Title of Invention

AZAINDOLE DERIVATIVES AS CFTR MODULATORS

Abstract The present invention relates to. modulators of ATP-Binding Cassette ("ABC") transporters or fragments thereof of formula (I), including Cystic F1-brosis Transmembrane Conductance Regulator ("CFTR"), compositions thereof, and methods therewith. The present invention also relates to methods of treating ABC transporter mediated diseases using such modulators.
Full Text AZAINDOLEDERIVATIVES AS CFTR MODULATORS
TECHNICAL FIELD OF THE INVENTION
[00100] The present invention relates to modulators of ATP-Binding Cassette
("ABC") transporters or fragments thereof, including Cystic Fibrosis Transmembrane
Conductance Regulator ("CFTR"), compositions thereof, and methods therewith. The present
invention also relates to methods of treating ABC transporter mediated diseases using such
modulators.
BACKGROUND OF THE INVENTION
[00101] ABC transporters are a family of membrane transporter proteins that regulate
the transport of a wide variety of pharmacological agents, potentially toxic drugs, and
xenobiotics, as well as anions. ABC transporters are homologous membrane proteins that bind
and use cellular adenosine triphosphate (ATP) for their specific activities. Some of these
transporters were discovered as multidrug resistance proteins (like the MDR1-P glycoprotein,
or the multidrug resistance protein, MRP 1), defending malignant cancer cells against
chemotherapeutic agents. To date, 48 ABC Transporters have been identified and grouped into
7 families based on their sequence identity and function.
[00102] ABC transporters regulate a variety of important physiological roles within
the body and provide defense against harmful environmental compounds. Because of this, they
represent important potential drug targets for the treatment of diseases associated with defects
in the transporter, prevention of drug transport out of the target cell, and intervention in other
diseases in which modulation of ABC transporter activity may be beneficial.
[00103] One member of the ABC transporter family commonly associated with
disease is the cAMP/ATP-mediated anion channel, CFTR. CFTR 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 repeate 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.
[00104] 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.
[00105] In patients with cystic fibrosis, 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.
[00106] 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, > 1000 disease
causing mutations in the CF gene have been identified rhttp:///vt'^-w.genet.sickkids.on.ca/cftr/).
The most prevalent mutation is a deletion of phenylalanine at position 508 of the CFTR amino
acid sequence, and is commonly referred to as AF508-CFTR. This mutation occurs in
approximately 70% of the cases of cystic fibrosis and is associated with a severe disease.
[00107] The deletion of residue 508 in AF508-CFTR prevents the nascent protein
from folding correctly. This results in the inability of the mutant protein to exit the
endoplasmic reticulum ("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 AF5O8-
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 AF508-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.
[00108] 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+ channel, ENaC, Na+/2C17K+ co-transporter, Na+-K+-ATPase pump and the basolateral
membrane K+ channels, that are responsible for the uptake of chloride into the cell.
[00109] 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+-K+-ATPase pump and Cl- channels expressed on the basolateral surface of the cell.
Secondary active transport of chloride from the luminal side leads to the accumulation of
intracellular chloride, which can then passively leave the cell via Cl" channels, resulting in a
vectorial transport. Arrangement of Na+/2C17K+ co-transporter, Na+-K+-ATPase pump and the
basolateral membrane K+ 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.
[00110] In addition to Cystic Fibrosis, modulation of CFTR activity may be beneficial
for other diseases not directly caused by mutations in CFTR, such as secretory diseases and
other protein folding diseases mediated by CFTR. These include, but are not limited to,
chronic obstructive pulmonary disease (COPD), dry eye disease, and Sjogren's Syndrome.
[00111] COPD is characterized by airflow limitation that is progressive and not fully
reversible. The airflow limitation is due to mucus hypersecretion, emphysema, and
bronchiolitis. Activators of mutant or wild-type CFTR offer a potential treatment of mucus
hypersecretion and impaired mucociliary clearance that is common in COPD. Specifically,

increasing anion secretion across CFTR may facilitate fluid transport into the airway surface
liquid to hydrate the mucus and optimized periciliary fluid viscosity. This would lead to
enhanced mucociliary clearance and a reduction in the symptoms associated with COPD. Dry
eye disease is characterized by a decrease in tear aqueous production and abnormal tear film
lipid, protein and mucin profiles. There are many causes of dry eye, some of which include age,
Lasik eye surgery, arthritis, medications, chemical/thermal burns, allergies, and diseases, such
as cystic fibrosis and Sjogrens's syndrome. Increasing anion secretion via CFTR would
enhance fluid transport from the corneal endothelial cells and secretory glands surrounding the
eye to increase corneal hydration. This would help to alleviate the symptoms associated with
dry eye disease. Sjogrens's syndrome is an autoimmune disease in which the immune system
attacks moisture-producing glands throughout the body, including the eye, mouth, skin,
respiratory tissue, liver, vagina, and gut. Symptoms, include, dry eye, mouth, and vagina, as
well as lung disease. The disease is also associated with rheumatoid arthritis, systemic lupus,
systemic sclerosis, and polymypositis/dermatomyositis. Defective protein trafficking is
believed to cause the disease, for which treatment options are limited. Modulators of CFTR
activity may hydrate the various organs afflicted by the disease and help to elevate the
associated symptoms.
[00112] As discussed above, it is believed that the deletion of residue 508 in AF508-
CFTR prevents the nascent protein from folding correctly, resulting in the inability of this
mutant protein to exit the ER, and traffic to the plasma membrane. As a result, insufficient
amounts of the mature protein are present at the plasma membrane and chloride transport
within epithelial tissues is significantly reduced. In fact, this cellular phenomenon of defective
ER processing of ABC transporters by the ER machinery, has been shown to be the underlying
basis not only for CF disease, but for a wide range of other isolated and inherited diseases. The
two ways that the ER machinery can malfunction is either by loss of coupling to ER export of
the proteins leading to degradation, or by the ER accumulation of these defective/misfolded
proteins [Aridor M, et al., Nature Med., 5(7), pp 745- 751 (1999); Shastry, B.S., et al,
Neurochem. International, 43, pp 1-7 (2003); Rutishauser, I, et al, Swiss Med Wkly, 132, pp
211-222 (2002); Morello, JP et al, TIPS, 21, pp. 466- 469 (2000); Bross P., et al, Human
Mut, 14, pp. 186-198 (1999)]. The diseases associated with the first class of ER malfunction
are cystic fibrosis (due to misfolded AF508-CFTR as discussed above), hereditary emphysema
(due to al-antitrypsin; non Piz variants), hereditary hemochromatosis, coagulation-fibrinolysis
deficiencies, such as protein C deficiency, Type 1 hereditary angioedema, lipid processing
deficiencies, such as familial hypercholesterolemia, Type 1 chylomicronemia,

abetalipoproteinemia, lysosomal storage diseases, such as I-cell disease/pseudo-Hurler,
mucopolysaccharidoses (due to lysosomal processing enzymes), Sandhof/Tay-Sachs (due to p-
hexosaminidase), Crigler-Najjar type II (due to UDP-glucuronyl-sialyc-transferase),
polyendocrinopathy/hyperinsulemia, diabetes mellitus (due to insulin receptor), Laron
dwarfism (due to growth hormone receptor), myleoperoxidase deficiency, primary
hypoparathyroidism (due to preproparathyroid hormone), melanoma (due to tyrosinase). The
diseases associated with the latter class of ER malfunction are glycanosis CDG type 1,
hereditary emphysema (due to ocl-antitrypsin (PiZ variant), congenital hyperthyroidism,
osteogenesis imperfecta (due to Type I, II, IV procollagen), hereditary hypofibrinogenemia
(due to fibrinogen), ACT deficiency (due to al-antichymotrypsin), diabetes insipidus (DI),
neurophyseal DI (due to vasopvessin hormone/V2-receptor), neprogenic DI (due to aquaporin
IT), Charcot-Marie Tooth syndrome (due to peripheral myelin protein 22), Perlizaeus-
Merzbacher disease, neurodegenerative diseases such as Alzheimer's disease ( due to (3APP and
presenilins), Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear plasy,
Pick's disease, several polyglutamine neurological disorders such as Huntington,
spinocerebullar ataxia type I, spinal and bulbar muscular atrophy, dentatorubal pallidoluysian,
and myotonic dystrophy, as well as spongiform encephalopathies, such as hereditary
Creutzfeldt-Jakob disease (due to prion protein processing defect), Fabry disease (due to
lysosomal a-galactosidase A), Straussler-Scheinker syndrome, chronic obstructive pulmonary
disease (COPD), dry eye disease, and Sjogren's Syndrome.
[00113] In addition to up-regulation of CFTR activity, reducing anion secretion by
CFTR modulators may be beneficial for the treatment of secretory diarrheas, in which epithelial
water transport is dramatically increased as a result of secretagogue activated chloride
transport. The mechanism involves elevation of cAMP and stimulation of CFTR.
[00114] Although there are numerous causes of diarrhea, the major consequences of
diarrheal diseases, resulting from excessive chloride transport are common to all, and include
dehydration, acidosis, impaired growth and death.
[00115] Acute and chronic diarrheas represent a major medical problem in many areas
of the world. Diarrhea is both a significant factor in malnutrition and the leading cause of death
(5,000,000 deaths/year) in children less than five years old.
[00116] Secretory diarrheas are also a dangerous condition in patients of acquired
immunodeficiency syndrome (AIDS) and chronic inflammatory bowel disease (IBD). Sixteen
million travelers to developing countries from industrialized nations every year develop

diarrhea, with the severity and number of cases of diarrhea varying depending on the country
and area of travel.
[00117] Diarrhea in barn animals and pets such as cows, pigs and horses, sheep, goats,
cats and dogs, also known as scours, is a major cause of death in these animals. Diarrhea can
result from any major transition, such as weaning or physical movement, as well as in response
to a variety of bacterial or viral infections and generally occurs within the first few hours of the
animal's life.
[00118] The most common diarrheal causing bacteria is enterotoxogenic E-coli
(ETEC) having the K99 pilus antigen. Common viral causes of diarrhea include rotavirus and
coronavirus. Other infectious agents include cryptosporidium, giardia lamblia, and salmonella,
among others.
[00119] Symptoms of rotaviral infection include excretion of watery feces,
dehydration and weakness. Coronavirus causes a more severe illness in the newborn animals,
and has a higher mortality rate than rotaviral infection. Often, however, a young animal may be
infected with more than one virus or with a combination of viral and bacterial microorganisms
at one time. This dramatically increases the severity of the disease.
[00120] Accordingly, there is a need for modulators of an ABC transporter activity,
and compositions thereof, that can be used to modulate the activity of the ABC transporter in
the cell membrane of a mammal.
[00121] There is a need for methods of treating ABC transporter mediated diseases
using such modulators of ABC transporter activity.
[00122] There is a need for methods of modulating an ABC transporter activity in an
ex vivo cell membrane of a mammal.
[00123] There is a need for modulators of CFTR activity that can be used to modulate
the activity of CFTR in the cell membrane of a mammal.
[00124] There is a need for methods of treating CFTR-mediated diseases using such
modulators of CFTR activity.
[00125] There is a need for methods of modulating CFTR activity in an ex vivo cell
membrane of a mammal.

SUMMARY OF THE INVENTION
[00126] It has now been found that compounds of this invention, and
pharmaceutically acceptable compositions thereof, are useful as modulators of ABC transporter
activity, particularly CFTR activity. These compounds have the general formula I:

or a pharmaceutically acceptable salt thereof, wherein Ar1, RN, ring A, ring B, X,
Rx, and x are described below.
[00127] These compounds and pharmaceutically acceptable compositions are useful
for treating or lessening the severity of a variety of diseases, disorders, or conditions, including,
but not limited to, cystic fibrosis, hereditary emphysema, hereditary hemochromatosis,
coagulation-fibrinolysis deficiencies, such as protein C deficiency, Type 1 hereditary
angioedema, lipid processing deficiencies, such as familial hypercholesterolemia, Type 1
chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, such as I-cell
disease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II,
polyendocrinopathy/hyperinsulemia, diabetes mellitus, laron dwarfism, myleoperoxidase
deficiency, primary hypoparathyroidism, melanoma, glycanosis CDG type 1, hereditary
emphysema, congenital hyperthyroidism, osteogenesis imperfecta, hereditary
hypofibrinogenemia, ACT deficiency, diabetes insipidus (di), neurophyseal di, neprogenic DI,
Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases
such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive
supranuclear plasy, Pick's disease, several polyglutamine neurological disorders asuch as
Huntington, spinocerebullar ataxia type I, spinal and bulbar muscular atrophy, dentatorubal
pallidoluysian, and myotonic dystrophy, as well as spongiform encephalopathies, such as
hereditary Creutzfeldt-Jakob disease, Fabry disease, Straussler-Scheinker syndrome, COPD,
dry-eye disease, and Sjogren's disease.

DETAILED DESCRIPTION OF THE INVENTION
1. General Description of Compounds of the Invention:
[00128] The present invention relates to compounds of formula I useful as modulators
of ABC transporter activity, particularly CFTR activity:

wherein:
each of G1, G2, G3, and G4 is independently selected from the group consisting of
CH and nitrogen, wherein one of G1, G2) G3, and G4 is nitrogen and the remainder of
Gi, G2, G3, and G4 each is CH;
Ar1 is attached to the N(RN) through G2 or G3;
Ar1 is optionally substituted with w occurrences of-WRw; and
RNis H,R2, or R3;
ring A is 3-7 membered monocyclic ring having 0-3 heteroatoms selected from the
group consisting of oxygen, sulfur, and nitrogen, wherein ring A is optionally
substituted with q occurrences of-Q-RQ;
ring B is optionally fused to a 5-7 membered ring selected from the group
consisting of cycloaliphatic, aryl, heterocyclic, and heteroaryl, wherein ring B, together
with said optionally fused ring, is optionally substituted with x occurrences of-XRX;
Q, W, or X is independently a bond or is independently an optionally substituted
(C1-C6) alkylidene chain wherein up to two methylene units of Q, W, or X are
optionally and independently replaced by -CO-, -CS-, -COCO-, -CONR'-, -CONRTsfR'-

, -C02-, -0C0-, -NR'CCV, -0-, -NR'CONR'-, -OCONR'-, -NR'NR', -NRTm'CO-,
-NR'CO-, -S-, -SO, -SO2-, -NR1-, -SO2NR'-, NR'SO2-, or -NR'SO2NR'-;
each RQ, Rw, and Rx is independently R1, R2, R3, R4, or R5;
R1 is independently R2, R3, or R6;
R1 is oxo, =NN(R6)2, =NN(R7)2, =NN(R6R7), R6, or ((Cl-C4)aliphatic)n-Y;
wherein n is 0 or 1; and
Y is halo, CN, N02, CF3, OCF3, OH, SR6, S(O)R6, SO2R6, NH2, NHR6, N(R6)2,
NR6R8, COOH, COOR6, or OR6; or

R2 is aliphatic, wherein each R2 is optionally substituted with up to 2 substituents
independently selected from the group consisting of R1, R4, and R5;
R3 is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, wherein R3 is optionally
substituted with up to 3 substituents, independently selected from the group consisting
of^.R2, R4, andR5;
R4 is OR5, OR6, OC(O)R6, OC(O)R5, OC(O)OR6, OC(O)OR5, OC(O)N(R6)2,
OC(O)N(R5)2, OC(O)N(R6R5), SR6, SR5, S(O)R6, S(O)R5, SO2R6, SO2R5, SO2N(R6)2,
SO2N(R5)2, SO2NR5R6, SO3R6, SO3RS, C(O)R5, C(O)OR5, C(O)R6, C(O)OR6,
C(O)N(R6)2, C(O)N(R5)2, C(O)N(R5R6), C(O)N(OR6)R6, C(O)N(OR5)R6,
C(O)N(OR6)R5, C(O)N(OR5)R5, C^OR^R6, CCNOR^R5, C(NOR5)R6, C(NOR5)R5,
N(R6)2, N(R5)2, N(R5R6), NR5C(O)R5, NR6C(O)R6, NR6C(O)R5, NR5C(O)R6,
NR6C(O)OR6, NR5C(O)OR6, NR6C(O)OR5, NR5C(O)OR5, NR6C(O)N(R6)2,
NR6C(O)NR5R6, NR6C(O)N(R5)2, NR5C(O)N(R6)2, NR5C(O)NR5R6, NR5C(O)N(R5)2,
NR6SO2R6, NR6SO2R5, NR5SO2R6,NR5SO2R5, NR6SO2N(R6)2, NR6SO2NR5R6,
NR6SO2N(R5)2, NR5SO2NR5R6, NR5SO2N(R5)2, N(OR6)R6, N(OR6)R5, N(OR5)R5, or
N(OR5)R6;

R is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, wherein R5 is optionally
substituted with up to 3 R1;
R6 is H or aliphatic, wherein R6 is optionally substituted with a R7;
R is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, and each R7 is
optionally substituted up to 2 substituents independently selected from the group
consisting of H, (Cl-C6)-straight or branched alkyl, (C2-C6) straight or branched
alkenyl oralkynyl, 1,2-methylenedioxy, 1,2-ethylenedioxy, and(CH2)n-Z;
Z is selected from the group consisting of halo, CN, NO2, CF3, OCF3, OH, S-
aliphatic, S(O)-aliphatic, SO2-aliphatic, NH2, NH-aliphatic, N(aliphatic)2,
N(aliphatic)R8, NHR8, COOH, C(O)O(-aliphatic), and O-aliphatic;
Q
R is an amino protecting group;
w is 0 to 5; and
each of x and q is independently 0-5.
2. Compounds and Definitions:
[00129] 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.
[00130] 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 ah, J.
Gen. Physiol. (1998): 111(3), 477-90.
[00131] 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, AF508 CFTR and G55 ID CFTR (see, e.g., http://www.gengt.sickkids.on.ca/cftr/. for
CFTR mutations).
[00132] The term "modulating" as used herein means increasing or decreasing by a
measurable amount.
[00133] For purposes of this invention, the chemical elements are identified in
accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and

Physics, 75th Ed. Additionally, general principles of organic chemistry are described in
"Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and
"March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, I, John Wiley
& Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.
[00134] 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°C or less, in
the absence of moisture or other chemically reactive conditions, for at least a week.
[00135] The term "aliphatic" or "aliphatic group", as used herein, means a straight-
chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is
completely saturated or that contains one or more units of unsaturation, or a monocyclic
hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more
units of unsaturation, but which is not aromatic (also referred to herein as "carbocycle"
"cycloaliphatic" or "cycloalkyl"), that has a single point of attachment to the rest of the
molecule. Unless otherwise specified, aliphatic groups contain 1-20 aliphatic carbon atoms. In
some embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In other
embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In still other embodiments,
aliphatic groups contain 1-6 aliphatic carbon atoms, and in yet other embodiments aliphatic
groups contain 1-4 aliphatic carbon atoms. In some embodiments, "cycloaliphatic" (or
"carbocycle" or "cycloalkyl") refers to a monocyclic C3-C8 hydrocarbon or bicyclic C8-C12

hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but
which is not aromatic, that has a single point of attachment to the rest of the molecule wherein
any individual ring in said bicyclic ring system has 3-7 members. Suitable aliphatic groups
include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl,
alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or
(cycloalkyl)alkenyl.
[00136] The term "heteroaliphatic", as used herein, means aliphatic groups wherein
one or two carbon atoms are independently replaced by one or more of oxygen, sulfur,
nitrogen, phosphorus, or silicon. Heteroaliphatic groups may be substituted or unsubstituted,
branched or unbranched, cyclic or acyclic, and include "heterocycle", "heterocyclyl",
"heterocycloaliphatic", or "heterocyclic" groups.
[00137] The term "heterocycle", "heterocyclyl", "heterocycloaliphatic", or
"heterocyclic" as used herein means non-aromatic, monocyclic, bicyclic, or tricyclic ring
systems in which one or a plurality of ring members is an independently selected heteroatom.
In some embodiments, the "heterocycle", "heterocyclyl", "heterocycloaliphatic", or
"heterocyclic" group has three to fourteen ring members in which one or more ring members is
a heteroatom independently selected from the group consisting of oxygen, sulfur, nitrogen, and
phosphorus, and each ring in the system contains 3 to 7 ring members.
[00138] The term "heteroatom" means one or more of boron, oxygen, sulfur, nitrogen,
phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon;
the quaternized form of any basic nitrogen or; a substirutable nitrogen of a heterocyclic ring, for
example N (as in 3,4-dihydro-2/f-pyrrolyl), NH (as in pyrrolidinyl) or NR+ (as in N-substituted
pyrrolidinyl)).
[00139] The term "unsaturated", as used herein, means that a moiety has one or more
units of unsaturation.
[00140] The term "alkoxy", or "thioalkyl", as used herein, refers to an alkyl group, as
previously defined, attached to the principal carbon chain through an oxygen ("alkoxy") or
sulfur ("thioalkyl") atom.
[00141] The terms "haloaliphatic" and "haloalkoxy" means aliphatic or alkoxy, as the
case may be, substituted with one or more halogen atoms. The term "halogen" means F, Cl, Br,
or I. Examples of haloaliphatic include -CHF2, -CH2F, -CF3, -CF2-, or perhaloalkyl, such as, -
CF2CF3.

[00142] The term "aryl" used alone or as part of a larger moiety as in "aralkyl",
"aralkoxy", or "aryloxyalkyl", refers to monocyclic, bicyclic, and tricyclic ring systems having
a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and
wherein each ring in the system contains 3 to 7 ring members. The term "aryl" may be used
interchangeably with the term "aryl ring". The term "aryl" also refers to heteroaryl ring
systems as defined hereinbelow.
[00143] The term "heteroaryl", used alone or as part of a larger moiety as in
"heteroaralkyl" or "heteroarylalkoxy", refers to monocyclic, bicyclic, and tricyclic ring systems
having a total of five to fourteen ring members, wherein at least one ring in the system is
aromatic, at least one ring in the system contains one or more heteroatoms, and wherein each
ring in the system contains 3 to 7 ring members. The term "heteroaryl" may be used
interchangeably with the term "heteroaryl ring" or the term "heteroaromatic".
[00144] An aryl (including aralkyl, aralkoxy, aryloxyalkyl and the like) or heteroaryl
(including heteroaralkyl and heteroarylalkoxy and the like) group may contain one or more
substituents. Suitable substituents on the unsaturated carbon atom of an aryl or heteroaryl
group are selected from the group consisting of halogen; -R°; -OR°; -SR°; 1,2-methylene-
dioxy; 1,2-ethylenedioxy; phenyl (Ph) optionally substituted with R°; -O(Ph) optionally
substituted with R°; -(CH2)i-2(Ph), optionally substituted with R°; -CH=CH(Ph), optionally
substituted with R°; -NO2; -CN; -N(R°)2; -NR°C(O)R°; -NR°C(O)N(R°)2; -NR°CO2R°;
-NR°NR°C(O)R°; -NR°NRoC(0)N(Ro)2; -NR°NRoC02Ro; -C(O)C(O)R°; -C(O)CH2C(O)R°; -
CO2R°; -C(O)R°; -C(O)N(R°)2; -OC(O)N(R°)2; -S(O)2R°; -SO2N(R°)2; -S(O)R°; -
NR°SO2N(R°)2; -NR°SO2R°; -C(=S)N(R°)2; -C(=NH)-N(R°)2; and-(CH2)0.2NHC(O)R°
wherein each independent occurrence of R° is selected from the group consisting of hydrogen,
optionally substituted Ci_6 aliphatic, an unsubstituted 5-6 membered heteroaryl or heterocyclic
ring, phenyl, -O(Ph), and -CH2(Ph), or, notwithstanding the definition above, two independent
occurrences of R°, on the same substituent or different substituents, taken together with the
atom(s) to which each R° group is bound, form a 3-8-membered cycloalkyl, heterocyclyl, aryl,
or heteroaryl ring having 0-3 heteroatoms independently selected from the group consisting of
nitrogen, oxygen, and sulfur. Optional substituents on the aliphatic group of R° are selected
from the group consisting of NH2, NH(C1-4aliphatic), N(C1-4aliphatic)2, halogen, C1-4aliphatic,
OH, O(C1-4aliphatic), NO2, CN, CO2H, CO2(C1-4aliphatic), 0(haloC1-4 aliphatic), and haloC1-4
aliphatic, wherein each of the foregoing C1-4aliphatic groups of R° is unsubstituted.

[00145] An aliphatic or heteroaliphatic group, or a non-aromatic heterocyclic ring
may contain one or more substituents. Suitable substituents on the saturated carbon of an
aliphatic or heteroaliphatic group, or of a non-aromatic heterocyclic ring are selected from the
group consisting of those listed above for the unsaturated carbon of an aryl or heteroaryl group
and additionally include the following: =0, =S, =NNHR*, =NN(R*)2, =NNHC(O)R*,
=NNHCO2(alkyl), =NNHSO2(alkyi), and =NR*, where each R* is independently selected from
the group consisting of hydrogen and an optionally substituted Ci-6 aliphatic. Optional
substituents on the aliphatic group of R are selected from the group consisting of NH2, NH(C1-4.
aliphatic), N(C1-4 aliphatic)2, halogen, C1-4 aliphatic, OH, O(C1-4 aliphatic), NO2, CN, CO2H,
CO2(C1-4 aliphatic), O(halo C1-4 aliphatic), and halo(C1-4 aliphatic), wherein each of the
foregoing C1-4aliphatic groups of R* is unsubstituted.
[00146] Optional substituents on the nitrogen of a non-aromatic heterocyclic ring are
selected from the group consisting of-R+, -NCR1)2, -C(O)R+, -CO2R+, -C(O)C(O)R+, -
C(O)CH2C(O)R+, -SO2R+, -SO2N(R+)2, -C(=S)N(R^)2, -C(=NH)-N(R% and -NR+SO2R+;
wherein R+ is hydrogen, an optionally substituted C1-6 aliphatic, optionally substituted phenyl,
optionally substituted -O(Ph), optionally substituted -CH2(Ph), optionally substituted -(CH2)i_
2(Ph); optionally substituted -CH=CH(Ph); or an unsubstituted 5-6 membered heteroaryl or
heterocyclic ring having one to four heteroatoms independently selected from the group
consisting of oxygen, nitrogen, and sulfur, or, notwithstanding the definition above, two
independent occurrences of R+, on the same substituent or different substituents, taken together
with the atom(s) to which each R+ group is bound, form a 3-8-membered cycloalkyl,
heterocyclyl, aryl, or heteroaryl ring having 0-3 heteroatoms independently selected from the
group consisting of nitrogen, oxygen, and sulfur. Optional substituents on the aliphatic group
or the phenyl ring of R+ are selected from the group consisting of NH2, NH(C1-4 aliphatic),
N(C1-4 aliphatic)2, halogen, C1-4 aliphatic, OH, O(C1-4 aliphatic), NO2, CN, CO2H, CO2(C1-4
aliphatic), O(halo C1-4 aliphatic), and halo(C1-4 aliphatic), wherein each of the foregoing C1-4
aliphatic groups of R+ is unsubstituted.
[00147] The term "alkylidene chain" refers to a straight or branched carbon chain that
may be fully saturated or have one or more units of unsaturation and has two points of
attachment to the rest of the molecule. The term "spirocycloalkylidene" refers to a carbocyclic
ring that may be fully saturated or have one or more units of unsaturation and has two points of
attachment from the same ring carbon atom to the rest of the molecule.

[00148] As detailed above, in some embodiments, two independent occurrences of R°
(or R+, or any other variable similarly defined herein), are taken together with the atom(s) to
which each variable is bound to form a 3-8-membered cycloalkyl, heterocyclyl, aryl, or
heteroaryl ring having 0-3 heteroatoms independently selected from the group consisting of
nitrogen, oxygen, and sulfur. Exemplary rings that are formed when two independent
occurrences of R° (or R+, or any other variable similarly defined herein) are taken together with
the atom(s) to which each variable is bound include, but are not limited to the following: a)
two independent occurrences of R° (or R+, or any other variable similarly defined herein) that
are bound to the same atom and are taken together with that atom to form a ring, for example,
N(R°)2, where both occurrences of R° are taken together with the nitrogen atom to form a
piperidin-1-yl, piperazin-1-yl, or morpholin-4-yl group; and b) two independent occurrences of
R° (or R+, or any other variable similarly defined herein) that are bound to different atoms and
are taken together with both of those atoms to form a ring, for example where a phenyl group is
i
formed when two independent occurrences of R° (or R+, or any other variable similarly defined
herein) are taken together with the atom(s) to which each variable is bound and that the
examples detailed above are not intended to be limiting.
[00149] Unless otherwise stated, structures depicted herein are also meant to include
all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of
the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E)
double bond isomers, and (Z) and (E) conformational isomers. Therefore, single
stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or
conformational) mixtures of the present compounds are within the scope of the invention.
Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the
scope of the invention. Additionally, unless otherwise stated, structures depicted herein are
also meant to include compounds that differ only in the presence of one or more isotopically
enriched atoms. For example, compounds having the present structures except for the
replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C- or

14C-enriched carbon are within the scope of this invention. Such compounds are useful, for
example, as analytical tools or probes in biological assays.
3. Description of Exemplary Compounds:
[00150] In one embodiment, Ar1 is an optionally substituted ring selected from the
group consisting of:

[00151] In some embodiments, Ar1 is an optionally substituted group selected from
Ar-i, Ar-ii, Ar-iii, and Ar-iv.
[00152] In some embodiments, Ar1 is an optionally substituted group attached to the
N(RN) nitrogen atom through atom G2 or G3.
[00153] In one embodiment, RN is hydrogen. In another embodiment, RN is an
optionally substituted C1-C6 aliphatic. Or, RN is C1-C4 alkyl. Exemplary embodiments
include methyl, ethyl, or i-propyl.
[00154] In some embodiments, ring A is an optionally substituted 3-7 membered
cycloaliphatic ring.
[00155] In other embodiments, ring A is an optionally substituted 3-7 membered ring
containing 1 heteroatom selected from the group consisting of O, NH, and S. Or, ring A
contains up two heteroatoms selected from the group consisting of O, S, and NH.
e-11 1 g 11


[00157] Ring A is preferably selected from the group consisting of a, b, c, d, and 1.
[00158] In one embodiment, ring B is fused to a 5-7 membered heterocyclic or
heteroaryl ring having up to 3 heteroatoms independently selected from the group consisting of
B, O, N, and S.
[00159] In another embodiment, ring B is fused to a 5-6 membered heterocyclic ring
having up to 3 heteroatoms independently selected from the group consisting of B, O, N, and S.
[00160] In another embodiment, ring B is fused to a 5-6 membered heteroaryl ring
having up to 3-heteroatoms independently selected from the group consisting of O, N, and S.
[00161] In yet another embodiment, ring B, together with said fused ring, is optionally
substituted with up to two Rx substituents.
[00162] In another embodiment, Rx substituent is R1.
[00163] In another embodiment, said ring fused to ring B is selected from the group
consisting of:



[00164] In some embodiments, the ring that is fused to ring B is selected from the
group consisting of i, ii, iii, viii, ix, x, xi, xii, xiii, and xvi. In some embodiments, the ring that
is fused to ring B is selected from the group consisting of i, ii, iii, ix, xi, xii, xiii and xvi. In
other embodiments, the ring that is fused to ring B is i. Or, the ring that is fused to ring B is ii.
Or, is iii.
[00165] According to another embodiment, R1 is R6, wherein R6 is straight chain or
branched (Cl-C6)alkyl or (C2-C6) alkenyl or alkynyl, optionally substituted with R7.
[00166] According to another embodiment, R1 is (C1-C4 aliphatic)n-Y, wherein n is 0
or 1, and Y is halo, CN, NO2) CF3, OCF3, OH, SR6, S(O)R6, SO2R6, NH2, NHR6, N(R6)2,
NR6R85 COOH, COOR6, or OR6.
[00167] According to another embodiment, R1 is selected from the group consisting of
halo, CF3, NH2, NH(C1-C4 alkyl), NHC(O)CH3, OH, O(C1-C4 alkyl), OPh, O-benzyl, S-(C1-
C4 alkyl), C1-C4 aliphatic, CN, SO2NH(C1-C4 alkyl), and SO2N(C1-C4 alkyl)2. According to
yet another embodiments, two R1, taken together, is selected from the group consisting of
methylenedioxy, difluoromethylenedioxy and ethylenedioxy.
[00168] According to another embodiment, R1 is selected from the group consisting of
methyl, n-propyl, i-propyl, t-butyl, cyclopropylmethyl, cyclopropyl, halo, CF3, NH2, NH(CH3),
NHC(O)CH3, OH, OCH3, OPh, O-benzyl, S-(C2H5), S-CH3, NO2, CN, SO2NH(n-propyl), and

SO2N(n-propyl)2. According to yet another embodiment, two R1, taken together, is selected
from the group consisting of methylenedioxy and difluoromethylenedioxy.
[00169] According to one embodiment, R2 is a straight chain or branched (Cl-
C6)alkyl or (C2-C6) alkenyl or alkynyl, optionally substituted with R1, R4, or R5. In certain
embodiments, R2 is a straight chain or branched (Cl-C4)alkyl or (C2-C4) alkenyl or alkynyl,
optionally substituted with R1, R4, or R5. According to other embodiments, R2 is a straight
chain or branched (Cl-C4)alkyl or (C2-C4) alkenyl or alkynyl.
[00170] According to one embodiment, R3 is a cycloaliphatic, aryl, heterocyclic, or
heteroaryl ring, wherein R3 is optionally substituted with up to 3 substituents, independently
selected from the group consisting of R1, R2, R4, and R5. In one embodiment, R3 is a C3-C8
cycloaliphatic optionally substituted with up to 3 substituents independently selected from R1,
R2, R4, and R5. Exemplary cycloaliphatics include cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, or cycloheptyl. In another embodiment, R3 is a C6-C10 aryl, optionally substituted
with up to 3 substituents, independently selected from R1, R2, R4, and R5. Exemplary aryl rings
include phenyl or naphthyl. In another embodiment, R3 is a C3-C8 heterocyclic, optionally
substituted with up to 3 substituents, independently selected from R1, R2, R4, and R5.
Exemplary heterocyclic rings include azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl,
morpholinyl, or thiomorpholinyl. In another embodiment, R3 is a C5-C10 heteroaryl ring,
optionally substituted with up to 3 substituents, independently selected from R1, R2, R4, and R5.
Exemplary heteroaryl rings include pyridyl, pyrazyl, triazinyl, furanyl, pyrrolyl, thiophenyl,
oxazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, imidazolyl, triazolyl, thiadiazolyl, pyrimidinyl.
quinolinyl, isoquinolinyl, benzofuranyl, benzothiophenyl, quinolinyl, isoquinolinyl,
benzofuranyl, benzothiophenyl, indolizinyl, indolyl, isoindolyl, indolinyl, indazolyl,
benzimidazolyl, benzothiazolyl, purinyl, cinnolinyl, phthalazine, quinazolinyl, quinaoxalinyl,
naphthylirinyl, or pteridinyl.
[00171] According to one embodiment, R4 is selected from the group consisting of
OR5, OR6, SR5, SR6, NR5COR5, NR5COR6, NR6COR5, andNR6COR6.
[00172] According to one embodiment, R5 is C5-C6 cycloalkyl, C6 or C10 aryl, C5-
C10 heteroaryl or C3-C7 heterocyclyl, optionally substituted with up to 2 R1. In certain
embodiments, R5 is an optionally substituted cyclohexyl, phenyl, C5-C6 heteroaryl, or C3-C6
heterocyclyl.
[00173] According to one embodiment, R6 is H.

[00174] According to another embodiment, R6 is a straight chain or branched (C1 -
C6)alkyl or (C2-C6 alkenyl) or alkynyl, optionally substituted with R7.
[00175] According to another embodiment, R6 is a straight chain or branched (Cl-
C6)alkyl or (C2-C6 alkenyl) or alkynyl.
[00176] According to one embodiment, R7 is C5-C6 cycloalkyl, phenyl, naphthyl, C5-
C10 heteroaryl or C3-C7 heterocyclyl, optionally substituted with straight chain or branched
(Cl-C6)alkyl or (C2-C6 alkenyl) or alkynyl. Or, R7 is C5-C6 cycloalkyl, phenyl, naphthyl, C5-
C10 heteroaryl or C3-C7 heterocyclyl, optionally substituted with methylenedioxy,
difluoromethylenedioxy, ethylenedioxy, or (CH2)n-Z. In certain embodiments, R7 is an
optionally substituted cyclohexyl, phenyl, C5-C6 heteroaryl, or C3-C6 heterocyclyl.
[00177] According to one embodiment, R8 is acetyl, arylsulfonyl or C1-C6
alkylsulfonyl.
[00178] In some embodiments, J is CH2. In other embodiments, J is CF2. Or, J is

[00179] In one embodiment, Q is a bond. Or, Q is an (Cl-C6)alkylidene chain. Or,
Q is an (C1-C6) alkylidene chain, wherein up to two methylene units therein are optionally and
independently replaced by -CO-, -CS-, -COCO-, -CONR1-, -CONRTSTR'-, -CO2-, -OCO-,
-NR'CO2-, -O-, -NR'CONR'-, -OCONR1-, -NR'NR1, -NR'NR'CO-, -NR'CO-, -S-, -SO, -SO2-, -
NR'-, -SO2NR'-, NR'SO2-, or -NR'SO2NR'-. In one embodiment, said up to two methylene
units therein are optionally and independently replaced by -CO-, -CONR'-, -CO2-, -OCO-,
-NR'CO2-, -O-, -NR'CONR'-, -OCONR'-, -NR'CO-, -S-, -SO, -SO2-, -NR'-, -SO2NR'-, NR'SO2-
, or -NR'SO2NR'-. Or, said up to two methylene units therein are optionally and independently
replaced by -CO-, -O-, -S-, -NR'-, -CO2-, or -SO2-.
[00180] In one embodiment, w is 0-3. In another embodiment, w is 1-3.
[00181] In some embodiments, W is a bond. In other embodiments, W is an
optionally substituted (C1-C6) alkylidene chain wherein up to two methylene units of W is
optionally and independently replaced by -CO-, -CONR'-, -CO2-, -OCO-, -NR'CO2-, -O-, -
NR'CONR'-, -OCONR'-, -NR'CO-, -S-, -SO, -SO2-, -NR'-, -SO2NR'-, NR'SO2-, or -
NR'SO2NR'-. Or, W is an optionally substituted (C1-C6) alkylidene chain wherein up to two

non-adjacent methylene unit of W is optionally replaced by -CONR'-, -CO2-, -O-, -S-, -SO2-, -
NR'-, or -SO2NR'-.
[00182] In some embodiments, Rw is independently R2 or R3.
[00183] In another embodiment, Rw is C1-C6 aliphatic optionally substituted with up
to four substituents selected from the group consisting of R1, R4, and R5.
[00184] In another embodiment, Rw is C6-C10 aryl optionally substituted with up to
five substituents selected from the group consisting of R1, R4, and R5.
[00185] In yet another embodiment, Rw is 3-10 membered monocyclic or bicyclic
heterocyclic ring optionally substituted with up to five substituents selected from the group
consisting of R1, R4, and R5.
[00186] In another embodiment, Rw is 5-10 membered monocyclic or bicyclic
heteroaryl ring optionally substituted with up to five substituents selected from the group
consisting of R1, R4, and R5.
[00187] In an alternative embodiment, x is 1-5. In some embodiments, x is 1; in
others, x is 2; in some others, x is 3; in yet others, x is 4; and in others, x is 5.
[00188] In some embodiments, X is a bond. In some other embodiments, X is an (Cl-
C6) alkylidene chain wherein one or two non-adjacent methylene units are optionally and
independently replaced by O, NR', S, SO2, COO, or CO. In some embodiments, Rx is R2 or R3.
[00189] In another embodiment, the present invention provides compounds of formula
II:


wherein one of G1, G2, G3, and G4 is nitrogen and the remainder of G1, G2, G3, and
G4 each is CH;
wherein Ar1 is attached to the N(RN) through G2 or G3;
Ar1 is optionally substituted with up to 3 Rw substituents, wherein each Rw is
independently selected from the group consisting of R1, R2, R3, and R4.
[00190] In one embodiment, Ar1 is attached through atom G2.
[00191] In other embodiments, Ar1 is attached through atom G3.
[00192] In another embodiment, the present invention provides compounds of formula
IIIA or formula HIB:

wherein Rx, X, x, m, RN, Gi, G2, G3, and G4 are defined above; and
each Rw is independently selected from the group consisting of R1, R2, R3, and R4.
[00193] In one embodiment of IIIA, Gi is N, each of G2 and G4 is CH, and G3 is C.
In another embodiment of IIIA, G2 is N, each of Gi and G4 is CH, and G3 is C. In yet another
embodiment of IIIA, G4 is N, each of Gi and G2 is CH, and G3 is C. In one embodiment of
IIIB, Gi is N, each of G3 and G4 is CH, and G2 is C. In another embodiment of IIIB, G3 is N,
each of Gi and G4 is CH, and G2 is C. In yet another embodiment of IIIB, G4 is N, each of Gi
and G3 is CH, and G2 is C.
[00194] In one embodiment, Rw is R6 or ((C1 -C4)aliphatic)n-Y;
n is 0 or 1; and
Y is halo, CN, NO2, CF3, OCF3, OH, SR6, S(O)R6, SO2R6, NH2, NHR6, N(R6)2,
NR6R8, COOH, COOR6, or OR6.
[00195] In one embodiment, Rw is C1-C6 aliphatic optionally substituted with up to
four substituents independently selected from the group consisting of R1, R4, and R5

[00196] In another embodiment, Rw is a C6-C10 aryl optionally substituted with up to
five substituents selected from the group consisting of R1, R4, and R5.
[00197] In yet another embodiment, Rw is a 3-10 membered monocyclic or bicyclic
heterocyclic ring optionally substituted with up to five substituents selected from the group
consisting of R1, R4, and R5.
[00198] In another embodiment, Rw is 5-10 membered monocyclic or bicyclic
heteroaryl ring optionally substituted with up to five substituents independently selected from
the group consisting of R1, R4, and R5.
[00199] In some embodiments, the present invention provides compounds of formula
IVA, formula IVB, or formula IVC:

wherein Rx, X, x, and Rw are defined above.
[00200] In one embodiment, Rw that is attached to carbon no. 2 is R2 or R3.
[00201] In some embodiments, Rw that is attached to carbon no. 2 is C1-C6 aliphatic
optionally substituted with up to four substituents selected from the group consisting of R1, R4,
and R5.
[00202] In some embodiments, Rw that is attached to carbon no. 2 is an optionally
substituted C1-C6 alkyl.
[00203] In some embodiments, Rw that is attached to carbon no. 2 is methyl, ethyl,
propyl, isopropyl, butyl, isobuyl, 1-methylcyclopropyl, or tert-butyl.
[00204] In some embodiments, Rw that is attached to carbon no. 2 is tert-butyl.
[00205] In some embodiments, Rwthat is attached to carbon no. 2 is ethyl.

[00206] In some embodiments, Rw that is attached to carbon no. 2 is 1-
methylcyclopropy 1.
[00207] In some embodiments, Rw that is attached to carbon no. 3 is H.
[00208] In some embodiments, the present invention provides compounds of formula
VA, formula VB, or formula VC:

[00209] wherein Rx, X, x, and Rw are defined above.
[00210] In some embodiments, W is an optionally substituted C1-C6 alkylidene.
[00211] In some embodiments, W is a C1-C6 alkylidene substituted with a hydroxy,
alkoxy, or amino group.
[00212] In some embodiments, W is a C1-C6 alkylidene substituted with a hydroxy
group.
[00213] In some embodiments, Rw is R4.
[00214] In some embodiments, Rw is OR6.
[00215] In some embodiments, Rw is OH.
[00216] In some embodiments, W is an optionally substituted C1-C6 alkylidene and
Rw is OR6.
[00217] In some embodiments, W is a C1-C6 alkylidene substituted with a hydroxyl,
alkoxy, or amino group, and Rw is OH.
[00218] In some embodiments -WRW is -C2H4OH or -CH2CH(OH)CH2OH.











a) R'"CHO, NaBH3CN, CF3CO2H; b) HOC-R", Pd(PPH3)2Cl2, Cul, Et3N; c) t-BuOK,
DMF; d) NH3 (aq)/CuSO4, autoclave.
[00232] In the schemes above, the radical R employed therein is a substiruent, e.g.,
Rw as defined hereinabove. One of skill in the art will readily appreciate that synthetic routes
suitable for various substituents of the present invention are such that the reaction conditions
and steps.
5. Uses, Formulation and Administration
Pharmaceutically acceptable compositions
[00233] As discussed above, the present invention provides compounds that are useful
as modulators of ABC transporters and thus are useful in the treatment of disease, disorders or
conditions such as Cystic fibrosis, Hereditary emphysema, Hereditary hemochromatosis,
Coagulation-Fibrinolysis deficiencies, such as Protein C deficiency, Type 1 hereditary
angioedema, Lipid processing deficiencies, such as Familial hypercholesterolemia, Type 1
chylomicronemia, Abetalipoproteinemia, Lysosomal storage diseases, such as I-cell

disease/Pseudo-Hurler, Mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II,
Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus, Laron dwarfism, Myleoperoxidase
deficiency, Primary hypoparathyroidism, Melanoma, Glycanosis CDG type 1, Hereditary
emphysema, Congenital hyperthyroidism, Osteogenesis imperfecta, Hereditary
hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI), Neurophyseal DI, Neprogenic
DI, Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerative
diseases such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,
Progressive supranuclear plasy, Pick's disease, several polyglutamine neurological disorders
asuch 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, and Sjogren's disease.
[00234] Accordingly, in another aspect of the present invention, pharmaceutically
acceptable compositions are provided, wherein these compositions comprise any of the
compounds as described herein, and optionally comprise a pharmaceutically acceptable carrier,
adjuvant or vehicle. In certain embodiments, these compositions optionally further comprise
one or more additional therapeutic agents.
[00235] It will also be appreciated that certain of the compounds of present invention
can exist in free form for treatment, or where appropriate, as a pharmaceutically acceptable
derivative thereof. According to the present invention, a pharmaceutically acceptable derivative
includes, but is not limited to, pharmaceutically acceptable salts, esters, salts of such esters, or
any other adduct or derivative which upon administration to a patient in need is capable of
providing, directly or indirectly, a compound as otherwise described herein, or a metabolite or
residue thereof.
[00236] 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. As used
herein, the term "inhibitorily active metabolite or residue thereof means that a metabolite or
residue thereof is also an inhibitor of an ATP-Binding Cassette Transporters.

[00237] Pharmaceutically acceptable salts are well known in the art. For example, S.
M. Berge, et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical
Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts
of the compounds of this invention include those derived from suitable inorganic and organic
acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are
salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic
acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic
acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using
other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts
include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,
butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,
gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate,
lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-
naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate,
sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts
derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and
N+(C1-4alkyl)4 salts. This invention also envisions the quaternization of any basic nitrogen-
containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible
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.
[00238] 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.
Uses of Compounds and Pharmaceutically Acceptable Compositions
[00239] In yet another aspect, the present invention provides a method of treating a
condition, disease, or disorder implicated by ABC transporter activity. In certain embodiments,
the present invention provides a method of treating a condition, disease, or disorder implicated
by a deficiency of ABC transporter activity, the method comprising administering a
composition comprising a compound of formula (I) to a subject, preferably a mammal, in need
thereof.
[00240] In certain preferred embodiments, the present invention provides a method of
treating cystic fibrosis, hereditary emphysema (due to al-antitrypsin; non Piz variants),
hereditary hemochromatosis, coagulation-fibrinolysis deficiencies, such as protein C
deficiency, Type 1 hereditary angioedema, lipid processing deficiencies, such as familial
hypercholesterolemia, Type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage

diseases, such as I-cell disease/pseudo-Hurler, mucopolysaccharidoses (due to lysosomal
processing enzymes), Sandhof/Tay-Sachs (due to P-hexosaminidase), Crigler-Najjar type II
(due to UDP-glucuronyl-sialyc-transferase), polyendocrinopathy/hyperinsulemia, diabetes
mellitus (due to insulin receptor), Laron dwarfism (due to growth hormone receptor),
myleoperoxidase deficiency, primary hypoparathyroidism (due to preproparathyroid hormone),
melanoma (due to tyrosinase). The diseases associated with the latter class of ER malfunction
are glycanosis CDG type 1, hereditary emphysema (due to a 1-antitrypsin (PiZ variant),
congenital hyperthyroidism, osteogenesis imperfecta (due to Type I, II, IV procollagen),
hereditary hypofibrinogenemia (due to fibrinogen), ACT deficiency (due to al-
antichymotrypsin), diabetes insipidus (DI), neurophyseal DI (due to vasopvessin hormone/V2-
receptor), neprogenic DI (due to aquaporin II), Charcot-Marie Tooth syndrome (due to
peripheral myelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerative diseases such
as Alzheimer's disease ( due to pAPP and presenilins), Parkinson's disease, amyotrophic lateral
sclerosis, progressive supranuclear plasy, Pick's disease, several polyglutamine neurological
disorders such as Huntington, spinocerebullar ataxia type I, spinal and bulbar muscular atrophy,
dentatorubal pallidoluysian, and myotonic dystrophy, as well as spongiform encephalopathies,
such as hereditary Creutzfeldt-Jakob disease (due to prion protein processing defect), Fabry
disease (due to lysosomal a-galactosidase A), Straussler-Scheinker syndrome, chronic
obstructive pulmonary disease (COPD), dry eye disease, and Sjogren's Syndrome, comprising
the step of administering to said mammal an effective amount of a composition comprising a
compound of formula (I), or a preferred embodiment thereof as set forth above.
[00241] According to an alternative preferred embodiment, the present invention
provides a method of treating cystic fibrosis comprising the step of administering to said
mammal a composition comprising the step of administering to said mammal an effective
amount of a composition comprising a compound of formula (I), or a preferred embodiment
thereof as set forth above.
[00242] 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 cystic fibrosis, hereditary emphysema (due to a 1-antitrypsin; non Piz
variants), hereditary hemochromatosis, coagulation-fibrinolysis deficiencies, such as protein C
deficiency, Type 1 hereditary angioedema, lipid processing deficiencies, such as familial
hypercholesterolemia, Type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage
diseases, such as I-cell disease/pseudo-Hurler, mucopolysaccharidoses (due to lysosomal

processing enzymes), Sandhof/Tay-Sachs (due to (3-hexosaminidase), Crigler-Najjar type II
(due to UDP-glucuronyl-sialyc-transferase), polyendocrinopathy/hyperinsulemia, diabetes
mellitus (due to insulin receptor), Laron dwarfism (due to growth hormone receptor),
myleoperoxidase deficiency, primary hypoparathyroidism (due to preproparathyroid hormone),
melanoma (due to tyrosinase). The diseases associated with the latter class of ER malfunction
are glycanosis CDG type 1, hereditary emphysema (due to al-antitrypsin (PiZ variant),
congenital hyperthyroidism, osteogenesis imperfecta (due to Type I, II, TV procollagen),
hereditary hypofibrinogenemia (due to fibrinogen), ACT deficiency (due to al-
antichymotrypsin), diabetes insipidus (DI), neurophyseal DI (due to vasopvessin hormone/V2-
receptor), neprogenic DI (due to aquaporin II), Charcot-Marie Tooth syndrome (due to
peripheral myelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerative diseases such
as Alzheimer's disease ( due to p\APP and presenilins), Parkinson's disease, amyotrophic lateral
sclerosis, progressive supranuclear plasy, Pick's disease, several polyglutamine neurological
disorders such as Huntington, spinocerebullar ataxia type I, spinal and bulbar muscular atrophy,
dentatorubal pallidoluysian, and myotonic dystrophy, as well as spongiform encephalopathies,
such as hereditary Creutzfeldt-Jakob disease (due to prion protein processing defect), Fabry
disease (due to lysosomal a-galactosidase A), Straussler-Scheinker syndrome, chronic
obstructive pulmonary disease (COPD), dry eye disease, and Sjogren's Syndrome.
[00243] The compounds and compositions, according to the method of the present
invention, may be administered using any amount and any route of administration effective for
treating or lessening the severity of one or more of cystic fibrosis, hereditary emphysema (due
to al-antitrypsin; non Piz variants), hereditary hemochromatosis, coagulation-fibrinolysis
deficiencies, such as protein C deficiency, Type 1 hereditary angioedema, lipid processing
deficiencies, such as familial hypercholesterolemia, Type 1 chylomicronemia,
abetalipoproteinemia, lysosomal storage diseases, such as I-cell disease/pseudo-Hurler,
mucopolysaccharidoses (due to lysosomal processing enzymes), Sandhof/Tay-Sachs (due to (3-
hexosaminidase), Crigler-Najjar type II (due to UDP-glucuronyl-sialyc-transferase),
polyendocrinopathy/hyperinsulemia, diabetes mellitus (due to insulin receptor), Laron
dwarfism (due to growth hormone receptor), myleoperoxidase deficiency, primary
hypoparathyroidism (due to preproparathyroid hormone), melanoma (due to tyrosinase). The
diseases associated with the latter class of ER malfunction are glycanosis CDG type 1,
hereditary emphysema (due to al-antitrypsin (PiZ variant), congenital hyperthyroidism,
osteogenesis imperfecta (due to Type I, II, TV procollagen), hereditary hypofibrinogenemia
(due to fibrinogen), ACT deficiency (due to al-antichymotrypsin), diabetes insipidus (DI),

neurophyseal DI (due to vasopvessin liormone/V2-receptor), neprogenic DI (due to aquaporin
II), Charcot-Marie Tooth syndrome (due to peripheral myelin protein 22), Perlizaeus-
Merzbacher disease, neurodegenerative diseases such as Alzheimer's disease ( due to pAPP and
presenilins), Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear plasy,
Pick's disease, several polyglutamine neurological disorders such as Huntington,
spinocerebullar ataxia type I, spinal and bulbar muscular atrophy, dentatorubal pallidoluysian,
and myotonic dystrophy, as well as spongiform encephalopathies, such as hereditary
Creutzfeldt-Jakob disease (due to prion protein processing defect), Fabry disease (due to
lysosomal a-galactosidase A), Straussler-Scheinker syndrome, chronic obstructive pulmonary
disease (COPD), dry eye disease, and Sjogren's Syndrome.
[00244] 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.
[00245] The pharmaceutically acceptable compositions of this invention can be
administered to humans and other animals orally, rectally, parenterally, intracisternally,
intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, as an
oral or nasal spray, or the like, depending on the severity of the infection being treated. In
certain embodiments, the compounds of the invention may be administered orally or
parenterally at dosage levels of about 0.01 mg/kg to about 50 mg/kg and preferably from about

1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain
the desired therapeutic effect.
[00246] 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.
[00247] 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.
[00248] 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.
[00249] 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.
[00250] 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.
[00251] 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.
[00252] 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.
[00253] 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.
[00254] 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.
[00255] As described generally above, the compounds of the invention are useful as
modulators of ABC transporters. Thus, without wishing to be bound by any particular theory,
the compounds and compositions are particularly useful for treating or lessening the severity of
a disease, condition, or disorder where hyperactivity or inactivity of ABC transporters is
implicated in the disease, condition, or disorder. When hyperactivity or inactivity of an ABC
transporter is implicated in a particular disease, condition, or disorder, the disease, condition, or
disorder may also be referred to as a "ABC transporter-mediated disease, condition or

disorder". Accordingly, in another aspect, the present invention provides a method for treating
or lessening the severity of a disease, condition, or disorder where hyperactivity or inactivity of
an ABC transporter is implicated in the disease state.
[00256] The activity of a compound utilized in this invention as a modulator of an
ABC transporter may be assayed according to methods described generally in the art and in the
Examples herein.
[00257] 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".
[00258] 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.
[00259] 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 US
Patents 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.
[00260] Another aspect of the invention relates to modulating ABC transporter
activity in a biological sample or a patient (e.g., in vitro or in vivo), which method comprises
administering to the patient, or contacting said biological sample with a compound of 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.
[00261] Modulation of ABC transporter activity in a biological sample is useful for a
variety of purposes that are known to one of skill in the art. Examples of such purposes
include, but are not limited to, the study of ABC transporters in biological and pathological
phenomena; and the comparative evaluation of new modulators of ABC transporters.
[00262] 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.
[00263] According to an alternative embodiment, the present invention provides a
method of increasing the number of functional ABC transporters in a membrane of a cell,
comprising the step of contacting said cell with a compound of formula (I). The term
"functional ABC transporter" as used herein means an ABC transporter that is capable of
transport activity. In preferred embodiments, said functional ABC transporter is CFTR.
[00264] According to another preferred embodiment, the activity of the ABC
transporter is measured by measuring the transmembrane voltage potential. Means for
measuring the voltage potential across a membrane in the biological sample may employ any of

the known methods in the art, such as optical membrane potential assay or other
electrophysiological methods.
[00265] 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).
[00266] These voltage sensitive assays are based on the change in fluorescence
resonant energy transfer (FRET) between the membrane-soluble, voltage-sensitive dye,
DiSBAC2(3), and a fluorescent phospholipid, CC2-DMPE, which is attached to the outer leaflet
of the plasma membrane and acts as a FRET donor. Changes in membrane potential (Vm)
cause the negatively charged DiSBAC2(3) to redistribute across the plasma membrane and the
amount of energy transfer from CC2-DMPE changes accordingly. The changes in fluorescence
emission can be monitored using VIPR™ n, which is an integrated liquid handler and
fluorescent detector designed to conduct cell-based screens in 96- or 384-well microtiter plates.
[00267] In another aspect the present invention provides a kit for use in measuring the
activity of a ABC transporter or a fragment thereof in a biological sample in vitro or in vivo
comprising (i) a composition comprising a compound of 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 ABC transporter or a fragment thereof. In one embodiment,
the kit further comprises instructions for a) contacting an additional composition with the
biological sample; b) measuring the activity of said ABC transporter or a fragment thereof in
the presence of said additional compound, and c) comparing the activity of the ABC transporter
in the presence of the additional compound with the density of the ABC transporter in the
presence of a composition of formula (I). In preferred embodiments, the kit is used to measure
the density of CFTR.
[00268] 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.


[0001] A mixture of benzo[l,3]dioxole-5-acetonitrile (5.10 g, 31.7 mmol), 1 -bromo-2-chloro-
ethane (9.00 mL, 109 mmol), and benzyltriethylammonium chloride (0.181 g, 0.795 mmol) was
heated at 70 °C and then 50% (wt./wt.) aqueous sodium hydroxide (26 mL) was slowly added
to the mixture. The reaction was stirred at 70 °C for 18 hours and then heated at 130 °C for 24
hours. The dark brown reaction mixture was diluted with water (400 mL) and extracted once
with an equal volume of ethyl acetate and once with an equal volume of dichloromethane. The
basic aqueous solution was acidified with concentrated hydrochloric acid to pH less than one
and the precipitate filtered and washed with 1 M hydrochloric acid. The solid material was
dissolved in dichloromethane (400 mL) and extracted twice with equal volumes of 1 M
hydrochloric acid and once with a saturated aqueous solution of sodium chloride. The organic
solution was dried over sodium sulfate and evaporated to dryness to give a white to slightly off-
white solid (5.23 g, 80%) ESI-MS m/z calc. 206.1, found 207.1 (M+l)+. Retention time of
2.37 minutes. *H NMR (400 MHz, DMSO-de) 5 1.07-1.11 (m, 2H), 1.38-1.42 (m, 2H), 5.98 (s,
2H), 6.79 (m, 2H), 6.88 (m, 1H), 12.26 (s, 1H).


Step a: 2,2-Difluoro-benzo[l,3j'dioxole-5-carboxylic acid methyl ester
[0002] A solution of 5-bromo-2,2-difluoro-benzo[l,3]dioxole (11.8 g, 50.0 mmol) and
tetrakis(triphenylphosphine)palladium (0) [Pd(PPH3)4, 5.78 g, 5.00 mmol] in methanol (20 mL)
containing acetonitrile (30 mL) and triethylamine (10 mL) was stirred under a carbon
monoxide atmosphere (55 PSI) at 75 °C (oil bath temperature) for 15 hours. The cooled
reaction mixture was filtered and the filtrate was evaporated to dryness. The residue was
purified by silica gel column chromatography to give crude 2,2-difluoro-benzo [1,3] dioxole-5-
carboxylic acid methyl ester (11.5 g), which was used directly in the next step.
Step b: (2,2-Difluoro-benzo[l,3]dioxol-5-yl)-methanol
[0003] Crude 2,2-difluoro-benzo[l,3]dioxole-5-carboxylic acid methyl ester (11.5 g) dissolved
in 20 mL of anhydrous tetrahydrofuran (THF) was slowly added to a suspension of lithium
aluminum hydride (4.10 g, 106 mmol) in anhydrous THF (100 mL) at 0 °C. The mixture was
then warmed to room temperature. After being stirred at room temperature for 1 hour, the
reaction mixture was cooled to 0 °C and treated with water (4.1 g), followed by sodium
hydroxide (10% aqueous solution, 4.1 mL). The resulting slurry was filtered and washed with
THF. The combined filtrate was evaporated to dryness and the residue was purified by silica
gel column chromatography to give (2,2-difluoro-benzo[l,3]dioxol-5-yl)-methanol (7.2 g, 38
mmol, 76 % over two steps) as a colorless oil.
Step c: 5-Chhromethyl-2,2-difluoro-benzo[l,3]dioxole
[0004] Thionyl chloride (45 g, 38 mmol) was slowly added to a solution of (2,2-difluoro-
benzo[l,3]dioxol-5-yl)-methanol (7.2 g, 38 mmol) in dichloromethane (200 mL) at 0 °C. The
resulting mixture was stirred overnight at room temperature and then evaporated to dryness.
The residue was partitioned between an aqueous solution of saturated sodium bicarbonate (100

mL) and dichloromethane (100 mL). The separated aqueous layer was extracted with
dichloromethane (150 mL). The organic layer was dried over sodium sulfate, filtered, and
evaporated to dryness to give crude 5-chloromethyl-2,2-difluoro-benzo[l,3]dioxole (4.4 g)
which was used directly in the next step.
Step d: (2,2-Difluoro-benzo[l,3]dioxol-5-yl)-acetonitrile
[0005] A mixture of crude 5-chloromethyl-2,2-difluoro-benzo[l,3]dioxole (4.4 g) and sodium
cyanide (1.36 g, 27.8 mmol) in dimethylsulfoxide (50 mL) was stirred at room temperature
overnight. The reaction mixture was poured into ice and extracted with ethyl acetate (300 mL).
The organic layer was dried over sodium sulfate and evaporated to dryness to give crude (2,2-
difluoro-benzo[l,3]dioxol-5-yl)-acetonitrile (3.3 g) which was used directly in the next step.
Step e: l-(2,2-Difluoro-benzo[l,3]dioxol-5-yl)-cyclopropanecarbonitrile
[0006J Sodium hydroxide (50% aqueous solution, 10 mL) was slowly added to a mixture of
crude (2,2-difluoro-benzo[l,3]dioxol-5-yl)-acetonitrile, benzyltriethylammonium chloride (3.00
g, 15.3 mmol), and l-bromo-2-chloroethane (4.9 g, 38 mmol) at 70 °C.
[00271] The mixture was stirred overnight at 70 °C before the reaction mixture was
diluted with water (30 mL) and extracted with ethyl acetate. The combined organic layers were
dried over sodium sulfate and evaporated to dryness to give crude l-(2,2-difluoro-
benzo[l,3]dioxol-5-yl)-cyclopropanecarbonitrile, which was used directly in the next step.
Stepf: l-(2,2-Difluoro-benzo[l,3]dioxol-5-yl)-cyclopropanecarboxylic acid
[0007] l-(2,2-Difluoro-benzo[l,3]dioxol-5-yl)-cyclopropanecarbonitrile (crude from the last
step) was refluxed in 10% aqueous sodium hydroxide (50 mL) for 2.5 hours. The cooled
reaction mixture was washed with ether (100 mL) and the aqueous phase was acidified to pH 2
with 2M hydrochloric acid. The precipitated solid was filtered to give 1 -(2,2-difluoro-
benzo[l,3]dioxol-5-yl)-cyclopropanecarboxylic acid as a white solid (0.15 g, 1.6% over four
steps). ESI-MS m/z calc. 242.04, found 241.58 (M+l)+; !H NMR (CDC13) 5 7.14-7.04 (m, 2H),
6.98-6.96 (m, 1H), 1.74-1.64 (m, 2H), 1.26-1.08 (m, 2H).
[0008] The following Table 3 contains a list of amine building blocks that were commercially
available, or prepared by one of the methods described below.
[00272] Table 3: Amine building blocks.



Step a: 6-Chloro-2-iodo-pyridin-3-ylamine
[0009] To a solution of 6-chloro-pyridin-3-ylamine (10.0 g, 77.8 mmol) in EtOH (150 mL) was
added Ag2SC>4 (12.1 g, 38.9 mmol) and I2 (23.7 g, 93.4 mmol) at room temperature. The
mixture was stirred at 20 °C overnight. The solvent was removed by evaporation under
vacuum. Water (100 mL) and EtOAc (200 mL) were added to the residue. The organic layer
was separated and the aqueous layer was extracted with EtOAc (100 mL * 3). The combined
organic layers were dried over anhydrous Na2SC>4 and evaporated under vacuum to give the
crude product, which was purified by column chromatography on silica gel (Petroleum
ether/Ethyl acetate 7:1) to give 6-chloro-2-iodo-pyridin-3-ylamine (17.1 g, 86%). !H NMR
(DMSO, 300 MHz) 5 7.16 (d, J= 8.4 Hz, 1 H), 7.01 (d, /= 8.4 Hz, 1 H), 5.57 (s, 2 H).

Step b: 6-Chloro-2-(3,3-dimethyl-but-l-ynyl)-pyridin-3-ylamine
[0010J To a solution of 6-chIoro-2-iodo-pyridin-3-ylamine (16.0 g, 62.7 mmol) in toluene (160
mL) and water (80 mL) were added Et3N (12.7 g, 125 mmol), Pd(PPH3)2Cl2 (2.2 g, 3.1 mmol),
Cul (238 mg, 1.3 mmol) and 3,3-dimethyl-but-l-yne (7.7 g, 94 mmol) successively under N2
atmosphere. The reaction mixture was heated at 70 °C for 3 hours and was allowed to cool to
room temperature. The resulting mixture was extracted with ethyl acetate (150 mL * 3). The
combined organic extracts were dried over anhydrous Na2SC>4 and evaporated under vacuum to
give 6-chloro-2-(3,3-dimethyl-but-l-ynyl)- pyridin-3-ylamine (11.5 g, 88%), which was used in
the next step without further purification.
Step c: 'N-[6-Chloro-2-(3,3-dimethyl-but-l-ynyl)-pyridin-3-ylJ-butyramide
fOOUJ To asolution of 6-chloro-2-(3,3-dimethyl-but-l-ynyl)-pyridin-3-ylamine (11.5 g, 55.2
mmol) and pyridine (13.1 g, 166 mmol) in CH2CI2 (150 mL) was added butyryl chloride (6.5 g,
61 mmol) dropwise at 0 °C. The mixture was allowed to warm to room temperature and was
stirred at this temperature overnight. Water (50 mL) was added dropwise at -0 °C. The
resulting mixture was extracted with ethyl acetate (100 mL * 3). The combined organic layers
were dried over anhydrous Na2SC>4 and evaporated under vacuum to give the crude iV-[6-
chloro-2-(3,3-dimethyl-but-l-ynyl)-pyridin-3-yl]-buryramide (16 g), which was used in the
next step without further purification. lH NMR (CDC13,300 MHz) 5 8.72 (d, /= 9.0 Hz, 1 H),
7.88 (brs, 1 H), 7.23 (d, /= 8.4 Hz, 1 H), 2.40 (d, J= 7.2 Hz, 2 H), 1.83-1.75 (m, 2 H), 1.40 (s,
9H), 1.04(d,J=7.2Hz, 3 H).
Step d: 2-tQrt-Butyl-5-chloro-lH-pyrrolo[3,2-bjpyridine
[0012] To asolution of crude Af-[6-chloro-2-(3,3-dimethyl-but-l-ynyl)-pyridin-3-yl]-
butyramide (16 g) in DMF (150 mL) was added f-BuOK (12.4 g, 110 mmol) at room
temperature. The mixture was heated at 70 °C for 1 hour. The solvent was removed by
evaporation under vacuum. Water (100 mL) and ethyl acetate (200 mL) were added. The
organic layer was separated and the aqueous layer was extracted with ethyl acetate (100 mL *
3). The combined organic layers were dried over anhydrous Na2SC>4 and evaporated under
vacuum to give the crude product, which was purified by column chromatography on silica gel
(petroleum ether/ ethyl acetate 10:1) to give 2-tert-butyl-5-chloro-lH-pyrrolo[3,2-b]pyridine
(10.8 g, two steps: 94%). lHNMR (CDC13,4OO MHz) 5 8.52 (brs, 1 H), 7.28 (d, J= 8.4 Hz, 1
H), 7.02 (d, /= 8.4 Hz, 1 H), 6.37 (d, J= 2.0 Hz, 1 H), 1.40 (s, 9 H).

Step e: 2-tert-Butyl-lH-pyrrolo[3,2-b]pyridin-5-amine
[0013] In a 500 mL autoclave, a solution of 2-ter?-butyl-5-chloro-lH-pyrrolo[3,2-b]pyridine
(5.0 g, 24 mmol) and CuSO4 5H2O (0.5 g, 2.0 mmol) in aqueous ammonia (200 mL) and
CH3OH (100 mL) was heated at 180 °C (at this temperature, the pressure in the autoclave was
about 2MPa) and stirred for 10 hours. The mixture was allowed to cool down to room
temperature. The solvent was removed by evaporation under vacuum. The resulting mixture
was extracted with ethyl acetate (100 mL x3). The combined organic layers were dried over
anhydrous Na2SO4 and evaporated under vacuum to give the crude product, which was purified
by the preparative HPLC to give 2-tert-butyl-lH-pyrrolo[3,2-b]pyridin-5-amine (1.15 g, 26%).
'H NMR (CDCI3,300 MHz) 5 10.63 (brs, 1 H), 7.35 (d, J= 8.7 Hz, 1 H), 6.23 (d, /= 8.7 Hz, 1
H), 5.86 (d, J= 1.5 Hz, 1 H), 5.40 (brs, 2 H), 1.29 (s, 9 H); MS (ESI;) m/e (M+H+): 190.2.

Step a: (6-Chloro-pyridin-3-yl)-carbamic acid tert-butyl ester
[0014] To a mixture of 6-chloropyridin-3-amine (30.0 g, 0.23 mol), DMAP (1 g) and Et3N
(41.7 g, 0.47 mol) in CH2C12 (200 mL) was added Boc2O (54.5 g, 0.25 mol) at 0 °C. The
mixture was allowed to warm to room temperature and stirred overnight. The mixture was
washed with saturated NaHCC>3 solution. The aqueous solution was extracted with
dichloromethane. The combined organics were washed with brine (100 mL), dried over
Na2SC>4 and evaporated under vacuum to give tert-butyl 6-chloropyridin-3-ylcarbamate (50.0 g,
94%), which was used directly in the next reaction. *H NMR (300 MHz, CDCI3) 5 8.23 (d, J=
2.7 Hz, 1 H), 7.97 (d, J= 6.9 Hz, 1 H), 7.27-7.24 (m, 1 H), 6.58 (s, 1 H), 1.52 (s, 9 H).
Step b: (6-Chloro-4-iodo-pyridin-3-yl)-carbamic acid tert-butyl ester
[0015] To a solution of TMEDA (1.45 g, 12.5 mmol) in dry Et2O (30 mL) was added dropwise
«-BuLi (5.0 mL, 12.5 mmol) at -78 °C. The mixture was stirred for 0.5 h at -78 °C. A
solution of (6-chloro-pyridin-3-yl)-carbamic acid tert-buty\ ester (1.14 g, 5.0 mmol) in dry Et2O
(10 mL) was added dropwise to the reaction mixture at -78 °C and the resultant mixture was

continued to stir for 1 h at -78 °C. A solution of I2 (1.52 g, 6.0 mmol) in dry Et2O (10 mL) was
added dropwised at -78 °C. The mixture was continued to stir for 1 h at this temperature. The
reaction was quenched with saturated aqueous NH4CI. The organic layer was separated and the
aqueous phase was extracted with ethyl acetate (50 mL x 3). The combined organic layers
were dried over anhydrous Na2SO4 and evaporated under reduced pressure to give a residue,
which was purified by column (petroleum ether/ ethyl acetate = 10/1) to obtain (6-chloro-4-
iodo-pyridin-3-yl)-carbamic acid tert-butyl ester (1.75 g, 30%). *H NMR (400 MHz, CDC13) 5
8.95 (br s, 1 H), 7.73 (s, 1 H), 6.64 (br s, 1 H), 1.54 (s, 1 H).
Step c: [6-Chloro-4-(3,3-dimethyl-but-l-ynyl)-pyridin-3-yl]-carbamic acid
tert-butyl ester
[0016] To a deoxygenated solution of (6-chloro-4-iodo-pyridin-3-yl)-carbamic acid terf-butyl
ester (23.3 g, 65.6 mmol), 3,3-dimethyl-but-l-yne (53.8 g, 0.656 mol), Cul (623 mg, 3.3 mmol)
and triethylamine (13.3 g, 0.13 mol) in toluene (150 mL) and water (50 mL) was added
Pd(PPH3)2Cl2 (2.30 g, 3.28 mmol) under N2. The mixture was heated at 70 °C and stirred for 24
hours. The solid was filtered off and washed with ethyl acetate (200 mL x 3). The filtrate was
evaporated under reduced pressure to obtain a residue, which was purified by column
(petroleum ether/ ethyl acetate = 10/1) to give [6-chloro-4-(3,3-dimethyl-but-l-ynyl)-pyridin-3-
yl]-carbamic acid tert-butyl ester (15.8 g, 78%). *H NMR (300 MHz, CDC13) 8 9.10 (br s, 1
H), 7.21 (s, 1 H), 6.98 (br s, 1 H), 1.53 (s, 9 H), 1.36 (s, 9 H).
Step d: 2-tQft-Butyl-5-chloro-lH-pyrrolo[2,3-cJpyridine
[0017] A mixture of [6-chloro-4-(3,3-dimethyl-but-l-ynyl)-pyridin-3-yl]-carbamic acid tert-
butyl ester (15.8 g, 51 mmol) and TBAF (26.6 g, 0.1 mol) in THF (200 mL) was heated at
reflux for 24 hours. After cooling, the mixture was poured into ice water and extracted with
CH2C12 (300 mL x 3). The combined organic layers were dried over anhydrous Na2SO4 and
evaporated under reduced pressure to obtain a residue, which was purified by column
chromatography (petroleum ether/ ethyl acetate = 10/1) to give 2-tert-butyl-5-chloro-lH-
pyrrolo[2,3-c]pyridine (9.2 g, 87%). !H NMR (300 MHz, CDC13) 8 9.15 (br s, 1 H), 8.43 (s, 1
H), 7.44 (s, 1 H), 6.25 (dd, J=0.6, 2.1 Hz, 1H), 1.42 (s, 9 H).
Step e: 2-tett-Butyl-lH-pyrrolo[2,3-c]pyridin-5-amine
[0018] To a solution of 2-tert-butyl-5-chloro-lH-pyrrolo[2,3-c]pyridine (5.0 g, 24 mmol) in
NH3.H2O (400 mL) was added CuSO4.5H2O (595 mg, 2.39 mmol). The mixture was heated at
200 °C (3 MPa pressure) for 24 h. After cooling, the mixture was extracted with CH2C12 (150

mL x 3). The combined organic layers were dried over anhydrous Na2SO4 and evaporated
under reduced pressure to give a residue, which was purified by column (petroleum ether/ ethyl
acetate = 10/1) to obtain 2-tert-butyl-lH-pyrrolo[2,3-c]pyridin-5-amine (1.2 g, 27%). *H NMR
(400 MHz, DMSO) 5 10.66 (br s, 1 H), 8.02 (s, 1 H), 6.39 (s, 1 H), 5.86 (d, J = 1.2 Hz, 1 H),
4.85 (br s, 2 H), 1.29 (s, 9 H).

Step a: 3-Bromo-5-nitropyridin-2-amine
[0019] To a solution of 5-nitro-pyridin-2-ylamine (30 g, 0.22 mol) in acetic acid (200 mL) at
10 °C was added Br2 (38 g, 0.24 mol) dropwise. After addition, the mixture was stirred at 20
°C for 30 min. The solid was filtered and then dissolved in ethyl acetate (200 mL). The
mixture was basified to pH 8-9 with saturated aqueous NaHCCh. The organic layer was
separated, and the aqueous layer was extracted with ethyl acetate (100 mL x 3). The combined
organic layers were washed with water, brine, dried over Na2SO4 and concentrated under
vacuum to afford 3-bromo-5-nitropyridin-2-amine (14.8 g, 32%). !H-NMR (CDC13, 400 MHz)
5 8.94 (d, J= 2.4 Hz, 1 H), 8.50 (d, J= 2.4 Hz, 1 H), 5.67 (brs, 2 H).
Step b: 3-(3,3-Dimethylbut-l-ynyl)-5-nitropyridin-2-amine
[0020] To a solution of 3-bromo-5-nitropyridin-2-amine (1.0 g, 4.6 mmol) in toluene/water (5
mL/2.5 mL), was added Et3N (1.2 mL, 9.2 mmol), Pd(PPH3)2Cl2 (0.3 g, 0.46 mmol), Cul (35
mg, 0.18 mmol) and 3,3-dimethyl-but-l-yne (0.75 g, 9.2 mmol) successively under N2
protection. The mixture was heated at 70 °C for 2.5 h. The solid was filtered and the organic
layer was separated. The aqueous layer was exacted with ethyl acetate (10 mL x 3). The
combined organic layers were washed with brine, dried over Na2SO4 and concentrated under
vacuum to afford 3-(3,3-dimethylbut-l-ynyl)-5-nitropyridin-2-amine (0.9 g, 90%). 'H-NMR
(CDCU, 400 MHz) 8 8.87 (d, J= 3.2 Hz, 1 H), 8.25 (d, J= 3.2 Hz, 1 H), 5.80 (brs, 2 H), 1.36
(s, 9 H).

Step c: 2-\zn-Butyl-5-nitro-lH-pyrrolo[2,3-bJpyridine
[0021] A solution of 3-(3,3-dimethylbut-l-ynyl)-5-nitropyridin-2-amine (0.4 g, 1.8 mmol) and
TBAF (1.9 g, 7.3 mmol) in THF (10 mL) was heated at reflux overnight. The reaction mixture
was concentrated to dryness under vacuum, and the residue was dissolved in ethyl acetate (20
mL). The organic layer was washed with water, brine, dried over Na2SO4 and concentrated
under vacuum to afford 2-rerr-butyl-5-nitro-lH-pyrrolo[2,3-b]pyridine (0.25 g, 63%). 'H-NMR
(CDC13, 400 MHz) 8 11.15 (bra, 1 H), 9.20 (s, J= 2.0 Hz, 1 H), 8.70 (d,J= 2.0 Hz, 1 H), 6.43
(d, J= 1.6 Hz, 1 H), 1.51 (s, 9 H).
Step d: 2-tert-Butyl- lH-pyrrolo[2,3-b]pyridin-5-amine
[0022] To a solution of 2-terr-butyl-5-nitro-lH-pyrrolo[2,3-b]pyridine (2.3 g, 0.01 mol) in
MeOH (50 mL) was added Raney Ni (0.23 g, 10%) under N2 protection. The mixture was
stirred under hydrogen atmosphere (1 atm) at 30 °C for 1 h. The catalyst was filtered off and
the filtrate was concentrated to dryness under vacuum. The residue was purified by column
chromatography on silica gel (petroleum ether/ ethyl acetate 1:2) to give 2-terf-butyl-lH-
pyrrolo[2,3-b]pyridin-5-amine (1.4 g, 70%). 'H-NMR (MeOD, 400 MHz) 5 7.71 (s, 1 H), 7.27
(s, 1 H), 5.99 (s, 1 H), 1.37(s, 9 H). MS (ESI) m/e (M+H^) 190.1.

Step a: (6-chloro-pyridin-3-yl)-carbamic acid tert-butyl ester
[0023] To a mixture of 6-chloro-pyridin-3-amine (30.0 g, 230 mmol), DMAP (1.0 g) and Et3N
(41.7 g, 470 mmol) in CH2C12 (200 mL) was added Boc2O (54.5 g, 250 mmol) at 0°C. The
reaction mixture was allowed to warm to the room temperature and stirred overnight. The
resulting mixture was washed with saturated NaHCO3 solution and brine (100 mL). The
organic layer was dried over anhydrous Na2SO4 and evaporated under vacuum. The residue
was purified by column chromatography on silica gel (petroleum ether/ethyl acetate 10/1) to
give tert-butyl 6-chloropyridin-3-yl-carbamate (40.0 g, 76%). lH-NMR (CDCI3,400 MHz) 5

8.23(d, J= 2.8 Hz, 1 H), 7.96 (d, /= 5.6 Hz, 1 H), 7.25(d, J= 5.6 Hz, 1H), 6.58 (brs, 1 H),
1.52(s,9H).
Step Z>: (6-chloro-4-iodo-pyridin-3-yl)-carbamic acid tert-butyl ester
[0024] To a solution of TMEDA (25.4 g, 219.3 mmol) in dry THF (300 mL) was added
dropwise n-BuLi (87.7 mL, 219.3mmol) at -78°C, the mixture was stirred for 0.5 h at this
temperature. A solution of (6-chloro-pyridin-3-yl)-carbamic acid tert-butyl ester (20 g, 87.7
mmol) in THF (170 mL) was added dropwise to the reaction mixture at -78 °C and the resulting
mixture was continued to stir for 1 h at -78 °C. Then a solution of I2 (26.7 g, 105.3 mmol) in
dry THF (170 mL) was added dropwise at -78 °C. After 1 h, the reaction was quenched with
sat. aqueous NH4C1 (300 mL). The organic layer was separated and the aqueous phase was
extracted with ethyl acetate (150 mL x 3). The combined organic layers were dried over
anhydrous Na2SO4 and concentrated under the reduced pressure. The residue was purified by
column chromatography on silica gel (Petroleum ether/Ethyl acetate, 10/1) to give (6-chloro-4-
iodo-pyridin-3-yl)-carbamic acid tert-butyl ester (7 g, 22.7%). ^-NMR (CDC13, 300 MHz) 5
8.95 (s, 1 H), 7.73 (s, 1 H), 6.64 (brs, 1 H), 1.54 (s, 9H).
Step c: tert-Butyl 4-(but-l-ynyl)-6-chloropyridin-3-ylcarbamate
[0025] To a deoxygenated solution of (6-chloro-4-iodo-pyridin-3-yl)-carbamic acid tert-butyl
ester (6.0 g, 16.9 mmol), 1-butyne (9 g, 169 mmol), Cul (160.1 mg, 0.84mmol) and
triethylamine (3.4 g, 33.2 mmol) in toluene (40 mL) and water (14 mL) was added
Pd(PPH3)2Cl2 (592 mg, 0.84 mmol) under N2 in a autoclave. The mixture was heated to 70 °C
and stirred for 24 h. The solid was filtered off and washed with ethyl acetate (60 mL x 3). The
filtrate was evaporated under reduced pressure and the residue was purified by column
chromatography on silica gel (petroleum ether/ethyl acetate, 10/1) to give tert-butyl 4-(but-l-
ynyl)-6-chloropyridin-3-ylcarbamate (3.5 g, 74%). 'H-NMR (CDC13, 300 MHz) 8 9.13 (s, 1
H), 7.22 (s, 1 H), 6.98 (s, 1 H), 2.53(q, J= 7.5 Hz, 2H), 1.54(s, 9H), 1.29 (t, /= 7.5 Hz, 3H).
Step d: 2-Ethyl-5-chloro-lH-pyrrolo[2, 3-cJpyridine
[0026] A mixture of tert-butyl 4-(but-l-ynyl)-6-chloropyridin-3-ylcarbamate (3.5 g, 12.5
mmol) and TBAF (6.65 g, 25 mmol) in THF (60 mL) was heated at reflux for 24 hours. After
cooling, the mixture was poured into ice water and extracted with CH2CI2 (100 mL * 3). The
combined organic layers was dried over anhydrous Na2SO4 and evaporated under reduced
pressure. The residue was purified by column chromatography on silica gel (petroleum
ether/ethyl acetate, 10/1) to give 2-ethyl-5-chloro-lH-pyrrolo[2, 3-c]pyridine (2.0 g, 89%). *H-

NMR (CDCI3, 300 MHz) 8 8.96 (brs, H), 8.46 (s, 1 H), 7.44 (s, 1 H), 6.24 (s, 1H), 2.89 (q, /=
7.5 Hz, 2H), 1.37 (t, J= 7.5 Hz, 3H).
Step e: 2-Ethyl-lH-pyrrolo[2,3 -cJpyridin-5-amine
[0027] A suspension of 2-ethyl-5-chloro-lH-pyrrolo[2, 3-c]pyridine (1.3 g, 7.19 mmol) in
EtOH (20 mL), CuSO4-5H2O (179 mg, 0.72 mmol) and NH3-H2O (60 ml) was added into an
autoclave (100 mL). The reaction was stirred at 200 °C and 2 MPa for 10 h. The reaction was
cooled to 25 °C and was quenched with water and extracted with ethyl acetate (100 mL x 3).
The combined organic layers were dried over anhydrous Na2SO4 and evaporated under reduced
pressure. The residue was purified by column chromatography on silica gel (petroleum
ether/ethyl acetate, 10/1) to give 2-ethyl-lH-pyrrolo[2,3 -c]pyridin-5-amine (190 mg, 16%).
'H-NMR (CDC13> 300 MHz) 5 10.71 (brs, H), 8.00 (s, 1 H), 6.39 (s, 1 H), 5.87 (s, 1H), 4.89 (br
s, 2 H), 2.65 (q, J= 7.5 Hz, 2H), 1.22 (t, /= 7.5 Hz, 3H).

Step a: 6-chloro-4-((l~methylcyclopropyl)ethynyl)pyridin-3-amine
[0028] To a solution of 6-chloro-4-iodopyridin-3-amine (7.0 g, 28 mmol) in Et3N (100 mL)
was added 1-ethynyl-l-methyl-cyclopropane (11.0 g, 137 mmol), Cul (0.53 g, 2.8 mmol) and
Pd(PPH3)2Cl2 (1.9 g, 2.8 mmol) under N2 atmosphere. The mixture was refluxed overnight and
quenched with H2O (100 mL). The organic layer was separated and the aqueous layer was
extracted with ethyl acetate (100 mL x 3). The combined organic layers were washed with
brine, dried over anhydrous Na2SO4 and purified by chromatography on silica gel (3% EtOAc
in Petroleum ether as eluant) to afford 6-chloro-4-((l-methylcyclopropyl)ethynyl)pyridin-3-
amine (3.0 g, 53%). !H-NMR (CDCb, 300 MHz) 5 7.84 (s, 1 H), 7.08 (s, 1 H), 4.11 (br s, 2 H),
1.37 (s, 3 H), 1.03 (t, /= 2.4, 2 H) 0.76 (t, J= 2.4, 2 H).

Step b: 5-chloro-2-(l-methylcyclopropyl)-lH-pyrrolo[2,3-cJpyridine
[0029] To a solution of 6-chloro-4-((l-methylcyclopropyl)ethynyl)pyridin-3-amine (3.0 g, 15
mmol) in DMF (50 mL) was added f-BuOK (3.3 g, 29 mmol) under N2 atmosphere. The
mixture was heated at 80 °C overnight and quenched with H2O (100 mL). The organic layer
was separated and the aqueous layer was extracted with ethyl acetate (50 mL x 3). The
combined organic layer was washed with brine, dried over anhydrous Na2SO4 and purified by
chromatography on silica gel (3% EtOAc in petroleum ether) to afford 5-chloro-2-(l-
methylcyclopropyl)-lH-pyrrolo[2,3-c]pyridine (2.2 g, 73%). !H-NMR (CDC13) 300 MHz) 5
9.79 (br s, 1 H), 8.37 (s, 1 H), 7.36 (s, 1 H), 6.16 (s, 1 H), 1.51 (s, 3 H), 1.09 (m, 2 H), 0.89 (m,
2H).
Step c: 2-(l-methylcyclopropyl)-lH-pyrrolo[2,3-c]pyridin-5-amine
[0030) In a 100 mL autoclave, a solution of 5-chloro-2-(l-methylcyclopropyl)-lH-pyrrolo[2,3-
c]pyridine (1.0 g, 4.9 mmol) and CUSO45H2O (100 mg, 0.4 mmol) in aqueous ammonia (60
mL) and EtOH (20 mL) was heated to 200 □ and stirred at this temperature for 8 hours. The
mixture was allowed to cool down to room temperature. The alcohol was removed under
vacuum. The resulting mixture was extracted with ethyl acetate (50 mL * 3). The combined
organic layer was dried over anhydrous Na2SO4 and purified by chromatography on silica gel
(2% CH3OH in dichloromethane as eluant) to afford 2-(l-methylcyclopropyl)-lH-pyrrolo[2,3-
c]pyridin-5-amine (250 mg, 27%). 'H-NMR (DMSO, 300 MHz) 5 7.96 (s, 1 H), 6.37 (s, 1 H),
5.88 (d, J= 1.2, 1 H), 5.01 (br s, 2 H), 1.41 (s, 3 H), 0.98 (t,J = 2.1,2 H), 0.80 (t, J= 2.1, 2 H).

Step a: Preparation of ethynylcyclobutane
[0031] n-BuLi was added to a solution of 6-chlorohex-l-yne (10.0 g, 86 mmol) in THF (100
mL) dropwise at -78°C. After being stirred for 20 min at -78°C, it was allowed to warm up to

40°C and was stirred for 3 days at that temperature. The reaction was quenched with saturated
aqueous solution of NH4CI and extracted with ether (3 x 50 mL). The combined extracts were
washed with brine, dried and the ether was removed by distillation to afford a solution of
ethynylcyclobutane in THF that was used in step b.
Step b: tert-Butyl 6-chloro-4-(cyclobutylethynyl)pyridin-3-ylcarbamate
[0032] To the solution of tert-butyl 6-chloro-4-iodopyridin-3-ylcarbamate (7.0 g, 19.8 mmol)
in Et3N (100 mL) was added the solution of ethynylcyclobutane in THF (prepared in step a),
Pd(PPH3)2Cl2 (1.8 g, 2.1 mmol) and Cul (400 mg, 2.1 mmol). The reaction mixture was stirred
at 25°C for 16h. The mixture was diluted with water and extracted with dichloromethane (3 x
100 mL). The extract was washed with brine, dried, concentrated in vacuo and purified by
chromatography on silica gel (5-10% ethyl acetate in petroleum ether as eluant) to afford tert-
butyl 6-chloro-4-(cyclobutylethynyl)pyridin-3-ylcarbamate (3.6 g, 60% yield). lH NMR (300
MHz, CDCI3) 5: 9.11 (br, s, 1 H), 7.22 (s 1 H), 6.97 (s, 1 H), 3.39-3.25 (m, 1 H), 2.48-1.98 (m,
6H),1.52(s,9H).
Step c: 5-chloro-2-cyclobutyl-lH-pyrrolo[2,3-cJpyridine
[0033] To the solution of tert-butyl 6-chloro-4-(cyclobutylethynyl)pyridin-3-ylcarbamate (2.6
g, 8.5 mmol) in DMF (50 mL) was added J-BuOK (1.9 g, 16 mmol). The reaction mixture was
stirred at 90°C for 2h. The mixture was diluted with water and extracted with dichloromethane
(3 x 50 mL). The extract was washed with brine, dried, concentrated in vacuo and purified by
chromatography on silica gel (5-10% ethyl acetate in petroleum ether as eluant) to afford the
pure product of 5-chloro-2-cyclobutyl-lH-pyrrolo[2,3-c]pyridine (0.9 g, 53% yield). [HNMR
(300 MHz, CDCb) 8: 8.66 (br, s, 1 H), 8.45 (s, 1H), 7.44 (s 1 H), 6.27 (s, 1 H), 3.75-3.52 (m, 1
H), 2.52-1.90 (m, 6 H).
Step d: 2-Cyclobutyl-lH-pyrrolo[2,3-c]pyridin-5-amine
[0034] To the solution of 5-chloro-2-cyclobutyl-lH-pyrrolo[2,3-c]pyridine (200 mg, 0.97
mmol) in EtOH (10 mL) and NH3H2O (30 mL) was added CuSO4-5H2O (30 mg, 0.12 mmol).
The reaction mixture was stirred under 3 MPa at 180°C for 16 h. The mixture was extracted
with ethyl acetate (3 x 30 mL). The extract was dried, concentrated in vacuo and purified by
chromatography on silica gel (5-10% MeOH in ethyl acetate as eluant) to afford the pure 2-
cyclobutyl-lH-pyrrolo[2,3-c]pyridin-5-amine (40 mg, 22% yield). !H NMR (300 MHz,

MeOH) 5: 8.02 (s, 1 H), 6.72 (s, 1H), 6.11 (s 1 H), 3.72-3.60 (m, 1 H), 2.47-1.90 (m, 6 H).
MS (ESI): mlz [M+H+] 188.1.

Step a: tert-Butyl 6-chloropyridin-3-yl-carbamate
[0035] To a mixture of 6-chloro-pyridin-3-amine (30.0 g, 230 mmol), DMAP (1.0 g) and Et3N
(41.7 g, 470 mmol) in CH2C12 (200 mL) was added Boc2O (54.5 g, 250 mmol) at 0°C. The
reaction mixture was allowed to warm to the room temperature and stirred overnight. The
resulting mixture was washed with saturated NaHCCb solution and brine (100 mL), the organic
layer was dried over anhydrous Na2SO4 and evaporated under vacuum, the residue was purified
by column chromatography on silica gel (petroleum ether/ ethyl acetate, 10/1) to give tert-buryl
6-chloropyridin-3-yl-carbamate (40.0 g, 76%). *H-NMR (CDC13, 400 MHz) 5 'H-NMR
(CDCI3, 400 MHz) 8.23(d, J= 2.8 Hz, 1 H), 7.98 (m, 1 H), 7.26 (d, J= 5.6 Hz, 1H), 6.51 (br s,
1H), 1.52(8, 9H).
Step b: tert-Butyl 6-chloro-4-iodopyridin-3-ylcarbamate
[0036] To a solution of TMEDA (25.4 g, 219.3 mmol) in dry THF (300 mL) was added
dropwise n-BuLi (87.7 mL, 219.3mmol) at -78°C, the mixture was stirred for 0.5 h at this
temperature. A solution of tert-buty\ 6-chloropyridin-3-yl-carbamate (20 g, 87.7 mmol) in THF
(170 mL) was added dropwise to the reaction mixture at -78 °C and the resuling mixture was

continued to stir for 1 h at -78 °C. Then a solution of I2 (26.7 g, 105.3 mmol) in dry THF (170
mL) was added dropwise at -78 °C. After 1 h, the reaction was quenched with sat. aqueous
NH4C1 (300 mL). The organic layer was separated and the aqueous phase was extracted with
EtOAc (150 mL x 3). The combined organic layers were dried over anhydrous Na2SO4 and
concentrated under reduced pressure to yield a residue that was purified by column
chromatography on silica gel [petroleum ether/ethyl acetate, 10/1) to give tert-butyl 6-chloro-4-
iodopyridin-3-ylcarbamate (10.0 g, 33%). XH-NMR (CDC13, 400 MHz) 5 8.95 (s, 1 H), 7.73 (s,
1 H), 6.64 (br s, 1 H), 1.53 (s, 9H).
Step c: 6-chloro-4-iodopyridin-3-amine
[0037] The solution of tert-butyl 6-chloro-4-iodopyridin-3-ylcarbamate (10.0 g, 28 mmol) in
3M HC1 (600 mL) was heated at 60 °C for 12 h. The mixture was allowed to cool to room
temperature and treated with sat. NaHCCh to pH=8. The aqueous layer was extracted with ethyl
acetate (100 mL x 3). The combined organic layers were washed with brine, dried over
anhydrous Na2SO4, concentrated and purified by chromatography on silica gel (10% ethyl
acetate in petroleum ether as eluant) to afford 6-chloro-4-iodopyridin-3-amine (6.6 g, 93%).
LH-NMR (CDCI3, 400 MHz) 5 7.81 (s, 1 H), 7.60 (s, 1 H), 4.13 (br s, 2 H).
Step d: 2-(6-Chloro-4-iodoyyridin-3-ylamino)ethanol
[0038] To a solution of 6-chloro-4-iodopyridin-3-amine (6.5 g, 25.5 mmol) in CH3OH (1500
mL) was added 2-(tert-butyldimethylsilyloxy) acetaldehyde (18.0 g, 103 mmol). Then
trifluoroacetic acid (150 mL) and NaBH3CN (8.0 g, 127 mmol) were added slowly at 0 °C. The
mixture was allowed to warm to 25°C and the stirring was continued for an additional 12 hours.
The mixture was concentrated under reduced pressure and treated with NaOH (3M) to pH=8.
The aqueous layer was extracted with ethyl acetate (100 mL x 3). The combined organic layers
were washed with brine, dried over anhydrous Na2SO4. The solvent was concentrated in vacuo
to afford crude product 2-(6-Chloro-4-iodopyridin-3-ylamino)ethanol (14.5 g), that was used in
the next step without further purification.
Step e: 2-(6-chloro-4-(3,3-dimethylbut-l-ynyl)pyridin-3-ylamino)ethanol
[0039] To a solution of 2-(6-chloro-4-iodopyridin-3-ylamino)ethanol (14.5 g, 49 mmol) in
Et3N (200 mL) were added 3,3-dimethyl-but-l-yne (12.0 g, 146 mol), Cul (0.9 g, 4.9 mmol)
and Pd(PPH3)Cl2 (3.4 g, 4.9 mmol) under N2 atmosphere. The mixture was refluxed overnight
and quenched with H2O (100 mL). The organic layer was separated and the aqueous layer was
extracted with ethyl acetate (100 mL x 3). The combined organic layers were washed with

brine, dried over anhydrous Na2SO4 and purified by chromatography on silica gel (3% ethyl
acetate in petroleum ether as eluant) to afford 2-(6-chloro-4-(3,3-dimethylbut-l-ynyl)pyridin-3-
ylamino)ethanol (3.6 g, 29 %). LH-NMR (CDC13, 300 MHz) 5 7.74 (s, 1 H), 7.11 (s, 1 H), 4.77
(br s, 1 H), 3.92-3.90 (m, 2 H), 3.78-3.36 (m, 2 H), 1.34 (s, 9 H).
Step f: 2-(2-tert-Butvl-5-chloro-lH-pyrrolo[2,3-c]pyridin-l-yl)ethanol
[0040] To a solution of 2-(6-chloro-4-(3,3-dimethylbut-l-ynyl)pyridin-3-ylamino)ethanol (3.6
g, 14.3 mmol) in DMF (100 mL) was added ?-BuOK (3.1 g, 28 mol) under N2 atmosphere. The
mixture was heated at 80 °C for 12 h and then was quenched with H2O (200 mL). The organic
layer was separated and the aqueous layer was extracted with EtOAc (150 mL x 3). The
combined organic layers were washed with brine, dried over anhydrous Na2SO4 and purified by
chromatography on silica gel (3% ethyl acetate in petroleum ether as eluant) to afford 2-{2-tert-
butyl-5-chloro-lH-pyrrolo[2,3-c]pyridin-l-yl)ethanol (1.6 g, 44 %). 'H-NMR (CDCI3, 300
MHz) 5 8.50 (s, 1 H), 7.39 (s, 1 H), 6.28 (s, 1 H), 4.55 (t, J= 6.6, 2 H), 4.05 (t, J= 6.6, 2 H),
1.49 (s, 9 H).
Step s: 2-(5-amino-2-tert-butvl-lH-vvrrolo[2,3-clvvridin-l-vl)ethanol
[0041] In a 100 mL autoclave, a mixture of 2-(2-ter£-butyl-5-chloro-lH-pyrrolo[2,3-c]pyridin-
l-yl)ethanol (350 mg, 1.39 mmol) and CUSO4.5H2O (35 mg, 1.4 mmol) in aqueous ammonia
(14 mL) and CH3OH (7 ml) was heated to 120 °C for 14 h. The mixture was allowed to cool
down to 25 °C. The methanol was removed by evaporation under vacuum and the resulting
mixture was extracted with ethyl acetate (50 mL x 3). The combined organic layers were
washed with brine, dried over anhydrous Na2SO4 and purified by chromatography on silica gel
(5% CH3OH in dichloromethane as eluant) to afford 2-(5-amino-2-tert-butyl-lH-pyrrolo[2,3-
c]pyridin-l-yl)ethanol (50 mg, 16 %). 1H-NMR (CDC13, 300 MHz) 5 8.22 (s, 1 H), 6.59 (s, 1
H), 6.08 (s, 1 H), 4.44 (t, J= 6.9 Hz, 2 H), 3.99 (t, /= 6.9 Hz, 2 H), 1.45 (s, 9 H).

[0042] 2-tert-Butyl-lH-pyrrolo[2,3-c]pyridin-5-amine (325 mg, 0.158 mmol) and 1-
(benzo[d][l,3]dioxol-5-yl)cyclopropanecarboxylic acid (300 mg, 1.58 mmol) were dissolved in

acetonitrile (10 mL) containing triethylamine (659 [xL, 0.470 mmol). 0-(7-Azabenzotriazol-l-
yty-N,N,N,N,-tetramethyluronium hexafluorophosphate (608 mg, 1.60 mmol) was added to the
mixture and the resulting solution was allowed to stir for 16 hours during which time a large
amount of precipitate formed. The reaction mixture was filtered and the filtercake was washed
with acetonitrile and then dried to yield l-(benzo[d][\,3]dioxol-5-yl)-N-(2-tert-buty\-lH-
pyrrolo[2,3-c]pyridin-5-yl)cyclopropanecarboxamide (397 mg, 67%). ESI-MS m/z calc. 377.2,
found; 378.5 (M+l)+; Retention time 1.44 minutes. !H NMR (400 MHz, DMSO) 11.26 (s, 1H),
8.21 (s, 1H), 8.06 (s, 1H), 7.83 (s, 1H), 7.13 (d, J= 1.5 Hz, 1H), 7.04 - 6.98 (m, 2H), 6.20 (d, /
= 1.0 Hz, 1H), 6.09 (s, 2H), 1.49-1.45 (m, 2H), 1.38 (s, 9H), 1.12-1.08 (m, 2H).

[0043] HATU (38 mg, 0.10 mmol) was added to a solution of l-(2,2-
difluorobenzo[cT|[l,3]dioxol-5-yl)cyclopropanecarboxylic acid (24 mg, 0.10 mmol), 2-(5-
amino-2-tert-butyl-lH-pyrrolo[2,3-c]pyridin-l-yl)ethanol (23 mg, 0.10 mmol) and
triethylamine (42 uL, 0.30 mmol) in DMF (1 mL). The mixture was stirred at room
temperature for 1 hour. The mixture was filtered and purified by reverse-phase HPLC (10 -
99% CH3CN - H2O with 0.035% TFA) to yield A^-(2-terr-butyl-l-(2-hydroxyethyl)-lH-
pyrrolo[2,3-c]pyridin-5-yl)-l-(2,2-difluorobenzo[fir|[l,3]dioxol-5-yl)cyclopropanecarboxamide.
ESI-MS m/z calc. 457.2, found 458.5 (M+l)+. Retention time 1.77 minutes. *H NMR (400
MHz, DMSO-tf J = 8.3 Hz, 1H), 7.35 (dd, J = 8.3, 1.7 Hz, 1H), 6.67 (s, 1H), 4.58 (t, J = 5.7 Hz, 2H), 3.76 (t, J =
5.7 Hz, 2H), 1.58 (m, 2H), 1.46 (s, 9H), 1.28 (m, 2H).
12. Preparation of N-(2-tert-butyl-lH-pyrrolo[3,2-b]pyridin-5-yl)-l-(2,2-
difluorobenzo[d] [1,3] dioxol-5-yl)cyclopropanecarboxamide


[0044] HATU (31 mg, 0.083 mmol) was added to a solution of 1-(2,2-
difJuorobenzo[rf][l,3]dioxol-5-yl)cyclopropanecarboxylic acid (18 mg, 0.075 mmol), 2-tert-
butyl-lH-pyrrolo[3,2-b]pyridin-5-amine (16 mg, 0.083 mmol) and triethylamine (21 uL, 0.15
mmol) in DMF (1 mL). The reaction was stirred at 60 °C for 18 h. The mixture was filtered
and purified by reverse-phase HPLC (10 - 99% CH3CN - H2O with 0.035% TFA) to yield N-
(2-terf-butyl-1 H-pyrrolo[3,2-b]pyridin-5-yl)-1 -(2,2-difluorobenzo [d] [ 1,3 ]dioxol-5-
yl)cyclopropanecarboxamide as the TFA salt. ESI-MS m/z calc. 413.2, found 414.1 (M+l)+.
Retention time 2.86 minutes.

[00273] HATU (31 mg, 0.083 mmol) was added to a solution of l-(2,2-
difluorobenzo[J][l,3]dioxol-5-yl)cyclopropanecarboxylic acid (18 mg, 0.075 mmol), 2-tert-
butyl-lH-pyrrolo[2,3-c]pyridin-5-amine (16 mg, 0.083 mmol) and triethylamine (21 uL, 0.15
mmol) in DMF (1 mL). The reaction was stirred at 60 °C for 18 h. The mixture was filtered
and purified by reverse-phase HPLC (10 - 99% CH3CN - H2O with 0.035% TFA) to yield N-
(2-tert-butyl-lH-pyrrolo[2,3-b]pyridin-5-yl)-l-(2,2-difluorobenzo[^][l,3]dioxol-5-
yl)cyclopropanecarboxamide as the TFA salt. ESI-MS m/z calc. 413.2, found 414.3 (M+l)+.
Retention time 3.25 minutes.






[00275] Assays for Detecting and Measuring AF508-CFTR Correction Properties of
Compounds
[00276] Membrane potential optical methods for assaying AF508-CFTR modulation
properties of compounds.
[00277] The assay utilizes fluorescent voltage sensing dyes to measure changes in
membrane potential using a fluorescent plate reader (e.g., FLIPR in, Molecular Devices, Inc.)
as a readout for increase in functional AF508-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.
[00278] Identification of Correction Compounds
[00279] To identify small molecules that correct the trafficking defect associated with
AF5O8-CFTR; a single-addition HTS assay format was developed. Assay Plates containing cells
are incubated for ~2-4 hours in tissue culture incubator at 37oC, 5%CO2, 90% humidity. Cells are
then ready for compound exposure after adhering to the bottom of the assay plates.
[00280] The cells were incubated in serum-free medium for 16-24 hrs in tissue culture
incubator at 37oC, 5%CCte, 90% humidity in the presence or absence (negative control) of test
compound. The cells were subsequently rinsed 3X with Krebs Ringers solution and loaded
with a voltage sensing redistribution dye. To activate AF5O8-CFTR, 10 uM forskolin and the
CFTR potentiator, genistein (20 uM), were added along with Cl"-free medium to each well. The
addition of Cl-free medium promoted Cl" efflux in response to AF508-CFTR activation and the
resulting membrane depolarization was optically monitored using voltage sensor dyes.
[00281] Identification of Potentiator Compounds
[00282] To identify potentiators of AF508-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 AF508 CFTR in temperature-corrected AF508 CFTR NIH 3T3 cells. The
driving force for the response is a Cl" ion gradient in conjunction with channel activation with
forskolin in a single liquid addition step using a fluoresecent 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.

[00283] Solutions:
[00284] Bath Solution #1: (in mM) NaCl 160, KC14.5, CaCl2 2, MgCl2 1, HEPES 10,
pH 7.4 with NaOH.
[00285] Chloride-free bath solution: Chloride salts in Bath Solution #1 are
substituted with gluconate salts.
[00286] Cell Culture
[00287] NIH3T3 mouse fibroblasts stably expressing AF508-CFTR are used for
optical measurements of membrane potential. The cells are maintained at 37 °C in 5% CO2
and 90 % humidity in Dulbecco's modified Eagle's medium supplemented with 2 mM
glutamine, 10 % fetal bovine serum, 1 X NEAA, p-ME, 1 X pen/strep, and 25 mM HEPES in
175 cm2 culture flasks. For all optical assays, the cells were seeded at -20,000/well in 384-well
matrigel-coated plates and cultured for 2 hrs at 37 °C before culturing at 27 °C for 24 hrs. for
the potentiator assay. For the correction assays, the cells are cultured at 27 °C or 37 °C with
and without compounds for 16 - 24 hours.
[00288] Electrophysiological Assays for assaying AF508-CFTR modulation
properties of compounds.
[00289] 1 .Ussing Chamber Assay
[00290] Ussing chamber experiments were performed on polarized airway epithelial
cells expressing AF508-CFTR to further characterize the AF508-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® Snapwell™ filters that were precoated 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 AF508-CFTR.
[00291] HBE grown on Costar® Snapwell™ cell culture inserts were mounted in an
Ussing chamber (Physiologic Instruments, Inc., San Diego, CA), and the transepithelial
resistance and short-circuit current in the presence of a basolateral to apical Cl" gradient (ISc)

were measured using a voltage-clamp system (Department of Bioengineering, University of
Iowa, IA). Briefly, HBE were examined under voltage-clamp recording conditions (Vhoid = 0
mV) at 37 °C. The basolateral solution contained (in mM) 145 NaCl, 0.83 K2HPO4, 3.3
KH2PO4) 1.2 MgCl2, 1.2 CaCl2, 10 Glucose, 10 HEPES (pH adjusted to 7.35 with NaOH) and
the apical solution contained (in mM) 145 NaGluconate, 1.2 MgCl2, 1.2 CaCl2, 10 glucose, 10
HEPES (pH adjusted to 7.35 with NaOH).
[00292] Identification of Correction Compounds
[00293] Typical protocol utilized a basolateral to apical membrane Cl" concentration
gradient. To set up this gradient, normal ringer was used on the basolateral membrane, whereas
apical NaCl was replaced by equimolar sodium gluconate (titrated to pH 7.4 with NaOH) to
give a large Cl" concentration gradient across the epithelium. All experiments were performed
with intact monolayers. To fully activate AF508-CFTR, forskolin (10 fiM), PDE inhibitor,
IBMX (100 yM) and CFTR potentiator, genistein (50 uM) were added to the apical side.
[00294] As observed in other cell types, incubation at low temperatures of FRT cells
and human bronchial epithelial cells isolated from diseased CF patients (CF-HBE)expressing
AF508-CFTR increases the functional density of CFTR in the plasma membrane. To determine
the activity of correction compounds, the cells were incubated with test compound for 24-48
hours at 37°C and were subsequently washed 3X prior to recording. The cAMP- and genistein-
mediated Isc in compound-treated cells was normalized to 37°C controls and expressed as
percentage activity of CFTR activity in wt-HBE. Preincubation of the cells with the correction
compound significantly increased the cAMP- and genistein-mediated Isc compared to the 37CC
controls.
[00295] Identification of Potentiator Compounds
[00296] Typical protocol utilized a basolateral to apical membrane Cl" 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" concentration gradient across the epithelium. Forskolin (10 \iM) and
all test compounds were added to the apical side of the cell culture inserts. The efficacy of the
putative AF508-CFTR potentiators was compared to that of the known potentiator, genistein.
[00297] 2. Patch-clamp Recordings
[00298] Total Cl" current in AF5O8-NIH3T3 cells was monitored using the perforated-
patch recording configuration as previously described (Rae, J., Cooper, K., Gates, P., &

Watsky, M. (1991)./ Neurosci. Methods 37, 15-26). Voltage-clamp recordings were
performed at 22 °C using an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.,
Foster City, CA). The pipette solution contained (in mM) 150 TV-methyl-D-glucamine
(NMDG)-Cl, 2 MgCl2, 2 CaCl2, 10 EGTA, 10 HEPES, and 240 ng/ml amphotericin-B (pH
adjusted to 7.35 with HC1). The extracellular medium contained (in mM) 150 NMDG-C1, 2
MgCl2, 2 CaCl2, 10 HEPES (pH adjusted to 7.35 with HC1). 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 AF508-CFTR, 10 |aM
forskolin and 20 (iM genistein were added to the bath and the current-voltage relation was
monitored every 30 sec.
[00299] Identification of Correction Compounds
[00300] To determine the activity of correction compounds for increasing the density
of functional AF508-CFTR in the plasma membrane, we used the above-described perforated-
patch-recording techniques to measure the current density following 24-hr treatment with the
correction compounds. To fully activate AF508-CFTR, 10 nM forskolin and 20uM genistein
were added to the cells. Under our recording conditions, the current density following 24-hr
incubation at 27°C was higher than that observed following 24-hr incubation at 37 °C. These
results are consistent with the known effects of low-temperature incubation on the density of
AF508-CFTR in the plasma membrane. To determine the effects of correction compounds on
CFTR current density, the cells were incubated with 10 nM of the test compound for 24 hours
at 37°C and the current density was compared to the 27°C and 37°C controls (% activity).
Prior to recording, the cells were washed 3X with extracellular recording medium to remove
any remaining test compound. Preincubation with 10 uM of correction compounds
significantly increased the cAMP- and genistein-dependent current compared to the 37°C
controls.
[00301] Identification of Potentiator Compounds
[00302] The ability of AF5O8-CFTR potentiators to increase the macroscopic AF508-
CFTR Cl" current (IAFSOS) in NIH3T3 cells stably expressing AF508-CFTR was also
investigated using perforated-patch-recording techniques. The potentiators identified from the
optical assays evoked a dose-dependent increase in IAFSOS 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 EQ (-28 mV).

[00303] Cell Culture
[00304] NIH3T3 mouse fibroblasts stably expressing AF508-CFTR are used for
whole-cell recordings. The cells are maintained at 37 °C in 5% CO2 and 90 % humidity in
Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 10 % fetal bovine
serum, 1 X NEAA, 0-ME, 1 X pen/strep, and 25 mM HEPES in 175 cm2 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 °C before use to test the activity of potentiators; and
incubated with or without the correction compound at 37 °C for measuring the activity of
correctors.
[00305] 3.Single-channel recordings
[00306] Gating activity of wt-CFTR and temperature-corrected AF508-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., LazdunsM, 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 CaCl2, 2 MgCl2, and 10 HEPES (pH adjusted to 7.35
with Tris base). The bath contained (in mM): 150 NMDG-C1, 2 MgCl2, 5 EGTA, 10 TES, and
14 Tris base (pH adjusted to 7.35 with HC1). After excision, both wt- and AF508-CFTR were
activated by adding 1 mM Mg-ATP, 75 nM of the catalytic subunit of cAMP-dependent protein
kinase (PKA; Promega Corp. Madison, WI), 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 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 AF5O8-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 (Po) were determined from 120 sec of channel activity. The Po was determined
using the Bio-Patch software or from the relationship Po = I/i(N), where I = mean current, i =
single-channel current amplitude, and N = number of active channels in patch.

[00307] Cell Culture
[00308] NIH3T3 mouse fibroblasts stably expressing AF5O8-CFTR are used for
excised-membrane patch-clamp recordings. The cells are maintained at 37 °C in 5% CO2 and
90 % humidity in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine,
10 % fetal bovine serum, 1 X NEAA, p-ME, 1 X pen/strep, and 25 mM HEPES in 175 cm2
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 °C before use.
[00309] The compounds of Table 1 were found to exhibit Correction activity as
measured in the assay described above.
[00310] Compounds of the invention are useful as modulators of ATP binding
cassette transporters. Using the procedures described above, the activities, i.e., EC50s, of
compounds of the present invention have been measured and are shown in Table 5.


CLAIMS
1. A compound having formula I:

I
or a pharmaceutically acceptable salt thereof, wherein:
Ar1 is:

wherein
each of Gi, G2, G3, and G4 is independently selected from the group consisting of CH
and nitrogen, wherein one of Gi, G2, G3, and G4 is nitrogen and the remainder of Gi, G2, G3,
and G4 each is CH;
Ar1 is attached to the N(RN) through G2 or G3;
Ar1 is optionally substituted with w occurrences of-WRW; and
RN is H, R2, or R3;
ring A is 3-7 membered monocyclic ring having 0-3 heteroatoms selected from the
group consisting of oxygen, sulfur, and nitrogen, wherein ring A is optionally substituted with q
occurrences of-Q-RQ;
ring B is optionally fused to a 5-7 membered ring selected from the group consisting of
cycloaliphatic, aryl, heterocyclic, and heteroaryl, wherein ring B, together with said optionally
fused ring, is optionally substituted with x occurrences of-XRX;
Q, W, or X is independently a bond or is independently an optionally substituted (Cl-
C6) alkylidene chain wherein up to two methylene units of Q, W, or X are optionally and
independently replaced by -CO-, -CS-, -COCO-, -CONR1-, -CONRTSfR1-, -CO2-, -OCO-,

-NR'CO2-, -0-, -NR'CONR'-, -OCONR'-, -NRNR', -NRTSDEt'CO-, -NR'CO-, -S-, -SO, -SO2-, -
NR'-, -SO2NR'-, NR!SO2-, or -NR'SO2NR'-;
each RQ, Rw, and Rx is independently R1, R2, R3, R4, or R5;
R' is independently R2, R3, or R6;
R1 is oxo, =NN(R6)2, =NN(R7)2, =NN(R6R7), R6, or ((Cl-C4)aliphatic)n-Y;
wherein n is 0 or 1; and

R2 is aliphatic, wherein each R2 is optionally substituted with up to 2 substituents
independently selected from the group consisting of R1, R4, and R5;
R3 is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, wherein R3 is optionally
substituted with up to 3 substituents, independently selected from the group consisting of R1,
R2, R4, and R5;
R4 is OR5, OR6, OC(O)R6, OC(O)R5, OC(O)OR6, OC(O)OR5, OC(O)N(R6)2,
OC(O)N(R5)2, OC(O)N(R6R5), SR6, SRS, S(O)R6, S(O)R5, SO2R6, SO2R5, SO2N(R6)2,
SO2N(R5)2, SO2NR5R6, SO3R6, SO3R5, C(O)R5, C(O)OR5, C(O)R6, C(O)OR6, C(O)N(R6)2,
C(O)N(R5)2, C(O)N(R5R6), C(O)N(OR6)R6, C(O)N(OR5)R6, C(O)N(OR6)R5, C(O)N(OR5)R5,
CfNOR^R6, CCNOR^R5, C(NOR5)R6, C(NOR5)R5, N(R6)2, N(R5)2, N^R6), NR5C(O)R5,
NR6C(O)R6, NR6C(O)R3, NR5C(O)R6, NR6C(O)OR6, NR5C(O)OR6, NR6C(O)OR5,
NR5C(O)OR5, NR6C(O)N(R6)2) NR6C(O)NR5R6, NR6C(O)N(R5)2, NR5C(O)N(R6)2,
NR5C(O)NR5R6, NR5C(O)N(R5)2, NR6SO2R6, NR6SO2R5, NR5SO2R6,NR5SO2R5,
NR6SO2N(R6)2, NR6SO2NR5R6, NR6SO2N(R5)2, NR5SO2NR5R6, NR5SO2N(R5)2, N(OR6)R6,
NCOR^R5, N(OR5)R5, or N(OR5)R6;

R5 is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, wherein R5 is optionally
substituted with up to 3 R1;
R6 is H or aliphatic, wherein R6 is optionally substituted with a R7;
R7 is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, and each R7 is optionally
substituted up to 2 substituents independently selected from the group consisting of H, (Cl-
C6)-straight or branched alkyl, (C2-C6) straight or branched alkenyl or alkynyl, 1,2-
methylenedioxy, 1,2-ethylenedioxy, and(CH2)n-Z;
Z is selected from the group consisting of halo, CN, NO2, CF3, OCF3, OH, S-aliphatic,
S(O)-aliphatic, SO2-aliphatic, NH2, NH-aliphatic, N(aliphatic)2, N(aliphatic)R8, NHR8, COOH,
C(O)O(-aliphatic), and O-aliphatic;
R8 is an amino protecting group;
w is 0 to 5; and
each of x and q is independently 0-5.
2. The compound according to claim 1, wherein Ar1 is an optionally substituted
ring selected from the group consisting of:

3. The compound according to claim 1, wherein w is 0-3.
4. The compound according to claim 1, wherein W is a bond.
5. The compound according to claim 1, wherein W is an optionally substituted
(C1-C6) alkylidene chain wherein up to two methylene units of W is optionally and
independently replaced by -CO-, -CONR'-, -CO2-, -OCO-, -NR'CO2-, -O-, -NR'CONR'-,
-OCONR1-, -NR'CO-, -S-, -SO, -SO2-, -NR'-, -SO2NR'-, NR'SO2-, or -NR'SO2NR'-.
6. The compound according to claim 1, wherein W is an optionally substituted
(C1-C6) alkylidene chain wherein up to two non-adjacent methylene unit of W is optionally
replaced by -CONR'-, -CO2-, -O-, -S-, -SO2-, -NR1-, or -SO2NR'-.

7. The compound according to claim 1, wherein Rwis independently R2 or R3.
8. The compound according to claim 1, wherein Rw is a C1-C6 aliphatic
optionally substituted with up to four substituents selected from the group consisting of R1, R4,
and Rs.
9. The compound according to claim 1, wherein Rw is C6-C10 aryl optionally
substituted with up to five substituents selected from the group consisting of R1, R4, and R5.
10. The compound according to claim 1, wherein Rw is 3-10 membered
monocyclic or bicyclic heterocyclic ring optionally substituted with up to four substituents
selected from the group consisting of R1, R4, and R5.
11. The compound according to claim 1, wherein Rw is 5-10 membered
monocyclic or bicyclic heteroaryl ring optionally substituted with up to five substituents
selected from the group consisting of R1, R4, and R5.
12. The compound according to claim 1, wherein -WRW is an optionally
substituted group selected from the group consisting of methyl, ethyl, propyl, iso-propyl, butyl,
iso-butyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
13. The compound according to claim 1, wherein x is 1-5.
14. The compound according to claim 1, wherein X is independently a bond.
15. The compound according to claim 1, wherein X is an (C1-C6) alkylidene
chain wherein one or two non-adjacent methylene units are optionally and independently
replaced by O, NR\ S, SO2, COO, or CO.
16. The compound according to claim 1 wherein Rx is R1, R2 or R3, or two Rx are
two adjacent R1 taken together to fon therein J is -CH2- or -CF2-.
17. The compound according to claim 1, wherein RN is hydrogen or a C1-C6
aliphatic.
18. The compound according to claim 1, wherein ring A is an optionally
substituted 3-7 membered cycloaliphatic ring.

19. The compound according to claim 1, wherein ring A is an optionally
sbustituted 3-7 membered ring containing 1 heteroatom selected from the group consisting of
O, NH, and S.
20. The compound according to claim 1, wherein ring A is selected from the group
consisting of:



wherein:
Rx, X, x, RN, G1; G2, G3, and G4 are defined above;
m is 0 to 4;
Ar1 is:
wherein Ar1 is attached to theN(RN) through G2 or G3; and
Ar1 is optionally substituted with up to 3 Rw substituents, wherein each Rw is
independently selected from the group consisting of R1, R2, R3, and R4.
22. The compound according to claim 1, wherein Ar1 is attached through atom G2.
23. The compound according to claim 1, wherein Ar1 is attached through atom G3.
24. The compound according to claim 1, wherein said compound has formula
IIIA, or formula IIIB:

wherein Rx, X, x, m, RN, G1, G2, G3, and G4 are defined above; and
each Rw is independently selected from the group consisting of R1, R2, R3, and R4.
25. The compound according to claim 24, wherein G3 in formula IIIA is a C.

26. The compound according to claim 24, wherein G2 in formula IIIB is a C.
27. The compound according to claim 1, wherein Rw is R6 or ((Cl-C4)aliphatic)n-
Y; n is 0 or 1; and Y is halo, CN, NO2, CF3, OCF3, OH, SR6, S(O)R6, SO2R6, NH2) NHR6,
N(R6)2, NR^8, COOH, COOR6, or OR6.
28. The compound according to claim 1, wherein Rw is a C1-C6 aliphatic
optionally substituted with up to four substituents selected from the group consisting of R1, R4,
and R5.
29. The compound according to claim 1, wherein Rw is a C6-C10 aryl optionally
substituted with up to five substituents selected from the group consisting of R1, R4, and R5.
30. The compound according to claim 1, wherein Rw is a 3-10 membered
monocyclic or bicyclic heterocyclic ring optionally substituted with up to four substituents
selected from the group consisting of R1, R4, and R5.
31. The compound according to claim 1, wherein Rw is 5-10 membered
monocyclic or bicyclic heteroaryl ring optionally substituted with up to five substituents
independently selected from the group consisting of R1, R4, and R5.
32. The compound according to claim 1, wherein ring B is fused to a 5-7
membered heterocyclic or heteroaryl ring having up to 3 heteroatoms independently selected
from the group consisting of O, N, and S.
33. The compound according to claim 1, wherein ring B is fused to a 5-6
membered heterocyclic ring having up to 3 heteroatoms independently selected from the group
consisting of O, N, and S.
34. The compound according to claim 1, wherein ring B is fused to a 5-6
membered heteroaryl ring having up to 3-heteroatoms independently selected from the group
consisting of O, N, and S.
35. The compound according to claim 1, wherein ring B, together with said fused
ring, is optionally substituted with up to two Rx substituents.
36. The compound according to claim 1, wherein said Rx substituent is R1.




39. The compound according to claim 38, wherein Rw that is attached to carbon
no. 2 is R2 or R3.
40. The compound according to claim 3 8, wherein Rw that is attached to carbon
no. 2 is C1-C6 aliphatic optionally substituted with up to four substituents selected from the
group consisting of R1, R4, and R5.
41. The compound according to claim 38, wherein Rw that is attached to carbon
no. 2 is an optionally substituted C1-C6 alkyl.
42. The compound according to claim 38, wherein Rw that is attached to carbon
no. 2 is methyl, ethyl, propyl, isopropyl, butyl, isobuyl, 1-methylcyclopropyl, or tert-butyl.
43. The compound according to claim 38, wherein Rw that is attached to carbon
no. 2 is tert-butyl.
44. The compound according to claim 38, wherein Rw that is attached to carbon
no. 3 is H.
45. The compound according to claim 1, wherein said compound has formula
VA, formula VB, or formula VC:


46. The compound according to claim 45, wherein W is an optionally substituted
C1-C6 alkylidene.
47. The compound according to claim 45, wherein W is a C1-C6 alkylidene
substituted with a hydroxy, alkoxy, or amino group.
48. The compound according to claim 45, wherein W is a C1-C6 alkylidene
substituted with a hydroxy group.
49. The compound according to claim 45, wherein Rw is R4.
50. The compound according to claim 45, wherein Rw is OR6.
51. The compound according to claim 45, wherein Rw is OH.
52. The compound according to claim 45, wherein W is an optionally substituted
C1-C6 alkylidene and Rw is OR6.
53. The compound according to claim 45, wherein W is a C1-C6 alkylidene
substituted with a hydroxyl, alkoxy, or amino group, and Rw is OH.
54. The compound according to claim 45, wherein -WRW is -C2H4OH or -
CH2CH(OH)CH2OH.
55. The compound according to claim 1, wherein the compound is selected from
Table 1 above.
56. A pharmaceutical composition comprising
(i) a compound according to claim 1; and
(ii) a pharmaceutic^ly acceptable carrier.
57. The composition of claim 56, further comprising an additional agent selected
from the group consisting of a mucolytic agent, bronchodialator, an anti-biotic, an anti-infective
agent, an anti-inflammatory agent, CFTR corrector, and a nutritional agent.
58. A method of modulating ABC transporters in a membrane of a cell, comprising
the step of contacting said cell with a compound according to claim 1.
59. The method of claim 58, wherein the ABC transporter is CFTR.

60. A kit for use in measuring the activity of a ABC transporter or a fragment
thereof in a biological sample in vitro or in vivo, comprising:
(i) a first composition comprising a compound according to claim 1; and
(ii) instructions for:
a) contacting the composition with the biological sample;
b) measuring activity of said ABC transporter or a fragment thereof.
61. The kit according to claim 60, further comprising instructions for
a) contacting an additional composition with the biological sample;
b) measuring the activity of said ABC transporter or a fragment thereof in the presence
of said additional compound, and
c) comparing the activity of the ABC transporter in the presence of the additional
compound with the density of the ABC transporter in the presence of said first composition.
62. The kit of claim 60, wherein the kit is used to measure the density of CFTR.
63. A method of treating a condition, disease, or disorder in a patient implicated by
ABC transporter activity, comprising the step of administering to said patient a compound
having formula I:


each of Gi, G2, G3, and G4 is independently selected from the group consisting of CH
and nitrogen, wherein one of Gi, G2, G3, and G4 is nitrogen and the remainder of Gi, G2) G3,
and G4 each is CH;
Ar1 is attached to the N(RN) through G2 or G3;
Ar1 is optionally substituted with w occurrences of-WRw; and
RN is H, R2, or R3;
ring A is 3-7 membered monocyclic ring having 0-3 heteroatoms selected from the
group consisting of oxygen, sulfur, and nitrogen, wherein ring A is optionally substituted with q
occurrences of-Q-RQ;
ring B is optionally fused to a 5-7 membered ring selected from the group consisting of
cycloaliphatic, aryl, heterocyclic, and heteroaryl, wherein ring B, together with said optionally
fused ring, is optionally substituted with x occurrences of-XRX;
Q, W, or X is independently a bond or is independently an optionally substituted (Cl-
C6) alkylidene chain wherein up to two methylene units of Q, W, or X are optionally and
independently replaced by -CO-, -CS-, -COCO-, -CONR'-, -CONRTSfR'-, -CO2-, -OCO-,
-NR'CO2-, -O-, -NR'CONR1-, -OCONR'-, -NR'NR1, -NR'NR'CO-, -NR'CO-, -S-, -SO, -SO2-, -
NR'-, -SO2NR'-, NR'SO2-, or -NR'SO2NR'-;
each RQ, Rw, and Rx is independently R1, R2, R3, R4, or R5;
R1 is independently R2, R3, or R6;
R1 is oxo, =NN(R6)2, =NN(R7)2) =NN(R6R7), R6, or ((Cl-C4)aliphatic)n-Y;
wherein n is 0 or 1; and
Y is halo, CN, NO2, CF3, OCF3) OH, SR6, S(O)R6, SO2R6, NH2, NHR6, N(R6)2, NR6R8,
COOH, COOR6, or OR6; or


R2 is aliphatic, wherein each R2 is optionally substituted with up to 2 substituents
independently selected from the group consisting of R1, R4, and R5;
R3 is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, wherein R3 is optionally
substituted with up to 3 substituents, independently selected from the group consisting of Rl,
R2,R4,andR5;
R4 is OR5, OR6, OC(O)R6, OC(O)R5, OC(O)OR6, OC(O)OR5, OC(O)N(R6)2,
OC(O)N(R5)2, OC(O)N(R6R5), SR6, SR5, S(O)R6, S(O)R5, SO2R6, SO2R5, SO2N(R6)2,
SO2N(R5)2, SO2NR5R6, SO3R6, SO3R5, C(O)R5, C(O)OR5, C(O)R6, C(O)OR6, C(O)N(R6)2,
C(O)N(R5)2, C(O)N(R5R6), C(O)N(OR6)R6, C(O)N(OR5)R6, C(O)N(OR6)R5, C(O)N(OR5)R5,
C(NOR6)R6, CCNOR^R5, C(NOR5)R6, C(NOR5)R5, N(R6)2, N(R5)2, N(R5R6), NR5C(O)R5,
NR6C(O)R6, NR6C(O)R5, NR5C(O)R6, NR6C(O)OR6, NR5C(O)OR6, NR6C(O)OR5,
NR5C(O)OR5, NR6C(O)N(R6)2, NR6C(O)NR5R6, NR6C(O)N(R5)2, NR5C(O)N(R6)2,
NR5C(O)NR5R6, NR5C(O)N(R5)2, NR6SO2R6, NR6SO2R5, NR5SO2R6,NR5SO2R5,
NR6SO2N(R6)2, NR6SO2NR5R6, NR6SO2N(R5)2, NR5SO2NR5R6, NR5SO2N(R5)2, N(OR6)R6,
NCOR^R5, N(OR5)R5, or N(OR5)R6;
R5 is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, wherein R5 is optionally
substituted with up to 3 R1;
R6 is H or aliphatic, wherein R6 is optionally substituted with a R7;
R7 is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, and each R7 is optionally
substituted up to 2 substituents independently selected from the group consisting of H, (Cl-
C6)-straight or branched alkyl, (C2-C6) straight or branched alkenyl or alkynyl, 1,2-
methylenedioxy, 1,2-ethylenedioxy, and (CH2)n-Z;
Z is selected from the group consisting of halo, CN, NO2, CF3, OCF3, OH, S-aliphatic,
S(O)-aliphatic, SO2-aliphatic, NH2, NH-aliphatic, N(aliphatic)2, N(aliphatic)R8, NHR8, COOH,
C(O)O(-aliphatic), and O-aliphatic;
R8 is an amino protecting group;
w is 0 to 5; and
each of x and q is independently 0-5.
64. The method according to claim 63, wherein Ar1 is an optionally substituted ring
selected from the group consisting of:


65. The method according to claim 63, wherein w is 0-3.
66. The method according to claim 63, wherein W is a bond.
67. The method according to claim 63, wherein W is an optionally substituted (Cl-
C6) alkylidene chain wherein up to two methylene units of W is optionally and independently
replaced by -CO-, -CONR'-, -CO2-, -OCO-, -NR'CO2-, -O-, -NR'CONR1-, -OCONR-, -NR'CO-
, -S-, -SO, -SO2-, -NR1-, -SO2NR'-, NR'SO2-, or -NR'S02NR'-.
68. The method according to claim 63, wherein W is an optionally substituted (Cl-
C6) alkylidene chain wherein up to two non-adjacent methylene unit of W is optionally
replaced by -CONR1-, -CO2-, -O-, -S-, -SO2-, -NR1-, or -SO2NR'-.
69. The method according to claim 63, wherein Rw is independently R2 or R3.
70. The method according to claim 63, wherein Rw is C1-C6 aliphatic optionally
substituted with up to four substituents selected from the group consisting of R1, R4, and R5.
71. The method according to claim 63, wherein Rw is C6-C10 aryl optionally
substituted with up to five substituents selected from the group consisting of R1, R4, and R5.
72. The method according to claim 63, wherein Rw is 3-10 membered monocyclic
or bicyclic heterocyclic ring optionally substituted with up to four substituents selected from
the group consisting of R1, R4, and R5.
73. The method according to claim 63, wherein Rw is 5-10 membered monocyclic
or bicyclic heteroaryl ring optionally substituted with up to five substituents selected from the
group consisting of R1, R4, and R5.
74. The method according to claim 63, wherein -WRW is an optionally substituted
group selected from the group consisting of methyl, ethyl, propyl, iso-propyl, butyl, iso-buryl,
tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

75. The method according to claim 63, wherein x is 1-5.
76. The method according to claim 63, wherein X is independently a bond.
77. The method according to claim 63, wherein X is an (C1-C6) alkylidene chain
wherein one or two non-adjacent methylene units are optionally and independently replaced by
O, NR', S, SO2, COO, or CO.
78. The method according to claim 63, whherein Rx is R1, R2 or R3, or two Rx are
two adjacent R1 taken together to form wherein J is -CH2- or -CF2-.
79. The method according to claim 63, wherein RN is hydrogen or a C1-C6
aliphatic.
80. The method according to claim 63, wherein ring A is an optionally substituted 3-
7 membered cycloaliphatic ring.
81. The method according to claim 63, wherein ring A is an optionally substituted
3-7 membered ring containing 1 heteroatom selected from the group consisting of O, NH, and
S.
82. The method according to claim 63, wherein ring A is selected from the group
consisting of:




The method according to claim 63, wherein Ari is attached through atom G2.
85. The method according to claim 63, wherein Ari is attached through atom G3.
86. The method according to claim 63, wherein said compound has formula IIIA, or
formula IIIB:

87. The method according to claim 86, wherein G3 in IIIA is a carbon.
88. The method according to claim 86, wherein G2 in IIIB is a carbon.
89. The method according to claim 63, wherein Rw is R6 or ((Cl-C4)aliphatic)n-Y;
n is 0 or 1; and Y is halo, CN, NO2, CF3, OCF3, OH, SR6, S(O)R6, SO2R6, NH2, NHR6, N(R6)2,
NR6R8, COOH, COOR6, or OR6.
90. The method according to claim 63, wherein Rw is a C1-C6 aliphatic optionally
substituted with up to four substituents selected from the group consisting of R1, R4, and R5.
91. The method according to claim 63, wherein Rw is a C6-C10 aryl optionally
substituted with up to five substituents selected from the group consisting of R1, R4, and R5.
92. The method according to claim 63, wherein Rw is a 3-10 membered monocyclic
or bicyclic heterocyclic ring optionally substituted with up to four substituents selected from
the group consisting of R1, R4, and R5.
93. The method according to claim 63, wherein Rw is 5-10 membered monocyclic
or bicyclic heteroaryl ring optionally substituted with up to five substituents independently
selected from the group consisting of R1, R4, and R5.

94. The method according to claim 63, wherein ring B is fused to a 5-7 membered
heterocyclic or heteroaryl ring having up to 3 heteroatoms independently selected from the
group consisting of O, N, and S.
95. The method according to claim 63, wherein ring B is fused to a 5-6 membered
heterocyclic ring having up to 3 heteroatoms independently selected from the group consisting
ofO,N, andS.
96. The method according to claim 63, wherein ring B is fused to a 5-6 membered
heteroaryl ring having up to 3-heteroatoms independently selected from the group consisting of
O, N, and S.
97. The method according to claim 63, wherein said ring B, together with said fused
ring, is optionally substituted with up to two Rx substituents.
98. The method according to claim 63, wherein said Rx substituent is R1.
99. The method according to claim 63, wherein said ring fused to ring B is selected
from the group consisting of:
ix, x, xi, xn,


101. The method according to claim 100, wherein R that is attached to carbon no. 2
is R2 or R3.
102. The method according to claim 100, wherein Rw that is attached to carbon no. 2
is C1-C6 aliphatic optionally substituted with up to four substituents selected from the group
consisting of R1, R4, and R5.
103. The method according to claim 100, wherein Rw that is attached to carbon no. 2
is an optionally substituted C1-C6 alkyl.
104. The method according to claim 100, wherein Rw that is attached to carbon no. 2
is methyl, ethyl, propyl, isopropyl, butyl, isobuyl, 1-methylcyclopropyl, or tert-butyl.
105. The method according to claim 100, wherein Rw that is attached to carbon no. 2
is tert-butyl.

106. The method according to claim 100, wherein Rw that is attached to carbon no. 3
isH.
107. The method according to claim 63, wherein said compound has formula VA,
formula VB, or formula VC:

108. The method according to claim 107, wherein W is an optionally substituted Cl-
C6 alkylidene.
109. The method according to claim 107, wherein W is a C1-C6 alkylidene
substituted with a hydroxy, alkoxy, or amino group.
110. The method according to claim 107, wherein W is a C1-C6 alkylidene
substituted with a hydroxyl group.
111. The method according to claim 107, wherein Rw is R4.
112. The method according to claim 107, wherein Rw is OR6.
113. The method according to claim 107, wherein Rw is OH.
114. The method according to claim 107, wherein W is an optionally substituted Cl-
C6 alkylidene and Rw is OR6.
115. The method according to claim 107, W is a C1-C6 alkylidene substituted with a
hydroxyl, alkoxy, or amino group, and Rw is OH.

116. The method according to claim 104, wherein -WRW is -C2H4OH or -
CH2CH(OH)CH2OH.
117. The method according to claim 63, wherein the compound is selected from
Table 1 above.
118. The method according to claim 63, wherein said ABC transporter is CFTR.
119. The method according to claim 63, wherein the compound is selected from
Table 1 above..
120. The method according to claim 63, wherein said condition, disease, or disorder
is selected from cystic fibrosis, 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-Najjartypell,
polyendocrinopathy/hyperinsulemia, diabetes mellitus, laron dwarfism, myleoperoxidase
deficiency, primary hypoparathyroidism, melanoma, glycanosis CDG type 1, hereditary
emphysema, congenital hyperthyroidism, osteogenesis imperfecta, hereditary
hypofibrinogenemia, ACT deficiency, diabetes insipidus (di), neurophyseal di, neprogenic DI,
Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases
such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive
supranuclear plasy, Pick's disease, several polyglutamine neurological disorders asuch as
Huntington, spinocerebullar ataxia type I, spinal and bulbar muscular atrophy, dentatorubal
pallidoluysian, and myotonic dystrophy, as well as spongiform encephalopathies, such as
hereditary Creutzfeldt-Jakob disease, Fabry disease, Straussler-Scheinker syndrome, COPD,
dry-eye disease, and Sjogren's disease.

The present invention relates to. modulators of ATP-Binding Cassette ("ABC") transporters or fragments thereof of formula (I), including Cystic F1-brosis Transmembrane Conductance Regulator ("CFTR"), compositions thereof, and
methods therewith. The present invention also relates to methods of treating ABC transporter mediated diseases using such modulators.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=YJKJl+qfPNerp2/avJxOig==&loc=wDBSZCsAt7zoiVrqcFJsRw==


Patent Number 268874
Indian Patent Application Number 1955/KOLNP/2009
PG Journal Number 39/2015
Publication Date 25-Sep-2015
Grant Date 21-Sep-2015
Date of Filing 25-May-2009
Name of Patentee VERTEX PHARMACEUTICALS INCORPORATED
Applicant Address 130 WAVERLY ST., CAMBRIDGE, MA 02139
Inventors:
# Inventor's Name Inventor's Address
1 HADIDA RUAH, SARA 2356 TORREY PINES #16, LA JOLLA, CA 92037
2 MILLER, MARK 5075 LA JOLLA BLVD., #9, SAN DIEGO, CA 92109
3 BEAR, BRIAN 5108 SPENCER COURT, OCEANSIDE, CA 92057
4 ZHOU, JINGLAN 4466 SHOREPOINTE WAY, SAN DIEGO, CA 92130
PCT International Classification Number A61P 3/00,A61P 25/00
PCT International Application Number PCT/US2007/083464
PCT International Filing date 2007-11-02
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/856,584 2006-11-03 U.S.A.