U.S. patent application number 14/985650 was filed with the patent office on 2016-08-04 for modulators of cystic fibrosis transmembrane conductance regulator.
This patent application is currently assigned to Vertex Pharmaceuticals Incorporated. The applicant listed for this patent is Vertex Pharmaceuticals Incorporated. Invention is credited to Martyn Curtis Botfield, Peter Diederik Jan Grootenhuis, Sara Sabina Hadida Ruah, Fredrick F. Van Goor, Xiaoqing Yang, Gregor Zlokarnik.
Application Number | 20160221952 14/985650 |
Document ID | / |
Family ID | 42136194 |
Filed Date | 2016-08-04 |
United States Patent
Application |
20160221952 |
Kind Code |
A1 |
Yang; Xiaoqing ; et
al. |
August 4, 2016 |
MODULATORS OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE
REGULATOR
Abstract
This invention relates to a compound of Formula I: ##STR00001##
or a pharmaceutically acceptable salt thereof, wherein R is COOH or
CH.sub.2OH.
Inventors: |
Yang; Xiaoqing; (San Diego,
CA) ; Hadida Ruah; Sara Sabina; (La Jolla, CA)
; Grootenhuis; Peter Diederik Jan; (San Diego, CA)
; Van Goor; Fredrick F.; (San Diego, CA) ;
Botfield; Martyn Curtis; (Concord, MA) ; Zlokarnik;
Gregor; (La Jolla, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vertex Pharmaceuticals Incorporated |
Boston |
MA |
US |
|
|
Assignee: |
Vertex Pharmaceuticals
Incorporated
Boston
MA
|
Family ID: |
42136194 |
Appl. No.: |
14/985650 |
Filed: |
December 31, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14310634 |
Jun 20, 2014 |
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14985650 |
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|
14084203 |
Nov 19, 2013 |
8796308 |
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14310634 |
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13237303 |
Sep 20, 2011 |
8614325 |
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14084203 |
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PCT/US2010/028062 |
Mar 19, 2010 |
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13237303 |
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61162130 |
Mar 20, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 15/10 20180101;
A61P 31/00 20180101; A61K 45/06 20130101; A61P 1/18 20180101; A61P
3/00 20180101; A61P 25/14 20180101; A61P 29/00 20180101; C07D
215/233 20130101; A61P 5/10 20180101; A61P 31/04 20180101; A61P
11/00 20180101; A61P 5/18 20180101; A61P 7/00 20180101; A61P 5/14
20180101; A61P 11/02 20180101; A61P 13/02 20180101; A61P 31/10
20180101; A61P 37/06 20180101; C07D 215/48 20130101; G01N 33/5041
20130101; G01N 2800/382 20130101; A61P 3/08 20180101; A61P 17/00
20180101; A61P 1/16 20180101; A61P 3/06 20180101; A61P 15/08
20180101; A61P 3/10 20180101; A61P 11/08 20180101; A61P 25/00
20180101; A61P 27/02 20180101; A61P 43/00 20180101; A61P 7/12
20180101; A61P 25/16 20180101; A61P 25/28 20180101; A61P 1/10
20180101; A61P 35/00 20180101; A61P 13/12 20180101; A61P 21/02
20180101; A61P 13/00 20180101; A61P 19/08 20180101; A61P 27/00
20180101; A61P 7/10 20180101; A61P 11/06 20180101; A61K 31/47
20130101; A61P 7/04 20180101; A61P 21/04 20180101; C07D 215/56
20130101; A61P 21/00 20180101; A61P 37/02 20180101; A61P 37/08
20180101; G01N 33/6872 20130101; A61P 3/02 20180101; G01N 2333/705
20130101 |
International
Class: |
C07D 215/56 20060101
C07D215/56; A61K 45/06 20060101 A61K045/06; G01N 33/68 20060101
G01N033/68; A61K 31/47 20060101 A61K031/47 |
Claims
1. A compound of Formula I: ##STR00083## or a pharmaceutically
acceptable salt thereof, wherein R is COOH or CH.sub.2OH.
2. A compound according to claim 1, wherein R is CH.sub.2OH.
3. A compound according to claim 1, wherein R is COOH.
4. (canceled)
5. (canceled)
6. A pharmaceutical composition comprising: i. a compound of
Formula I according to claim 1 or a pharmaceutically acceptable
salt thereof; and ii. a pharmaceutically acceptable carrier or
adjuvant.
7. The pharmaceutical composition according to claim 6, wherein the
compound of Formula I has the structure: ##STR00084##
8. The pharmaceutical composition according to claim 6, wherein the
compound of Formula I has the structure: ##STR00085##
9. The pharmaceutical composition according to claim 6, further
comprising an additional agent selected from a mucolytic agent, a
bronchodilator, an antibiotic, an anti-invective agent, an
anti-inflammatory agent, a CFTR modulator, or a nutritional
agent.
10. A method of modulating CFTR activity in a biological sample
comprising the step of contacting said biological sample with a
compound of Formula I according to claim 1 or a pharmaceutically
acceptable salt thereof.
11. The method of modulating CFTR activity in a biological sample
according to claim 10, wherein the compound of Formula I has the
structure: ##STR00086##
12. The method of modulating CFTR activity in a biological sample
according to claim 10, wherein the compound of Formula I has the
structure: ##STR00087##
13. A method of treating or lessening the severity of a disease in
a patient comprising administering to said patient an effective
amount of a compound of Formula I according to claim 1 or a
pharmaceutically acceptable salt thereof, wherein said disease is
selected from cystic fibrosis, asthma, smoke induced COPD, chronic
bronchitis, rhinosinusitis, constipation, pancreatitis, pancreatic
insufficiency, male infertility caused by congenital bilateral
absence of the vas deferens (CBAVD), mild pulmonary disease,
idiopathic pancreatitis, allergic bronchopulmonary aspergillosis
(ABPA), liver 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, 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.
14. The method of claim 13, wherein the compound of Formula I has
the structure: ##STR00088##
15. The method of claim 13, wherein the compound of Formula I has
the structure: ##STR00089##
16. The method according to claim 13, wherein said disease is
cystic fibrosis.
17. A kit for use in measuring the activity of CFTR or a fragment
thereof in a biological sample in vitro or in vivo, comprising: i.
a composition comprising a compound of Formula I or a
pharmaceutically acceptable salt thereof according to claim 1; ii.
and instructions for: a. contacting the composition with the
biological sample; and b. measuring the activity of said CFTR or a
fragment thereof.
18. The kit of claim 17, further comprising instructions for: i.
contacting an additional compound with the biological sample; ii.
measuring the activity of said CFTR or a fragment thereof in the
presence of said additional compound; and iii. comparing the
activity of the CFTR or a fragment thereof in the presence of the
additional compound with the activity of the CFTR or a fragment
thereof in the presence of a composition of Formula I.
19. The kit according to claim 18, wherein the step of comparing
the activity of said CFTR or a fragment thereof provides a measure
of the density of said CFTR or a fragment thereof.
20. The kit of claim 17, wherein the compound of Formula I has the
structure: ##STR00090##
21. The kit of claim 17, wherein the compound of Formula I has the
structure: ##STR00091##
Description
CLAIM OF PRIORITY
[0001] This application is a continuation of PCT Application No.
PCT/US2010/028062 filed Mar. 19, 2010, which claims the priority of
U.S. Application No. 61/162,130 filed Mar. 20, 2009, which is
incorporated by reference herein in its entirety.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to modulators of cystic
fibrosis transmembrane conductance regulator ("CFTR"), compositions
thereof, and methods therewith. The present invention also relates
to methods of treating diseases using modulators of CFTR.
BACKGROUND OF THE INVENTION
[0003] Cystic fibrosis (CF) is a recessive genetic disease that
affects approximately 30,000 children and adults in the United
States and approximately 30,000 children and adults in Europe.
Despite progress in the treatment of CF, there is no cure.
[0004] CF is caused by mutations in the cystic fibrosis
transmembrane conductance regulator (CFTR) gene that encodes an
epithelial chloride ion channel responsible for aiding in the
regulation of salt and water absorption and secretion in various
tissues. Small molecule drugs, known as potentiators that increase
the probability of CFTR channel opening represent one potential
therapeutic strategy to treat CF.
[0005] Specifically, CFTR is a cAMP/ATP-mediated anion channel that
is expressed in a variety of cells types, including absorptive and
secretory epithelia cells, where it regulates anion flux across the
membrane, as well as the activity of other ion channels and
proteins. In epithelia cells, normal functioning of CFTR is
critical for the maintenance of electrolyte transport throughout
the body, including respiratory and digestive tissue. CFTR is
composed of approximately 1480 amino acids that encode a protein
made up of a tandem repeat of transmembrane domains, each
containing six transmembrane helices and a nucleotide binding
domain. The two transmembrane domains are linked by a large, polar,
regulatory (R)-domain with multiple phosphorylation sites that
regulate channel activity and cellular trafficking.
[0006] The gene encoding 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 causes mutations in
CFTR resulting in cystic fibrosis ("CF"), the most common fatal
genetic disease in humans. Cystic fibrosis affects approximately
one in every 2,500 infants 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 debilitating and fatal effects of CF, including
chronic lung disease.
[0007] In patients with CF, mutations in CFTR endogenously
expressed in respiratory epithelia leads to reduced apical anion
secretion causing an imbalance in ion and fluid transport. The
resulting decrease in anion transport contributes to enhanced mucus
accumulation in the lung and the accompanying microbial infections
that ultimately cause death in CF patients. In addition to
respiratory disease, CF patients typically suffer from
gastrointestinal problems and pancreatic insufficiency that, if
left untreated, results in death. In addition, the majority of
males with cystic fibrosis are infertile and fertility is decreased
among females with cystic fibrosis. 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).
To date, over 1000 disease causing mutations in the CF gene have
been identified (http://www.genet.sickkids.on.ca/cftr/app). The
most prevalent mutation is a deletion of phenylalanine at position
508 of the CFTR amino acid sequence, and is commonly referred to as
.DELTA.F508-CFTR. This mutation occurs in approximately 70% of the
cases of cystic fibrosis and is associated with a severe
disease.
[0009] The deletion of residue 508 in .DELTA.F508-CFTR prevents the
nascent protein from folding correctly. This results in the
inability of the mutant protein to exit the ER, and traffic to the
plasma membrane. As a result, the number of channels present in the
membrane is far less than observed in cells expressing wild-type
CFTR. In addition to impaired trafficking, the mutation results in
defective channel gating. Together, the reduced number of channels
in the membrane and the defective gating lead to reduced anion
transport across epithelia leading to defective ion and fluid
transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studies
have shown, however, that the reduced numbers of .DELTA.F508-CFTR
in the membrane are functional, albeit less than wild-type CFTR.
(Dalemans et al. (1991), Nature Lond. 354: 526-528; Denning et al.,
supra; Pasyk and Foskett (1995), J. Cell. Biochem. 270: 12347-50).
In addition to .DELTA.F508-CFTR, other disease causing mutations in
CFTR that result in defective trafficking, synthesis, and/or
channel gating could be up- or down-regulated to alter anion
secretion and modify disease progression and/or severity.
[0010] Although CFTR transports a variety of molecules in addition
to anions, it is clear that this role (the transport of anions)
represents one element in an important mechanism of 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 ENaC and CFTR present on the apical
membrane and the Na.sup.+--K.sup.+-ATPase pump and Cl ion 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] 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.
[0013] Accordingly, there is a need for modulators of CFTR
activity, and compositions thereof, which can be used to modulate
the activity of the CFTR in the cell membrane of a mammal.
[0014] There is a need for methods of treating diseases caused by
mutation in CFTR using such modulators of CFTR activity.
[0015] There is a need for methods of modulating CFTR activity in
an ex vivo cell membrane of a mammal.
SUMMARY OF THE INVENTION
[0016] In one aspect, this invention relates to a compound of
Formula I:
##STR00002##
or a pharmaceutically acceptable salt thereof, wherein R is COOH or
CH.sub.2OH.
[0017] In one embodiment, the compound has the structure:
##STR00003##
or a pharmaceutically acceptable salt thereof.
[0018] In another embodiment, the compound has the structure:
##STR00004##
or a pharmaceutically acceptable salt thereof.
[0019] In one aspect, this invention relates to a pharmaceutical
composition including a compound of Formula I, or a
pharmaceutically acceptable salt thereof, wherein R is defined as
above, and a pharmaceutically acceptable carrier or adjuvant.
[0020] In one embodiment of the pharmaceutical composition, the
compound has the structure:
##STR00005##
or a pharmaceutically acceptable salt thereof.
[0021] In another embodiment of the pharmaceutical composition, the
compound has the structure:
##STR00006##
or a pharmaceutically acceptable salt thereof.
[0022] Embodiments of this aspect may also include a pharmaceutical
composition containing an additional agent selected from a
mucolytic agent, a bronchodilator, an antibiotic, an anti-infective
agent, an anti-inflammatory agent, a CFTR modulator, or a
nutritional agent.
[0023] In one aspect, this invention includes a method of
modulating CFTR activity in a biological sample comprising the step
of contacting said biological sample with a compound of Formula I,
or a pharmaceutically acceptable salt thereof, wherein R is defined
as above.
[0024] In one embodiment of the method of modulating CFTR activity,
the compound has the structure:
##STR00007##
or a pharmaceutically acceptable salt thereof.
[0025] In another embodiment of the method of modulating CFTR
activity, the compound has the structure:
##STR00008##
or a pharmaceutically acceptable salt thereof.
[0026] In another aspect, the invention also provides a method of
treating or lessening the severity of a disease in a patient
comprising administering to said patient one of the compositions as
defined herein, and said disease is selected from cystic fibrosis,
asthma, smoke induced COPD, chronic bronchitis, rhinosinusitis,
constipation, pancreatitis, pancreatic insufficiency, male
infertility caused by congenital bilateral absence of the vas
deferens (CBAVD), mild pulmonary disease, idiopathic pancreatitis,
allergic bronchopulmonary aspergillosis (ABPA), liver 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, 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's, 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, Osteoporosis,
Osteopenia, bone healing and bone growth (including bone repair,
bone regeneration, reducing bone resorption and increasing bone
deposition), Gorham's Syndrome, chloride channelopathies such as
myotonia congenita (Thomson and Becker forms), Bartter's syndrome
type III, Dent's disease, epilepsy, hyperekplexia, lysosomal
storage disease, Angelman syndrome, and Primary Ciliary Dyskinesia
(PCD), a term for inherited disorders of the structure and/or
function of cilia, including PCD with situs inversus (also known as
Kartagener syndrome), PCD without situs inversus and ciliary
aplasia.
[0027] In another embodiment of the method of treating or lessening
the severity of a disease, the disease is selected from CF, COPD,
smoke induced COPD, pancreatitis, and rhinosinusitis.
[0028] In certain embodiments, the disease is cystic fibrosis.
[0029] In one embodiment of the method of treating or lessening the
severity of a disease, the compound has the structure:
##STR00009##
or a pharmaceutically acceptable salt thereof.
[0030] In another embodiment of the method of treating or lessening
the severity of a disease, the compound has the structure:
##STR00010##
or a pharmaceutically acceptable salt thereof.
[0031] In another aspect, the present invention provides a kit for
use in measuring the activity of CFTR or a fragment thereof in a
biological sample in vitro or in vivo comprising (i) a composition
comprising a compound of Formula I or any of the above embodiments;
and (ii) instructions for a) contacting the composition with the
biological sample and b) measuring activity of said CFTR or a
fragment thereof.
[0032] In one embodiment of this aspect, the kit further comprises
instructions for a) contacting an additional composition with the
biological sample; b) measuring the activity of said CFTR or a
fragment thereof in the presence of said additional compound, and
c) comparing the activity of the CFTR or a fragment thereof in the
presence of the additional compound with the density of the CFTR or
a fragment thereof in the presence of a composition of Formula
I.
[0033] In preferred embodiments, the kit is used to measure the
density of CFTR or a fragment thereof.
[0034] In a further preferred embodiment, the kit is used to
measure the density of said CFTR or a fragment thereof and the step
of comparing the activity of said CFTR or a fragment thereof
provides a measure of the density of said CFTR or a fragment
thereof.
[0035] In one embodiment of the kit for use in measuring the
activity of CFTR, the compound has the structure:
##STR00011##
or a pharmaceutically acceptable salt thereof.
[0036] In another embodiment of the kit for use in measuring the
activity of CFTR, the compound has the structure:
##STR00012##
or a pharmaceutically acceptable salt thereof.
[0037] The compounds of Formula I provide advantageous properties
such as, but not limited to, increased polarity, favorable aqueous
solubility, lower distribution volume and lower tissue
penetration.
DETAILED DESCRIPTION
Definitions
[0038] Compounds of this invention include those described
generally above, and are further illustrated by the classes,
subclasses, and species disclosed herein. As used herein, the
following definitions shall apply unless otherwise indicated.
[0039] 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.
[0040] The term "CFTR" as used herein means cystic fibrosis
transmembrane conductance regulator or a mutation thereof capable
of regulator activity, including, but not limited to, .DELTA.F508
CFTR and G551D CFTR (see, e.g.,
http://www.genet.sickkids.on.ca/cftr/, for CFTR mutations).
[0041] The term "modulating" as used herein means increasing or
decreasing by a measurable amount.
[0042] 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, 75.sup.th 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.
[0043] 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.
[0044] The term "protecting group" (PG) as used herein, represents
those groups intended to protect a functional group, such as, for
example, an alcohol, amine, carboxyl, carbonyl, etc., against
undesirable reactions during synthetic procedures. Commonly used
protecting groups are disclosed in Greene and Wuts, Protective
Groups in Organic Synthesis, 3.sup.rd Edition (John Wiley &
Sons, New York, 1999), which is incorporated herein by reference.
Examples of nitrogen protecting groups include acyl, aroyl, or
carbamyl groups such as formyl, acetyl, propionyl, pivaloyl,
t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl,
trichloroacetyl, phthalyl, o-nitrophenoxyacetyl,
.alpha.-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl,
4-nitrobenzoyl and chiral auxiliaries such as protected or
unprotected D, L or D, L-amino acids such as alanine, leucine,
phenylalanine and the like; sulfonyl groups such as
benzenesulfonyl, p-toluenesulfonyl and the like; carbamate groups
such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,
p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,
2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,
3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl,
2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,
2-nitro-4,5-dimethoxybenzyloxycarbonyl,
3,4,5-trimethoxybenzyloxycarbonyl,
1-(p-biphenylyl)-1-methylethoxycarbonyl,
.alpha.,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl,
benzhydryloxycarbonyl, t-butyloxycarbonyl,
diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,
methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl,
phenoxycarbonyl, 4-nitrophenoxy carbonyl,
fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,
adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and
the like, arylalkyl groups such as benzyl, triphenylmethyl,
benzyloxymethyl and the like and silyl groups such as
trimethylsilyl and the like. Preferred N-protecting groups are
tert-butyloxycarbonyl (Boc).
[0045] Examples of useful protecting groups for acids are
substituted alkyl esters such as 9-fluorenylmethyl, methoxymethyl,
methylthiomethyl, tetrahydropyranyl, tetrahydrofuranyl,
methoxyethoxymethyl, 2-(trimethylsilyl)ethoxymethyl,
benzyloxymethyl, pivaloyloxymethyl, phenylacetoxymethyl,
triisopropropylsysilylmethyl, cyanomethyl, acetol, phenacyl,
substituted phenacyl esters, 2,2,2-trichloroethyl, 2-haloethyl,
w-chloroalkyl, 2-(trimethylsilyl)ethyl, 2-methylthioethyl, t-butyl,
3-methyl-3-pentyl, dicyclopropylmethyl, cyclopentyl, cyclohexyl,
allyl, methallyl, cynnamyl, phenyl, silyl esters, benzyl and
substituted benzyl esters, 2,6-dialkylphenyl esters such as
pentafluorophenyl, 2,6-dialkylpyhenyl. Preferred protecting groups
for acids are methyl or ethyl esters.
[0046] Methods of adding (a process generally referred to as
"protection") and removing (process generally referred to as
"deprotection") such amine and acid protecting groups are
well-known in the art and available, for example in P. J.
Kocienski, Protecting Groups, Thieme, 1994, which is hereby
incorporated by reference in its entirety and in Greene and Wuts,
Protective Groups in Organic Synthesis, 3.sup.rd Edition (John
Wiley & Sons, New York, 1999).
[0047] Unless otherwise stated, all tautomeric forms of the
compounds of the invention are within the scope of the invention.
That is, compounds of Formula I may exist as tautomers:
##STR00013##
[0048] 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, probes in biological assays or as therapeutic
agents.
[0049] Examples of suitable solvents are, but not limited to,
water, methanol, dichloromethane (DCM), acetonitrile,
dimethylformamide (DMF), ethyl acetate (EtOAc), isopropyl alcohol
(IPA), isopropyl acetate (IPAc), tetrahydrofuran (THF), methyl
ethyl ketone (MEK), t-butanol and N-methyl pyrrolidone (NMP).
[0050] Examples of suitable coupling agents are, but not limited
to, 1-(3-(dimethylamino)propyl)-3-ethyl-carbodiimide hydrochloride
(EDCI), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBT),
2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate (HATU), 2-chloro-1,3-dimethyl-2-imidazolium
tetrafluoroborate,
1-H-benzotriazolium-1-[bis(dimethylamino)methylene]-5-chlorohexafluoropho-
sphate (HCTU), 2-chloro-4,6-dimethoxy-1,3,5-triazine, and 2-propane
phosphonic anhydride (T3P.RTM.).
[0051] Examples of suitable bases are, but not limited to,
K.sub.2CO.sub.3, N-Methylmorpholine (NMM), triethylamine (TEA),
diisopropyl-ethyl amine (DIEA), pyridine, potassium hydroxide,
sodium hydroxide, and sodium methoxide.
[0052] Compounds
[0053] In one embodiment, the invention includes a compound of
Formula I:
##STR00014##
or a pharmaceutically acceptable salt thereof, wherein R is COOH or
CH.sub.2OH.
[0054] In some embodiments, R is CH.sub.2OH.
[0055] In some embodiments, R is COOH.
[0056] In another embodiment, the invention includes a compound of
the structure:
##STR00015##
or a pharmaceutically acceptable salt thereof.
[0057] In another embodiment, the invention includes a compound of
the structure:
##STR00016##
or a pharmaceutically acceptable salt thereof.
[0058] In one embodiment, the invention includes a pharmaceutical
composition comprising a compound of Formula I, and a
pharmaceutically acceptable carrier or adjuvant.
[0059] In another embodiment, the invention includes a
pharmaceutical composition comprising a compound of the
structure:
##STR00017##
and a pharmaceutically acceptable carrier or adjuvant.
[0060] In another embodiment, the invention includes a
pharmaceutical composition comprising a compound of the
structure:
##STR00018##
and a pharmaceutically acceptable carrier or adjuvant.
[0061] In a another embodiment, the pharmaceutical composition
further comprises an additional agent selected from a mucolytic
agent, a bronchodilator, an antibiotic, an anti-invective agent, an
anti-inflammatory agent, a CFTR modulator, or a nutritional
agent.
III. General Synthesis
[0062] Compounds of Formula I can be synthesized according to
Scheme 1.
##STR00019##
[0063] In Scheme 1, anilines of Formula II, wherein R and OH
optionally and independently bear protecting groups thereon, are
reacted with carboxylic acid intermediates of Formula III under
coupling conditions to form compounds of Formula IV. Compounds of
Formula IV that bear one or more protecting groups can then be
deprotected to provide derivatives of Formula I.
[0064] The coupling reaction described in Scheme 1 can be achieved
by dissolving the reactants in a suitable solvent, treating the
resulting solution with a suitable coupling reagent optionally in
the presence of a suitable base.
[0065] Quinoline derivatives of Formula III can be synthesized
according to Scheme 2.
##STR00020##
[0066] Anilines of Formula II, wherein R is --CH.sub.2OH can be
synthesized according to Scheme 3.
##STR00021## ##STR00022## ##STR00023##
[0067] Anilines of Formula II, wherein R is --COOH can be
synthesized according to Scheme 4.
##STR00024## ##STR00025## ##STR00026##
[0068] Uses and Methods of Use
[0069] Pharmaceutically Acceptable Compositions
[0070] In one 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.
[0071] 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 or a
prodrug thereof. According to the present invention, a
pharmaceutically acceptable derivative or a prodrug 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 thereof is capable of
providing, directly or indirectly, a compound as otherwise
described herein, or a metabolite or residue thereof.
[0072] As used herein, the term "pharmaceutically acceptable salt"
refers to those salts which are, within the scope of sound medical
judgment, 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.
[0073] Pharmaceutically acceptable salts are well known in the art.
For example, S. M. Berge, et al. describes 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, edisylate
(ethanedisulfonate), 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.sup.+(C.sub.1-4alkyl).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.
[0074] 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.
[0075] Uses of Compounds and Pharmaceutically Acceptable
Compositions
[0076] In yet another aspect, the present invention provides a
method of treating, or lessening the severity of a condition,
disease, or disorder implicated by CFTR mutation. In certain
embodiments, the present invention provides a method of treating a
condition, disease, or disorder implicated by a deficiency of the
CFTR activity, the method comprising administering a composition
comprising a compound of Formula I to a subject, preferably a
mammal, in need thereof.
[0077] In another aspect, the invention also provides a method of
treating or lessening the severity of a disease in a patient
comprising administering to said patient one of the compositions as
defined herein, and said disease is selected from cystic fibrosis,
asthma, smoke induced COPD, chronic bronchitis, rhinosinusitis,
constipation, pancreatitis, pancreatic insufficiency, male
infertility caused by congenital bilateral absence of the vas
deferens (CBAVD), mild pulmonary disease, idiopathic pancreatitis,
allergic bronchopulmonary aspergillosis (ABPA), liver 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, 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's, 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, Osteoporosis,
Osteopenia, bone healing and bone growth (including bone repair,
bone regeneration, reducing bone resorption and increasing bone
deposition), Gorham's Syndrome, chloride channelopathies such as
myotonia congenita (Thomson and Becker forms), Bartter's syndrome
type III, Dent's disease, hyperekplexia, epilepsy, hyperekplexia,
lysosomal storage disease, Angelman syndrome, and Primary Ciliary
Dyskinesia (PCD), a term for inherited disorders of the structure
and/or function of cilia, including PCD with situs inversus (also
known as Kartagener syndrome), PCD without situs inversus and
ciliary aplasia.
[0078] In some embodiments, the method includes treating or
lessening the severity of cystic fibrosis in a patient comprising
administering to said patient one of the compositions as defined
herein. In certain embodiments, the patient possesses mutant forms
of human CFTR. In other embodiments, the patient possesses one or
more of the following mutations .DELTA.F508, R117H, and G551D of
human CFTR. In one embodiment, the method includes treating or
lessening the severity of cystic fibrosis in a patient possessing
the .DELTA.F508 mutation of human CFTR comprising administering to
said patient one of the compositions as defined herein. In one
embodiment, the method includes treating or lessening the severity
of cystic fibrosis in a patient possessing the G551D mutation of
human CFTR comprising administering to said patient one of the
compositions as defined herein. In one embodiment, the method
includes treating or lessening the severity of cystic fibrosis in a
patient possessing the .DELTA.F508 mutation of human CFTR on at
least one allele comprising administering to said patient one of
the compositions as defined herein. In one embodiment, the method
includes treating or lessening the severity of cystic fibrosis in a
patient possessing the .DELTA.F508 mutation of human CFTR on both
alleles comprising administering to said patient one of the
compositions as defined herein. In one embodiment, the method
includes treating or lessening the severity of cystic fibrosis in a
patient possessing the G551D mutation of human CFTR on at least one
allele comprising administering to said patient one of the
compositions as defined herein. In one embodiment, the method
includes treating or lessening the severity of cystic fibrosis in a
patient possessing the G551D mutation of human CFTR on both alleles
comprising administering to said patient one of the compositions as
defined herein.
[0079] In some embodiments, the method includes lessening the
severity of cystic fibrosis in a patient comprising administering
to said patient one of the compositions as defined herein. In
certain embodiments, the patient possesses mutant forms of human
CFTR. In other embodiments, the patient possesses one or more of
the following mutations .DELTA.F508, R117H, and G551D of human
CFTR. In one embodiment, the method includes lessening the severity
of cystic fibrosis in a patient possessing the .DELTA.F508 mutation
of human CFTR comprising administering to said patient one of the
compositions as defined herein. In one embodiment, the method
includes lessening the severity of cystic fibrosis in a patient
possessing the G551D mutation of human CFTR comprising
administering to said patient one of the compositions as defined
herein. In one embodiment, the method includes lessening the
severity of cystic fibrosis in a patient possessing the .DELTA.F508
mutation of human CFTR on at least one allele comprising
administering to said patient one of the compositions as defined
herein. In one embodiment, the method includes lessening the
severity of cystic fibrosis in a patient possessing the .DELTA.F508
mutation of human CFTR on both alleles comprising administering to
said patient one of the compositions as defined herein. In one
embodiment, the method includes lessening the severity of cystic
fibrosis in a patient possessing the G551D mutation of human CFTR
on at least one allele comprising administering to said patient one
of the compositions as defined herein. In one embodiment, the
method includes lessening the severity of cystic fibrosis in a
patient possessing the G551D mutation of human CFTR on both alleles
comprising administering to said patient one of the compositions as
defined herein.
[0080] In some aspects, the invention provides a method of treating
or lessening the severity of Osteoporosis in a patient comprising
administering to said patient compound of Formula 1 or a
pharmaceutically acceptable salt thereof.
[0081] In certain embodiments, the method of treating or lessening
the severity of Osteoporosis in a patient comprises administering
to said patient a pharmaceutical composition as described
herein.
[0082] In some aspects, the invention provides a method of treating
or lessening the severity of Osteopenia in a patient comprising
administering to said patient compound of Formula 1 or a
pharmaceutically acceptable salt thereof.
[0083] In certain embodiments, the method of treating or lessening
the severity of Osteopenia in a patient comprises administering to
said patient a pharmaceutical composition as described herein.
[0084] In some aspects, the invention provides a method of bone
healing and/or bone repair in a patient comprising administering to
said patient compound of Formula 1 or a pharmaceutically acceptable
salt thereof.
[0085] In certain embodiments, the method of bone healing and/or
bone repair in a patient comprises administering to said patient a
pharmaceutical composition as described herein.
[0086] In some aspects, the invention provides a method of reducing
bone resorption in a patient comprising administering to said
patient compound of Formula 1 or a pharmaceutically acceptable salt
thereof.
[0087] In some aspects, the invention provides a method of
increasing bone deposition in a patient comprising administering to
said patient compound of Formula 1 or a pharmaceutically acceptable
salt thereof.
[0088] In certain embodiments, the method of increasing bone
deposition in a patient comprises administering to said patient a
pharmaceutical composition as described herein.
[0089] In some aspects, the invention provides a method of treating
or lessening the severity of COPD in a patient comprising
administering to said patient compound of Formula 1 or a
pharmaceutically acceptable salt thereof.
[0090] In certain embodiments, the method of treating or lessening
the severity of COPD in a patient comprises administering to said
patient a pharmaceutical composition as described herein.
[0091] In some aspects, the invention provides a method of treating
or lessening the severity of smoke induced COPD in a patient
comprising administering to said patient compound of Formula 1 or a
pharmaceutically acceptable salt thereof.
[0092] In certain embodiments, the method of treating or lessening
the severity of smoke induced COPD in a patient comprises
administering to said patient a pharmaceutical composition as
described herein.
[0093] In some aspects, the invention provides a method of treating
or lessening the severity of chronic bronchitis in a patient
comprising administering to said patient compound of Formula 1 or a
pharmaceutically acceptable salt thereof.
[0094] In certain embodiments, the method of treating or lessening
the severity of chronic bronchitis in a patient comprises
administering to said patient a pharmaceutical composition as
described herein.
[0095] 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 a compound of the present invention.
[0096] According to the invention an "effective amount" of the
compound or pharmaceutically acceptable composition is that amount
effective for treating or lessening the severity of one or more of
the diseases, disorders or conditions as recited above.
[0097] Another aspect of the present invention provides a method of
administering a pharmaceutical composition by orally administering
to a patient at least once per day the composition comprising a
compound of Formula 1. In one embodiment, the method comprises
administering a pharmaceutical composition comprising a compound of
Formula 1 every 24 hours. In another embodiment, the method
comprises administering a pharmaceutical composition comprising a
compound of Formula 1 every 12 hours. In a further embodiment, the
method comprises administering a pharmaceutical composition
comprising a compound of Formula 1 three times per day. In still a
further embodiment, the method comprises administering a
pharmaceutical composition comprising a compound of Formula 1 every
4 hours.
[0098] 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 the diseases, disorders or conditions as
recited above.
[0099] In certain embodiments, the compounds and compositions of
the present invention are useful for treating or lessening the
severity of cystic fibrosis in patients who exhibit residual CFTR
activity in the apical membrane of respiratory and non-respiratory
epithelia. The presence of residual CFTR activity at the epithelial
surface can be readily detected using methods known in the art,
e.g., standard electrophysiological, biochemical, or histochemical
techniques. Such methods identify CFTR activity using in vivo or ex
vivo electrophysiological techniques, measurement of sweat or
salivary Cl.sup.- concentrations, or ex vivo biochemical or
histochemical techniques to monitor cell surface density. Using
such methods, residual CFTR activity can be readily detected in
patients heterozygous or homozygous for a variety of different
mutations, including patients homozygous or heterozygous for the
most common mutation, .DELTA.F508.
[0100] In another embodiment, the compounds and compositions of the
present invention are useful for treating or lessening the severity
of cystic fibrosis in patients who have residual CFTR activity
induced or augmented using pharmacological methods or gene therapy.
Such methods increase the amount of CFTR present at the cell
surface, thereby inducing a hitherto absent CFTR activity in a
patient or augmenting the existing level of residual CFTR activity
in a patient.
[0101] In one embodiment, the compounds and compositions of the
present invention are useful for treating or lessening the severity
of cystic fibrosis in patients within certain genotypes exhibiting
residual CFTR activity, e.g., class III mutations (impaired
regulation or gating), class IV mutations (altered conductance), or
class V mutations (reduced synthesis) (Lee R. Choo-Kang, Pamela L.,
Zeitlin, Type I, II, III, IV, and V cystic fibrosis Transmembrane
Conductance Regulator Defects and Opportunities of Therapy; Current
Opinion in Pulmonary Medicine 6:521-529, 2000). Other patient
genotypes that exhibit residual CFTR activity include patients
homozygous for one of these classes or heterozygous with any other
class of mutations, including class I mutations, class II
mutations, or a mutation that lacks classification.
[0102] In one embodiment, the compounds and compositions of the
present invention are useful for treating or lessening the severity
of cystic fibrosis in patients within certain clinical phenotypes,
e.g., a moderate to mild clinical phenotype that typically
correlates with the amount of residual CFTR activity in the apical
membrane of epithelia. Such phenotypes include patients exhibiting
pancreatic insufficiency or patients diagnosed with idiopathic
pancreatitis and congenital bilateral absence of the vas deferens,
or mild lung disease.
[0103] 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.
[0104] 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, drops or
patch), 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 0.5 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.
[0105] 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.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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 polyethylene
glycols and the like.
[0112] 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.
[0113] 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.
[0114] The activity of a compound utilized in this invention as a
modulator of CFTR may be assayed according to methods described
generally in the art and in the Examples herein.
[0115] 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."
[0116] In one embodiment, the additional agent is selected from a
mucolytic agent, bronchodialator, an anti-biotic, an anti-infective
agent, an anti-inflammatory agent, a CFTR modulator other than a
compound of the present invention, or a nutritional agent.
[0117] In one embodiment, the additional agent is an antibiotic.
Exemplary antibiotics useful herein include tobramycin, including
tobramycin inhaled powder (TIP), azithromycin, aztreonam, including
the aerosolized form of aztreonam, amikacin, including liposomal
formulations thereof, ciprofloxacin, including formulations thereof
suitable for administration by inhalation, levoflaxacin, including
aerosolized formulations thereof, and combinations of two
antibiotics, e.g., fosfomycin and tobramycin.
[0118] In another embodiment, the additional agent is a mucolyte.
Exemplary mucolytes useful herein includes Pulmozyme.RTM..
[0119] In another embodiment, the additional agent is a
bronchodialator. Exemplary bronchodilators include albuterol,
metaprotenerol sulfate, pirbuterol acetate, salmeterol, or
tetrabuline sulfate.
[0120] In another embodiment, the additional agent is effective in
restoring lung airway surface liquid. Such agents improve the
movement of salt in and out of cells, allowing mucus in the lung
airway to be more hydrated and, therefore, cleared more easily.
Exemplary such agents include hypertonic saline, denufosol
tetrasodium ([[(3S,
5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyp-
hosphoryl] [[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,
4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]hy-
drogen phosphate), or bronchitol (inhaled formulation of
mannitol).
[0121] In another embodiment, the additional agent is an
anti-inflammatory agent, i.e., an agent that can reduce the
inflammation in the lungs. Exemplary such agents useful herein
include ibuprofen, docosahexanoic acid (DHA), sildenafil, inhaled
glutathione, pioglitazone, hydroxychloroquine, or simavastatin.
[0122] In another embodiment, the additional agent is a CFTR
modulator other than compound 1, i.e., an agent that has the effect
of modulating CFTR activity. Exemplary such agents include ataluren
("PTC124.RTM."; 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic
acid), sinapultide, lancovutide, depelestat (a human recombinant
neutrophil elastase inhibitor), cobiprostone (7-{(2R, 4aR, 5R,
7aR)-2-[(3S)-1,1-difluoro-3-methylpentyl]-2-hydroxy-6-oxooctahydrocyclope-
nta[b]pyran-5-yl}heptanoic acid), or
(3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)
cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid. In
another embodiment, the additional agent is
(3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)
cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid.
[0123] In another embodiment, the additional agent is a nutritional
agent. Exemplary such agents include pancrelipase (pancreating
enzyme replacement), including Pancrease.RTM., Pancreacarb.RTM.,
Ultrase.RTM., or Creon.RTM., Liprotomase.RTM. (formerly
Trizytek.RTM.), Aquadeks.RTM., or glutathione inhalation. In one
embodiment, the additional nutritional agent is pancrelipase.
[0124] 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.
[0125] 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.
[0126] Another aspect of the invention relates to modulating CFTR
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 Formula I 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.
[0127] Modulation of CFTR 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 CFTR in biological and pathological phenomena; and the
comparative evaluation of new modulators of CFTR.
[0128] 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 Formula I. 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.
[0129] According to an alternative embodiment, the present
invention provides a method of increasing the number of functional
CFTR in a membrane of a cell, comprising the step of contacting
said cell with a compound of Formula I.
[0130] According to another preferred embodiment, the activity of
the CFTR 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.
[0131] 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).
[0132] 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 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.
[0133] In one embodiment, the present invention provides a method
of modulating CFTR activity in a biological sample comprising the
step of contacting said biological sample with a compound of
Formula I, or a pharmaceutically acceptable salt thereof, wherein R
is defined as above.
[0134] In one embodiment, the present invention provides a method
of modulating CFTR activity in a biological sample comprising the
step of contacting said biological sample with a compound of the
structure:
##STR00027##
or a pharmaceutically acceptable salt thereof.
[0135] In one embodiment, the present invention provides a method
of modulating CFTR activity in a biological sample comprising the
step of contacting said biological sample with a compound of the
structure:
##STR00028##
or a pharmaceutically acceptable salt thereof.
[0136] In one embodiment, the present invention provides a method
of treating or lessening the severity of a disease in a patient
comprising administering to said patient an effective amount of a
compound of Formula I, or a pharmaceutically acceptable salt
thereof, wherein R is defined as above, and said disease is
selected from cystic fibrosis, asthma, smoke induced COPD, chronic
bronchitis, rhinosinusitis, constipation, pancreatitis, pancreatic
insufficiency, male infertility caused by congenital bilateral
absence of the vas deferens (CBAVD), mild pulmonary disease,
idiopathic pancreatitis, allergic bronchopulmonary aspergillosis
(ABPA), liver 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, 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.
[0137] In one embodiment, the method includes treating or lessening
the severity of a disease in a patient by administering to said
patient an effective amount of a compound having the structure:
##STR00029##
or a pharmaceutically acceptable salt thereof.
[0138] In another embodiment, the method includes treating or
lessening the severity of a disease in a patient by administering
to said patient an effective amount of a compound having the
structure:
##STR00030##
or a pharmaceutically acceptable salt thereof.
[0139] In one further embodiment, the disease is cystic
fibrosis.
[0140] In another aspect the present invention provides a kit for
use in measuring the activity of CFTR or a fragment thereof in a
biological sample in vitro or in vivo comprising (i) a composition
comprising a compound of Formula I or any of the above embodiments;
and (ii) instructions for a) contacting the composition with the
biological sample and b) measuring activity of said CFTR or a
fragment thereof.
[0141] In one embodiment, the kit further comprises instructions
for a) contacting an additional composition with the biological
sample; b) measuring the activity of said CFTR or a fragment
thereof in the presence of said additional compound, and c)
comparing the activity of the CFTR in the presence of the
additional compound with the density of the CFTR in the presence of
a composition of Formula I.
[0142] In preferred embodiments, the kit is used to measure the
density of CFTR.
[0143] In one embodiment, the kit includes a composition comprising
a compound having the structure:
##STR00031##
or a pharmaceutically acceptable salt thereof.
[0144] In one embodiment, the kit includes a composition comprising
a compound having the structure:
##STR00032##
or a pharmaceutically acceptable salt thereof.
[0145] 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
Preparation 1: Total Synthesis of
4-oxo-1,4-dihydroquinoline-3-carboxylic acid (26)
##STR00033##
[0146] Procedure for the preparation of ethyl
4-oxo-1,4-dihydroquinoline-3-carboxylate (25)
##STR00034##
[0148] Compound 23 (4.77 g, 47.7 mmol) was added dropwise to
compound 22 (10 g, 46.3 mmol) with subsurface N.sub.2 flow to drive
out ethanol below 30.degree. C. for 0.5 hours. The solution was
then heated to 100-110.degree. C. and stirred for 2.5 hours. After
cooling the mixture to below 60.degree. C., diphenyl ether was
added. The resulting solution was added dropwise to diphenyl ether
that had been heated to 228-232.degree. C. for 1.5 hours with
subsurface N.sub.2 flow to drive out ethanol. The mixture was
stirred at 228-232.degree. C. for another 2 hours, cooled to below
100.degree. C. and then heptane was added to precipitate the
product. The resulting slurry was stirred at 30.degree. C. for 0.5
hours. The solids were then filtrated, and the cake was washed with
heptane and dried in vacuo to give compound 25 as brown solid.
.sup.1H NMR (DMSO-d.sub.6; 400 MHz) .delta. 12.25 (s), .delta. 8.49
(d), .delta. 8.10 (m), .delta. 7.64 (m), .delta. 7.55 (m), .delta.
7.34 (m), .delta. 4.16 (q), .delta. 1.23 (t).
Procedure for the preparation of
4-oxo-1,4-dihydroquinoline-3-carboxylic acid (26)
##STR00035##
[0149] Method 1
[0150] Compound 25 (1.0 eq) was suspended in a solution of HCl
(10.0 eq) and H.sub.2O (11.6 vol). The slurry was heated to
85-90.degree. C., although alternative temperatures are also
suitable for this hydrolysis step. For example, the hydrolysis can
alternatively be performed at a temperature of from about 75 to
about 100.degree. C. In some instances, the hydrolysis is performed
at a temperature of from about 80 to about 95.degree. C. In others,
the hydrolysis step is performed at a temperature of from about 82
to about 93.degree. C. (e.g., from about 82.5 to about 92.5.degree.
C. or from about 86 to about 89.degree. C.). After stifling at
85-90.degree. C. for approximately 6.5 hours, the reaction was
sampled for reaction completion. Stirring may be performed under
any of the temperatures suited for the hydrolysis. The solution was
then cooled to 20-25.degree. C. and filtered. The reactor/cake was
rinsed with H.sub.2O (2 vol.times.2). The cake was then washed with
2 vol H.sub.2O until the pH.gtoreq.3.0. The cake was then dried
under vacuum at 60.degree. C. to give compound 26.
Method 2
[0151] Compound 25 (11.3 g, 52 mmol) was added to a mixture of 10%
NaOH (aq) (10 mL) and ethanol (100 mL). The solution was heated to
reflux for 16 hours, cooled to 20-25.degree. C. and then the pH was
adjusted to 2-3 with 8% HCl. The mixture was then stirred for 0.5
hours and filtered. The cake was washed with water (50 mL) and then
dried in vacuo to give compound 26 as a brown solid. .sup.1H NMR
(DMSO-d.sub.6; 400 MHz) .delta. 15.33 (s), .delta. 13.39 (s),
.delta. 8.87 (s), .delta. 8.26 (m), .delta. 7.87 (m), .delta. 7.80
(m), .delta. 7.56 (m).
Example 1
Total synthesis of
N-(2-tert-butyl-5-hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo--
1,4-dihydroquinoline-3-carboxamide (27)
[0152] The overall scheme of the synthesis of compound 27 is shown
below, followed by the procedure for the synthesis of each
synthetic intermediate.
##STR00036## ##STR00037## ##STR00038##
Procedure for the preparation of 2-hydroxy-5-tert-butylbenzaldehyde
(2)
##STR00039##
[0154] To a stirred solution of compound 1 (700 g, 4.66 mol) in
CH.sub.3CN (7.0 L) was added MgCl.sub.2 (887 g, 9.32 mol),
Para-Formaldehyde (1190 g) and TEA (2.5 L, 17.9 mol) under N.sub.2.
The mixture was heated to reflux for 5 hours. After cooling to room
temperature, 2 L ice water was added to the mixture, followed by 6
L of 3 M HCl (aq). The suspension was left stifling until the
solution became clear. The organic layer was separated and the
aqueous layer was extracted with MTBE (3 L.times.3). The organic
layers were combined and concentrated to dryness. The residue was
dissolved in MTBE (4000 mL), washed with water (1000 mL.times.2)
and brine (1000 mL), dried over anhydrous Na.sub.2SO.sub.4,
filtered, then concentrated to give compound 2 as a light-yellow
solid which was used in the next reaction without further drying or
purification. .sup.1H NMR (CDCl.sub.3; 400 MHz) .delta. 10.86 (s),
.delta. 9.89 (s), .delta. 7.59 (m), .delta. 7.51 (d), .delta. 6.94
(d), .delta. 10.61 (s).
Procedure for the preparation of
2-(benzyloxy)-5-tert-butylbenzaldehyde (3)
##STR00040##
[0156] To a stirred solution of compound 2 (614.5 g, 3.33 mol) in
DMF (3.5 L) was added K.sub.2CO.sub.3 (953 g, 6.90 mol) and benzyl
chloride (480 g, 3.80 mol). The mixture was heated to 90.degree. C.
and left stifling for 3 hours. The suspension was cooled to room
temperature, then MTBE (2 L) was added, followed by water (12 L).
The mixture was then stirred for 10 minutes and the aqueous layer
was separated and extracted with MTBE (2 L.times.3). The organic
layers were combined and washed with water (2 L.times.2) and brine
(1.5 L.times.1) and concentrated to give compound 3 as a
light-yellow solid. .sup.1H NMR (DMSO-d.sub.6; 400 MHz) .delta.
10.42 (s), .delta. 7.71 (m), .delta. 7.51 (m), .delta. 7.43 (m),
.delta. 7.35 (m), .delta. 7.24 (m), .delta. 5.27 (s), .delta. 1.26
(s).
Procedure for the preparation of 2-(benzyloxy)-5-tert-butylbenzyl
alcohol (4)
##STR00041##
[0158] To a stirred suspension of compound 3 (974 g, 3.63 mol) in
MeOH (4000 mL) was slowly added NaBH.sub.4 (121 g, 3.20 mol) at
0-20.degree. C. The solution was left stifling at 15.degree. C. for
3 hours, and then cooled to 0.degree. C. 2N HCl (aq) (1300 mL) was
added dropwise at below 20.degree. C. The solution was then
filtered and evaporated to dryness, and the residue was dissolved
in MTBE (5 L). The solution was then washed with water (2
L.times.2) and brine (1.5 L.times.1). Evaporation of the solvent
gave compound 4 as a light-yellow solid which was used in the next
reaction without further purification. .sup.1H NMR (DMSO-d.sub.6;
400 MHz) .delta. 7.40 (m), .delta. 7.32 (m), .delta. 7.17 (m),
.delta. 6.91 (m), .delta. 5.09 (s), .delta. 5.00 (t), .delta. 4.56
(d), .delta. 1.26 (s).
Procedure for the preparation of 2-(benzyloxy)-5-tert-butylbenzyl
chloride (5)
##STR00042##
[0160] To a stirred solution of compound 4 (963 g, 3.56 mol) in
anhydrous DCM (2000 mL) was added slowly SOCl.sub.2 (535 g, 4.5
mol) at 0.degree. C. The mixture was stirred at 20.degree. C. for 2
hours, then concentrated in vacuo to give compound 5 as an oil,
which was used in the next reaction without further drying or
purification.
Procedure for the preparation of 2-(benzyloxy)-5-tert-butylbenzyl
nitrile (6)
##STR00043##
[0162] To a stirred solution of compound 5 (1045 g, 3.54 mol) in
anhydrous DMF (1000 mL) was added KCN (733 g, 11.3 mol). The
mixture was stirred at 35.degree. C. for 24 hours, then poured into
water (10 L). Ethyl acetate (4 L) was added and the mixture was
stirred for 30 minutes. The organic layer was then separated and
the aqueous layer was extracted with ethyl acetate (3000
mL.times.2). The organic layers were combined and washed with water
(4 L.times.2) and brine (3 L.times.1), then concentrated in vacuo
to give compound 6 as a yellow solid. .sup.1H NMR (DMSO-d.sub.6;
400 MHz) .delta. 7.51 (m), .delta. 7.37 (m), 7.02 (d), .delta. 5.17
(s), .delta. 3.88 (s), 1.26 (s).
Procedure for the preparation of
2-(2-(benzyloxy)-5-tert-butylphenyl)-2-methylpropanenitrile (7)
##STR00044##
[0164] To a stirred suspension of NaH (86 g, 2.15 mol, 60% in
mineral oil) in DMF (1000 mL) was added dropwise a solution of
compound 6 (100.0 g, 0.358 mol) in DMF (500 mL) at 20.degree. C.
After stifling for 30 minutes, MeI (205 g, 1.44 mol) in DMF (500
mL) was added dropwise at below 30.degree. C. during a period of 2
hours. The suspension was stirred for 1.5 hours at 25-30.degree.
C., then ice (100 g) was added slowly until no gas was generated.
The pH was adjusted to approximately 7 by the slow addition of 2N
HCl. The mixture was diluted with water (4 L) and MTBE (2 L). The
organic layer was separated and the aqueous layer was extracted
with MTBE (500 mL.times.2). The combined organic layers were washed
with water and brine, dried over Na.sub.2SO.sub.4, filtered, and
then concentrated in vacuo to give compound 7 as a white solid.
.sup.1H NMR (DMSO-d.sub.6; 400 MHz) .delta. 7.56 (m), .delta. 7.40
(m), .delta. 7.34 (m), .delta. 7.10 (d), .delta. 5.21 (s), .delta.
1.73 (s), .delta. 1.27 (s).
Procedure for the preparation of
2-(2-(benzyloxy)-5-tert-butylphenyl)-2-methylpropanal (8)
##STR00045##
[0166] To a stirred solution of compound 7 (20 g, 0.065 mol) in
toluene (300 mL), was added drop wise DIBAH (80 mL, 1 M in toluene)
at about -60 to -50.degree. C. After stifling for 2 hours, 6 N HCl
(300 mL) was added to the reaction mixture and stirring was
continued for 30 minutes. The organic layer was then separated,
washed with 2 N HCl followed by a NaHCO.sub.3 solution, then a
brine solution, dried over Na.sub.2SO.sub.4 and concentrated in
vacuo to afford the compound 8 as an oil. The product was used in
the next reaction without further purification. .sup.1H NMR
(CDCl.sub.3; 400 MHz) .delta. 9.61 (s), .delta. 7.36 (m), .delta.
7.25 (m), .delta. 6.87 (m), .delta. 5.06 (m), .delta. 1.43 (s),
.delta. 1.33 (s).
Procedure for the preparation of
2-(2-(benzyloxy)-5-tert-butylphenyl)-2-methylpropan-1-ol (9)
##STR00046##
[0168] To a stirred solution of compound 8 (9.21 g, 0.030 mol) in
MeOH (150 mL) was added slowly NaBH.sub.4 (2.3 g, 0.061 mol) at
0.degree. C. After the mixture was stirred at 20.degree. C. for 3
hours, 12 mL of 6 N HCl was added, and the mixture was stirred for
an additional 30 minutes. The solution was then concentrated to
about one-quarter of the original volume and extracted with EtOAc.
The organic layer was separated and washed with water and brine,
dried with Na.sub.2SO.sub.4, filtered, and then concentrated in
vacuo to afford compound 9 as a white solid. .sup.1H NMR
(DMSO-d.sub.6; 400 MHz) .delta. 7.47 (m), .delta. 7.42 (m), .delta.
7.34 (m), .delta. 7.28 (m), .delta. 7.16 (m), .delta. 6.94 (m),
.delta. 5.08 (s), .delta. 4.45 (t), .delta. 3.64 (d), .delta. 1.28
(s), .delta. 1.25 (s).
Procedure for the preparation of
2-(2-hydroxy-5-tert-butylphenyl)-2-methylpropan-1-ol (10)
##STR00047##
[0170] Pd(OH).sub.2 (1 g) and compound 9 (9.26 g, 0.030 mol) in
MeOH (200 mL) were stirred under hydrogen at 20-30 psi pressure for
16-18 hours. The mixture was then filtered through Celite.RTM., and
the filtrate was concentrated to give compound 10 as a white solid.
.sup.1H NMR (DMSO-d.sub.6; 400 MHz) .delta. 9.16 (s), .delta. 7.16
(d), .delta. 7.00 (m), .delta. 6.65 (m), .delta. 4.71 (t), .delta.
3.62 (d), .delta. 1.27 (s), .delta. 1.22 (s).
Procedure for the preparation of
1-((methylcaroboxy)oxy)-2-(1-((methylcaroboxy)oxy)-2-methylpropan-2-yl)-4-
-tert-butyl benzene (11)
##STR00048##
[0172] To a stirred solution of compound 10 (23.2 g, 0.10 mol),
DMAP (1.44 g) and DIEA (72.8 g, 0.56 mol) in anhydrous DCM (720 mL)
was added dropwise methyl chloroformate (43.5 g, 0.46 mol) in DCM
(160 mL) at 0.degree. C. After the mixture was stirred at
20.degree. C. for 16 hours, it was washed with water, 1 N HCl and
brine, dried with MgSO.sub.4 and concentrated in vacuo. The residue
was purified using column chromatography on silica gel (1:20
EtOAc:Petroleum ether) to give compound 11 as a white solid.
.sup.1H NMR (DMSO-d.sub.6; 400 MHz) .delta. 7.32 (m), .delta. 7.10
(d), .delta. 4.26 (s), .delta. 3.84 (s), .delta. 3.64 (s), .delta.
1.31 (s), .delta. 1.28 (s).
Procedure for preparation of
1-((methylcaroboxy)oxy)-2-(1-((methylcaroboxy)oxy)-2-methylpropan-2-yl)-4-
-tert-butyl-5-nitro benzene (12)
##STR00049##
[0174] To a stirred solution of compound 11 (32 g, 0.095 mol) in
DCM (550 mL) was added dropwise 98% H.sub.2SO.sub.4 (43 g, 0.43
mol) at 0.degree. C. After stifling for 20 minutes at 0.degree. C.,
65% HNO.sub.3 (16.2 g, 0.17 mol) was added to the mixture dropwise
at 0.degree. C. The mixture was then stirred at 1-10.degree. C. for
4 hours and then ice-water (200 mL) was added. The aqueous layer
was separated and extracted with DCM (200 mL.times.3) and the
combined organic layers were washed with water (aq), NaHCO.sub.3
and brine, then dried with MgSO.sub.4 and concentrated in vacuo.
The residue was purified via column chromatography on silica gel
(1:20 EtOAc:Petroleum ether) to afford crude compound 12 as an
oil.
Procedure for the preparation of
2-tert-butyl-5-((methylcaroboxy)oxy)-4-(1-((methylcaroboxy)oxy)-2-methylp-
ropan-2-yl) aniline (13)
##STR00050##
[0176] Pd/C (2.6 g) and compound 12 (14 g, crude) were stirred in
MeOH (420 mL) at room temperature under hydrogen at 20-30 psi
pressure for 16-18 hours. The mixture was then filtered with
Kieselguhr.RTM., and the filtrate was concentrated in vacuo. The
residue was purified via column chromatography on silica gel (1:10
EtOAc:Petroleum ether) to give compound 13 as a gray solid. .sup.1H
NMR (CDCl.sub.3; 400 MHz) .delta. 7.26 (s), .delta. 7.19 (s),
.delta. 4.26 (s), .delta. 3.89 (s), .delta. 3.74 (s), .delta. 1.40
(s), .delta. 1.35 (s).
Procedure for the preparation of
N-(2-tert-butyl-5-((methylcaroboxy)oxy)-4-(1-((methylcaroboxy)oxy)-2-meth-
ylpropan-2-yl)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide
(14)
##STR00051##
[0178] To a stirred solution of compound 26 (5.0 g, 0.026 mol) in
anhydrous DMF (120 mL) was added EDCI (5.6 g, 0.029 mol), HOBT (3.8
g, 0.028 mol) and DIEA (6.6 g, 0.051 mol) at 0.degree. C. After
stifling for 1 hour, the mixture was added dropwise a solution of
compound 13 (3.0 g, 0.008 mol) in DCM (30 ml) at 0.degree. C. The
mixture was stirred at 25.degree. C. for 72 hours, and then was
concentrated in vacuo. The residue was dissolved in EtOAc (225 mL)
and washed with water (120 mL.times.1), 1N HCl (120 mL) and brine,
dried with Na.sub.2SO.sub.4 and concentrated in vacuo. The residue
was purified via column chromatography on silica gel (1:1
EtOAc:Petroleum ether) to give compound 14 as a white solid.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 12.34 (s, 1H), 11.58 (s,
1H), 9.07 (s, 1H), 8.42 (d, 1H), 7.66 (s, 1H), 7.51 (s, 1H), 7.47
(s, 1H), 7.39 (s, 1H), 6.72 (s, 1H), 4.34 (s, 2H), 3.82 (s, 3H),
3.74 (s, 3H), 1.41 (s, 9H), 1.40 (s, 6H).
Procedure for the preparation of
N-(2-tert-butyl-5-hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo--
1,4-dihydroquinoline-3-carboxamide (27)
##STR00052##
[0180] To a stirred solution of KOH (1.2 g, 0.02 mol) in MeOH (80
mL) was added compound 14 (1.9 g, 0.0036 mol) at 0.degree. C. After
stirring for 2-3 hours at 5-15.degree. C., the mixture was
concentrated to dryness. The residue was then triturated in water
(10 mL), filtered, washed with DCM and dried in vacuo for 24 hours
to give compound 27 as a white solid. .sup.1H NMR (DMSO-d.sub.6;
400 MHz) .delta. 12.77 (s), .delta. 8.86 (s), .delta. 8.20 (d),
.delta. 7.55 (d), .delta. 7.42 (t), .delta. 7.16 (q), .delta. 7.02
(s), .delta. 6.85 (m), .delta. 3.55 (s), .delta. 1.55 (s), .delta.
1.35 (s), .delta. 1.27 (s). MS Found (M+H) 409.2.
Example 2
Alternative Total Synthesis of
N-(2-tert-butyl-5-hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo--
1,4-dihydroquinoline-3-carboxamide (27)
##STR00053##
[0181] Procedure for the preparation of methyl
2-(5-tert-butyl-2-hydroxy-4-nitrophenyl)-2-methylpropanoate
(38)
##STR00054##
[0183] A mixture of 2-bromo-4-tert-butyl-5-nitrophenol (15.00 g,
54.72 mmol), bis(tri-tert-butylphospine)palladium(0) (1.422 g,
2.783 mmol), zinc fluoride (2.82 g, 27.27 mmol), methyl
trimethylsilyl dimethylketene acetal (MTDA) (19.35 g, 111.0 mmol),
and dimethylformamide (150 mL) was heated at 70.degree. C. for 18
h. The mixture was cooled to room temperature and diluted with
water. After stifling for one hour, the aqueous phase was extracted
with MTBE. The organic phase was dried in vacuo to afford the crude
product as a brown solid. Purification of the product was
accomplished by trituration in n-heptane. .sup.1H-NMR (400 MHZ,
DMSO-d6) .delta. 10.38 (s, 1H); 7.37 (s, 1H); 6.79 (s, 1H); 3.54
(s, 3H); 1.45 (s, 6H); 1.32 (s, 9H)
Procedure for the preparation of
4-tert-butyl-2-(1-hydroxy-2-methylpropan-2-yl)-5-nitrophenol
(39)
##STR00055##
[0185] A 1M solution of lithium aluminum hydride in THF (11.80 mL,
11.80 mmol) was added to a solution of methyl
2-(5-tert-butyl-2-hydroxy-4-nitrophenyl)-2-methylpropanoate (5.36
g, 18.15 mmol) in THF (50 mL). The mixture was stirred at ambient
temperature for 3 h, and then diluted with methanol. The mixture
was acidified with 1N HCl (pH 1-2) and the aqueous phase was
extracted with MTBE. The organic phase was dried in vacuo to afford
4-tert-butyl-2-(1-hydroxy-2-methylpropan-2-yl)-5-nitrophenol which
was used without further purification in the next step. .sup.1H-NMR
(400 MHZ, DMSO-d6) .delta. 10.12 (s, 1H); 7.37 (s, 1H); 6.80 (s,
1H); 4.77 (s, 1H); 3.69-3.65 (m, 2H); 1.30 (s, 9H); 1.29 (s,
6H)
Procedure for the preparation of
4-tert-butyl-2-(2-methoxycarbonyloxy-1,1-dimethyl-ethyl)-5-nitro-phenyl]
methyl carbonate (12)
##STR00056##
[0187] To a solution of
4-tert-butyl-2-(1-hydroxy-2-methylpropan-2-yl)-5-nitrophenol (1.92
g, 7.18 mmol), triethylamine (1.745 g, 17.24 mmol), and
dimethylaminopyridine (87.74 mg, 0.718 mmol) in dichloromethane (30
mL) at 0.degree. C. was slowly charged ethylchloroformate (2.376 g,
25.14 mmol), keeping the temperature below 5.degree. C. After the
addition, the mixture was allowed to warm to ambient temperature
and was stirred until HPLC showed complete conversion of the
starting material (2-8 h). The reaction mixture was diluted with
water and acidified with 1N HCl (pH 1-2). The aqueous phase was
extracted with DCM and the combined organics dried in vacuo. The
crude amber semi-solid was re-crystallized from methanol and
dichloromethane to give the title compound as a yellow crystalline
solid. .sup.1H-NMR (400 MHZ, DMSO-d6) .delta. 7.67 (s, 1H); 7.52
(s, 1H); 4.30 (s, 2H); 3.86 (s, 3H); 3.64 (s, 3H); 1.35 (s, 9H);
1.35 (s, 6H)
Procedure for the preparation of
5-amino-4-tert-butyl-2-(2-methoxycarbonyloxy-1,1-dimethyl-ethyl)phenyl]
methyl carbonate (13)
##STR00057##
[0189] A mixture of
[4-tert-butyl-2-(2-methoxycarbonyloxy-1,1-dimethyl-ethyl)-5-nitro-phenyl]
methyl carbonate (1.27 g, 3.313 mmol) and Pd/C (75 mg, 0.035 mmol)
in methanol (50 mL) was purged with nitrogen. After purging the
flask with hydrogen, the mixture was hydrogenated for 18 hours at
ambient temperature and pressure. The solution was filtered through
Celite.RTM. and dried in vacuo to obtain the product as a solid.
.sup.1H-NMR (400 MHZ, DMSO-d6) .delta. 6.99 (s, 1H); 6.39 (s, 1H);
4.92 (s, 2H); 4.13 (s, 2H); 3.82 (s, 3H); 3.65 (s, 3H); 1.32 (s,
9H); 1.23 (s, 6H)
Procedure for the preparation of
N-(2-tert-butyl-5-hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo--
1,4-dihydroquinoline-3-carboxamide (27)
##STR00058##
[0191] To a mixture of
[5-amino-4-tert-butyl-2-(2-methoxycarbonyloxy-1,1-dimethyl-ethyl)phenyl]
methyl carbonate (103 mg, 0.29 mmol),
4-oxo-1,4-dihydroquinoline-3-carboxylic acid (50 mg, 0.26 mmol),
and pyridine (42 mg, 0.53 mmol) in 2-MeTHF (3.0 mL) was charged T3P
as a 50 wt % solution in 2-MeTHF (286 mg, 0.45 mmol). The mixture
was heated to 50.degree. C. for 18 h. After cooling to ambient
temperature, the mixture was diluted with water. The organic phase
was separated and again washed with water. Sodium methoxide (39 mg,
0.72 mmol) was charged to the organic phase and the solution
stirred for 2 hours. The reaction was quenched with 1N HCl, and
after separating the phases, the organic phase was washed with 0.1N
HCl. The organic phase was than dried in vacuo to yield Compound 27
as a solid. The .sup.1H-NMR spectrum was consistent with that
reported above.
Example 3
Total Synthesis of
2-(5-tert-butyl-2-hydroxy-4-(4-oxo-1,4-dihydroquinoline-3-carboxamido)phe-
nyl)-2-methylpropanoic acid (28)
##STR00059## ##STR00060## ##STR00061##
[0192] Procedure for the preparation of
2-(5-tert-butyl-2-hydroxyphenyl)-2-methylpropanenitrile (15)
##STR00062##
[0194] Pd(OH).sub.2/C (2.0 g) and compound 7 (20.0 g, 0.104 mol)
were stirred in MeOH (150 mL) at room temperature under hydrogen at
10 psi pressure for 16-18 hours. The mixture was then filtered
through a pad of Celite.RTM., and the filtrate was concentrated to
give compound 15, which was used in the next reaction without
further purification. .sup.1H NMR (DMSO-d.sub.6; 400 MHz) .delta.
9.83 (s), .delta. 7.24 (s), .delta. 7.18 (m), .delta. 6.80 (m),
.delta. 1.71 (s), .delta. 1.24 (s).
Procedure for the preparation of
4-tert-butyl-2-(2-cyanopropan-2-yl)phenyl methyl carbonate (16)
##STR00063##
[0196] To a stirred mixture of compound 15 (126.6 g, 0.564 mol),
DMAP (6.0 g) and DIEA (188 g, 1.46 mol) in anhydrous DCM (1500 mL)
was added dropwise methyl chloroformate (110 g, 1.17 mol) in
anhydrous DCM (300 mL) at 0.degree. C. within 2 hours. After
stirring for 12 hours at 0.degree. C., ice-water (1.5 L) was added
and the mixture was stirred at 0.degree. C. for 30 minutes. The
organic layer was separated and washed with 1 N HCl, water, and
brine. The DCM solution was dried over MgSO.sub.4 and concentrated
in vacuo to give compound 16 as a yellow solid. .sup.1H NMR
(DMSO-d.sub.6; 400 MHz) .delta. 7.47 (m), .delta. 7.39 (d), .delta.
7.24 (d), .delta. 3.84 (s), .delta. 1.71 (s), .delta. 1.30 (s).
Procedure for the preparation of
2-(1-amino-2-methyl-1-oxopropan-2-yl)-4-tert-butyl-5-nitrophenyl
methyl carbonate (17)
##STR00064##
[0198] To a stirred mixture of compound 16 (10.0 g, 36.3 mmol) and
KNO.sub.3 (5.51 g, 54.5 mmol) in DCM (1000 mL) was added dropwise
98% H.sub.2SO.sub.4 (145.4 g, 1.45 mol) at 0.degree. C. The mixture
was stirred at 30.degree. C. for 4 days. The H.sub.2SO.sub.4 layer
was then separated and poured into ice-water (50 g) and then
extracted with DCM (100 mL.times.3). The combined organic layers
were washed with water, aqueous NaHCO.sub.3 solution and brine,
then dried over MgSO.sub.4 and concentrated in vacuo. The residue
was purified via column chromatography on silica gel (Petroleum
ether/EtOAc 20:1.fwdarw.10:1.fwdarw.5:1.fwdarw.3:1) to give
compound 17 as a yellow solid. .sup.1H NMR (CDCl.sub.3; 400 MHz)
.delta. 8.05 (s), .delta. 7.74 (s), .delta. 7.61 (s), .delta. 7.32
(s), .delta. 5.32 (s), .delta. 3.91 (s), .delta. 3.92 (s), .delta.
1.62 (s), .delta. 1.59 (s), .delta. 1.42 (s), .delta. 1.38 (s).
Procedure for the preparation of
2-(5-tert-butyl-2-hydroxy-4-nitrophenyl)-2-methylpropanoic acid
(18)
##STR00065##
[0200] To a mixture of compound 17 (7.3 g, 21.6 mmol) in methanol
(180 mL) was added water (18 mL) and NaOH (8.64 g, 216 mmol). The
solution was heated and maintained at reflux for 3 days. The
solvent was evaporated in vacuo and the residue was dissolved in
140 mL of water. Then the solution was acidified to pH 2 by the
addition of 2N HCl. The aqueous phase was extracted with ethyl
acetate (100 mL.times.3), and the combined organic phases were
washed with water and brine, dried over anhydrous Na.sub.2SO.sub.4
and then concentrated to give compound 18 as a yellow solid, which
was used in the next reaction without further purification.
Procedure for the preparation of
5-tert-butyl-3,3-dimethyl-6-nitrobenzofuran-2(3H)-one (19)
##STR00066##
[0202] To a solution of compound 18 (7.10 g, 25.2 mmol) in 710 mL
of anhydrous THF was added EDCI (14.5 g, 75.6 mmol). The resulting
suspension was left stirring at 30.degree. C. overnight. The
precipitate was filtered and thoroughly washed with DCM. The
filtrate was concentrated to dryness and the residue was dissolved
in DCM (100 mL). The solution was washed with water (50 mL.times.2)
and brine (50 mL.times.1). The DCM layer was then dried over
anhydrous Na.sub.2SO.sub.4 and concentrated to give the crude
product, which was purified via column chromatography on silica gel
(Petroleum ether/EtOAc 200:1.fwdarw.100:1.fwdarw.50:1) to give
compound 19 as a white solid. .sup.1H NMR (CDCl.sub.3; 400 MHz)
.delta. 7.36 (s), .delta. 7.10 (s), .delta. 1.53 (s), .delta. 1.41
(s).
Procedure for the preparation of
6-amino-5-tert-butyl-3,3-dimethylbenzofuran-2(3H)-one (20)
##STR00067##
[0204] Pd/C (1.50 g) and compound 19 (3.00 g, 1.14 mmol) were
suspended in THF (1500 mL) at 25.degree. C. under hydrogen at 30
psi for 4 hours. The mixture was then filtered through a pad of
Celite.RTM., and the filtrate was concentrated in vacuo to give
compound 20 as a white solid. .sup.1H NMR (DMSO-d.sub.6; 400 MHz)
.delta. 7.05 (s), .delta. 6.49 (s), .delta. 5.01 (s), .delta. 1.35
(s), .delta. 1.33 (s).
Procedure for the preparation of
N-(5-tert-butyl-3,3-dimethyl-2-oxo-2,3-dihydrobenzofuran-6-yl)-4-oxo-1,4--
dihydroquinoline-3-carboxamide (21)
##STR00068##
[0206] A suspension of HATU (17.6 g, 46.3 mol) and compound 26
(8.36 g, 44.2 mmol) in anhydrous acetonitrile (1 L) was stirred at
room temperature for 1 hour. Compound 20 (3.40 g, 14.6 mmol) was
added to the suspension, and then DIEA (11.5 g, 89.0 mmol) was
added dropwise. The mixture was stirred at 45.degree. C. for 4
days. The resulting precipitate was filtered and thoroughly washed
with DCM. The filtrate was concentrated to dryness and the residue
was dissolved in DCM (200 mL) and washed with 1N HCl (200
mL.times.2) followed by 5% aqueous NaHCO.sub.3 (200 mL.times.3) and
then brine (200 mL.times.1). The mixture was then dried over
Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was
purified via column chromatography on silica gel
(CH.sub.2Cl.sub.2/MeOH 100:1.fwdarw.50:1) to give compound 21 as a
light yellow solid. .sup.1H-NMR (400 MHZ, DMSO-d6) .delta. 12.96 (d
J 6.4 Hz, 1H); 12.1 (s, 1H); 8.9 (d, J 6.4 Hz, 1H); 8.33 (d, J 8
Hz, 1H); 7.84-7.75 (m, 2H); 7.55-7.48 (m, 3H); 1.47 (s, 6H); 1.45
(s, 9H).
Procedure for the preparation of
2-(5-tert-butyl-2-hydroxy-4-(4-oxo-1,4-dihydroquinoline-3-carboxamido)phe-
nyl)-2-methylpropanoic acid (28)
##STR00069##
[0208] To a stirred solution of compound 21 (0.9 g, 2.45 mmol) in
MeOH (50 mL) was added NaOH (1.5 g, 37.5 mmol) at 0.degree. C.
After stirring for 16 hours at 40.degree. C., the solvent was
evaporated in vacuo, then the residue was dissolved in H.sub.2O (50
ml). The precipitate was filtered and the filtrate was washed with
DCM (100 mL.times.1) and ethyl acetate (100 mL.times.1). The
aqueous layer was acidified with 2N HCl to pH 1-2. The precipitate
was filtered and washed with H.sub.2O (80 mL) and heptane (50 mL).
It was dried in vacuo to give compound 28 as a white solid. .sup.1H
NMR (DMSO-d.sub.6; 400 MHz) .delta. 12.85 (s), .delta. 11.84 (s),
.delta. 11.77 (s), .delta. 9.39 (s), .delta. 8.86 (s), .delta. 8.33
(s), .delta. 7.79 (m), .delta. 7.52 (m), .delta. 7.18 (s), .delta.
7.09 (s), .delta. 1.44 (s), .delta. 1.40 (s). MS found (M+H)
423.08
Example 4
Second alternative Synthesis of
N-(2-tert-butyl-5-hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo--
1,4-dihydroquinoline-3-carboxamide (27)
##STR00070##
[0210] A 3-neck 50 mL round bottom flask was equipped with magnetic
stirrer, nitrogen bubbler and thermocouple. Compound 21 (514 mg,
1.27 mmol) and 2-MeTHF (4 mL) are charged to the flask. The
reaction mixture was stirred at room temperature. Lithium aluminum
hydride (204 mg, 6.6 mmol) was added as solid until 100% conversion
is achieved, which was monitored using HPLC. Potassium sodium
2,3-dihydroxybutanedioate tetrahydrate salt (50 mL of a 400 g/L
solution) and MTBE (50 mL) were added to the reaction mixture. The
resulting solution was stirred for 15 minutes and then let sit for
15 min. The organic layer was separated and the pH of the aqueous
layer was adjusted to a pH of about 6-7 by adding tartaric acid.
The aqueous layer was extracted with MTBE. The organic layer was
concentrated and dried under high vacuum to provide the title
compound as an off-white powder. The .sup.1H-NMR spectrum was
consistent with that reported above.
Example 5
Alternative Total synthesis of
2-(5-tert-butyl-2-hydroxy-4-(4-oxo-1,4-dihydroquinoline-3-carboxamido)phe-
nyl)-2-methylpropanoic acid (28)
##STR00071## ##STR00072##
[0211] Procedure for the preparation of Carbonic acid 2-bromide,
4-tertbutyl phenyl ester methyl ester (35)
##STR00073##
[0213] A 3-neck 2 L round bottom flask was equipped with mechanical
stirrer, nitrogen bubbler and thermocouple. 2-Bromo-4-tertbutyl
phenol (50 g, 211.7 mmol) was added followed by DCM (1.75 L), DMAP
(1.29 g, 10.58 mmol) and Et.sub.3N (44.3 mL, 317.6 mmol). The
reaction mixture was cooled down to 0.degree. C. Methyl
chloroformate (19.62 mL, 254 mmol) was added drop-wise to the
reaction mixture. The mixture was allowed to warm to room
temperature while stirring overnight. When the reaction was
complete, the mixture was filtered via sintered funnel. The
filtrate was transferred into 1 L separatory funnel. To quench, 1N
HCl (300 mL) was added to filtrate and the organic layer was
separated. The organic layer was then washed with a mixture of 291
mL saturated NaHCO.sub.3 and 100 mL water. The layers were
separated, and the aqueous layer was determined to have a pH of
about 8. The organic layer was concentrated and dried under high
vacuum for about 16 hours to give the title compound as a clear
yellow oil, which was used in the next step without further
purification. .sup.1H-NMR (400 MHz. DMSO-d6) 7.66 (d, J 2.0 Hz,
1H), 7.46 (dd, J 8.4, 2.0 Hz, 1H), 7.32 (d, J 8.4 Hz, 1H), 3.86 (s,
3H), 1.28 (s, 9H)
Procedure for the preparation of
(2-bromo-4-tert-butyl-5-nitro-phenyl) methyl carbonate (36)
##STR00074##
[0215] A 3-neck 2 L round bottom flask was equipped with mechanical
stirrer, nitrogen bubbler and thermocouple. Compound 35 (176 g,
612.9 mmol) and concentrated sulfuric acid (264 mL) were charged to
the flask. The reaction mixture was cooled to -5.degree.
C.-0.degree. C. Nitric acid (28.6 mL, 612.9 mmol) was added
drop-wise and the reaction mixture was stirred at 0.degree. C. for
2 hours. When complete, water (264 mL) was added followed by MTBE
(264 mL). The solution was stirred for 15 minutes, then let stand
for 15 minutes. The organic layer was separated, concentrated and
dried under high vacuum to give the title compound as a dark brown
oil, which was used in the next step without further purification.
.sup.1H-NMR (400 MHz. DMSO-d6) 7.96 (s, 1H), 7.92 (s, 1H), 3.89 (s,
3H), 1.34 (s, 9H)
Procedure for the preparation of
2-bromo-4-tert-butyl-5-nitro-phenol (37)
##STR00075##
[0217] (2-Bromo-4-tert-butyl-5-nitro-phenyl)methyl carbonate (72.9
g, 219.5 mmol) was charged to a reactor and DCM (291.6 mL) was
added. The yellow reaction solution was cooled using an ice bath.
Sodium methoxide (67.04 g, 69.11 mL of 5.4 M, 373.2 mmol) was added
portion-wise at 2.2-6.9.degree. C. After complete addition, the
reaction was slowly warmed to ambient temperature. When complete,
the reaction was cooled to 0.degree. C. and quenched with 1M HCl
(373.2 mL, 373.2 mmol). The biphasic mixture was stirred for 20 min
and transferred to a separatory funnel. The organic layer was
separated and washed with water (300 mL) followed by brine (300
ml). The organic layer was concentrated and the crude product dried
under high vacuum. The product was further purified using
Supercritical Fluid Chromatography (SFC) separation on a Berger
MultiGram III (Mettler Toledo AutoChem, Newark Del.). The method
conditions were 20% methanol at 250 mL/min on a PPU column (30*150)
from Princeton Chromatography, 100 bar, 35 C, 220 nm. An injection
of 3.5 mL of a 55-70 mg/mL solution was injected. The data was
collected using SFC ProNTo software. The purified product received
from SFC purification was a methanol solvate. To remove the
methanol, an azeotropic distillation was performed. The dark yellow
solid, 2-bromo, 4-tertbuyl, 5-nitro phenol methanol solvate, (111.3
g, 59.9 mmol) was charged to a 1 L round bottom flask, followed by
heptane (500 mL). The slurry is heated to 64.degree. C. to obtain a
clear solution. The solvent was distilled under reduced pressure
(649 mbar) for 30 minutes and then stripped to dryness. This
procedure was repeated three times until no MeOH was detected by
.sup.1H-NMR. The product was dried under high vacuum for 16 hours
to give the product as a dark yellow semi solid. .sup.1H-NMR (400
MHZ, DMSO-d6) .delta. 11.2 (bs, OH), 7.69 (s, 1H); 7.03 (s, 1H);
1.30 (s, 9H)
Procedure for the preparation of
5-tert-butyl-3,3-dimethyl-6-nitrobenzofuran-2(3H)-one (19)
##STR00076##
[0219] Difluorozinc (6.093 g, 58.92 mmol) was added to a round
bottomed flask, which was flushed with nitrogen.
Pd(tBu.sub.3P).sub.2 (2 g, 3.835 mmol) was then added under
nitrogen stream. 2-Bromo-4-tert-butyl-5-nitro-phenol (16.15 g,
58.92 mmol) dissolved in DMF (80.75 mL) was then added to the
flask. The reaction mixture was an orange suspension.
(1-Methoxy-2-methyl-prop-1-enoxy)trimethylsilane (21.61 g, 25.13
mL, 117.8 mmol) was added to the mixture and the resulting mixture
was heated to 80.degree. C. and stirred for 16 h. When complete,
the reaction mixture was cooled to ambient temperature and filtered
through Celite.RTM.. The filter cake was washed with MTBE (536.0
mL) and water (893.3 mL) was added to the filtrate. The mixture was
stirred for 15 min and settled for another 15 min. The layers were
separated and 0.5M HCl (500 mL, 250.0 mmol) was added to the
organic phase. The layers were separated and the organic layer was
washed with water (500 mL). The layers were separated and the
organic layer was washed with NaCl (500 mL; 8 wt %). The organic
layer was separated and the solvent removed in vacuo. The crude
product was obtained as a brown crystalline solid and was then
purified through a silica plug, using hexane:MTBE 20:1-10:1 as an
eluent. The fractions containing product were combined and the
solvent removed in vacuo to give the pure product as a white
crystalline solid. .sup.1H-NMR (400 MHZ, DMSO-d6) .delta. 7.80 (s,
1H); 7.62 (s, 1H); 1.49 (s, 6H); 1.34 (s, 9H)
Procedure for the preparation of
6-amino-5-tert-butyl-3,3-dimethylbenzofuran-2(3H)-one (20)
##STR00077##
[0221] Palladium on carbon (wet; 5 wt %) was placed into a round
bottomed flask under nitrogen flow.
5-Tert-butyl-3,3-dimethyl-6-nitro-benzofuran-2-one (4.7 g, 17.85
mmol) was then added to the vessel. Methanol (120 mL) was then
carefully charged to the vessel under nitrogen atmosphere. The
vessel was then purged with N.sub.2, evacuated, then charged with
hydrogen gas. The vessel was evacuated and re-charged with hydrogen
gas, and then a continuous hydrogen gas stream was introduced.
After completion, the reaction was filtered through Celite.RTM. and
the cake was washed with MeOH (300 ml). The solvent was removed in
vacuo and the product dried under high vacuum to give a white
crystalline solid. .sup.1H-NMR (400 MHZ, DMSO-d6) .delta. 7.05 (s,
1H); 6.48 (s, 1H); 5.02 (s, 2H, NH.sub.2); 1.34 (s, 6H); 1.30 (s,
9H)
Procedure for the preparation of
N-(5-tert-butyl-3,3-dimethyl-2-oxo-2,3-dihydrobenzofuran-6-yl)-4-oxo-1,4--
dihydroquinoline-3-carboxamide (21)
##STR00078##
[0223] A reaction vessel was charged with compound 26 (2.926 g,
15.43 mmol), Compound 20 (4.32 g, 18.52 mmol), 2-MeTHF (35.99 mL),
and subsequently 50% T3P in 2-MeTHF (13.36 g, 21.00 mmol). Pyridine
(2.441 g, 2.496 mL, 30.86 mmol) was added and the suspension heated
at 47.5.degree. C..+-.5.degree. C. for 18 h. After completion, the
reaction was cooled to ambient temperature and 2-MeTHF (36) and
water (30 ml) were added. The layers were split and the organic
layer was washed with 10 wt % citric acid solution (30 ml), water
(30 ml) and twice with NaHCO.sub.3 (20 ml). The organic layer was
washed with brine (50 ml), separated and the solvent removed in
vacuo. The crude product was dissolved in MTBE (100 ml) and hexane
(200 ml) was added as an anti-solvent. A solid precipitated and the
resulting slurry was stirred for two hours. The solid was collected
by suction filtration and the cake was washed with hexane. The
resulting product was dried in a vacuum oven at 55.degree. C. with
nitrogen bleed to give the title compound as a beige solid.
.sup.1H-NMR (400 MHZ, DMSO-d6) .delta. 12.96 (d J 6.4 Hz, 1H); 12.1
(s, 1H); 8.9 (d, J 6.4 Hz, 1H); 8.33 (d, J 8 Hz, 1H); 7.84-7.75 (m,
2H); 7.55-7.48 (m, 3H); 1.47 (s, 6H); 1.45 (s, 9H).
Procedure for the preparation of
2-(5-tert-butyl-2-hydroxy-4-(4-oxo-1,4-dihydroquinoline-3-carboxamido)phe-
nyl)-2-methylpropanoic acid (28)
##STR00079##
[0225] Compound 26 (81.30 mg, 0.4288 mmol) and Compound 20 (110 mg,
0.4715 mmol) were charged to a round bottomed flask. 2-MeTHF (1 mL)
followed by 50% T3P in 2-MeTHF (371.4 mg, 0.5836 mmol) and pyridine
(67.84 mg, 69.37 .mu.L, 0.8576 mmol) in 2-MeTHF were then added.
The suspension was heated at 47.5.degree. C..+-.5.degree. C.
overnight. After completion, the reaction was cooled to ambient
temperature. 2-MeTHF (1.014 mL) and water (811.2 .mu.L) were added.
The layers were separated and the organic layer was washed with
water (811.2 .mu.L) and twice with NaHCO.sub.3 (2 mL). The organic
layer was transferred into a round bottomed flask. LiOH (38.6 mg,
0.9 mmol) dissolved in water (2 mL) was added and the reaction was
heated to 45.degree. C. After completion, the layers were separated
and the organic layer was discarded. The aqueous layer was cooled
with an ice bath and hydrochloric acid (10.72 mL of 1.0 M, 10.72
mmol) was added to the solution until the pH reached a pH of about
3-4. The aqueous layer was extracted twice with 2-MeTHF (5 ml), and
the organic layers were combined and washed with brine (5 ml). The
organic layer was separated and the solvent removed in vacuo. The
resulting solid was dried in a vacuum oven with nitrogen bleed at
50.degree. C. to give the title compound. .sup.1H-NMR (400 MHZ,
DMSO-d6) .delta. 12.89 (d, J 6.8 Hz, 1H); 11.84 (s, 1H); 11.74 (s,
1H); 9.36 (s, 1H); 8.87-8.61 (d, J 6.4 Hz, 1H); 8.34-8.32 (d, J 9.1
Hz 1H); 7.83-7.745 (m, 2H); 7.17-7.09 (m, 1H); 7.17 (s, 1H); 7.09
(s, 1H); 1.43 (s, 6H); 1.40 (s, 9H)
Example 6
Procedure for the biosynthesis of
N-(2-tert-butyl-5-hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo--
1,4-dihydroquinoline-3-carboxamide (27) and
2-(5-tert-butyl-2-hydroxy-4-(4-oxo-1,4-dihydroquinoline-3-carboxamido)phe-
nyl)-2-methylpropanoic acid (28)
##STR00080##
[0227] Streptomyces rimosus (DSM 40260) was purchased from DSMZ as
frozen culture. This culture was used to inoculate agar slants,
which were maintained and stored at 4.degree. C. Yeast extract-malt
extract-peptone (YMP) media containing yeast extract (4 g/L), malt
extract (10 g/L) and soya flour (5 g/L) was prepared and sterilized
at 130.degree. C. for 60 minutes. Five flasks containing 1 L of YMP
media were directly inoculated with S. rimosus from the agar
slants. The culture was allowed to grow for 2-3 days at 30.degree.
C. with gentle agitation of approximately 100 rpm. Under these
conditions, two growth types have been observed, either a cloudy
solution or spherical particulates which aggregate at the bottom of
the flask. The latter growth type has been shown to result in
higher conversions to Compound 27. The cells were then spun down,
harvested and resuspended in two flasks containing 1 L of 0.1 M
potassium phosphate buffer, pH 7.0. 5.0 g of Compound 34 in 50 mL
N,N-dimethylformamide (DMF) were added to the flasks. The reactions
proceeded for 24 hours at 30.degree. C. with gentle agitation of
about 100 rpm at which point conversions of 7.59% Compound 27 and
1.17% Compound 28 were indicated by HPLC.
[0228] Both flasks were combined, centrifuged at 3500 rpm for 10
minutes, and re-suspended in 500 mL of methanol. This suspension
was stirred vigorously for 30 minutes and then spun down again at
6000 rpm for 10 minutes. The organic layer was collected and the
process was repeated two times. The methanol extracts were
concentrated in vacuo to yield 2.50 g, 1.57 g and 1.11 g of solid
material, respectively. The solids from these extracts were shown
to contain 74.78-91.96% Compound 34, 7.66-19.73% Compound 27 and
0.39-5.49% Compound 28. In an effort to cull off a portion of
Compound 34 from the bio-oxidation products, the solids from the
first two extractions were combined, suspended in 250 mL methanol,
agitated vigorously for 1 hour and vacuum filtered. While Compounds
27 and 28 were enriched in the filtrate (22.09 and 6.14%,
respectively), the solids still also contained Compound 27 (8.96%)
and Compound 28 (0.50%).
[0229] The methanol filtrate containing approximately 2.2 g of
dissolved solids was adsorbed onto 4.5 g of silica and purified by
flash chromatography using a gradient of 100% dichloromethane to
88:12 dichloromethane/methanol. Fractions containing Compound 27
were concentrated in vacuo and further dried via freeze-drying to
obtain 130 mg of Compound 27 (98.5% purity by HPLC). A fraction
containing impure Compound 28 was also concentrated in vacuo to
yield less than 10 mg of solid.
[0230] The cell pellet was re-suspended in 500 mL methanol and
homogenized in a BeadBeater to break apart the cells and recover
any remaining product. The organic layer was obtained by
centrifuging the homogenized suspension at 6000 rpm for 10 minutes.
This was added to the solid obtained from the third extraction and
the filtered solids from the slurry enrichment of the first two
extractions and slurried at reflux overnight. The slurry was then
cooled and suction filtered to obtain 1.99 g of solid. The solid
was re-dissolved in 300 mL methanol which was then adsorbed onto
approximately 5 g of silica and purified by flash chromatography
using a gradient of 100% dichloromethane to 94:6
dichloromethane/methanol to provide 820 mg of solid containing
Compound 34 and Compound 27 as well as other impurities. This was
re-columned using a more gradual solvent gradient (100% DCM up to a
mixture of 6% MeOH/94% DCM) to obtain an additional 89 mg of
Compound 27. The .sup.1H-NMR spectrum was consistent with that
reported above.
Example 7
General Procedure to Test Solubility at pH 7.4
[0231] A high throughput shake flask assay was used to determine
solubility of compounds in pH 7.4 buffer. To calculate the
concentration of compounds in solution, two conditions per compound
were run: 300 uM in 100% DMSO and 200 uM in pH 7.4 phosphate buffer
with 2% DMSO present. Each sample was left to shake overnight then
injected onto HPLC-UV to determine peak area using the following
conditions: Phenomenex 00A-4251-B0--30.times.2.00 mm Luna 3u C18(2)
100 A column; 0.8 mL/min flow rate; 20 uL injection volume; HPLC
grade water with 0.1% formic acid and HPLC grade acetonitrile with
0.1% formic acid mobile phases; peak area determined at 254 nm.
Solubility in uM was calculated using the following equation:
conc.=(peak area pH 7.4)/(peak area 300 uM DMSO standard
condition)*300 uM concentration of standard condition. Peaks of
interest were identified in buffer conditions based on retention
time (RT) of the largest area peak in the 300 uM DMSO standard
condition.
Activity Assays
Example 8
General Procedure for Activity Assays
[0232] Assays for Detecting and Measuring .DELTA.F508-CFTR
Potentiation Properties of Compounds
[0233] Membrane Potential Optical Methods for Assaying
.DELTA.F508-CFTR Modulation Properties of Compounds
[0234] The assay utilizes fluorescent voltage sensing dyes to
measure changes in membrane potential using a fluorescent plate
reader (e.g., FLIPR III, Molecular Devices, Inc.) as a readout for
increase in functional .DELTA.F508-CFTR in NIH 3T3 cells. The
driving force for the response is the creation of a chloride ion
gradient in conjunction with channel activation by a single liquid
addition step after the cells have previously been treated with
compounds and subsequently loaded with a voltage sensing dye.
[0235] Identification of Potentiator Compounds
[0236] To identify potentiators of .DELTA.F508-CFTR, a
double-addition HTS assay format was developed. This HTS assay
utilizes fluorescent voltage sensing dyes to measure changes in
membrane potential on the FLIPR III as a measurement for increase
in gating (conductance) of .DELTA.F508 CFTR in
temperature-corrected .DELTA.F508 CFTR NIH 3T3 cells. The driving
force for the response is a Cl.sup.- ion gradient in conjunction
with channel activation with forskolin in a single liquid addition
step using a fluorescent plate reader such as FLIPR III after the
cells have previously been treated with potentiator compounds (or
DMSO vehicle control) and subsequently loaded with a redistribution
dye.
[0237] Solutions [0238] 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. [0239]
Chloride-free bath solution: Chloride salts in Bath Solution #1 are
substituted with gluconate salts.
[0240] Cell Culture
[0241] 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 .about.20,000/well
in 384-well matrigel-coated plates and cultured for 2 hrs at
37.degree. C. before culturing at 27.degree. C. for 24 hours. 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.
[0242] 1. Ussing Chamber Assay
[0243] Ussing chamber experiments were performed on polarized
airway epithelial cells expressing .DELTA.F508-CFTR to further
characterize the .DELTA.F508-CFTR modulators identified in the
optical assays. Non-CF and CF airway epithelia were isolated from
bronchial tissue, cultured as previously described (Galietta, L. J.
V., Lantero, S., Gazzolo, A., Sacco, O., Romano, L., Rossi, G. A.,
& Zegarra-Moran, O. (1998) In Vitro Cell. Dev. Biol. 34,
478-481), and plated onto Costar.RTM. Snapwell.TM. filters that
were pre-coated with NIH3T3-conditioned media. After four days the
apical media was removed and the cells were grown at an air liquid
interface for >14 days prior to use. This resulted in a
monolayer of fully differentiated columnar cells that were
ciliated, features that are characteristic of airway epithelia.
Non-CF HBE were isolated from non-smokers that did not have any
known lung disease. CF-HBE were isolated from patients homozygous
for .DELTA.F508-CFTR.
[0244] HBE grown on Costar.RTM. Snapwell.TM. cell culture inserts
were mounted in an Ussing chamber (Physiologic Instruments, Inc.,
San Diego, Calif.), and the transepithelial resistance and
short-circuit current in the presence of a basolateral to apical
Cl.sup.- gradient (I.sub.SC) were measured using a voltage-clamp
system (Department of Bioengineering, University of Iowa, IA).
Briefly, HBE were examined under voltage-clamp recording conditions
(V.sub.hold=0 mV) at 37.degree. C. The basolateral solution
contained (in mM) 145 NaCl, 0.83 K.sub.2HPO.sub.4, 3.3
KH.sub.2PO.sub.4, 1.2 MgCl.sub.2, 1.2 CaCl.sub.2, 10 Glucose, 10
HEPES (pH adjusted to 7.35 with NaOH) and the apical solution
contained (in mM) 145 NaGluconate, 1.2 MgCl.sub.2, 1.2 CaCl.sub.2,
10 glucose, 10 HEPES (pH adjusted to 7.35 with NaOH).
[0245] Identification of Potentiator Compounds
[0246] 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, 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. Forskolin (10 .mu.M) and all test compounds were added
to the apical side of the cell culture inserts. The efficacy of the
putative .DELTA.F508-CFTR potentiators was compared to that of the
known potentiator, genistein.
[0247] 2. Patch-Clamp Recordings
[0248] Total Cl.sup.- current in .DELTA.F508-NIH3T3 cells was
monitored using the perforated-patch recording configuration as
previously described (Rae, J., Cooper, K., Gates, P., & Watsky,
M. (1991) J. Neurosci. Methods 37, 15-26). Voltage-clamp recordings
were performed at 22.degree. C. using an Axopatch 200B patch-clamp
amplifier (Axon Instruments Inc., Foster City, Calif.). The pipette
solution contained (in mM) 150 N-methyl-D-glucamine (NMDG)-Cl, 2
MgCl.sub.2, 2 CaCl.sub.2, 10 EGTA, 10 HEPES, and 240 .mu.g/ml
amphotericin-B (pH adjusted to 7.35 with HCl). The extracellular
medium contained (in mM) 150 NMDG-Cl, 2 MgCl.sub.2, 2 CaCl.sub.2,
10 HEPES (pH adjusted to 7.35 with HCl). 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.). To activate .DELTA.F508-CFTR, 10 .mu.M forskolin
and 20 .mu.M genistein were added to the bath and the
current-voltage relation was monitored every 30 sec.
[0249] Identification of Potentiator Compounds
[0250] 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.DELTA..sub.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).
[0251] Cell Culture
[0252] 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.
[0253] 3. Single-Channel Recordings
[0254] Gating activity of wt-CFTR and temperature-corrected
.DELTA.F508-CFTR expressed in NIH3T3 cells was observed using
excised inside-out membrane patch recordings as previously
described (Dalemans, W., Barbry, P., Champigny, G., Jallat, S.,
Dott, K., Dreyer, D., Crystal, R. G., Pavirani, A., Lecocq, J-P.,
Lazdunski, M. (1991) Nature 354, 526-528) using an Axopatch 200B
patch-clamp amplifier (Axon Instruments Inc.). The pipette
contained (in mM): 150 NMDG, 150 aspartic acid, 5 CaCl.sub.2, 2
MgCl.sub.2, and 10 HEPES (pH adjusted to 7.35 with Tris base). The
bath contained (in mM): 150 NMDG-Cl, 2 MgCl.sub.2, 5 EGTA, 10 TES,
and 14 Tris base (pH adjusted to 7.35 with HCl). After excision,
both wt- and .DELTA.F508-CFTR were activated by adding 1 mM Mg-ATP,
75 nM of the catalytic subunit of cAMP-dependent protein kinase
(PKA; Promega Corp. Madison, Wis.), and 10 mM NaF to inhibit
protein phosphatases, which prevented current rundown. The pipette
potential was maintained at 80 mV. 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.
[0255] Cell Culture
[0256] 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.
[0257] Compounds 27 and 28 were tested for solubility using the
procedure given in Example 3 and for activity using the procedure
given in the assays of Example 4. The results are shown in Table 1.
The compound activity is illustrated with "+++" if EC.sub.50 (or
IC.sub.50) was measured to be less than 2.0 .mu.M, "++" if activity
was measured to be from 2 .mu.M to 5.0 .mu.M, "+" if activity was
measured to be greater than 5.0 .mu.M, and "-" if no data was
available. The efficacy is illustrated with "+++" if efficacy was
calculated to be greater than 100%, "++" if efficacy was calculated
to be from 100% to 25%, "+" if efficacy was calculated to be less
than 25%, and "-" if no data was available.
TABLE-US-00001 TABLE 1 Examples of activities and efficacies of the
compounds of Formula I Structure ##STR00081## ##STR00082## HTSF
Solubility @ pH = 7.4 87 uM >200 uM Optical EC.sub.50 +++ ++
.DELTA.508-HBE +++ +++ EC.sub.50 .DELTA.508/G551D-HBE +++ ++
EC.sub.50 HLM/RLM ++ +++ (% remain. @ 30 min) CaV 1.2 IC.sub.50 +
-
OTHER EMBODIMENTS
[0258] All publications and patents referred to in this disclosure
are incorporated herein by reference to the same extent as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. Should the
meaning of the terms in any of the patents or publications
incorporated by reference conflict with the meaning of the terms
used in this disclosure, the meaning of the terms in this
disclosure are intended to be controlling. Furthermore, the
foregoing discussion discloses and describes merely exemplary
embodiments of the present invention. One skilled in the art will
readily recognize from such discussion and from the accompanying
drawings and claims, that various changes, modifications and
variations can be made therein without departing from the spirit
and scope of the invention as defined in the following claims.
* * * * *
References