, is a
major cause of death in these animals. Diarrhea can result from any
major transition, such as weaning or physical movement, as well as
in response to a variety of bacterial or viral infections and
generally occurs within the first few hours of the animal's
life.
[0021] The most common diarrheal causing bacteria is
enterotoxogenic E-coli (ETEC) having the K.sub.99 pilus antigen.
Common viral causes of diarrhea include rotavirus and coronavirus.
Other infectious agents include cryptosporidium, giardia lamblia,
and salmonella, among others.
[0022] Symptoms of rotaviral infection include excretion of watery
feces, dehydration and weakness. Coronavirus causes a more severe
illness in the newborn animals, and has a higher mortality rate
than rotaviral infection. Often, however, a young animal may be
infected with more than one virus or with a combination of viral
and bacterial microorganisms at one time. This dramatically
increases the severity of the disease.
[0023] Accordingly, there is a need for modulators of an ABC
transporter activity, and compositions thereof, that can be used to
modulate the activity of the ABC transporter in the cell membrane
of a mammal.
[0024] There is a need for methods of treating ABC transporter
mediated diseases using such modulators of ABC transporter
activity.
[0025] There is a need for methods of modulating an ABC transporter
activity in an ex vivo cell membrane of a mammal.
[0026] There is a need for modulators of CFTR activity that can be
used to modulate the activity of CFTR in the cell membrane of a
mammal.
[0027] There is a need for methods of treating CFTR-mediated
diseases using such modulators of CFTR activity.
[0028] There is a need for methods of modulating CFTR activity in,
an ex vivo cell membrane of a mammal.
[0029] There is a need for modulators that enhance the activity
and/or function of CFTR in the plasma membrane.
SUMMARY OF THE INVENTION
[0030] It has now been found that compounds of this invention, and
pharmaceutically acceptable compositions thereof, are useful as
modulators of ABC transporter activity. These compounds have the
general Formula I:
##STR00002##
or a pharmaceutically acceptable salt thereof, wherein R.sup.B, n,
B, R.sup.C, R.sup.D, R.sup.E, A, and Z are described generally and
in classes and subclasses below.
[0031] These compounds and pharmaceutically acceptable compositions
are useful for treating or lessening the severity of a variety of
diseases, disorders, or conditions, including, but not limited to,
cystic fibrosis, hereditary emphysema, hereditary hemochromatosis,
coagulation-cibrinolysis deficiencies, such as protein C
deficiency, Type 1 hereditary angioedema, lipid processing
deficiencies, such as familial hypercholesterolemia, Type 1
chylomicronemia, abetalipoproteinemia, lysosomal storage diseases,
such as I-cell disease/pseudo-Hurler, secretory diarrhea or
polycystic kidney disease, mucopolysaccharidoses,
Sandhof/Tay-Sachs, Crigler-Najjar type II,
polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron
dwarfism, myleoperoxidase deficiency, primary hypoparathyroidism,
melanoma, glycanosis CDG type 1, hereditary emphysema, congenital
hyperthyroidism, osteogenesis imperfecta, hereditary
hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),
neurophyseal DI, neprogenic DI, Charcot-Marie Tooth syndrome,
Perlizaeus-Merzbacher disease, neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, amyotrophic lateral
sclerosis, progressive supranuclear plasy, Pick's disease, several
polyglutamine neurological disorders such as Huntington,
spinocerebullar ataxia type I, spinal and bulbar muscular atrophy,
dentatorubal pallidoluysian, and myotonic dystrophy, as well as
spongiform encephalopathies, such as hereditary Creutzfeldt-Jakob
disease (due to prion protein processing defect), Fabry disease,
Straussler-Scheinker syndrome, COPD, dry eye disease, or Sjogren's
disease.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0032] The term "ABC-transporter" as used herein means an
ABC-transporter protein or a fragment thereof comprising at least
one binding domain, wherein said protein or fragment thereof is
present in vivo or in vitro. The term "binding domain" as used
herein means a domain on the ABC-transporter that can bind to a
modulator. See, e.g., Hwang, T. C. et al., J. Gen. Physiol. (1998):
111(3), 477-90.
[0033] The term "CFTR" as used herein means cystic fibrosis
transmembrane conductance regulator or a mutation thereof capable
of regulator activity in part or full, including, but not limited
to, .DELTA.F508 CFTR and G551D CFTR (see, e.g.,
http://www.genet.sickkids.on.ca/cftr/, for CFTR mutations).
[0034] The term "COPD" as used herein means chronic obstructive
pulmonary disease and comprises chronic obstructive bronchitis, and
emphysema.
[0035] The term "modulating" as used herein means increasing or
decreasing by a measurable amount.
[0036] For purposes of this invention, the chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 75th Ed.
Additionally, general principles of organic chemistry are described
in "Organic Chemistry", Thomas Sorrell, University Science Books,
Sausalito: 1999, and "March's Advanced Organic Chemistry", 5.sup.th
Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New
York: 2001, the entire contents of which are hereby incorporated by
reference.
[0037] As described herein, compounds of the invention may
optionally be substituted with one or more substituents, such as
are illustrated generally above, or as exemplified by particular
classes, subclasses, and species of the invention. It will be
appreciated that the phrase "optionally substituted" is used
interchangeably with the phrase "substituted or unsubstituted." In
general, the term "substituted", whether preceded by the term
"optionally" or not, refers to the replacement of hydrogen radicals
in a given structure with the radical of a specified substituent.
Unless otherwise indicated, an optionally substituted group may
have a substituent at each substitutable position of the group, and
when more than one position in any given structure may be
substituted with more than one substituent selected from a
specified group, the substituent may be either the same or
different at every position. Combinations of substituents
envisioned by this invention are preferably those that result in
the formation of stable or chemically feasible compounds. The term
"stable", as used herein, refers to compounds that are not
substantially altered when subjected to conditions to allow for
their production, detection, and preferably their recovery,
purification, and use for one or more of the purposes disclosed
herein. In some embodiments, a stable compound or chemically
feasible compound is one that is not substantially altered when
kept at a temperature of 40.degree. C. or less, in the absence of
moisture or other chemically reactive conditions, for at least a
week.
[0038] The term "aliphatic" or "aliphatic group", as used herein,
means a straight-chain (i.e., unbranched) or branched, substituted
or unsubstituted hydrocarbon chain that is completely saturated or
that contains one or more units of unsaturation, or a monocyclic,
bicyclic, or tricyclic hydrocarbon that is completely saturated or
that contains one or more units of unsaturation, but which is not
aromatic (also referred to herein as "carbocycle" "cycloaliphatic"
or "cycloalkyl"), that has a single point of attachment to the rest
of the molecule. Unless otherwise specified, aliphatic groups
contain 1-20 aliphatic carbon atoms, i.e., ((C1-C20)alkyl). In some
embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms,
i.e., ((C1-C10)alkyl). In other embodiments, aliphatic groups
contain 1-8 aliphatic carbon atoms, i.e., ((C1-C8)alkyl. In still
other embodiments, aliphatic groups contain 1-6 aliphatic carbon
atoms, i.e., ((C1-C6)alkyl, and in yet other embodiments aliphatic
groups contain 1-4 aliphatic carbon atoms, i.e., ((C1-C4)alkyl. In
some embodiments, "cycloaliphatic" (or "carbocycle" or
"cycloalkyl") refers to a monocyclic C3-C8 hydrocarbon or bicyclic
or tricyclic C8-C12 hydrocarbon that is completely saturated or
that contains one or more units of unsaturation, but which is not
aromatic, that has a single point of attachment to the rest of the
molecule wherein any individual ring in said bicyclic ring system
has 3-7 members. Suitable aliphatic groups include, but are not
limited to, linear or branched, substituted or unsubstituted alkyl,
alkenyl, alkynyl groups and hybrids thereof such as
(cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
[0039] The term "heteroaliphatic", as used herein, means aliphatic
groups wherein one or two carbon atoms are independently replaced
by one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon.
Heteroaliphatic groups may be substituted or unsubstituted,
branched or unbranched, cyclic or acyclic, and include
"heterocycle", "heterocyclyl", "heterocycloaliphatic", or
"heterocyclic" groups.
[0040] The term "heterocycle", "heterocyclyl",
"heterocycloaliphatic", or "heterocyclic" as used herein means
non-aromatic, monocyclic, bicyclic, or tricyclic ring systems in
which one or more ring members is an independently selected
heteroatom. In some embodiments, the "heterocycle", "heterocyclyl",
"heterocycloaliphatic", or "heterocyclic" group has three to
fourteen ring members in which one or more ring members is a
heteroatom independently selected from oxygen, sulfur, nitrogen, or
phosphorus, and each ring in the system contains 3 to 7 ring
members.
[0041] The term "heteroatom" means one or more of oxygen, sulfur,
nitrogen, phosphorus, or silicon (including, any oxidized form of
nitrogen, sulfur, phosphorus, or silicon; the quaternized form of
any basic nitrogen or a substitutable nitrogen of a heterocyclic
ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in
pyrrolidinyl) or NR.sup.+ (as in N-substituted pyrrolidinyl)).
[0042] The term "unsaturated", as used herein, means that a moiety
has one or more units of unsaturation.
[0043] The term "alkoxy", or "thioalkyl", as used herein, refers to
an alkyl group, as previously defined, attached to the principal
carbon chain through an oxygen ("alkoxy") or sulfur ("thioalkyl")
atom.
[0044] The terms "haloalkyl", "haloalkenyl" and "haloalkoxy" means
alkyl, alkenyl or alkoxy, as the case may be, substituted with one
or more halogen atoms. The term "halogen" means F, Cl, Br, or
I.
[0045] The term "aryl" used alone or as part of a larger moiety as
in "aralkyl", "aralkoxy", or "aryloxyalkyl", refers to monocyclic,
bicyclic, and tricyclic ring systems having a total of five to
fourteen ring members, wherein at least one ring in the system is
aromatic and wherein each ring in the system contains 3 to 7 ring
members. The term "aryl" may be used interchangeably with the term
"aryl ring". The term "aryl" also refers to heteroaryl ring systems
as defined hereinbelow.
[0046] The term "heteroaryl", used alone or as part of a larger
moiety as in "heteroaralkyl" or "heteroarylalkoxy", refers to
monocyclic, bicyclic, and tricyclic ring systems having a total of
five to fourteen ring members, wherein at least one ring in the
system is aromatic, at least one ring in the system contains one or
more heteroatoms, and wherein each ring in the system contains 3 to
7 ring members. The term "heteroaryl" may be used interchangeably
with the term "heteroaryl ring" or the term "heteroaromatic".
[0047] An aryl (including aralkyl, aralkoxy, aryloxyalkyl and the
like) or heteroaryl (including heteroaralkyl and heteroarylalkoxy
and the like) group may contain one or more substituents. Suitable
substituents on the unsaturated carbon atom of an aryl or
heteroaryl group are selected from halogen; --R.sup.o; --OR.sup.o;
--SR.sup.o; 1,2-methylene-dioxy; 1,2-ethylenedioxy; phenyl (Ph)
optionally substituted with R.sup.o; --O(Ph) optionally substituted
with R.sup.o; --(CH.sub.2).sub.1-2(Ph), optionally substituted with
R.sup.o; --CH.dbd.CH(Ph), optionally substituted with R.sup.o;
--NO.sub.2; --CN; --N(R.sup.o).sub.2; --NR.sup.oC(O)R.sup.o;
--NR.sup.oC(O)N(R.sup.o).sub.2; --NR.sup.oCO.sub.2R.sup.o;
--NR.sup.oNR.sup.oC(O)R.sup.o;
--NR.sup.oNR.sup.oC(O)N(R.sup.o).sub.2;
--NR.sup.oNR.sup.oCO.sub.2R.sup.o; --C(O)C(O)R.sup.o;
--C(O)CH.sub.2C(O)R.sup.o; --CO.sub.2R.sup.o; --C(O)R.sup.o;
--C(O)N(R.sup.o).sub.2; --OC(O)N(R.sup.o).sub.2;
--S(O).sub.2R.sup.o; --SO.sub.2N(R.sup.o).sub.2; --S(O)R.sup.o;
--NR.sup.oSO.sub.2N(R.sup.o).sub.2; --NR.sup.oSO.sub.2R.sup.o;
(.dbd.S)N(R.sup.o).sub.2; --C(.dbd.NH)--N)(R.sup.o).sub.2; or
--(CH.sub.2).sub.0-2NHC(O)R.sup.o wherein each independent
occurrence of R.sup.o is selected from hydrogen, optionally
substituted C.sub.1-6 aliphatic, an unsubstituted 5-6 membered
heteroaryl or heterocyclic ring, phenyl, --O(Ph), or
--CH.sub.2(Ph), or, notwithstanding the definition above, two
independent occurrences of R.sup.o, on the same substituent or
different substituents, taken together with the atom(s) to which
each R.sup.o group is bound, form a 3- to 8-membered cycloalkyl,
heterocyclyl, aryl, or heteroaryl ring having 0-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. Optional
substituents on the aliphatic group of R.sup.o are selected from
NH.sub.2, NH(C.sub.1-4aliphatic), N(C.sub.1-4aliphatic).sub.2,
halogen, C.sub.1-4aliphatic, OH, O(C.sub.1-4aliphatic), NO.sub.2,
CN, CO.sub.2H, CO.sub.2(C.sub.1-4aliphatic),
O(haloC.sub.1-4aliphatic), or haloC.sub.1-4aliphatic, wherein each
of the foregoing C.sub.1-4aliphatic groups of R.sup.o is
unsubstituted.
[0048] An aliphatic or heteroaliphatic group, or a non-aromatic
heterocyclic ring may contain one or more substituents. Suitable
substituents on the saturated carbon of an aliphatic or
heteroaliphatic group, or of a non-aromatic heterocyclic ring are
selected from those listed above for the unsaturated carbon of an
aryl or heteroaryl group and additionally include the following:
.dbd.O, .dbd.S, .dbd.NNHR*, .dbd.NN(R*).sub.2, .dbd.NNHC(O)R*,
.dbd.NNHCO.sub.2(alkyl), .dbd.NNHSO.sub.2(alkyl), or .dbd.NR*,
where each R* is independently selected from hydrogen or an
optionally substituted C.sub.1-6 aliphatic. Optional substituents
on the aliphatic group of R* are selected from NH.sub.2,
NH(C.sub.1-4 aliphatic), N(C1-C4 aliphatic).sub.2, halogen,
(C1-C4)aliphatic, OH, O((C1-C4)aliphatic), NO.sub.2, CN, CO.sub.2H,
CO.sub.2((C1-C4)aliphatic), O(halo(C1-C4)aliphatic), or
halo((C1-C4)aliphatic), wherein each of the foregoing
(C1-C4)aliphatic groups of R* is unsubstituted.
[0049] Optional substituents on the nitrogen of a non-aromatic
heterocyclic ring are selected from --R.sup.+, --N(R.sup.+).sub.2,
--C(O)R.sup.+, --CO.sub.2R.sup.+, --C(O)C(O)R.sup.+,
--C(O)CH.sub.2C(O)R.sup.+, --SO.sub.2R.sup.+,
--SO.sub.2N(R.sup.+).sub.2, --C(.dbd.S)N(R.sup.+).sub.2,
--C(.dbd.NH)--N(R.sup.+).sub.2, or --NR.sup.+SO.sub.2R.sup.+;
wherein R.sup.+ is hydrogen, an optionally substituted C.sub.1-6
aliphatic, optionally substituted phenyl, optionally substituted
--O(Ph), optionally substituted --CH.sub.2(Ph), optionally
substituted --(CH.sub.2).sub.1-2(Ph); optionally substituted
--CH.dbd.CH(Ph); or an unsubstituted 5-6 membered heteroaryl or
heterocyclic ring having one to four heteroatoms independently
selected from oxygen, nitrogen, or sulfur, or, notwithstanding the
definition above, two independent occurrences of R.sup.+, on the
same substituent or different substituents, taken together with the
atom(s) to which each R.sup.+ group is bound, form a 3- to
8-membered cycloalkyl, heterocyclyl, aryl, or heteroaryl ring
having 0-3 heteroatoms independently selected from nitrogen,
oxygen, or sulfur. Optional substituents on the aliphatic group or
the phenyl ring of R.sup.+ are selected from NH.sub.2,
NH((C1-C4)aliphatic), N((C1-C4)aliphatic).sub.2, halogen,
(C1-C4)aliphatic, OH, O((C1-C4)aliphatic), NO.sub.2, CN, CO.sub.2H,
CO.sub.2((C1-C4)aliphatic), O(halo(C1-C4)aliphatic), or
halo((C1-C4)aliphatic), wherein each of the foregoing
C.sub.1-4aliphatic groups of R.sup.+ is unsubstituted.
[0050] The term "alkylidene chain" refers to a straight or branched
carbon chain that may be fully saturated or have one or more units
of unsaturation and has two points of attachment to the rest of the
molecule.
[0051] As detailed above, in some embodiments, two independent
occurrences of R.sup.o (or R.sup.+, or any other variable similarly
defined herein), are taken together with the atom(s) to which each
variable is bound to form a 3-8-membered cycloalkyl, heterocyclyl,
aryl, or heteroaryl ring having 0-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur. Exemplary rings that are
formed when two independent occurrences of R.sup.o (or R.sup.+, or
any other variable similarly defined herein) are taken together
with the atom(s) to which each variable is bound include, but are
not limited to the following: a) two independent occurrences of
R.sup.o (or R.sup.+, or any other variable similarly defined
herein) that are bound to the same atom and are taken together with
that atom to form a ring, for example, N(R.sup.o).sub.2, where both
occurrences of R.sup.o are taken together with the nitrogen atom to
form a piperidin-1-yl, piperazin-1-yl, or morpholin-4-yl group; and
b) two independent occurrences of R.sup.o (or R.sup.+, or any other
variable similarly defined herein) that are bound to different
atoms and are taken together with both of those atoms to form a
ring, for example where a phenyl group is substituted with two
occurrences of OR.sup.o
##STR00003##
these two occurrences of R.sup.o are taken together with the oxygen
atoms to which they are bound to form a fused 6-membered oxygen
containing ring:
##STR00004##
It will be appreciated that a variety of other rings can be formed
when two independent occurrences of R.sup.o (or R.sup.+, or any
other variable similarly defined herein) are taken together with
the atom(s) to which each variable is bound and that the examples
detailed above are not intended to be limiting.
[0052] Unless otherwise stated, structures depicted herein are also
meant to include all isomeric (e.g., enantiomeric, diastereomeric,
and geometric (or conformational)) forms of the structure; for
example, the R and S configurations for each asymmetric center, (Z)
and (E) double bond isomers, and (Z) and (E) conformational
isomers. Therefore, single stereochemical isomers as well as
enantiomeric, diastereomeric, and geometric (or conformational)
mixtures of the present compounds are within the scope of the
invention. Unless otherwise stated, all tautomeric forms of the
compounds of the invention are within the scope of the invention.
Additionally, unless otherwise stated, structures depicted herein
are also meant to include compounds that differ only in the
presence of one or more isotopically enriched atoms. For example,
compounds having the present structures except for the replacement
of hydrogen by deuterium or tritium, or the replacement of a carbon
by a .sup.13C- or .sup.14C-enriched carbon are within the scope of
this invention. Such compounds are useful, for example, as
analytical tools or probes in biological assays.
2. General Description of the Invention
[0053] The present invention relates to compounds of formula I:
##STR00005##
or a pharmaceutically acceptable salt thereof, wherein: [0054] A is
C(O), or SO.sub.2; [0055] R.sup.C and R.sup.D are independently
selected from H, (C1-C4)alkyl, and aryl, or may be taken together
to form a (C3-C8)cycloalkyl or heterocyclic; [0056] R.sup.E is H,
(C1-C4)alkyl optionally substituted with a substituent selected
from CN, NO.sub.2, CF.sub.3, OCF.sub.3, OH, SR.sup.6, S(O)R.sup.6,
SO.sub.2R.sup.6, COOH, COOR.sup.6, OR.sup.6 or phenyl optionally
substituted with R.sup.Z; [0057] B is aryl or heterocyclic; [0058]
Z is
[0058] ##STR00006## [0059] wherein, [0060] L is (C1-C6)alkylidene,
--O--((C1-C6)alkylidene), ((C1-6)alkylidene)--O--, or a bond,
wherein up to two carbon atoms in said alkylidene in L are
independently replaced with O, S, or N; [0061] W is aryl,
heterocyclic, or (C5-C7)cycloalkyl; m and n are independently 0 to
5; and R.sup.B and R.sup.Z are independently selected from R.sup.1,
R.sup.2, R.sup.3, R.sup.4, [0062] or R.sup.5, wherein: [0063]
R.sup.1 is oxo, R.sup.6 or ((C1-C4)aliphatic).sub.n-Y; [0064] n is
0 or 1; [0065] Y is halo, CN, NO.sub.2, CF.sub.3, OCF.sub.3, OH,
SR.sup.6, S(O)R.sup.6, SO.sub.2R.sup.6, NH.sub.2, NHR.sup.6,
N(R.sup.6).sub.2, NR.sup.6R.sup.8, N(R.sup.8).sub.2, COOH,
COOR.sup.6 or OR.sup.6; or two R.sup.1 on adjacent ring atoms,
taken together, form 1,2-methylenedioxy or 1,2-ethylenedioxy;
[0066] R.sup.2 is aliphatic, wherein each R.sup.2 optionally
comprises up to 2 substituents independently selected from R.sup.1,
R.sup.4, or R.sup.5; [0067] R.sup.3 is a cycloaliphatic, aryl,
heterocyclic, or heteroaryl ring optionally comprising up to 3
substituents, independently selected from R.sup.1, R.sup.2, R.sup.4
or R.sup.5; [0068] R.sup.4 is OR.sup.5, OR.sup.6, OC(O)R.sup.6,
OC(O)R.sup.5, OC(O)OR.sup.6, OC(O)OR.sup.5, OC(O)N(R.sup.6).sub.2,
OC(O)N(R.sup.5).sub.2, OC(O)N(R.sup.6R.sup.5), SR.sup.6, SR.sup.5,
S(O)R.sup.6, S(O)R.sup.5, SO.sub.2R.sup.6, SO.sub.2R.sup.5,
SO.sub.2N(R.sup.6).sub.2, SO.sub.2N(R.sup.5).sub.2,
SO.sub.2NR.sup.5R.sup.6, SO.sub.3R.sup.6, SO.sub.3R.sup.5,
C(O)R.sup.5, C(O)OR.sup.5, C(O)R.sup.6, C(O)OR.sup.6,
C(O)N(R.sup.6).sub.2, C(O)N(R.sup.5).sub.2, C(O)N(R.sup.5R.sup.6),
C(O)N(OR.sup.6)R.sup.6, C(O)N(OR.sup.5)R.sup.6,
C(O)N(OR.sup.6)R.sup.5, C(O)N(OR.sup.5)R.sup.5,
C(NOR.sup.6)R.sup.6, C(NOR.sup.6)R.sup.5, C(NOR.sup.5)R.sup.6,
C(NOR.sup.5)R.sup.5, N(R.sup.6).sub.2, N(R.sup.5).sub.2,
N(R.sup.5R.sup.6), NR.sup.5C(O)R.sup.5, NR.sup.6C(O)R.sup.6,
NR.sup.5C(O)R.sup.6, NR.sup.6C(O)R.sup.5, NR.sup.6C(O)OR.sup.6,
NR.sup.5C(O)OR.sup.6, NR.sup.6C(O)OR.sup.5, NR.sup.5C(O)OR.sup.5,
NR.sup.6C(O)N(R.sup.6).sub.2, NR.sup.6C(O)NR.sup.5R.sup.6,
NR.sup.6C(O)N(R.sup.5).sub.2, NR.sup.5C(O)N(R.sup.6).sub.2,
NR.sup.5C(O)NR.sup.5R.sup.6, NR.sup.5C(O)N(R.sup.5).sub.2,
NR.sup.6SO.sub.2R.sup.6, NR.sup.6SO.sub.2R.sup.5,
NR.sup.5SO.sub.2R.sup.5, NR.sup.5SO.sub.2R.sup.6,
NR.sup.6SO.sub.2N(R.sup.6).sub.2, NR.sup.5SO.sub.2N(R.sup.6).sub.2,
NR.sup.6SO.sub.2NR.sup.5R.sup.6, NR.sup.6SO.sub.2N(R.sup.5).sub.2,
NR.sup.5SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2N(R.sup.5).sub.2,
N(OR.sup.6)R.sup.6, N(OR.sup.6)R.sup.5, N(OR.sup.5)R.sup.5, or
N(OR.sup.5)R.sup.6; [0069] R.sup.5 is a cycloaliphatic, aryl,
heterocyclic, or heteroaryl ring, optionally comprising up to 3
R.sup.1 substituents; [0070] R.sup.6 is H or aliphatic, wherein
R.sup.6 optionally comprises a R.sup.7 substituent; [0071] R.sup.7
is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, and
each R.sup.7 optionally comprises up to 2 substituents
independently chosen from H, (C.sub.1-C.sub.6)-straight or branched
alkyl, (C.sub.2-C.sub.6) straight or branched alkenyl or alkynyl,
1,2-methylenedioxy, 1,2-ethylenedioxy, or (CH.sub.2).sub.n-Q;
[0072] Q is selected from halo, CN, NO.sub.2, CF.sub.3, OCF.sub.3,
OH, S-aliphatic, S(O)-aliphatic, SO.sub.2-aliphatic, NH.sub.2,
NH(aliphatic), N(aliphatic).sub.2, N(aliphatic)R.sup.8, NHR.sup.8,
N(R.sup.8).sub.2, COOH, C(O)O-(aliphatic), or O-aliphatic; and
R.sup.8 is an amino protecting group.
[0073] The term "amino protecting group" refers to a suitable
chemical group that may be attached to a nitrogen atom. The term
"protecting" refers to when the designated amino group is attached
to a suitable chemical group (e.g., capping group). Examples of
suitable amino capping groups are described in T. W. Greene et al.,
Protective Groups in Organic Synthesis, 3d. Ed., John Wiley and
Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents
for Organic Synthesis, John Wiley and Sons (1994); L. Paquette, ed.
Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons
(1995) and are exemplified in certain of the specific compounds
used in this invention.
[0074] In certain other embodiments in the compounds of formula I:
[0075] a) when A is C(O), L is a bond, Z is phenyl, R.sup.C and
R.sup.D taken together is cyclopentyl, B is phenyl, then R.sup.B
and R.sup.Z are not methoxy; [0076] b) when A is C(O), L is a bond,
Z is phenyl, R.sup.C and R.sup.D taken together is cyclopropyl, B
is phenyl, then R.sup.B is not hydrogen; [0077] c) when A is C(O),
L is a bond, Z is benzofuranyl, R.sup.C and R.sup.D taken together
is cyclopentyl, B is phenyl, then R.sup.B is not methoxy; [0078] d)
when A is C(O), L is a bond, Z is phenyl, R.sup.C and R.sup.D taken
together is cyclopentyl, B is phenyl, R.sup.B is hydrogen, then
R.sup.Z is not chloro; [0079] e) when A is C(O), L is a bond, Z is
furanyl, R.sup.C and R.sup.D taken together is cyclopentyl, B is
phenyl, R.sup.B is methoxy, then R.sup.z is not bromo; [0080] f)
when A is C(O), L is a bond, Z is furanyl, R.sup.C and R.sup.D
taken together is cyclopentyl, B is phenyl, then R.sup.B is not
hydrogen; and [0081] g) when A is C(O), L is a bond, Z is phenyl,
R.sup.C and R.sup.D taken together is cyclohexyl, B is phenyl,
R.sup.B is methoxy, n is 2, R.sup.Z is nitro, then m is not 2.
[0082] In certain embodiments of compounds of the present
invention, when R.sup.C and R.sup.D each is methyl, R.sup.E is
hydrogen, and A is carbonyl, then the following compounds are
excluded:
TABLE-US-00001 Ring B with R.sup.B & n Z ##STR00007##
Pyrazol-3-yl, 2,3-dimethyl- furan-5-yl, or pyrazin-2(1H)- one-5-yl
Phenyl, 4-chlorophenyl, or 3,4-chlorophenyl ##STR00008##
4-chlorophenyl ##STR00009##
[0083] In an alternative embodiment, the present invention provides
a compound of formula II:
##STR00010##
or a pharmaceutically acceptable salt thereof, wherein: [0084] A is
C(O) or SO.sub.2; [0085] R.sup.C and R.sup.D taken together form a
3-6 membered cycloalkyl ring or 4-pyranyl ring; [0086] R.sup.E is
H, (C1-C4)alkyl optionally substituted with a substituent selected
from (C1-C4)alyl selected CN, NO.sub.2, CF.sub.3, OCF.sub.3, OH,
SR.sup.6, S(O)R.sup.6, SO.sub.2R.sup.6, COOH, COOR.sup.6, OR.sup.6
or phenyl optionally substituted with R.sup.Z; [0087] B is phenyl;
[0088] Z is
[0088] ##STR00011## [0089] wherein, [0090] L is a bond; [0091] W is
a 5-14 membered monocyclic, bicyclic, or tricyclic heterocyclic or
heteroaryl ring; [0092] m and n are independently 0 to 5; or [0093]
Z is diphenylmethyl wherein each phenyl has up to 5 R.sup.Z is
substituents; and [0094] R.sup.B and R.sup.Z are independently
selected from R.sup.1, R.sup.2, R.sup.3, [0095] R.sup.4, or
R.sup.5, wherein: [0096] R.sup.1 is oxo, R.sup.6 or
((C1-C4)aliphatic).sub.n-Y; [0097] n is 0 or 1; [0098] Y is halo,
CN, NO.sub.2, CF.sub.3, OCF.sub.3, OH, SR.sup.6, S(O)R.sup.6,
SO.sub.2R.sup.6, NH.sub.2, NHR.sup.6, N(R.sup.6).sub.2,
NR.sup.6R.sup.8, COOH, COOR.sup.6 or OR.sup.6; or two R.sup.1 on
adjacent ring atoms, taken together, form 1,2-methylenedioxy or
1,2-ethylenedioxy; [0099] R.sup.2 is aliphatic, wherein each
R.sup.2 optionally comprises up to 2 substituents independently
selected from R.sup.1, R.sup.4, or R.sup.5; [0100] R.sup.3 is a
cycloaliphatic, aryl, heterocyclic, or heteroaryl ring optionally
comprising up to 3 substituents, independently selected from
R.sup.1, R.sup.2, R.sup.4 or R.sup.5; [0101] R.sup.4 is OR.sup.5,
OR.sup.6, OC(O)R.sup.6, OC(O)R.sup.5, OC(O)OR.sup.6, OC(O)OR.sup.5,
OC(O)N(R.sup.6).sub.2, OC(O)N(R.sup.5).sub.2,
OC(O)N(R.sup.6R.sup.5), SR.sup.6, SR.sup.5, S(O)R.sup.6,
S(O)R.sup.5, SO.sub.2R.sup.6, SO.sub.2R.sup.5,
SO.sub.2N(R.sup.6).sub.2, SO.sub.2N(R.sup.5).sub.2,
SO.sub.2NR.sup.5R.sup.6, SO.sub.3R.sup.6, SO.sub.3R.sup.5,
C(O)R.sup.5, C(O)OR.sup.5, C(O)R.sup.6, C(O)OR.sup.6,
C(O)N(R.sup.6).sub.2, C(O)N(R.sup.5).sub.2, C(O)N(R.sup.5R.sup.6),
C(O)N(OR.sup.6)R.sup.6, C(O)N(OR.sup.5)R.sup.6,
C(O)N(OR.sup.6)R.sup.5, C(O)N(OR.sup.5)R.sup.5,
C(NOR.sup.6)R.sup.6, C(NOR.sup.6)R.sup.5, C(NOR.sup.5)R.sup.6,
C(NOR.sup.5)R.sup.5, N(R.sup.6).sub.2, N(R.sup.5).sub.2,
N(R.sup.5R.sup.6), NR.sup.5C(O)R.sup.5, NR.sup.6C(O)R.sup.6,
NR.sup.5C(O)R.sup.6, NR.sup.6C(O)R.sup.5, NR.sup.6C(O)OR.sup.6,
NR.sup.5C(O)OR.sup.6, NR.sup.6C(O)OR.sup.5, NR.sup.5C(O)OR.sup.5,
NR.sup.6C(O)N(R.sup.6).sub.2, NR.sup.6C(O)NR.sup.5R.sup.6,
NR.sup.6C(O)N(R.sup.5).sub.2, NR.sup.5C(O)N(R.sup.6).sub.2,
NR.sup.5C(O)NR.sup.5R.sup.6, NR.sup.5C(O)N(R.sup.5).sub.2,
NR.sup.6SO.sub.2R.sup.6, NR.sup.6SO.sub.2R.sup.5,
NR.sup.5SO.sub.2R.sup.5, NR.sup.5SO.sub.2R.sup.6,
NR.sup.6SO.sub.2N(R.sup.6).sub.2, NR.sup.5SO.sub.2N(R.sup.6).sub.2,
NR.sup.6SO.sub.2NR.sup.5R.sup.6, NR.sup.6SO.sub.2N(R.sup.5).sub.2,
NR.sup.5SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2N(R.sup.5).sub.2,
N(OR.sup.6)O, N(OR.sup.6)R.sup.5, N(OR.sup.5)R.sup.5, or
N(OR.sup.5)R.sup.6; [0102] R.sup.5 is a cycloaliphatic, aryl,
heterocyclic, or heteroaryl ring, optionally comprising up to 3
R.sup.1 substituents; [0103] R.sup.6 is H or aliphatic, wherein
R.sup.6 optionally comprises a R.sup.7 substituent; [0104] R.sup.7
is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, and
each R.sup.7 optionally comprises up to 2 substituents
independently chosen from H, (C.sub.1-C.sub.6)-straight or branched
alkyl, (C.sub.2-C.sub.6) straight or branched alkenyl or alkynyl,
1,2-methylenedioxy, 1,2-ethylenedioxy, or (CH.sub.2).sub.n-Q;
[0105] Q is selected from halo, CN, NO.sub.2, CF.sub.3, OCF.sub.3,
OH, S-aliphatic, S(O)-aliphatic, SO.sub.2-aliphatic, NH.sub.2,
NH(aliphatic), N(aliphatic).sub.2, N(aliphatic)R.sup.8, NHR.sup.8,
N(R.sup.8).sub.2, COOH, C(O)O-(aliphatic), or O-aliphatic; and
[0106] R.sup.8 is an amino protecting group, provided that:
[0107] (i) when R.sup.C and R.sup.D taken together form a 4-pyran
ring, R.sup.E is hydrogen, A is C(O), and ring W together with
R.sup.Z and m is 2-amino-pyrazin-3-yl, then ring B together with
(R.sup.B).sub.n is not phenyl, 4-methylphenyl, 4-chlorophenyl,
3-fluorophenyl, 4-methoxyphenyl, 2,4-difluorophenyl, or
4-fluorophenyl;
[0108] (ii) when R.sup.C and R.sup.D taken together form a
cyclohexyl ring, R.sup.E is hydrogen, A is C(O), and L is
2-methoxy-pyridin-3-yl, then ring B together with (R.sup.B).sub.n
is not phenyl;
[0109] (iii) when R.sup.C and R.sup.D taken together form a
cyclobutyl ring, R.sup.E is hydrogen, A is C(O), and ring W
together with R.sup.Z and m is
2,5,7,8-tetramethyl-6-hydroxy-2H-1-benzopyran-2-yl, then ring B
together with (R.sup.B).sub.n is not
4-[(imino-thien-2-ylmethyl)amino]phenyl;
[0110] (iv) when R.sup.C and R.sup.D taken together form a
cyclopropyl ring, R.sup.E is hydrogen, A is C(O), and ring W
together with R.sup.Z and m is
2,5-dihydro-4-hydroxy-1-methyl-5-oxo-1H-pyrrol-3-yl, then ring B
together with (R.sup.B).sub.n is not phenyl;
[0111] (v) when R.sup.C and R.sup.D taken together form a
cyclopropyl ring, R.sup.E is hydrogen, A is C(O), and ring W
together with R.sup.Z and m is
2,3,4,9-tetrahydro-3-[(3'-(2,6-diisopropyl)-ureido]-1H-carbazol--
3-yl, then ring B together with (R.sup.8).sub.n is not
4-chlorophenyl;
[0112] (vi) when R.sup.C and R.sup.D taken together form a
cyclopropyl ring, R.sup.E is hydrogen, A is C(O), and ring W
together with R.sup.Z and m is 9,10-dihydro-9-oxo-acridin-3-yl,
then ring B together with (R.sup.B).sub.n is not
4-chlorophenyl;
[0113] (vii) when R.sup.E is hydrogen and A is C(O), then the
following compounds are excluded:
TABLE-US-00002 R.sup.C & R.sup.D together ring W together with
R.sup.Z and m ring B with R.sup.B & n 4-pyran ##STR00012##
phenyl 4-pyran diphenylmethyl phenyl cyclobutyl ##STR00013## phenyl
cyclopentyl benzofuran-2-yl 3,4-dimethoxyphenyl cyclopropyl
##STR00014## 4-chlorophenyl cyclopropyl ##STR00015## phenyl 4-pyran
or cyclohexyl diphenylmethyl 3,4-dimethoxyphenyl 4-pyran 2-furanyl
4-methoxyphenyl 4-pyran 5-bromo-2-furanyl phenyl cyclopentyl
##STR00016## phenyl 4-pyran 1,4-benzodioxin-2-yl phenyl 4-pyran
4,5-dimethyl-furan-2-yl phenyl cyclohexyl benzofuran-2-yl
3,4-dimethoxyphenyl cyclopentyl diphenylmethyl 3,4-dimethoxyphenyl
cyclopentyl ##STR00017## phenyl cyclopentyl 5-bromo-furan-2-yl
3,4-dimethoxyphenyl cyclopentyl ##STR00018## phenyl 4-pyran
2-furanyl, 5-ethyl-furan-2-yl, 2- phenyl thienyl, cyclopentyl
furanyl phenyl cyclopentyl ##STR00019## phenyl ##STR00020##
4-pyranyl 2-benzofuranyl phenyl 4-pyranyl 5-bromofuran-2-yl
4-methoxyphenyl cyclopentyl 5-bromofuran-2-yl phenyl 4-pyranyl
2-thienyl 4-methoxyphenyl 4-pyranyl diphenylmethyl 4-methoxyphenyl
cyclopentyl 2-benzofuranyl phenyl 4-pyranyl 2-benzofuranyl
3,4-dimethoxyphenyl cyclopentyl 1-phenyl-1-(4-isobutoxy- phenyl
phenyl)-methyl cyclopentyl 1,4-benzodioxin-2-yl
3,4-dimethoxyphenyl
[0114] (viii) when R.sup.C and R.sup.D taken together form a
cyclopentyl ring, R.sup.E is hydrogen, A is C(O), and ring W
together with R.sup.Z and m is diphenylmethyl, then ring B together
with (R.sup.B).sub.n is not phenyl, 4-ethoxyphenyl, 4-butoxyphenyl,
4-isobutoxyphenyl, or 4-methoxyphenyl.
[0115] In one embodiment of the present invention, A is C(O). Or, A
is SO.sub.2.
[0116] In one embodiment, R.sup.E is hydrogen. Or, R.sup.E is C1-C4
alkyl.
[0117] In another embodiment, R.sup.C and R.sup.D, taken together,
form a 4-pyranyl ring.
[0118] In another embodiment, R.sup.C and R.sup.D, taken together,
form a 3-6 membered cycloalkyl ring. In one embodiment, R.sup.C and
R.sup.D, taken together, form a 5-6 membered cycloalkyl ring.
[0119] In another embodiment, R.sup.C and R.sup.D, taken together,
form a 5-membered cycloalkyl ring. Or, R.sup.C and R.sup.D, taken
together, form a 6-membered cycloalkyl ring.
[0120] In another embodiment, W is an optionally substituted
indolyl, benzofuranyl, or benzothienyl. Or, W is indol-2-yl or
indol-3-yl. Or W is benzofuran-2-yl. Or, W is benzothien-2-yl.
[0121] In another embodiment, W is an optionally substituted
pyrazolyl or indazolyl.
[0122] In another embodiment, W is an optionally substituted
pyrazol-3-yl or pyrazol-4-yl. Or, W is an optionally substituted
indazol-3-yl.
[0123] In another embodiment, W is an optionally substituted
phenyl.
[0124] In another embodiment, W is an optionally substituted
six-membered heteroaromatic ring having up to three heteroatoms
selected from O, S, or N. In certain embodiments, W is pyridyl.
[0125] In another embodiment, Z is diphenylmethyl.
[0126] In certain embodiments, W is an optionally substituted ring
selected from furanyl, thienyl, isoxazolyl, or pyrrolyl.
[0127] In another embodiment, W is an optionally substituted 10-12
membered bicyclic, heteroaromatic ring. In certain embodiments, W
is an optionally substituted ring selected from quinolinyl or
cinnolinyl.
[0128] In one embodiment of the present invention, R.sup.C and
R.sup.D each is methyl.
[0129] According to a preferred embodiment, R.sup.8 is acetyl,
arylsulfonyl or alkylsulfonyl.
[0130] Another embodiment of the present invention provides a
method of treating an ABC transporter mediated disease in a mammal,
comprising the step of administering to said mammal a composition
comprising a compound of the present invention or a
pharmaceutically acceptable salt thereof.
[0131] A preferred aspect of the present embodiment is where the
ABC transporter mediated disease is selected from cystic fibrosis,
hereditary emphysema, hereditary hemochromatosis,
coagulation-cibrinolysis deficiencies, such as protein C
deficiency, Type 1 hereditary angioedema, lipid processing
deficiencies, such as familial hypercholesterolemia, Type 1
chylomicronemia, abetalipoproteinemia, lysosomal storage diseases,
such as I-cell disease/Pseudo-Hurler, secretory diarrhea or
polycystic kidney disease, mucopolysaccharidoses,
Sandhof/Tay-Sachs, Crigler-Najjar type II,
polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron
dwarfism, myleoperoxidase deficiency, primary hypoparathyroidism,
melanoma, glycanosis CDG type 1, hereditary emphysema, congenital
hyperthyroidism, osteogenesis imperfecta, hereditary
hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),
neurophyseal DI, Neprogenic DI, Charcot-Marie Tooth syndrome,
Perlizaeus-Merzbacher disease, neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, Amyotrophic lateral
sclerosis, progressive supranuclear plasy, Pick's disease, several
polyglutamine neurological disorders such as Huntington,
Spinocerebullar ataxia type I, spinal and bulbar muscular atrophy,
Dentatorubal pallidoluysian, and Myotonic dystrophy, as well as
Spongiform encephalopathies, such as Hereditary Creutzfeldt-Jakob
disease (due to Prion protein processing defect), Fabry disease,
Straussler-Scheinker syndrome, COPD, dry eye disease, or Sjogren's
disease.
[0132] An especially preferred method is where the disease is
CF.
[0133] Another embodiment of the present invention provides a
pharmaceutical composition comprising:
[0134] a. a compound of the present invention;
[0135] b. a pharmaceutically acceptable carrier; and
[0136] c. an additional agent selected from a mucolytic agent,
bronchodialator, an antibiotic, an anti-infective agent, an
anti-inflammatory agent, CFTR modulator other than a compound of
the present invention, or a nutritional agent.
[0137] Another embodiment of the present invention provides a
method of modulating ABC transporter activity, comprising the step
of contacting said ABC transporter with a compound of the present
invention.
[0138] A preferred aspect of this embodiment is where the ABC
transporter or a fragment thereof is in vivo. Another preferred
aspect of this embodiment is where the ABC transporter or a
fragment, thereof is in vitro. Another preferred aspect of this
embodiment is where the ABC transporter is CFTR.
[0139] According to an alternative embodiment, the present
invention provides a method of increasing the number of functional
ABC transporters in a membrane of a cell, comprising the step of
contacting said cell with a compound of formula (I). The term
"functional ABC transporter" as used herein means an ABC
transporter that is capable of transport activity.
[0140] According to a preferred embodiment, said functional ABC
transporter is CFTR.
[0141] Another embodiment of the present invention provides a kit
for use in measuring the activity of a ABC transporter or a
fragment thereof in a biological sample in vitro or in vivo,
comprising:
[0142] a. a composition comprising a compound of the present
invention; and
[0143] b. instructions for: [0144] i) contacting said composition
with the biological sample; and [0145] ii) measuring activity of
said ABC transporter or fragment thereof.
[0146] A preferred aspect of this embodiment is where the ABC
transporter is CFTR.
3. Description of Exemplary Compounds
[0147] As described generally above, for compounds of the
invention, R.sup.C and R.sup.D are independently selected from H,
C.sub.1-4alkyl, and aryl, or may be taken together to form a
(C3-C8)cycloalkyl; B is aryl; and Z is (C1-C6)alkyl, aryl,
(C1-C4alkyl)aryl, C5-C7cycloalkyl, or ((C1-4)alkyl)
C.sub.5-7cycloalkyl.
[0148] A preferred embodiment of the present invention is where A
is C(O), and L is a bond.
[0149] A preferred embodiment of the present invention is where
R.sup.C and R.sup.D taken together form (C3-C8)cycloalkyl.
[0150] A particularly preferred embodiment of the present invention
is where R.sup.C and R.sup.D taken together form cyclopentyl.
[0151] Another particularly preferred embodiment of the present
invention is where R.sup.C and R.sup.D taken together form
cyclohexyl.
[0152] A preferred embodiment of the present invention is where
R.sup.C and R.sup.D taken together form a heterocyclic.
[0153] A particularly preferred embodiment of the present invention
is where R.sup.C and R.sup.D taken together form pyranyl. In
certain embodiments, R.sup.C and R.sup.D taken together form
4-pyranyl.
[0154] Yet another particularly preferred embodiment of the present
invention is where R.sup.C and R.sup.D are H.
[0155] Yet another particularly preferred embodiment of the present
invention is where R.sup.C and R.sup.D are methyl.
[0156] A preferred embodiment of the present invention is where B
is aryl.
[0157] A particularly preferred embodiment of the present invention
is where B is phenyl.
[0158] A preferred embodiment of the present invention is where Z
is aryl.
[0159] Another preferred embodiment of the present invention is
where Z is pyridinyl.
[0160] Another preferred embodiment of the present invention is
where Z is phenyl.
[0161] Another preferred embodiment of the present invention is
where Z is benzofuran.
[0162] Another preferred embodiment of the present invention is
where Z is benzothiophenyl.
[0163] Another preferred embodiment of the present invention is
where Z is indolyl.
[0164] Another preferred embodiment of the present invention is
where Z is pyrazolyl.
[0165] Another preferred embodiment of the present invention is
where Z is furanyl.
[0166] Another preferred embodiment of the present invention is
where Z is quinolinyl.
[0167] Another preferred embodiment of the present invention is
where Z is isoquinolinyl.
[0168] Another preferred embodiment of the present invention is
where Z is cinnolinyl.
[0169] An especially preferred embodiment of the present invention
is where Z is benzofuranyl, and R.sup.C and R.sup.D taken together
form cyclohexyl.
[0170] Another especially preferred embodiment is where B is a
substituted phenyl.
[0171] An especially preferred embodiment of the present invention
is a compound where Z is benzofuranyl, R.sup.C and R.sup.D taken
together form cyclohexyl, and B is a substituted phenyl as
represented by formula II:
##STR00021##
or a pharmaceutically acceptable salt thereof; wherein: [0172]
R.sup.Z is independently selected from (C1-C4)alkyl, (C1-C4)alkoxy,
and halo, particularly methyl, methoxy, F, or Cl; [0173] n is 0 to
4; [0174] R.sup.B is independently selected from halo, and
(C1-C4)alkoxy; and [0175] m is 0 to 5.
[0176] In a preferred embodiment of the compound of formula III, m
is 3, and R.sup.B is fluoro or a methoxy moiety. Exemplary
compounds of the present invention are shown below in Table 1:
TABLE-US-00003 TABLE 1 Cmpd # Compound 1 ##STR00022## 2
##STR00023## 3 ##STR00024## 4 ##STR00025## 5 ##STR00026## 6
##STR00027## 7 ##STR00028## 8 ##STR00029## 9 ##STR00030## 10
##STR00031## 11 ##STR00032## 12 ##STR00033## 13 ##STR00034## 14
##STR00035## 15 ##STR00036## 16 ##STR00037## 17 ##STR00038## 18
##STR00039## 19 ##STR00040## 20 ##STR00041## 21 ##STR00042## 22
##STR00043## 23 ##STR00044## 24 ##STR00045## 25 ##STR00046## 26
##STR00047## 27 ##STR00048## 28 ##STR00049## 29 ##STR00050## 30
##STR00051## 31 ##STR00052## 32 ##STR00053## 33 ##STR00054## 34
##STR00055## 35 ##STR00056## 36 ##STR00057## 37 ##STR00058## 38
##STR00059## 39 ##STR00060## 40 ##STR00061## 41 ##STR00062## 42
##STR00063## 43 ##STR00064## 44 ##STR00065## 45 ##STR00066## 46
##STR00067## 47 ##STR00068## 48 ##STR00069## 49 ##STR00070## 50
##STR00071## 51 ##STR00072## 52 ##STR00073## 53 ##STR00074## 54
##STR00075## 55 ##STR00076## 56 ##STR00077## 57 ##STR00078## 58
##STR00079## 59 ##STR00080## 60 ##STR00081## 61 ##STR00082## 62
##STR00083## 63 ##STR00084## 64 ##STR00085## 65 ##STR00086## 66
##STR00087## 67 ##STR00088## 68 ##STR00089## 69 ##STR00090## 70
##STR00091## 71 ##STR00092## 72 ##STR00093## 73 ##STR00094## 74
##STR00095## 75 ##STR00096## 76 ##STR00097## 77 ##STR00098## 78
##STR00099## 79 ##STR00100## 80 ##STR00101## 81 ##STR00102## 82
##STR00103## 83 ##STR00104## 84 ##STR00105## 85 ##STR00106## 86
##STR00107## 87 ##STR00108## 88 ##STR00109## 89 ##STR00110## 90
##STR00111## 91 ##STR00112## 92 ##STR00113## 93 ##STR00114## 94
##STR00115## 95 ##STR00116## 96 ##STR00117## 97 ##STR00118## 98
##STR00119## 99 ##STR00120## 100 ##STR00121## 101 ##STR00122## 102
##STR00123## 103 ##STR00124## 104 ##STR00125## 105 ##STR00126## 106
##STR00127## 107 ##STR00128## 108 ##STR00129##
[0177] The compounds of the present invention can be prepared by
methods well known in the art. An exemplary method of producing
compounds of the present invention is shown below in Schemes
1-3.
[0178] Scheme 1 below illustrates an exemplary method for producing
amine intermediates for compounds of the present invention wherein
R.sup.C and R.sup.D cyclize to form a ring:
##STR00130##
[0179] An optionally substituted (R*) 2-phenylacetonitrile is
reacted with an appropriate dihalo-alkyl compound, sodium hydride
or the like, in THF or a similar solvent. The resulting spiro
compound is reacted with lithium aluminum hydride or similar
reducing reagent to provide the desired cyclic amine.
[0180] Scheme 2 below illustrates an exemplary method for producing
amine intermediates for the present invention wherein R.sup.C and
R.sup.D do not cyclize to form a ring.
##STR00131##
[0181] Scheme 3 below illustrates an exemplary method for producing
certain compounds of the present invention using the amine
intermediates of, e.g., Scheme 1 and Scheme 2 above.
##STR00132##
[0182] According to another preferred embodiment, the ABC
transporter mediated disease is selected from Cystic fibrosis,
COPD, Asthma, chronic pancreatitis, pneumonia, polycystic kidney
disease, Hereditary emphysema, Hereditary hemochromatosis,
Coagulation-Fibrinolysis deficiencies, such as Protein C
deficiency, Type 1 hereditary angioedema, Lipid processing
deficiencies, such as Familial hypercholesterolemia, Type 1
chylomicronemia, Abetalipoproteinemia, Lysosomal storage diseases,
such as I-cell disease/Pseudo-Hurler, Mucopolysaccharidoses,
Sandhof/Tay-Sachs, Crigler-Najjar type II,
Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus, Laron
dwarfism, Myleoperoxidase deficiency, Primary hypoparathyroidism,
Melanoma. The diseases associated with the latter class of ER
malfunction are Glycanosis CDG type 1, Hereditary emphysema,
Congenital hyperthyroidism, Osteogenesis imperfecta, Hereditary
hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),
Neurophyseal DI, Neprogenic DI, Charcot-Marie Tooth syndrome,
Perlizaeus-Merzbacheri disease, neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, Amyotrophic lateral
sclerosis, Progressive supranuclear plasy, Pick's disease, several
polyglutamine neurological disorders such as Huntington,
Spinocerebullar ataxia type I, Spinal and bulbar muscular atrophy,
Dentatorubal pallidoluysian, and Myotonic dystrophy, as well as
Spongiform encephalopathies, such as Hereditary Creutzfeldt-Jakob
disease (due to Prion protein processing defect), Fabry disease and
Straussler-Scheinker syndrome.
[0183] Most preferably, the ABC transporter mediated disease is
cystic fibrosis.
[0184] Another embodiment of the present invention provides a
method of treating a disease selected from Cystic fibrosis, COPD,
asthma, chronic pancreatitis, pneumonia, polycytic kidney disease,
Hereditary emphysema, Hereditary hemochromatosis,
Coagulation-Fibrinolysis deficiencies, such as Protein C
deficiency, Type 1 hereditary angioedema, Lipid processing
deficiencies, such as Familial hypercholesterolemia, Type 1
chylomicronemia, Abetalipoproteinemia, Lysosomal storage diseases,
such as I-cell disease/Pseudo-Hurler, Mucopolysaccharidoses,
Sandhof/Tay-Sachs, Crigler-Najjar type II,
Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus, Laron
dwarfism, Myleoperoxidase deficiency, Primary hypoparathyroidism,
Melanoma, Glycanosis CDG type 1, Hereditary emphysema, Congenital
hyperthyroidism, Osteogenesis imperfecta, Hereditary
hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),
Neurophyseal DI, Neprogenic DI, Charcot-Marie Tooth syndrome,
Perlizaeus-Merzbacher disease, neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, Amyotrophic lateral
sclerosis, Progressive supranuclear plasy, Pick's disease, several
polyglutamine neurological disorders such as Huntington,
Spinocerebullar ataxia type I, Spinal and bulbar muscular atrophy,
Dentatorubal pallidoluysian, and Myotonic dystrophy, as well as
Spongiform encephalopathies, such as Hereditary Creutzfeldt-Jakob
disease, Fabry disease, or Straussler-Scheinker syndrome comprising
the step of administering to a mammal an effective amount of a
composition comprising a compound according to the present
invention.
[0185] According to a more preferred embodiment, the disease so
treated is selected, from Tangier's disease, stargardt disease 1,
age related macular dystrophy 2, retinintis pigmentosa, bare
lymphocyte syndrome, PFIC-3, anemia, progressive intrahepatic
cholestasis-2, Dublin-Johnson syndrome, Pseudoxanthoma elasticum,
cystic fibrosis, familial persistent hyperinsulinemic hyproglycemia
of infancy, adrenolecukodystrophy, sitosterolemia, chronic
obstructive pulmonary disease, asthma, disseminated bronchiectasis,
chronic pancreatitis, male infertility, emphysema, or
pneumonia.
[0186] According to another more preferred embodiment, the ABC
transporter mediated disease is secretory diarrhea, COPD, or
polycystic kidney disease in a mammal.
[0187] According to an alternative preferred embodiment, the
present invention provides a method of treating cystic fibrosis or
secretory diahrrea comprising the step of administering to said
mammal a composition comprising the step of administering to said
mammal a composition comprising a compound of the present
invention, or a preferred embodiment thereof as set forth above.
Most preferably, said disease is cystic fibrosis.
[0188] According to an alternative preferred embodiment, the
present invention provides a method of modulating CFTR activity in
a cell membrane ("potentiating") of a mammal in need thereof,
comprising the step of administering to said mammal a composition
comprising a compound of the present invention as defined
above.
[0189] The preferred embodiments of the compounds of the present
invention useful in potentiating the activity of CFTR include the
preferred embodiments of the present invention described above.
[0190] According to an alternative embodiment, the present
invention provides a method of increasing the number of functional
ABC transporters in a membrane of a cell, comprising the step of
contacting said cell with a compound of the present invention. The
term "functional ABC transporter" as used herein means an ABC
transporter that is capable of transport activity.
[0191] According to a preferred embodiment, said functional ABC
transporter is CFTR.
[0192] The preferred embodiments of compounds of the present
invention useful in increasing the number of functional ABC
transporters include preferred embodiments of compound of the
present invention as described above.
[0193] According to another embodiment, the present invention
provides a method of modulating activity of an anion channel in
vitro or in vivo, comprising the step of contacting said channel
with a compound of the present invention. Preferably, said anion
channel is a chloride channel or a bicarbonate channel. More
preferably, said anion channel is a chloride channel.
[0194] According to yet another embodiment, the present invention
provides a method of treating an anion channel mediated disease in
a mammal, comprising the step of administering to said mammal a
composition comprising a compound according to the present
invention.
[0195] According to another embodiment, the present invention
provides a pharmaceutical composition comprising:
[0196] (i) a compound of the present invention as described
above;
[0197] (ii) a pharmaceutically acceptable carrier; and
[0198] (iii) an additional agent selected from a mucolytic agent,
bronchodialator, an anti-biotic, an anti-infective agent, an
anti-inflammatory agent, CFTR modulator other than a compound of
the present invention, or a nutritional agent.
[0199] Preferred embodiments of compounds the present invention in
the above pharmaceutical composition are those as described
above.
[0200] According to another embodiment, the present invention
provides a kit for use in measuring the activity of a ABC
transporter or a fragment thereof in a biological sample in vitro
or in vivo, comprising:
[0201] (i) a composition comprising a compound of the present
invention; and
[0202] (ii) instructions for:
[0203] a) contacting the composition with the biological
sample;
[0204] b) measuring activity of said ABC transporter or a fragment
thereof.
[0205] According to a preferred embodiment, the kit is useful in
measuring the activity of CFTR.
4. General Synthetic Methodology
[0206] The compounds of this invention may be prepared in general
by methods known to those skilled in the art for analogous
compounds, as illustrated by the general schemes below, and the
preparative examples that follow. Starting materials are
commercially available from typical chemical reagent supply
companies, such as, Aldrich Chemicals Co., Sigma Chemical Company,
ChemBridge Corporation, and the like. Compounds that are not
commercially available can be prepared by those of ordinary skill
in art following procedures set forth in references such as,
"Fieser and Fieser's Reagents for Organic Synthesis", Volumes 1-15,
John Wiley and Sons, 1991; "Rodd's Chemistry of Carbon Compounds",
Volumes 1-5 and Supplementals, Elservier Science Publishers, 1989;
and "Organic Reactions", Volumes 1-40, John Wiley and Sons,
1991.
[0207] Generally, the compounds of the present invention are
prepared by the formation of an amide functionally between an
optionally substituted carboxylic acid or acid chloride, and an
optionally substituted primary amine.
[0208] Unless otherwise stated, structures depicted herein are also
meant to include all stereochemical forms of the structure; i.e.,
the R and S configurations for each asymmetric center. Therefore,
single stereochemical isomers as well as enantiomeric and
diastereomeric mixtures of the present compounds are within the
scope of the invention. Unless otherwise stated, structures
depicted herein are also meant to include compounds that differ
only in the presence of one or more isotopically enriched atoms.
For example, compounds having the present structures except for the
replacement of a hydrogen by a deuterium or tritium, or the
replacement of a carbon by a .sup.13C- or .sup.14C-enriched carbon
are within the scope of this invention. Such compounds are useful,
for example, as analytical tools or probes in biological
assays.
5. Uses, Formulation and Administration
Pharmaceutically Acceptable Compositions
[0209] As discussed above, the present invention provides compounds
that are useful as modulators of ABC transporters and thus are
useful in treating a disease selected from Cystic fibrosis, COPD,
chronic pancreatitis, pneumonia, polycystic kidney disease,
Hereditary emphysema, Hereditary hemochromatosis,
Coagulation-Fibrinolysis deficiencies, such as Protein C
deficiency, Type 1 hereditary angioedema, Lipid processing
deficiencies, such as Familial hypercholesterolemia, Type 1
chylomicronemia, Abetalipoproteinemia, Lysosomal storage diseases,
such as I-cell disease/Pseudo-Hurler, Mucopolysaccharidoses,
Sandhof/Tay-Sachs, Crigler-Najjar type II,
Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus, Laron
dwarfism, Myleoperoxidase deficiency, Primary hypoparathyroidism,
Melanoma, Glycanosis CDG type 1, Hereditary emphysema, Congenital
hyperthyroidism, Osteogenesis imperfecta, Hereditary
hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),
Neurophyseal DI, Neprogenic DI, Charcot-Marie Tooth syndrome,
Perlizaeus-Merzbacher disease, neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, Amyotrophic lateral
sclerosis, Progressive supranuclear plasy, Pick's disease, several
polyglutamine neurological disorders such as Huntington,
Spinocerebullar ataxia type I, Spinal and bulbar muscular atrophy,
Dentatorubal pallidoluysian, and Myotonic dystrophy, as well as
Spongiform encephalopathies, such as Hereditary Creutzfeldt-Jakob
disease, Fabry disease, or Straussler-Scheinker syndrome
comprising.
[0210] Accordingly, in another aspect of the present invention,
pharmaceutically acceptable compositions are provided, wherein
these compositions comprise any of the compounds as described
herein, and optionally comprise a pharmaceutically acceptable
carrier, adjuvant or vehicle. In, certain embodiments, these
compositions optionally further comprise one or more additional
therapeutic agents.
[0211] It will also be appreciated that certain of the compounds of
present invention can exist in free form for treatment, or where
appropriate, as a pharmaceutically acceptable derivative thereof.
According to the present invention, a pharmaceutically acceptable
derivative includes, but is not limited to, pharmaceutically
acceptable salts, esters, salts of such esters, or any other adduct
or derivative which upon administration to a patient in need is
capable of providing, directly or indirectly, a compound as
otherwise described herein, or a metabolite or residue thereof.
[0212] As used herein, the term "pharmaceutically acceptable salt"
refers to those salts which are, within the scope of sound medical
judgement, suitable for use in contact with the tissues of humans
and lower animals without undue toxicity, irritation, allergic
response and the like, and are commensurate with a reasonable
benefit/risk ratio. A "pharmaceutically acceptable salt" means any
non-toxic salt or salt of an ester of a compound of this invention
that, upon administration to a recipient, is capable of providing,
either directly or indirectly, a compound of this invention or an
inhibitorily active metabolite or residue thereof.
[0213] Pharmaceutically acceptable salts are well known in the art.
For example, S. M. Berge, et al. describe pharmaceutically
acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66,
1-19, incorporated herein by reference. Pharmaceutically acceptable
salts of the compounds of this invention include those derived from
suitable inorganic and organic acids and bases. Examples of
pharmaceutically acceptable, nontoxic acid addition salts are salts
of an amino group formed with inorganic acids such as hydrochloric
acid, hydrobromic acid, phosphoric acid, sulfuric acid and
perchloric acid or with organic acids such as acetic acid, oxalic
acid, maleic acid, tartaric acid, citric acid, succinic acid or
malonic acid or by using other methods used in the art such as ion
exchange. Other pharmaceutically acceptable salts include adipate,
alginate, ascorbate, aspartate, benzenesulfonate, benzoate,
bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Salts derived from appropriate bases include alkali metal, alkaline
earth metal, ammonium and N+(C1-4alkyl).sub.4 salts. This invention
also envisions the quaternization of any basic nitrogen-containing
groups of the compounds disclosed herein. Water or oil-soluble or
dispersable products may be obtained by such quaternization.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.
[0214] As described above, the pharmaceutically acceptable
compositions of the present invention additionally comprise a
pharmaceutically acceptable carrier, adjuvant, or vehicle, which,
as used herein, includes any and all solvents, diluents, or other
liquid vehicle, dispersion or suspension aids, surface active
agents, isotonic agents, thickening or emulsifying agents,
preservatives, solid binders, lubricants and the like, as suited to
the particular dosage form desired. Remington's Pharmaceutical
Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co.,
Easton, Pa., 1980) discloses various carriers used in formulating
pharmaceutically acceptable compositions and known techniques for
the preparation thereof. Except insofar as any conventional carrier
medium is incompatible with the compounds of the invention, such as
by producing any undesirable biological effect or otherwise
interacting in a deleterious manner with any other component(s) of
the pharmaceutically acceptable composition, its use is
contemplated to be within the scope of this invention. Some
examples of materials which can serve as pharmaceutically
acceptable carriers include, but are not limited to, ion
exchangers, alumina, aluminum stearate, lecithin, serum proteins,
such as human serum albumin, buffer substances such as phosphates,
glycine, sorbic acid, or potassium sorbate, partial glyceride
mixtures of saturated vegetable fatty acids, water, salts or
electrolytes, such as protamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium chloride, zinc
salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, wool fat, sugars such
as lactose, glucose and sucrose; starches such as corn starch and
potato starch; cellulose and its derivatives such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients such as cocoa
butter and suppository waxes; oils such as peanut oil, cottonseed
oil; safflower oil; sesame oil; olive oil; corn oil and soybean
oil; glycols; such a propylene glycol or polyethylene glycol;
esters such as ethyl oleate and ethyl laurate; agar; buffering
agents such as magnesium hydroxide and aluminum hydroxide; alginic
acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl
alcohol, and phosphate buffer solutions, as well as other non-toxic
compatible lubricants such as sodium lauryl sulfate and magnesium
stearate, as well as coloring agents, releasing agents, coating
agents, sweetening, flavoring and perfuming agents, preservatives
and antioxidants can also be present in the composition, according
to the judgment of the formulator.
6. Uses of Compounds and Pharmaceutically Acceptable
Compositions
[0215] In yet another aspect, the present invention provides a
method of treating a condition, disease, or disorder implicated by
ABC transporter activity. In certain embodiments, the present
invention provides a method of treating a condition, disease, or
disorder implicated by a deficiency of ABC transporter activity,
the method comprising administering a composition comprising a
compound of Formula I to a subject, preferably a mammal, in need
thereof.
[0216] In certain preferred embodiments, the present invention
provides a method of treating cystic fibrosis, hereditary
emphysema, hereditary hemochromatosis, coagulation-cibrinolysis
deficiencies, such as protein C deficiency, Type 1 hereditary
angioedema, lipid processing deficiencies, such as familial
hypercholesterolemia, Type 1 chylomicronemia, abetalipoproteinemia,
lysosomal storage diseases, such as I-cell disease/pseudo-Hurler,
secretory diarrhea or polycystic kidney disease,
mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II,
polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron
dwarfism, myleoperoxidase deficiency, primary hypoparathyroidism,
melanoma, glycanosis CDG type 1, hereditary emphysema, congenital
hyperthyroidism, osteogenesis imperfecta, hereditary
hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),
neurophyseal DI, neprogenic DI, Charcot-Marie Tooth syndrome,
Perlizaeus-Merzbacher disease, neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, amyotrophic lateral
sclerosis, progressive supranuclear plasy, Pick's disease, several
polyglutamine neurological disorders such as Huntington,
spinocerebullar ataxia type I, spinal and bulbar muscular atrophy,
dentatorubal pallidoluysian, and myotonic dystrophy, as well as
spongiform encephalopathies, such as hereditary Creutzfeldt-Jakob
disease (due to prion protein processing defect), Fabry disease,
Straussler-Scheinker syndrome, COPD, dry eye disease, or Sjogren's
disease, comprising the step of administering to said mammal an
effective amount of a composition comprising a compound of Formula
I, or a preferred embodiment thereof as set forth above.
[0217] According to an alternative preferred embodiment, the
present invention provides a method of treating cystic fibrosis
comprising the step of administering to said mammal a composition
comprising the step of administering to said mammal an effective
amount of a composition comprising a compound of the present
invention.
[0218] According to the invention an "effective amount" the
compound or pharmaceutically acceptable composition is that amount
effective for treating or lessening the severity of one or more of
cystic fibrosis, hereditary emphysema, hereditary hemochromatosis,
coagulation-cibrinolysis deficiencies, such as protein C
deficiency, Type 1 hereditary angioedema, lipid processing
deficiencies, such as familial hypercholesterolemia, Type 1
chylomicronemia, abetalipoproteinemia, lysosomal storage diseases,
such as I-cell disease/pseudo-Hurler, secretory diarrhea
or'polycystic kidney disease, mucopolysaccharidoses,
Sandhof/Tay-Sachs, Crigler-Najjar type II,
polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron
dwarfism, myleoperoxidase deficiency, primary hypoparathyroidism,
melanoma, glycanosis CDG type 1, hereditary emphysema, congenital
hyperthyroidism, osteogenesis imperfecta, hereditary
hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),
neurophyseal DI, neprogenic DI, Charcot-Marie Tooth syndrome,
Perlizaeus-Merzbacher disease, neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, amyotrophic lateral
sclerosis, progressive supranuclear plasy, Pick's disease, several
polyglutamine neurological disorders such as Huntington,
spinocerebullar ataxia type I, spinal and bulbar muscular atrophy,
dentatorubal pallidoluysian, and myotonic dystrophy, as well as
spongiform encephalopathies, such as hereditary Creutzfeldt-Jakob
disease (due to prion protein processing defect), Fabry disease,
Straussler-Scheinker syndrome, COPD, dry eye disease, or Sjogren's
disease.
[0219] The compounds and compositions, according to the method of
the present invention, may be administered using any amount and any
route of administration effective for treating or lessening the
severity of one or more of cystic fibrosis, hereditary emphysema,
hereditary hemochromatosis, coagulation-cibrinolysis deficiencies,
such as protein C deficiency, Type 1 hereditary angioedema, lipid
processing deficiencies, such as familial hypercholesterolemia,
Type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage
diseases, such as I-cell disease/pseudo-Hurler, secretory diarrhea
or polycystic kidney disease, mucopolysaccharidoses,
Sandhof/Tay-Sachs, Crigler-Najjar type II,
polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron
dwarfism, myleoperoxidase deficiency, primary hypoparathyroidism,
melanoma, glycanosis CDG type 1, hereditary emphysema, congenital
hyperthyroidism, osteogenesis imperfecta, hereditary
hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),
neurophyseal DI, neprogenic DI, Charcot-Marie Tooth syndrome,
Perlizaeus-Merzbacher disease, neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, amyotrophic lateral
sclerosis, progressive supranuclear plasy, Pick's disease, several
polyglutamine neurological disorders such as Huntington,
spinocerebullar ataxia type I, spinal and bulbar muscular atrophy,
dentatorubal pallidoluysian, and myotonic dystrophy, as well as
spongiform encephalopathies, such as hereditary Creutzfeldt-Jakob
disease (due to prion protein processing defect), Fabry disease,
Straussler-Scheinker syndrome, COPD, dry eye disease, or Sjogren's
disease. The exact amount required will vary from subject to
subject, depending on the species, age, and general condition of
the subject, the severity of the infection, the particular agent,
its mode of administration, and the like. The compounds of the
invention are preferably formulated in dosage unit form for ease of
administration and uniformity of dosage. The expression "dosage
unit form" as used herein refers to a physically discrete unit of
agent appropriate for the patient to be treated. It will be
understood, however, that the total daily usage of the compounds
and compositions of the present invention will be decided by the
attending physician within the scope of sound medical judgment. The
specific effective dose level for any particular patient or
organism will depend upon a variety of factors including the
disorder being treated and the severity of the disorder; the
activity of the specific compound employed; the specific
composition employed; the age, body weight, general health, sex and
diet of the patient; the time of administration, route of
administration, and rate of excretion of the specific compound
employed; the duration of the treatment; drugs used in combination
or coincidental with the specific compound employed, and like
factors well known in the medical arts. The term "patient", as used
herein, means an animal, preferably a mammal, and most preferably a
human.
[0220] The pharmaceutically acceptable compositions of this
invention can be administered to humans and other animals orally,
rectally, parenterally, intracisternally, intravaginally,
intraperitoneally, topically (as by powders, ointments, or drops),
bucally, as an oral or nasal spray, or the like, depending on the
severity of the infection being treated. In certain embodiments,
the compounds of the invention may be administered orally or
parenterally at dosage levels of about 0.01 mg/kg to about 50 mg/kg
and preferably from about 1 mg/kg to about 25 mg/kg, of subject
body weight per day, one or more times a day, to obtain the desired
therapeutic effect.
[0221] Liquid dosage forms for oral administration include, but are
not limited to, pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active compounds, the liquid dosage forms may
contain inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavoring, and perfuming agents.
[0222] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables.
[0223] The injectable formulations can be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0224] In order to prolong the effect of a compound of the present
invention, it is often desirable to slow the absorption of the
compound from subcutaneous or intramuscular injection. This may be
accomplished by the use of a liquid suspension of crystalline or
amorphous material with poor water solubility. The rate of
absorption of the compound then depends upon its rate of
dissolution that, in turn, may depend upon crystal size and
crystalline form. Alternatively, delayed absorption of a
parenterally administered compound form is accomplished by
dissolving or suspending the compound in an oil vehicle. Injectable
depot forms are made by forming microencapsule matrices of the
compound in biodegradable polymers such as
polylactide-polyglycolide. Depending upon the ratio of compound to
polymer and the nature of the particular polymer employed, the rate
of compound release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the compound in liposomes or microemulsions that are
compatible with body tissues.
[0225] Compositions for rectal or vaginal administration are
preferably suppositories which can be prepared by mixing the
compounds of this invention with suitable non-irritating excipients
or carriers such as cocoa butter, polyethylene glycol or a
suppository wax which are solid at ambient temperature but liquid
at body temperature and therefore melt in the rectum or vaginal
cavity and release the active compound.
[0226] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active compound is mixed with at least one inert,
pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol, and silicic acid,
b) binders such as, for example, carboxymethylcellulose, alginates,
gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants
such as glycerol, d) disintegrating agents such as agar-agar,
calcium carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate, e) solution retarding agents such
as paraffin, f) absorption accelerators such as quaternary ammonium
compounds, g) wetting agents such as, for example, cetyl alcohol
and glycerol monostearate, h) absorbents such as kaolin and
bentonite clay, and i) lubricants such as talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof. In the case of capsules, tablets and
pills, the dosage form may also comprise buffering agents.
[0227] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings well known in the pharmaceutical formulating art. They may
optionally contain opacifying agents and can also be of a
composition that they release the active ingredient(s) only, or
preferentially, in a certain part of the intestinal tract,
optionally, in a delayed manner. Examples of embedding compositions
that can be used include polymeric substances and waxes. Solid
compositions of a similar type may also be employed as fillers in
soft and hard-filled gelatin capsules using such excipients as
lactose or milk sugar as well as high molecular weight polethylene
glycols and the like.
[0228] The active compounds can also be in microencapsulated form
with one or more excipients as noted above. The solid dosage forms
of tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings and other coatings well known in the
pharmaceutical formulating art. In such solid dosage forms the
active compound may be admixed with at least one inert diluent such
as sucrose, lactose or starch. Such dosage forms may also comprise,
as is normal practice, additional substances other than inert
diluents, e.g., tableting lubricants and other tableting aids such
a magnesium stearate and microcrystalline cellulose. In the case of
capsules, tablets and pills, the dosage forms may also comprise
buffering agents. They may optionally contain opacifying agents and
can also be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain part of the
intestinal tract, optionally, in a delayed manner. Examples of
embedding compositions that can be used include polymeric
substances and waxes.
[0229] Dosage forms for topical or transdermal administration of a
compound of this invention include ointments, pastes, creams,
lotions, gels, powders, solutions, sprays, inhalants or patches.
The active component is admixed under sterile conditions with a
pharmaceutically acceptable carrier and any needed preservatives or
buffers as may be required. Ophthalmic formulation, eardrops, and
eye drops are also contemplated as being within the scope of this
invention. Additionally, the present invention contemplates the use
of transdermal patches, which have the added advantage of providing
controlled delivery of a compound to the body. Such dosage forms
are prepared by dissolving or dispensing the compound in the proper
medium. Absorption enhancers can also be used to increase the flux
of the compound across the skin. The rate can be controlled by
either providing a rate controlling membrane or by dispersing the
compound in a polymer matrix or gel.
[0230] As described generally above, the compounds of the invention
are useful as modulators of ABC transporters. Thus, without wishing
to be bound by any particular theory, the compounds and
compositions are particularly useful for treating or lessening the
severity of a disease, condition, or disorder where hyperactivity
or inactivity of ABC transporters is implicated in the disease,
condition, or disorder. When hyperactivity or inactivity of an ABC
transporter is implicated in a particular disease, condition, or
disorder, the disease, condition, or disorder may also be referred
to as a "ABC transporter-mediated disease, condition or disorder".
Accordingly, in another aspect, the present invention provides a
method for treating or lessening the severity of a disease,
condition, or disorder where hyperactivity or inactivity of an ABC
transporter is implicated in the disease state.
[0231] The activity of a compound utilized in this invention as a
modulator of an ABC transporter may be assayed according to methods
described generally in the art and in the Examples herein.
[0232] It will also be appreciated that the compounds and
pharmaceutically acceptable compositions of the present invention
can be employed in combination therapies, that is, the compounds
and pharmaceutically acceptable compositions can be administered
concurrently with, prior to, or subsequent to, one or more other
desired therapeutics or medical procedures. The particular
combination of therapies (therapeutics or procedures) to employ in
a combination regimen will take into account compatibility of the
desired therapeutics and/or procedures and the desired therapeutic
effect to be achieved. It will also be appreciated that the
therapies employed may achieve a desired effect for the same
disorder (for example, an inventive compound may be administered
concurrently with another agent used to treat the same disorder),
or they may achieve different effects (e.g., control of any adverse
effects). As used herein, additional therapeutic agents that are;
normally administered to treat or prevent a particular, disease, or
condition, are known as "appropriate for the disease, or condition,
being treated".
[0233] The amount of additional therapeutic agent present in the
compositions of this invention will be no more than the amount that
would normally be administered in a composition comprising that
therapeutic agent as the only active agent. Preferably the amount
of additional therapeutic agent in the presently disclosed
compositions will range from about 50% to 100% of the amount
normally present in a composition comprising that agent as the only
therapeutically active agent.
[0234] The compounds of this invention or pharmaceutically
acceptable compositions thereof may also be incorporated into
compositions for coating an implantable medical device, such as
prostheses, artificial valves, vascular grafts, stents and
catheters. Accordingly, the present invention, in another aspect,
includes a composition for coating an implantable device comprising
a compound of the present invention as described generally above,
and in classes and subclasses herein, and a carrier suitable for
coating said implantable device. In still another aspect, the
present invention includes an implantable device coated with a
composition comprising a compound of the present invention as
described generally above, and in classes and subclasses herein,
and a carrier suitable for coating said implantable device.
Suitable coatings and the general preparation of coated implantable
devices are described in U.S. Pat. Nos. 6,099,562; 5,886,026; and
5,304,121. The coatings are typically biocompatible polymeric
materials such as a hydrogel polymer, polymethyldisiloxane,
polycaprolactone, polyethylene glycol, polylactic acid, ethylene
vinyl acetate, and mixtures thereof. The coatings may optionally be
further covered by a suitable topcoat of fluorosilicone,
polysaccarides, polyethylene glycol, phospholipids or combinations
thereof to impart controlled release characteristics in the
composition.
[0235] Another aspect of the invention relates to modulating ABC
transporter activity in a biological sample or a patient (e.g., in
vitro or in vivo), which method comprises administering to the
patient, or contacting said biological sample with a compound of
the present invention or a composition comprising said compound.
The term "biological sample", as used herein, includes, without
limitation, cell cultures or extracts thereof; biopsied material
obtained from a mammal or extracts thereof; and blood, saliva,
urine, feces, semen, tears, or other body fluids or extracts
thereof.
[0236] Modulation of ABC transporter activity in a biological
sample is useful for a variety of purposes that are known to one of
skill in the art. Examples of such purposes include, but are not
limited to, the study of ABC transporters in biological and
pathological phenomena; and the comparative evaluation of new
modulators of ABC transporters.
[0237] In yet another embodiment, a method of modulating activity
of an anion channel in vitro or in vivo, is provided comprising the
step of contacting said channel with a compound of the present
invention. In preferred embodiments, the anion channel is a
chloride channel or a bicarbonate channel. In other preferred
embodiments, the anion channel is a chloride channel.
[0238] According to an alternative embodiment, the present
invention provides a method of increasing the number of functional
ABC transporters in a membrane of a cell, comprising the step of
contacting said cell with a compound of the present invention. The
term "functional ABC transporter" as used herein means an ABC
transporter that is capable of transport activity. In preferred
embodiments, said functional ABC transporter is CFTR.
[0239] According to another preferred embodiment, the activity of
the ABC transporter is measured by measuring the transmembrane
voltage potential. Means for measuring the voltage potential across
a membrane in the biological sample may employ any of the known
methods in the art, such as optical membrane potential assay or
other electrophysiological methods.
[0240] The optical membrane potential assay utilizes
voltage-sensitive FRET sensors described by Gonzalez and Tsien
(See, Gonzalez, J. E. and R. Y. Tsien (1995) "Voltage sensing by
fluorescence resonance energy transfer in single cells" Biophys J
69(4): 1272-80, and Gonzalez, J. E. and R. Y. Tsien (1997)
"Improved indicators of cell membrane potential that use
fluorescence resonance energy transfer" Chem Biol 4(4): 269-77) in
combination with instrumentation for measuring fluorescence changes
such as the Voltage/Ion Probe Reader (VIPR) (See, Gonzalez, J. E.,
K. Oades, et al. (1999) "Cell-based assays and instrumentation for
screening ion-channel targets" Drug Discov Today 4(9):
431-439).
[0241] These voltage sensitive assays are based on the change in
fluorescence resonant energy transfer (FRET) between the
membrane-soluble, voltage-sensitive dye, DiSBAC2(3), and a
fluorescent phospholipid, CC2-DMPE, which is attached to the outer
leaflet of the plasma membrane and acts as a FRET donor. Changes in
membrane potential (Vm) cause the negatively charged DiSBAC2(3) to
redistribute across the plasma membrane and the amount of energy
transfer from CC2-DMPE changes accordingly. The changes in
fluorescence emission can be monitored using VIPR.TM. II, which is
an integrated liquid handler and fluorescent detector designed to
conduct cell-based screens in 96- or 384-well microtiter
plates.
[0242] In another aspect the present invention provides a kit for
use in measuring the activity of a ABC transporter or a fragment
thereof in a biological sample in vitro or in vivo comprising (i) a
composition comprising a compound of the present invention; and
(ii) instructions for a) contacting the composition with the
biological sample and b) measuring activity of said ABC transporter
or a fragment thereof. In one embodiment, the kit further comprises
instructions for a) contacting an additional composition with the
biological sample; b) measuring the activity of said ABC
transporter or a fragment thereof in the presence of said
additional compound, and c) comparing the activity of the ABC
transporter in the presence of the additional compound with the
density of the ABC transporter in the presence of a composition of
the present invention. In preferred embodiments, the kit is used to
measure the density of CFTR.
[0243] In order that the invention described herein may be more
fully understood, the following examples are set forth. It should
be understood that these examples are for illustrative purposes
only and are not to be construed as limiting this invention in any
manner.
EXAMPLES
General Procedures
[0244] All reagents and solvents were used as received without
further purification. Thin layer chromatography was performed on
glass-backed silica gel 60 plates pre-coated with a fluorescent dye
from EM Science. Mass spectrometry was performed in the positive
mode on a PE SCIEX EX150 mass spectrometer. Purity was determined
by the observed total ion current, and the ultraviolet absorption
at 220 nm and 254 nm.
Example 1
Preparation of Certain Exemplary Amines
C-[1-(3,4-Dimethoxy-phenyl)-cyclopentyl]-methylamine
[0245] (3,4-Dimethoxy-phenyl)-acetonitrile (5.00 g, 28.2 mmol) was
dissolved in 60 mL of anhydrous tetrahydrofuran in a 250 mL round
bottom flask. Sodium hydride (2.03 g, 84.6 mmol) was slowly added
and the reaction mixture was warmed to 50-60.degree. C.
1,4-Dichlorobutane (4.30 g, 33.9 mmol) was then added and the
reaction mixture was heated to reflux for 16 hours. An additional
aliquot of 1,4-dichlorobutane (4.30 g, 33.9 mmol) was added and the
reaction mixture was refluxed for an additional 24 hours. The
reaction mixture was cooled to room temperature and quenched with
the slow addition of methanol. The reaction mixture was evaporated
to dryness and purified by column chromatography on silica gel to
yield a pale yellow oil (1.61 g, 6.98 mmol, 24.8%). The resulting
1-(3,4-dimethoxy-phenyl)-cyclopentanecarbonitrile (363 mg, 1.57
mmol) was dissolved in dry ether (4 mL) and cooled to 0.degree. C.
under an atmosphere of nitrogen. Lithium aluminum hydride (1.57 mL,
1M in ether) was slowly added and the reaction mixture was allowed
to warm to room temperature and stirred for 16 hours. The reaction
mixture was quenched with the slow addition of methanol. The
reaction mixture was washed with a saturated aqueous sodium
chloride solution, separated, and evaporated to dryness to give a
colorless oil (356 mg, 1.38 mmol, 87.9%).ESI-MS m/z calc. 235.3.
found 236.2 (M+1).sup.+. Retention time of 1.64 minutes.
[(2-(3,4-Dimethoxy-phenyl)-2-methyl]-propylamine
[0246] Starting from 3,4-dimethoxyphenyl-acetonitrile (1 g, 5.64
mmol) and following a procedure similar to the one reported for the
preparation of
C-[1-(3,4-Dimethoxy-phenyl)-cyclopentyl]-methylamine, 250 mg (21%
yield, 2 steps) of [2-(3,4-Dimethoxy-phenyl)-2-methyl]-propylamine
were obtained as a colorless oil. ESI-MS m/z calc. 209.3. found 210
(M+1).sup.+. 1H NMR (400 MHz; CDCl.sub.3) .delta. 1.26 (s, 6H),
2.76 (s, 2H), 3.78 (s, 3H), 3.79 (s, 3H), 6.3-6.82 (m, 3H).
[C-(1-(3,4-Dimethoxy-phenyl)-cyclohexyl]-methylamine
[0247] (3,4-Dimethoxy-phenyl)-acetonitrile (5.00 g, 28.2 mmol) was
dissolved in 60 mL of anhydrous tetrahydrofuran in a 250 mL round
bottom flask. Sodium hydride (2.03 g, 84.6 mmol) was slowly added
and the reaction mixture was warmed to 50-60.degree. C.
1,4-Dichloropentane (4.78 g, 33.9 mmol) was then added and the
reaction mixture was heated to reflux for 16 hours. The reaction
mixture was cooled to room temperature and quenched with the slow
addition of methanol. The reaction mixture was evaporated to
dryness and purified by column chromatography on silica gel to
yield a pale yellow oil (3.65 g, 14.9 mmol, 52.8%). The resulting
1-(3,4-dimethoxy-phenyl)-cyclohexanecarbonitrile (2.00 g, 8.15
mmol) was dissolved in dry ether (40 mL) and cooled to 0.degree. C.
under an atmosphere of nitrogen. Lithium aluminum hydride (8.15 mL,
1M in ether) was slowly added and the reaction mixture was allowed
to warm to room temperature and stirred for 16 hours. The reaction
mixture was cooled to 0.degree. C. and quenched with 0.34 mL water,
0.34 mL of 15% sodium hydroxide, and then an additional 1.4 mL of
water. The reaction mixture was then filtered through celite,
washed with water and a saturated aqueous sodium chloride solution.
The filtrate was evaporated to dryness to give a colorless oil
(1.91 g, 7.66 mmol, 94.0%). ESI-MS m/z calc. 249.2. found 250.2
(M+1).sup.+. Retention time of 1.76 minutes.
Example 2
Preparation of Exemplary Compounds of Formula I
Benzofuran-2-carboxylic acid
[1-(3,4-dimethoxy-phenyl)-cyclopentylmethyl]-amide
[0248] C-[1-(3,4-Dimethoxy-phenyl)-cyclopentyl]-methylamine (141
mg, 0.600 mmol) was dissolved in anhydrous 1,4-dioxane (2 mL)
containing triethylamine (167 .mu.L, 1.20 mmol).
Benzofuran-2-carbonyl chloride (108 mg, 0.600 mmol) was then added
and the reaction mixture was allowed to stir for 16 hours. The
reaction mixture was filtered, evaporated to dryness, and purified
by column chromatography on silica gel using a gradient of 5-50%
ethyl acetate in hexanes. The pure fractions were combined and
evaporated to dryness to yield a white solid (0.1775 g, 0.4678
mmol, 78.0%) ESI-MS m/z calc. 379.5. found 380.2 (M+1).sup.+.
Retention time of 3.36 minutes. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 1.57-2.03 (m, 8H), 3.46 (d, J=6.4 Hz, 2H), 3.72 (s, 6H),
6.81-6.89 (m, 3H), 7.31 (t, J=7.5 Hz, 1H), 7.44 (t, J=7.8 Hz, 1H),
7.48 (s, 1H), 7.60 (d, J=8.4 Hz, 1H), 7.75 (d, J=8.4 Hz, 1H), 7.94
(t, J=6.3 Hz, 1H). .sup.13C NMR (100 MHz, DMSO-d.sub.6) .delta.
23.1, 35.3, 47.2, 51.8, 55.5, 109.2, 111.5, 111.6, 111.7, 118.8,
122.6, 123.6, 126.7, 127.1, 139.3, 147.1, 148.3, 149.1, 154.1,
158.2.
Benzofuran-2-carboxylic acid
[1-(3,4-dimethoxy-phenyl)-cyclohexylmethyl]-amide
[0249] C-[1-(3,4-Dimethoxy-phenyl)-cyclohexyl]-methylamine (276 mg,
1.12 mmol) and Benzofuran-2-carbonyl chloride (223 mg, 1.23 mmol)
were dissolved in 6 mL of 1,4-dioxane containing triethylamine (312
.mu.L, 2.24 mmol) at 0.degree. C. The reaction mixture was
evaporated to dryness, redissolved in dichloromethane, and
extracted with 1M hydrochloric acid, 1M sodium hydroxide, and a
saturated aqueous solution of sodium chloride. The organic layer
dried over sodium sulfate and evaporated to dryness. The crude
product was then purified by column chromatography on silica gel
using a gradient of 0-20% ethyl acetate in hexanes. The pure
fractions were combined and evaporated to dryness to yield a white
solid (195 mg, 0.496 mmol, 44.2%). ESI-MS m/z calc. 393.2. found
394.2 (M+1).sup.+. Retention time of 2.96 minutes. .sup.1H NMR (400
MHz, CD.sub.3CN) .delta. 1.45-2.20 (m, 10H), 3.48 (d, J=6.5 Hz,
2H), 3.80 (s, 3H), 3.83 (s, 3H), 6.68 (s, 1H), 6.92-7.02 (m, 3H),
7.30-7.38 (m, 2H), 7.42-7.49 (m, 1H), 7.51-7.55 (m, 1H), 7.72 (d,
J=7.8 Hz, 1H)
Quinoline-2-carboxylic acid
[1-(3,4-dimethoxy-phenyl)-cyclopentylmethyl]-amide
[0250] Quinoline-2-carboxylic acid (0.502 g, 2.90 mmol) and
C-[1-(3,4-Dimethoxy-phenyl)-cyclopentyl]-methylamine (0.678 g, 2.90
mmol) were dissolved in acetonitrile (20 mL) containing
triethylamine (894 .mu.L, 6.38 mmol).
O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (1.54 g, 4.06 mmol) was added and the solution
was allowed to stir for 16 hours. The reaction mixture was
evaporated to dryness and purified by column chromatography on
silica gel using a gradient of 0-40% ethyl acetate in hexanes. The
pure fractions were combined and evaporated to dryness to yield a
white solid (0.426 g, 1.09 mmol, 37.7%). ESI-MS m/z calc. 390.2.
found 391.2 (M+1).sup.+. Retention time of 3.94 minutes. .sup.1H
NMR (400 MHz, CD.sub.3CN) .delta. 1.69-2.15 (m, 8H), 3.62 (d,
J=30.1 Hz, 2H), 3.77 (s, 3H), 3.90 (s, 3H), 6.91-7.03 (m, 3H), 7.69
(t, J=8.1 Hz, 1H), 7.83 (t, J=7.7 Hz, 1H), 7.99 (t, J=8.8 Hz, 2H),
8.08 (s, 1H), 8.18 (d, J=8.5 Hz, 1H), 8.44 (d, J=8.5 Hz, 1H)
2-Fluoro-N-phenethyl-nicotinamide
[0251] 2-Fluoro-nicotinic acid (0.793 g, 5.59 mmol) and
phenethylamine (0.705 mL, 5.59 mmol) were dissolved in acetonitrile
(20 mL) containing triethylamine (1.56 mL, 11.2 mmol).
O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (2.97 g, 7.83 mmol) was added and the solution
was allowed to stir for 16 hours. The reaction mixture was
evaporated to dryness and purified by column chromatography on
silica gel using a gradient of 0-40% ethyl acetate in hexanes. The
pure fractions were combined and evaporated to dryness to yield a
white solid (0.196 g, 0.802 mmol, 14.3%). ESI-MS m/z calc. 244.1.
found 245.2 (M+1).sup.+. Retention time of 2.76 minutes.
2-Butoxy-N-phenethyl-nicotinamide
[0252] 2-Fluoro-N-phenethyl-nicotinamide (196 mg, 0.802 mmol),
n-butanol (700 .mu.L, 7.65 mmol) and potassium
bis(trimethylsilyl)amide (2.5 mL, 0.5 M in toluene) were combined
and stirred for 5 minutes at room temperature. The reaction mixture
was evaporated to dryness and purified by column chromatography on
silica gel using a gradient of 0-20% ethyl acetate in hexanes. The
pure fractions were combined and evaporated to dryness to yield a
colorless oil (207 mg, 0.694 mmol, 86.5%). ESI-MS m/z calc. 298.2.
found 299.2 (M+1).sup.+. Retention time of 3.46 minutes. H NMR (400
MHz, CD.sub.3CN) .delta., 0.95 (t, J=7.4 Hz, 3H), 1.34-1.44 (m,
2H), 1.63-1.70 (m, 2H), 2.91 (t, J=7.0 Hz, 2H), 3.65-3.73 (m, 2H),
4.41 (t, J=6.7 Hz, 2H), 7.08 (dd, J=7.5, 4.8 Hz, 1H), 7.23-7.37 (m,
5H), 8.01 (s, 1H), 8.25 (dd, J=4.8, 2.0 Hz, 1H), 8.39 (dd, J=7.5,
2.0 Hz, 1H).
Benzofuran-2-carboxylic acid
[2-(3,4-dimethoxy-phenyl)-2-methyl-propyl]-amide
[0253] 2-(3,4-Dimethoxy-phenyl)-2-methyl-propylamine (106 mg, 0.506
mmol) and benzofuran-2-carbonyl chloride (90.7 mg, 0.502 mmol) were
dissolved in 2 mL of 1,4-dioxane containing triethylamine (139
.mu.L, 1.00 mmol). The reaction mixture was stirred for 15 hours,
evaporated to dryness, redissolved in dichloromethane, and
extracted with 1M hydrochloric acid, 1M sodium hydroxide, and a
saturated aqueous solution of sodium chloride. The organic layer
dried over sodium sulfate and evaporated to dryness. The crude
product was then purified by column chromatography on silica gel
using a gradient of 1-30% ethyl acetate in hexanes. The pure
fractions were combined and evaporated to dryness to yield a white
solid (153 mg, 0.433 mmol, 86.3%). ESI-MS m/z calc. 353.2. found
354.2 (M+1).sup.+. Retention time of 2.99 minutes. .sup.1H NMR (400
MHz, CD.sub.3CN) .delta. 1.38 (s, 6H), 3.60 (s, 2H), 3.81 (s, 3H),
3.83 (s, 3H), 6.85-7.06 (m, 4H), 7.31-7.39 (m, 2H), 7.46 (t, J=8.4
Hz, 1H), 7.52-7.56 (m, 1H), 7.72 (d, J=8.2 Hz, 1H).
Benzofuran-2-carboxylic acid
[1-(3,4,5-trimethoxy-phenyl)-cyclopentylmethyl]-amide
[0254] C-[1-(3,4,5-Trimethoxy-phenyl)-cyclopentyl]-methylamine
(53.1 mg, 0.200 mmol) and benzofuran-2-carbonyl chloride (36.1 mg,
0.200 mmol) were dissolved in 2 mL of 1,4-dioxane containing
triethylamine (84 .mu.L, 0.60 mmol). The reaction mixture was
stirred for 15 hours, evaporated to dryness, and purified by
reverse phase preparative liquid chromatography to yield the pure
product (9.59 mg, 0.0234 mmol, 11.7%). ESI-MS m/z calc. 409.2.
found 410.4 (M+1).sup.+. Retention time of 3.29 minutes.
Benzofuran-2-carboxylic acid
(1-benzo[1,3]dioxol-5-yl-cyclopentylmethyl)-amide
[0255] C-(1-Benzo[1,3]dioxol-5-yl-cyclopentyl)-methylamine (43.8
mg, 0.200 mmol) and benzofuran-2-carbonyl chloride (36.1 mg, 0.200
mmol) were dissolved in 2 mL of 1,4-dioxane containing
triethylamine (83.6 .mu.L, 0.600 mmol). The reaction mixture was
stirred for 15 hours, evaporated to dryness, and purified by
reverse phase preparative liquid chromatography to yield the pure
product (13.0 mg, 0.0358 mmol, 17.8%). ESI-MS m/z calc. 363.2.
found 364.2 (M+1).sup.+. Retention time of 4.22 minutes.
2-Cyclopentyloxy-N-phenethyl-nicotinamide
[0256] 2-Fluoro-nicotinic acid (84.7 mg, 0.600 mmol, cyclopentanol
(51.6 mg, 0.600 mmol) and potassium bis(trimethylsilyl)amide (478
mg, 2.40 mmol) were combined in 0.6 mL of N,N-dimethylformamide and
subjected to microwave irradiation for 3 minutes at 180.degree. C.
Phenethylamine (72.7 mg, 0.600 mmol) and
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (304 mg, 0.800 mmol) was added and the solution
was allowed to stir for 16 hours. The mixture was then purified by
reverse phase preparative liquid chromatography to yield the pure
product (2.9 mg, 0.0093 mmol, 1.6%) ESI-MS m/z calc. 310.2. found
311.2 (M+1).sup.+. Retention time of 3.40 minutes.
N-[2-(3,4-Dimethoxy-phenyl)-ethyl]-N-methyl-2-(3-methyl-butoxy)-nicotinami-
de
[0257] 2-Fluoro-nicotinic acid (84.7 mg, 0.600 mmol,
3-methyl-butan-1-ol (52.9 mg, 0.600 mmol) and potassium
bis(trimethylsilyl)amide (478 mg, 2.40 mmol) were combined in 0.6
mL of N,N-dimethylformamide and subjected to microwave irradiation
for 3 minutes at 180.degree. C.
[2-(3,4-Dimethoxy-phenyl)-ethyl]-methyl-amine (117 mg, 0.600 mmol)
and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (304 mg, 0.800 mmol) was added and the solution
was allowed to stir for 16 hours. The mixture was then purified by
reverse phase preparative liquid chromatography to yield the pure
product (1.5 mg, 0.0039 mmol, 0.65%) ESI-MS m/z calc. 386.2. found
387.4 (M+1).sup.+. Retention time of 2.98 minutes.
1H-Indazole-3-carboxylic acid
[1-(3,4-dimethoxy-phenyl)-cyclopentylmethyl]-amide
[0258] 1H-Indazole-3-carboxylic acid (32.4 mg, 0.200 mmol) and
C-[1-(3,4-Dimethoxy-phenyl)-cyclopentyl]-methylamine (47.1 g, 0.200
mmol) were dissolved in acetonitrile (1 mL) containing
triethylamine (83.6 .mu.L, 0.600 mmol).
O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (76.0 mg, 0.200 mmol) was added and the
solution was allowed to stir for 16 hours. The mixture was then
purified by reverse phase preparative liquid chromatography to
yield the pure product (7.47 mg, 0.0197 mmol, 9.84%) ESI-MS m/z
calc. 379.2. found 380.4 (M+1).sup.+. Retention time of 3.02
minutes.
4-Benzyl-N-[1-(3,4-dimethoxy-phenyl)-cyclopentylmethyl]-benzamide
[0259] 4-Benzyl-benzoic acid (21.2 mg, 0.100 mmol) and
C-[1-(3,4-Dimethoxy-phenyl)-cyclopentyl]-methylamine (23.5 g, 0.100
mmol) were dissolved in acetonitrile (1 mL) containing
triethylamine (41.8 .mu.L, 0.300 mmol).
O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (38.0 mg, 0.100 mmol) was added and the
solution was allowed to stir for 16 hours. The mixture was then
purified by reverse phase preparative liquid chromatography to
yield the pure product (24.1 mg, 0.0561 mmol, 56.1%) ESI-MS m/z
calc. 429.2. found 430.4 (M+1).sup.+. Retention time of 3.75
minutes.
N-[1-(3,4-Dimethoxy-phenyl)-cyclopentylmethyl]-2,2-diphenyl-acetamide
[0260] Diphenyl-acetic acid (42.4 mg, 0.200 mmol) and
C-[1-(3,4-Dimethoxy-phenyl)-cyclopentyl]-methylamine (47.1 g, 0.200
mmol) were dissolved in acetonitrile (1 mL) containing
triethylamine (83.6 .mu.L, 0.600 mmol).
O-(7-Azabenzotriazol-1-yl)-N,N,N', N'-tetramethyluronium
hexafluorophosphate (76.0 mg, 0.200 mmol) was added and the
solution was allowed to stir for 16 hours. The mixture was then
purified by reverse phase preparative liquid chromatography to
yield the pure product (13.3 mg, 0.0310 mmol, 15.5%) ESI-MS m/z
calc. 429.2. found 430.2 (M+1).sup.+. Retention time of 3.47
minutes.
2-Methyl-5-phenyl-furan-3-carboxylic acid
[1-(3,4-dimethoxy-phenyl)-cyclopentylmethyl]-amide
[0261] 2-Methyl-5-phenyl-furan-3-carboxylic acid (40.4 mg, 0.200
mmol) and C-[1-(3,4-Dimethoxy-phenyl)-cyclopentyl]-methylamine
(47.1 g, 0.200 mmol) were dissolved in acetonitrile (1 mL)
containing triethylamine (83.6 .mu.L, 0.600 mmol).
O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (76.0 mg, 0.200 mmol) was added and the
solution was allowed to stir for 16 hours. The mixture was then
purified by reverse phase preparative liquid chromatography to
yield the pure product (17.0 mg, 0.0405 mmol, 20.3%) ESI-MS m/z
calc. 419.2. found 420.4 (M+1).sup.+. Retention time of 3.62
minutes.
N-[1-(3,4-Dimethoxy-phenyl)-cyclopentylmethyl]-2-phenylsulfanyl-acetamide
[0262] Phenylsulfanyl-acetic acid (33.6 mg, 0.200 mmol) and
C-[1-(3,4-Dimethoxy-phenyl)-cyclopentyl]-methylamine (47.1 g, 0.200
mmol) were dissolved in acetonitrile (1 mL) containing
triethylamine (83.6 .mu.L, 0.600 mmol).
O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (76.0 mg, 0.200 mmol) was added and the
solution was allowed to stir for 16 hours. The mixture was then
purified by reverse phase preparative liquid chromatography to
yield the pure product (11.3 mg, 0.0293 mmol, 14.6%) ESI-MS m/z
calc. 385.2. found 386.0 (M+1).sup.+. Retention time of 3.12
minutes.
N-[1-(3,4-Dimethoxy-phenyl)-cyclopentylmethyl]-3-phenyl-propionamide
[0263] 3-Phenyl-propionic acid (30.0 mg, 0.200 mmol) and
C-[1-(3,4-Dimethoxy-phenyl)-cyclopentyl]-methylamine (47.1 g, 0.200
mmol) were dissolved in acetonitrile (1 mL) containing
triethylamine (83.6 .mu.L, 0.600 mmol).
O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (76.0 mg, 0.200 mmol) was added and the
solution was allowed to stir for 16 hours. The mixture was then
purified by reverse phase preparative liquid chromatography to
yield the pure product (0.19 mg, 0.0517 mmol, 25.8%) ESI-MS m/z
calc. 367.2. found 367.5 (M+1).sup.+. Retention time of 3.05
minutes.
[0264] The characterization data for certain compounds of the
present invention are shown below in Table 2.
TABLE-US-00004 TABLE 2 LC-MS Cmpd # ((M + 1).sup.+) LC-RT (min) 7
379.50 3.90 17 319.20 3.03 27 394.20 2.96 34 354.20 3.70 35 364.20
4.22 36 359.20 2.51 37 345.00 2.80 38 317.20 2.36 39 345.20 2.81 40
243.20 3.17 41 371.20 2.81 42 373.40 2.64 43 373.20 3.22 44 331.40
2.60 45 299.20 3.40 46 359.00 3.00 47 387.40 3.03 48 359.00 3.03 49
257.00 2.65 50 271.20 2.93 51 299.20 3.38 52 313.00 3.60 53 285.00
3.17 54 359.20 2.55 55 373.20 2.83 56 385.40 2.86 57 331.40 2.13 58
345.20 2.40 59 398.40 2.81 60 414.40 3.00 61 412.20 2.76 62 428.40
2.93 63 338.00 3.07 64 354.20 3.25 65 452.20 3.37 66 468.20 3.57 67
391.20 3.27 68 393.20 3.42 69 391.00 2.50 70 391.00 2.24 71 534.40
3.57 72 490.20 3.87 73 454.40 3.90 74 485.40 3.63 75 414.40 3.53 76
420.00 3.02 77 474.40 3.70 78 392.00 2.58 79 380.40 3.02 80 483.20
3.35 81 391.00 3.45 82 396.20 3.38 83 386.00 3.12 84 430.20 3.47 85
368.20 3.05 86 447.40 3.45 87 420.40 3.62 88 379.40 2.83 89 410.40
3.25 90 311.20 3.40 91 433.40 3.27 92 425.40 3.60 93 457.40 3.72 94
463.40 3.23 95 455.40 3.53 96 378.40 2.23 97 432.60 2.83 98 393.40
2.60 99 407.40 2.95 100 379.40 2.68 101 358.20 3.05 102 331.40 2.65
103 421.20 3.20 104 359.20 2.70 105 345.00 2.91 106 410.40 3.29 107
481.20 2.75 108 430.40 3.75
Example 3
Preparation of Additional Compounds of Formula I
[0265] Following the procedures taught in the specification and the
preceding Examples, the following compounds of Formula I can be
prepared.
##STR00133## ##STR00134## [0266] 3-1:
5-methoxy-N-((1-(3,4-dimethoxyphenyl)-cyclohexyl)methyl)benzofuran-2-carb-
oxamide [0267] 3-2:
N-((1-(2-fluoro-3,4-dimethoxyphenyl)cyclohexyl)methyl)-benzofuran-2-carbo-
xamide [0268] 3-3:
N-((1-(2-fluoro-4,5-dimethoxyphenyl)cyclohexyl)methyl)-benzofuran-2-carbo-
xamide [0269] 3-4:
N-((1-(4-ethoxy-3-methoxyphenyl)cyclohexyl)methyl)-benzofuran-2-carboxami-
de [0270] 3-5:
5-fluoro-N-((1-(3,4-dimethoxyphenyl)cyclohexyl)-methyl)benzofuran-2-carbo-
xamide [0271] 3-6:
N-((1-(3,4-dimethoxyphenyl)cyclohexyl)-methyl)benzo[b]thiophene-2-carboxa-
mide [0272] 3-7:
5-chloro-N-((1-(3,4-dimethoxyphenyl)-cyclohexyl)methyl)benzofuran-2-carbo-
xamide [0273] 3-8:
N-((1-(3-fluoro-4,5-dimethoxyphenyl)cyclohexyl)methyl)-benzofuran-2-carbo-
xamide [0274] 3-9:
N-((1-(3-ethoxy-4-methoxyphenyl)cyclohexyl)methyl)-benzofuran-2-carboxami-
de
Example 4
[0275] Assays for Detecting and Measuring .DELTA.F508-CFTR
Correction Properties of Compounds
Membrane Potential Optical Methods for Assaying .DELTA.F508-CFTR
Modulation Properties of Compounds.
[0276] The optical membrane potential assay utilized
voltage-sensitive FRET sensors described by Gonzalez and Tsien
(See, Gonzalez, J. E. and R. Y. Tsien (1995) "Voltage sensing by
fluorescence resonance energy transfer in single cells" Biophys J
69(4): 1272-80, and Gonzalez, J. E. and R. Y. Tsien (1997)
"Improved indicators of cell membrane potential that use
fluorescence resonance energy transfer" Chem Biol 4(4): 269-77) in
combination with instrumentation for measuring fluorescence changes
such as the Voltage/Ion Probe Reader (VIPR) (See, Gonzalez, J. E.,
K. Oades, et al. (1999) "Cell-based assays and instrumentation for
screening ion-channel targets" Drug Discov Today 4(9):
431-439).
[0277] These voltage sensitive assays are based on the change in
fluorescence resonant energy transfer (FRET) between the
membrane-soluble, voltage-sensitive dye, DiSBAC.sub.2(3), and a
fluorescent phospholipid, CC2-DMPE, which is attached to the outer
leaflet of the plasma membrane and acts as a FRET donor. Changes in
membrane potential (V.sub.m) cause the negatively charged
DiSBAC.sub.2(3) to redistribute across the plasma membrane and the
amount of energy transfer from CC2-DMPE changes accordingly. The
changes in fluorescence emission were monitored using VIPR.TM. II,
which is an integrated liquid handler and fluorescent detector
designed to conduct cell-based screens in 96- or 384-well
microtiter plates.
Identification of Correction Compounds
[0278] To identify small molecules that correct the trafficking
defect associated with .DELTA.F508-CFTR; a single-addition HTS
assay format was developed. The cells were incubated in serum-free
medium for 16 hrs at 37.degree. C. in the presence or absence
(negative control) of test compound. As a positive control, cells
plated in 384-well plates were incubated for 16 hrs at 27.degree.
C. to "temperature-correct" .DELTA.F508-CFTR. The cells were
subsequently rinsed 3.times. with Krebs Ringers solution and loaded
with the voltage-sensitive dyes. To activate .DELTA.F508-CFTR, 10
.mu.M forskolin and the CFTR potentiator, genistein (20 .mu.M),
were added along with Cl.sup.--free medium to each well. The
addition of Cl.sup.--free medium promoted Cl.sup.- efflux in
response to .DELTA.F508-CFTR activation and the resulting membrane
depolarization was optically monitored using the FRET-based
voltage-sensor dyes.
Identification of Potentiator Compounds
[0279] To identify potentiators of .DELTA.F508-CFTR, a
double-addition HTS assay format was developed. During the first
addition, a Cl.sup.--free medium with or without test compound was
added to each well. After 22 sec, a second addition of
Cl.sup.--free medium containing 2-10 .mu.M forskolin was added to
activate \F508-CFTR. The extracellular Cl.sup.- concentration
following both additions was 28 mM, which promoted Cl.sup.- efflux
in response to .DELTA.F508-CFTR activation and the resulting
membrane depolarization was optically monitored using the
FRET-based voltage-sensor dyes.
Solutions
[0280] Bath Solution #1: (in mM) NaCl 160, KCl 4.5, CaCl.sub.2 2,
MgCl.sub.2 1, HEPES 10, pH 7.4 with NaOH. [0281] Chloride-free bath
solution: Chloride salts in Bath Solution #1 are substituted with
gluconate salts. [0282] CC2-DMPE: Prepared as a 10 mM stock
solution in DMSO and stored at -20.degree. C. [0283]
DiSBAC.sub.2(3): Prepared as a 10 mM stock in DMSO and stored at
-20.degree. C.
Cell Culture
[0284] NIH3T3 mouse fibroblasts stably expressing .DELTA.F508-CFTR
are used for optical measurements of membrane potential. The cells
are maintained at 37.degree. C. in 5% CO.sub.2 and 90% humidity in
Dulbecco's modified Eagle's medium supplemented with 2 mM
glutamine, 10% fetal bovine serum, 1.times. NEAA, .beta.-ME,
1.times. pen/strep, and 25 mM HEPES in 175 cm.sup.2 culture flasks.
For all optical assays, the cells were seeded at 30,000/well in
384-well matrigel-coated plates and cultured for 2 hrs at
37.degree. C. before culturing at 27.degree. C. for 24 hrs. for the
potentiator assay. For the correction assays, the cells are
cultured at 27.degree. C. or 37.degree. C. with and without
compounds for 16-24 hours)
Electrophysiological Assays for Assaying .DELTA.F508-CFTR
Modulation Properties of Compounds
1. Ussing Chamber Assay
[0285] Ussing chamber experiments were performed on polarized
epithelial cells expressing .DELTA.F508-CFTR to further
characterize the .DELTA.F508-CFTR modulators identified in the
optical assays. FRT.sup..DELTA.F508-CFTR epithelial cells grown on
Costar Snapwell cell culture inserts were mounted in an Ussing
chamber (Physiologic Instruments, Inc., San Diego, Calif.), and the
monolayers were continuously short-circuited using a Voltage-clamp
System (Department of Bioengineering, University of Iowa, Iowa,
and, Physiologic Instruments, Inc., San Diego, Calif.).
Transepithelial resistance was measured by applying a 2-mV pulse.
Under these conditions, the FRT epithelia demonstrated resistances
of 4 K.OMEGA./cm.sup.2 or more. The solutions were maintained at
27.degree. C. and bubbled with air. The electrode offset potential
and fluid resistance were corrected using a cell-free insert. Under
these conditions, the current reflects the flow of Cl.sup.- through
.DELTA.F508-CFTR expressed in the apical membrane. The I.sub.SC was
digitally acquired using an MP100A-CE interface and AcqKnowledge
software (v3.2.6; BIOPAC Systems, Santa Barbara, Calif.).
Identification of Correction Compounds
[0286] Typical protocol utilized a basolateral to apical membrane
Cl.sup.- concentration gradient. To set up this gradient, normal
ringer was used on the basolateral membrane, whereas apical NaCl
was replaced by equimolar sodium gluconate (titrated to pH 7.4 with
NaOH) to give a large Cl.sup.- concentration gradient across the
epithelium. All experiments were performed with intact monolayers.
To fully activate .DELTA.F508-CFTR, forskolin (10 .mu.M) and the
PDE inhibitor, IBMX (100 .mu.M), were applied followed by the
addition of the CFTR potentiator, genistein (50 .mu.M).
[0287] As observed in other cell types, incubation at low
temperatures of FRT cells stably expressing .DELTA.F508-CFTR
increases the functional density of CFTR in the plasma membrane. To
determine the activity of correction compounds, the cells were
incubated with 10 .mu.M of the test compound for 24 hours at
37.degree. C. and were subsequently washed 3.times. prior to
recording. The cAMP- and genistein-mediated I.sub.SC in
compound-treated cells was normalized to the 27.degree. C. and
37.degree. C. controls and expressed as percentage activity.
Preincubation of the cells with the correction compound
significantly increased the cAMP- and genistein-mediated I.sub.SC
compared to the 37.degree. C. controls.
Identification of Potentiator Compounds
[0288] Typical protocol utilized a basolateral to apical membrane
Cl.sup.- concentration gradient. To set up this gradient, normal
ringers was used on the basolateral membrane and was permeabilized
with nystatin (360 .mu.g/ml), whereas apical NaCl was replaced by
equimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give a
large Cl.sup.- concentration gradient across the epithelium. All
experiments were performed 30 min after nystatin permeabilization.
Forskolin (10 .mu.M) and all test compounds were added to both
sides of the cell culture inserts. The efficacy of the putative
.DELTA.F508-CFTR potentiators was compared to that of the known
potentiator, genistein.
Solutions
[0289] Basolateral solution (in mM): NaCl (135), CaCl.sub.2 (1.2),
MgCl.sub.2 (1.2), K.sub.2HPO.sub.4 (2.4), KHPO.sub.4 (0.6),
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (10),
and dextrose (10). The solution was titrated to pH 7.4 with NaOH.
[0290] Apical solution (in mM): Same as basolateral solution with
NaCl replaced with Na Gluconate (135).
Cell Culture
[0291] Fisher rat epithelial (FRT) cells expressing
.DELTA.F508-CFTR (FRT.sup..DELTA.F508-CFTR) were used for Ussing
chamber experiments for the putative .DELTA.F508-CFTR modulators
identified from our optical assays. The cells were cultured on
Costar Snapwell cell culture inserts and cultured for five days at
37.degree. C. and 5% CO.sub.2 in Coon's modified Ham's F-12 medium
supplemented with 5% fetal calf serum, 100 U/ml penicillin, and 100
.mu.g/ml streptomycin. Prior to use for characterizing the
potentiator activity of compounds, the cells were incubated at
27.degree. C. for 16-48 hrs to correct for the .DELTA.F508-CFTR. To
determine the activity of corrections compounds, the cells were
incubated at 27.degree. C. or 37.degree. C. with and without the
compounds for 24 hours.
2. Whole-Cell Recordings
[0292] The macroscopic .DELTA.F508-CFTR current (I.sub..DELTA.F508)
in temperature- and test compound-corrected NIH3T3 cells stably
expressing .DELTA.F508-CFTR were monitored using the
perforated-patch, whole-cell recording. Briefly, voltage-clamp
recordings of I.sub..DELTA.F508 were performed at room temperature
using an Axopatch 200B patch-clamp amplifier (Axon Instruments
Inc., Foster City, Calif.). All recordings were acquired at a
sampling frequency of 10 kHz and low-pass filtered at 1 kHz.
Pipettes had a resistance of 5-6 M.OMEGA. when filled with the
intracellular solution. Under these recording conditions, the
calculated reversal potential for Cl.sup.- (E.sub.Cl) at room
temperature was -28 mV. All recordings had a seal resistance >20
G.OMEGA. and a series resistance <15 M.OMEGA.. Pulse generation,
data acquisition, and analysis were performed using a PC equipped
with a Digidata 1320 A/D interface in conjunction with Clampex 8
(Axon Instruments Inc.). The bath contained <250 .mu.l of saline
and was continuously perifused at a rate of 2 ml/min using a
gravity-driven perfusion system.
Identification of Correction Compounds
[0293] To determine the activity of correction compounds for
increasing the density of functional .DELTA.F508-CFTR in the plasma
membrane, we used the above-described perforated-patch-recording
techniques to measure the current density following
[0294] 24-hr treatment with the correction compounds. To fully
activate .DELTA.F508-CFTR, 10 .mu.M forskolin and 20 .mu.l
genistein were added to the cells. Under our recording conditions,
the current density following 24-hr incubation at 27.degree. C. was
higher than that observed following 24-hr incubation at 37.degree.
C. These results are consistent with the known effects of
low-temperature incubation on the density of .DELTA.F508-CFTR in
the plasma membrane. To determine the effects of correction
compounds on CFTR current density, the cells were incubated with 10
.mu.M of the test compound for 24 hours at 37.degree. C. and the
current density was compared to the 27.degree. C. and 37.degree. C.
controls (% activity). Prior to recording, the cells were washed
3.times. with extracellular recording medium to remove any
remaining test compound. Preincubation with 10 .mu.M of correction
compounds significantly increased the cAMP- and genistein-dependent
current compared to the 37.degree. C. controls.
Identification of Potentiator Compounds
[0295] The ability of .DELTA.F508-CFTR potentiators to increase the
macroscopic .DELTA.F508-CFTR Cl.sup.- current (I.sub..DELTA.F508)
in NIH3T3 cells stably expressing .DELTA.F508-CFTR was also
investigated using perforated-patch-recording techniques. The
potentiators identified from the optical assays evoked a
dose-dependent increase in I.sub..DELTA.F508 with similar potency
and efficacy observed in the optical assays. In all cells examined,
the reversal potential before and during potentiator application
was around -30 mV, which is the calculated E.sub.Cl (-28 mV).
Solutions
[0296] Intracellular solution (in mM): Cs-aspartate (90), CsCl
(50), MgCl.sub.2 (1), HEPES (10), and 240 .mu.g/ml amphotericin-B
(pH adjusted to 7.35 with CsOH). [0297] Extracellular solution (in
mM): N-methyl-D-glucamine (NMDG)-Cl (150), MgCl.sub.2 (2),
CaCl.sub.2 (2), HEPES (10) (pH adjusted to 7.35 with HCl).
Cell Culture
[0298] NIH3T3 mouse fibroblasts stably expressing .DELTA.F508-CFTR
are used for whole-cell recordings. The cells are maintained at
37.degree. C. in 5% CO.sub.2 and 90% humidity in Dulbecco's
modified Eagle's medium supplemented with 2 mM glutamine, 10% fetal
bovine serum, 1.times. NEAA, .beta.-ME, 1.times. pen/strep, and 25
mM HEPES in 175 cm.sup.2 culture flasks. For whole-cell recordings,
2,500-5,000 cells were seeded on poly-L-lysine-coated glass
coverslips and cultured for 24-48 hrs at 27.degree. C. before use
to test the activity of potentiators; and incubated with or without
the correction compound at 37.degree. C. for measuring the activity
of correctors.
3. Single-Channel Recordings
[0299] The single-channel activities of temperature-corrected
.DELTA.F508-CFTR stably expressed in NIH3T3 cells and activities of
potentiator compounds were observed using excised inside-out
membrane patch. Briefly, voltage-clamp recordings of single-channel
activity were performed at room temperature with an Axopatch 200B
patch-clamp amplifier (Axon Instruments Inc.). All recordings were
acquired at a sampling frequency of 10 kHz and low-pass filtered at
400 Hz. Patch pipettes were fabricated from Corning Kovar Sealing
#7052 glass (World Precision Instruments, Inc., Sarasota, Fla.) and
had a resistance of 5-8 M.OMEGA. when filled with the extracellular
solution. The .DELTA.F508-CFTR was activated after excision, by
adding 1 mM Mg-ATP, and 75 nM of the cAMP-dependent protein kinase,
catalytic subunit (PKA; Promega Corp. Madison, Wis.). After channel
activity stabilized, the patch was perifused using a gravity-driven
microperfusion system. The inflow was placed adjacent to the patch,
resulting in complete solution exchange within 1-2 sec. To maintain
.DELTA.F508-CFTR activity during the rapid perifusion, the
nonspecific phosphatase inhibitor F.sup.- (10 mM NaF) was added to
the bath solution. Under these recording conditions, channel
activity remained constant throughout the duration of the patch
recording (up to 60 min). Currents produced by positive charge
moving from the intra- to extracellular solutions (anions moving in
the opposite direction) are shown as positive currents. The pipette
potential (V.sub.p) was maintained at 80 mV.
[0300] Channel activity was analyzed from membrane patches
containing .ltoreq.2 active channels. The maximum number of
simultaneous openings determined the number of active channels
during the course of an experiment. To determine the single-channel
current amplitude, the data recorded from 120 sec of
.DELTA.F508-CFTR activity was filtered "off-line" at 100 Hz and
then used to construct all-point amplitude histograms that were
fitted with multigaussian functions using Bio-Patch Analysis
software (Bio-Logic Comp. France). The total microscopic current
and open probability (P.sub.o) were determined from 120 sec of
channel activity. The P.sub.o was determined using the Bio-Patch
software or from the relationship P.sub.o=I/i(N), where I=mean
current, i=single-channel current amplitude, and N=number of active
channels in patch.
Solutions
[0301] Extracellular solution (in mM): NMDG (150), aspartic acid
(150), CaCl.sub.2 (5), MgCl.sub.2 (2), and HEPES (10) (pH adjusted
to 7.35 with Tris base). [0302] Intracellular solution (in mM):
NMDG-Cl (150), MgCl.sub.2 (2), EGTA (5), TES (10), and Tris base
(14) (pH adjusted to 7.35 with HCl).
Cell Culture
[0303] NIH3T3 mouse fibroblasts stably expressing .DELTA.F508-CFTR
are used for excised-membrane patch-clamp recordings. The cells are
maintained at 37.degree. C. in 5% CO.sub.2 and 90% humidity in
Dulbecco's modified Eagle's medium supplemented with 2 mM
glutamine, 10% fetal bovine serum, 1.times. NEAA, .beta.-ME,
1.times.pen/strep, and 25 mM HEPES in 175 cm.sup.2 culture flasks.
For single channel recordings, 2,500-5,000 cells were seeded on
poly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at
27.degree. C. before use.
[0304] Compounds of the invention demonstrated activity as
modulators of ATP binding cassette transporters, specifically
CFTR.
Example 5
cAMP Measurements of Certain Compounds
[0305] This example shows that certain compounds with similar
structures have varying effects on cAMP (adenosine 3, 5-cyclic
monophosphate) levels. ABC Transfer proteins, and CFTR in
particular are cAMP regulated ion channels. Ideally, a modulator
compound of such a protein should not cause a change in cAMP
levels.
[0306] In the following example, the effect on cAMP levels by three
structurally similar compounds from Table 1 were determined using
the cAMP levels from 20 .mu.M forskolin as the normalized reference
measure.
Tropix.RTM. Assay for Measurement of cAMP
[0307] The level of cAMP in FRT cells following 0.5 .mu.M forskolin
or test compound application was determined using a commercially
available chemiluminiscent immunoassay system for mammalian cells
called Tropix.RTM. (Applied Biosystems, Bedford, Mass.). Briefly,
FRT cells were incubated for 15 minutes with a test compound in the
presence and absence of 0.5 .mu.M forskolin. The compounds were
aspirated and the cells were then lysed and transferred along with
the lysis buffer to a 96-well Tropix.RTM. ELISA plate. A cAMP-Alk
Phos conjugate is then added to the assay plate, followed by the
addition of cAMP anti-body. After several wash and aspiration
steps, Sapphire blue II solution is added and the fluorescence
emission is read on the Topcount fluorescence reader, and the cAMP
concentrations were determined using a cAMP standard curve that was
present in each plate.
Results
[0308] The compound
N-((1-(3,4-dimethoxyphenyl)-cyclopentyl)methyl)benzofuran-2-carboxamide,
(Table 1, Compound 49), has previously been reported in the
literature as a potentiator of .DELTA.F508-CFTR (J Biol Chem;
277(40): 37235-41, 2002). The authors have shown that the mechanism
of this activation was via the rise in cellular cAMP. This compound
was shown to increase cAMP content alone and also potentiated the
cAMP elevation educed by the low concentration (0.5 .mu.M) of
forskolin, similar to that found for 20 .mu.M forskolin. We were
able to produce similar results in our Tropix.RTM. system. Compound
49 alone, generated an average of 40.3.+-.3.5% of cAMP produced by
20 .mu.M of forskolin, which was a significant increase compared
with the DMSO control, 24.8.+-.3.9% of 20 .mu.M forskolin, n=4,
p<0.05. In the presence of 0.5 .mu.M forskolin, compound 49
generated an average of 92.3.+-.2.7% of cAMP produced by 20 .mu.M
of forskolin, which was also a significant increase compared with
the 0.5 .mu.M forskolin control, 45.9.+-.3.0% of 20 .mu.M
forskolin, n=4, p<0.05.
[0309] It has been surprisingly found that compounds with similar
structures show statistically significant varying levels of
activities in this cAMP assay. For example, the compound
N-(2-(3,4-dimethoxyphenyl)-2-methylpropyl)benzofuran-2-carboxamide
(Table 1, Compound 29) alone, generated an average of 24.4.+-.1.1%
of cAMP produced by 20 .mu.M of forskolin, which was not a
significant increase compared with the DMSO control 24.8.+-.3.9% of
20 .mu.M forskolin, n=4. But in the presence of 0.5 .mu.M
forskolin, Compound 29 generated an average of 61.5.+-.1.8% of cAMP
produced by 20 .mu.M of forskolin, which was a significant increase
compared with the 0.5 .mu.M forskolin control, 45.9.+-.3.0% of 20
.mu.M forskolin, n=4, p<0.05.
[0310] In comparison, the compound
N-((1-(3,4-dimethoxyphenyl)cyclohexyl)methyl)benzofuran-2-carboxamide,
Table 1, Compound 21, by itself generated an average of 7.9.+-.1.1%
of cAMP produced by 20 .mu.M of forskolin, which was not a
significant increase compared with the DMSO control of 8.4.+-.2.8%
of 20 .mu.M forskolin, n=4. Surprisingly, Compound 21 in the
presence of 0.5 .mu.M forskolin generated an average of
27.1.+-.1.8% of cAMP produced by 20 .mu.M of forskolin, which was
also not a significant increase compared with the 0.5 .mu.M
forskolin control, 32.2.+-.3.2% of 20 .mu.M forskolin, n=4.
[0311] The example teaches that compounds can have potentiator
activity without having an accompanying increase in cAMP
concentrations.
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References