WO2014186704A2 - Novel compounds for the treatment of cystic fibrosis - Google Patents

Abstract

The present invention is directed to novel compounds, pharmaceutical compositions comprising such compounds, and the methods of making and using the same. These compounds are useful as modulators of Cystic Fibrosis Transmembrane Conductor Regulator (CFTR). The present invention also relates to methods of treating or lessening the severity of cystic fibrosis in a patient. These compounds may be used alone or in combination with one or more secondary active agents.

Description

NOVEL COMPOUNDS FOR THE TREATMENT OF CYSTIC FIBROSIS

FIELD OF THE INVENTION

[0001] The present invention is directed to novel compounds, pharmaceutical compositions comprising such compounds, and the methods of making and using the same. These compounds are useful as modulators of Cystic Fibrosis Transmembrane Conductor Regulator (CFTR). The present invention also relates to methods of treating or lessening the severity of cystic fibrosis in a patient. These compounds may be used alone or in

combination with one or more secondary active agents.

BACKGROUND

[0002] Cystic fibrosis (CF) is one of the most common lethal genetic diseases in

Caucasians. Approximately one in 3,500 children in the US is born with CF each year. It is a disease that affects all racial and ethnic groups, but is more common among Caucasians. An estimated 30,000 American adults and children have CF (70,000 worldwide), and the median predicted age of survival is 36.8 years (CFF Registry Report 2011, Cystic Fibrosis

Foundation, Bethesda, MD). CF is an autosomal recessive hereditary disease caused by a mutation in the gene for the cystic fibrosis transmembrane regulator (CFTR) protein. More than 1,000 disease-associated mutations have been discovered in the CFTR gene with the most common mutation being a deletion of the amino acid phenylalanine at position 508 (F508del). This defect is present in 70% of CF patients. The CFTR protein is located on the apical membrane and is responsible for chloride transport across epithelial cells on mucosal surfaces. Currently there is no curative treatment for CF; therefore, new therapies are needed for the disease.

[0003] There is a significant need for novel compounds and methods for treating or lessening the severity of cystic fibrosis in a patient. The present invention satisfies these needs.

SUMMARY

[0004] The present invention provides compounds that are modulators of the CFTR protein. In particular, provided are compounds having the structure depicted below (Formula 1), or a pharmaceutically acceptable salt, stereoisomer, prodrug, metabolite thereof.

Formula 1

wherein

Cy is selected from

A is selected from -COOH, hydroxyl, -CH₂OH, -CH₂CH₂OH, tetrazole, -NHC(0)R_x, - NHC(0)OR_x, -NHC(0)N(CH₃)₂, and -S0₂R_x;

R_x is selected from the group consisting of methyl, ethyl, i-propyl, and n-propyl;

Ri is selected from

halogen, hydroxyl, cyano, NR₆R7,

optionally substituted Ci-C₆ alkyl group wherein substituents are selected from cyano, hydroxyl, and halogen,

optionally substituted C C₆ alkyl group having one methylene unit replaced by an oxygen atom, wherein substituents are selected from cyano, hydroxyl, and halogen,

and two Ri groups taken together to form a 4-7 membered saturated, partially saturated,

or aromatic ring with up to 3 ring atoms independently selected from O, NR₆, and

S and wherein the fused ring may optionally be substituted by one or more halogen or CrC₃ alkyl;

R₂ and R₃ are each independently of one another are selected from

hydrogen, fluoro, hydroxyl, cyano, C₂-C₆ alkenyl, C₂-C₆ alkynyl,

optionally substituted Ci-C₆ alkyl group, wherein substitutions are selected from cyano, hydroxyl, C₃-C₆ cycloalkyl, C₃-C₆ heterocycloalkyl, and halogen,

optionally substituted C -C alkyl group having one methylene unit replaced by an oxygen atom, wherein substituents are selected from cyano, hydroxyl, C₃-C₆ cycloalkyl, C₃-C₆ heterocycloalkyl, and halogen,

a C3-C₆ cycloalkyl group, in which a methylene unit in the cyclic moiety may optionally

be replaced by a -NR₆ - group, an oxygen, or a sulphur atom, and optionally the

cycloalkyl groups and heterocycloalkyl groups may be substituted by halogen, and

provided that R₂ and R3 cannot both be hydrogen;

each R₄ and R5 are independently selected from

halogen, cyano, hydroxyl, NR₆R₇,

optionally substituted C -C alkyl wherein substitutions are selected from halogen, cyano,

and hydroxyl, and

optionally substituted Ci-C₆ alkyl having one methylene unit replaced by an oxygen atom

wherein substitutions are selected from cyano, hydroxyl, and halogen;

R₆ and R₇ are independently selected from hydrogen and Q-C4 alkyl;

m is selected from 0, 1, 2, and 3;

n is selected from 0, 1, 2, and 3; and

o is selected from 0, 1, and 2.

[0005] The present invention also provides methods for treating or lessening the severity of CF, alone or in combination with one or more secondary active agents. Also encompassed by the invention are pharmaceutical compositions comprising at least one compound and at least one pharmaceutically acceptable carrier. Also encompassed by the invention are pharmaceutical compositions comprising at least one compound for the treatment of cystic fibrosis.

[0006] The compositions of the present invention can be prepared in any suitable pharmaceutically acceptable dosage form.

[0007] The methods of the invention encompass administration with one or more secondary active agents. Such administration can be sequential or in a combination composition. [0008] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publicly available publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control.

[0009] Both the foregoing summary and the following detailed description are exemplary and explanatory and are intended to provide further details of the compositions and methods as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description.

DETAILED DESCRIPTION [0010] A. Overview of the Invention

[0011] Cystic fibrosis (CF) is a lethal genetic disease affecting 70,000 people worldwide. Approximately one in 3,500 children in the US is born with CF each year. It is a disease that affects all racial and ethnic groups, but is more common among Caucasians. An estimated 30,000 American adults and children have CF, and the median predicted age of survival is 36.8 years (CFF Registry Report 2011, Cystic Fibrosis Foundation, Bethesda, MD). CF is an autosomal recessive hereditary disease caused by a mutation in the gene for the cystic fibrosis transmembrane regulator (CFTR) protein. CFTR aids the regulation of epithelial salt and water transport in multiple organs, including the lung, pancreas, liver, and intestinal tract. Clinical manifestations of CF include abnormal sweat electrolytes, chronic and progressive respiratory disease, exocrine pancreatic dysfunction, and infertility; however, it is Sung disease that is the primary cause of morbidity and mortality. In the lung, the loss of CFTR mediated CI^" secretion is believed to cause airway surface dehydration due to both a decrease in CFTR-mediated CF and fluid secretion and a secondary increase in epithelial Na⁺ channel (ENaC)-mediated Na'^" and fluid absorption. This imbalance results in dehydration of the airway surface, and likely contributes to the deleterious cascade of mucus accumulation, infection, inflammation, and destruction that c aracterizes CF lung disease. The

accumulation of mucus leads to plugging in the passageways in the lung and other organs, such as the pancreas.

[0012] Current therapies to treat CF lung disease, including mucolytics, antibiotics, anti-inflammatory agents, anti-infectives and nutritional agents, target the downstream disease consequences that are secondary to the loss of CFTR function. Since the median predicted survival age is currently about 37 years, there is a large medical need for more efficacious therapies that address the underlying defect of CF.

[0013] To address this need, there has been increased interest in small -molecule therapies that increase CFTR function because such an approach could address the consequences of CFTR dysfunction as well as slow the progression of the disease. Such therapies are broadly classified as CFTR modulators and include CFTR activators, potentiators, correctors, and antagonists. CFTR activators act on their own to stimulate CFTR-mediated ion transport and include agents that increase cAMP levels, such as β- adrenergic agonists, adenylate cyclase activators, and phosphodiesterase inhibitors. CFTR potentiators act in the presence of endogenous or pharmacological CFTR activators to increase the channel gating activity of cell-surface localized CFTR., resulting in enhanced ion transport, CFTR correctors act by increasing the delivery and amount of functional CFTR protein to the cell surface, resulting in enhanced ion transport. Depending on the molecular consequence of the mutation and disease severity, CFTR activators, potentiators, and correctors may be coadministered to maximize clinical efficacy or therapeutic window, if needed. CFTR antagonists act by decreasing CFTR-mediated ion transport and are being developed for the treatment of polycystic kidney disease and cholera-induced secretory diarrhea.

[0014] There are many (>1500) different gene mutations for CF. Mutations affecting the CFTR gene cause a large variety of defects including altered CFTR channel gating (class III mutations such as G551D and G1349D) or impaired CFTR protein maturation (class II mutations such as F508del). Therefore, compounds increasing CFTR-dependent chloride transport are potentially useful as drugs to treat CF patients. In particular, pharmacological activators of CFTR, called potentiators, are useful to overcome the gating defect caused by class III CF mutations. Conversely, other compounds, called correctors, may help the F508del-CFTR protein to escape the endoplasmic reticulum and reach the plasma membrane. Potentiators are also useful for F508del. Indeed, this mutation causes also a gating defect, although less severe than that of classical class III mutations. On the other hand, CFTR inhibitors are characterized by decreased CFTR activity.

[0015] The most common mutation, F508del-CFTR (class II), results from a 3 base pair deletion that leads to the deletion of phenylalanine at position 508 of the full-length protein. The resulting F508del-CFTR protein is unstable and susceptible to rapid degradation in the 26S proteosome, with little if any F508del-CFTR at the plasma membrane. In the lungs of CF patients, the lack of transport of chloride and accompanying water across the airway epithelium and excessive sodium reabsorption leads to dehydrated airway surface fluid, impaired mucociliary clearance, infection and inflammation. Increasing the amount of F508-CFTR that reaches the plasma membrane, or otherwise improving its function, offers the potential to improve the hydration of the airway surface fluid and reverse part of the underlying pathophysiology.

[0016] The compounds of the present invention may provide a novel therapeutic strategy in cystic fibrosis (CF).

[0017] B. Novel Compounds

[0018] 1. Inventive Compounds

[0019] The present invention provides compounds that are modulators of CFTR. In particular, provided are compounds having the structure depicted below (Formula 1), or a pharmaceutically acceptable salt, stereoisomer, prodrug, metabolite thereof.

[0020]

Formula 1

wherein

Cy is selected from

A is selected from -COOH, hydroxyl, -CH₂OH, -CH₂CH₂OH, tetrazole, -NHC(0)R_x, - NHC(0)OR_x, -NHC(0)N(CH₃)₂, and -S0₂R_x;

R_x is selected from the group consisting of methyl, ethyl, i-propyl, and n-propyl;

Ri is selected from

halogen, hydroxyl, cyano, NR₆R7,

optionally substituted C Ce alkyl group wherein substituents are selected from cyano, hydroxyl, and halogen,

optionally substituted C -C alkyl group having one methylene unit replaced by an oxygen atom, wherein substituents are selected from cyano, hydroxyl, and halogen,

and two R groups taken together to form a 4-7 membered saturated, partially saturated,

or aromatic ring with up to 3 ring atoms independently selected from O, NR₆, and

S and wherein the fused ring may optionally be substituted by one or more halogen or Q-C3 alkyl;

R₂ and R₃ are each independently of one another are selected from

hydrogen, fluoro, hydroxyl, cyano, C₂-C₆ alkenyl, C₂-C₆ alkynyl,

optionally substituted C -C alkyl group, wherein substitutions are selected from cyano,

hydroxyl, C₃-C₆ cycloalkyl, C₃-C₆ heterocycloalkyl, and halogen,

optionally substituted CrC₆ alkyl group having one methylene unit replaced by an oxygen atom, wherein substituents are selected from cyano, hydroxyl, C₃-C₆ cycloalkyl, C₃-C₆ heterocycloalkyl, and halogen,

a C₃-C₆ cycloalkyl group, in which a methylene unit in the cyclic moiety may optionally

be replaced by a -NR₆ - group, an oxygen, or a sulphur atom, and optionally the

cycloalkyl groups and heterocycloalkyl groups may be substituted by halogen, and

provided that R₂ and R cannot both be hydrogen;

each R₄ and R5 are independently selected from

halogen, cyano, hydroxyl, NR₆R₇,

optionally substituted CrC₆ alkyl wherein substitutions are selected from halogen, cyano,

and hydroxyl, and

optionally substituted C C₆ alkyl having one methylene unit replaced by an oxygen atom

wherein substitutions are selected from cyano, hydroxyl, and halogen;

R₆ and R₇ are independently selected from hydrogen and C C₄ alkyl; m is selected from 0, 1, 2, and 3;

n is selected from 0, 1, 2, and 3; and

o is selected from 0, 1, and 2.

[0021] In one embodiment, the compound of Formula 1 includes compounds wherein

Cy is selected from

A is selected from -COOH, hydroxyl, -CH₂OH, -CH₂CH₂OH, and tetrazole;

Ri is selected from

halogen, hydroxyl, cyano, NR₆R7,

optionally substituted C -C alkyl group wherein substituents are selected from cyano, hydroxyl, and halogen,

optionally substituted C C₆ alkyl group having one methylene unit replaced by an oxygen atom, wherein substituents are selected from cyano, hydroxyl, and halogen,

and two R groups taken together to form a 4-7 membered saturated, partially saturated,

or aromatic ring with up to 3 ring atoms independently selected from O, NR₆, and

S and wherein the fused ring may optionally be substituted by one or more halogen or CrC₃ alkyl;

R₂ and R₃ are each independently of one another are selected from

hydrogen, fluoro, hydroxyl, cyano, C₂-C₆ alkenyl, C₂-C₆ alkynyl,

optionally substituted CrC₆ alkyl group, wherein substitutions are selected from cyano,

hydroxyl, C₃-C₆ cycloalkyl, C₃-C₆ heterocycloalkyl, and halogen, optionally substituted C -C alkyl group having one methylene unit replaced by an oxygen atom, wherein substituents are selected from cyano, hydroxyl, C₃-C₆ cycloalkyl, C₃-C₆ heterocycloalkyl, and halogen,

a C₃-C₆ cycloalkyl group, in which a methylene unit in the cyclic moiety may optionally

be replaced by a -NR₆ - group, an oxygen, or a sulphur atom, and optionally the

cycloalkyl groups and heterocycloalkyl groups may be substituted by halogen, and

provided that R₂ and R₃ cannot both be hydrogen;

each R₄ and R5 are independently selected from

halogen, cyano, hydroxyl, NR₆R₇,

optionally substituted CrC₆ alkyl wherein substitutions are selected from halogen, cyano,

and hydroxyl, and

optionally substituted C -C alkyl having one methylene unit replaced by an oxygen atom

wherein substitutions are selected from cyano, hydroxyl, and halogen;

R₆ and R₇ are independently selected from hydrogen and C C₄ alkyl;

m is selected from 0, 1, 2, and 3;

n is selected from 0, 1, 2, and 3; and

o is selected from 0, 1, and 2.

[0022] ment, Cy is selected from

[0023] In another embodiment, Cy is selected from one of the following thiophenes:

[0024] embodiment, Cy is selected from

[0025] In another embodiment, Cy is selected from one of the following thiazoles:

In another embodiment, Cy is selected from one of the following saturated

[0027] In another embodiment, Cy is selected from

[0028] In one embodiment A is selected from -COOH, hydroxyl, -CH₂OH, -

CH₂CH₂OH, and tetrazole.

[0029] In one embodiment, A is -COOH.

[0030] In one embodiment, A is selected from -CH₂OH and -CH₂CH₂OH.

[0031] In one embodiment, A is selected from tetrazole.

[0032] In one embodiment, A is hydroxyl.

[0033] In one embodiment, Ri is selected from optionally substituted C -C alkyl wherein substitutions are selected from cyano, hydroxyl, and halogen. In one embodiment, the CrC₆ alkyl group is wholly or partly substituted by fluorine atoms.

[0034] In one embodiment, Ri is optionally substituted Ci-C₃ alkyl. Possible substitutions include cyano, hydroxyl, and halogen. For example, in this embodiment, Ri can be selected from methyl, ethyl, i-propyl, n-propyl. In another embodiment, the Ci-C₃ alkyl group is wholly or partly substituted by fluorine atoms. In this embodiment Ri can be selected from CF₃, CHF₂, CH₂F, CH₂CF₃, CF₂CF₃.

[0035] In another embodiment, Ri is optionally substituted C C₆ alkyl having one methylene unit replaced with an O, wherein possible substitutions include cyano, hydroxyl, and halogen. In another embodiment, the O containing C -C alkyl group is wholly or partly substituted by fluorine atoms.

[0036] In one embodiment, Ri is optionally substituted C₁-C4 alkyl having one methylene unit replaced with an O. Possible substitutions include cyano, hydroxyl, and halogen. In this embodiment, Ri can be selected from methoxy, ethoxy, n-propoxy, i- propoxy, n-butoxy, ethoxy methyl, methoxy ethyl, methoxy methyl. In another embodiment, the O containing C₁-C4 alkyl group may optionally be wholly or partly substituted by fluorine atoms. In this embodiment Ri can be selected from -OCF₃, -OCHF₂, -OCH₂F, -OCH₂CF₃, -

[0037] In one embodiment, Ri is selected from F, CI, Br, hydroxyl, cyano, NR₆R7, hydroxyl, methyl, ethyl, n-propyl, i-propyl, n-butyl, CF₃, CHF₂, CH₂F, CH₂CF₃, -OCH , and -OCH₂CH₃.

[0038] In another embodiment, Ri is selected from F, CI, Br, methyl, ethyl, CF₃,

CHF₂, CH₂F, CH₂CF₃, -OCH₃, and -OCH₂CH₃.

[0039] In another embodiment, Ri is selected from F and CI.

[0040] In one embodiment, two Ri groups taken together form a 4-7 membered saturated, partially saturated, or aromatic ring with up to 3 ring atoms independently selected from O, NR₆, and S and wherein the fused ring may optionally be substituted by one or more halogen or C₁-C3 alkyl.

[0041] In one embodiment, the bi-cyclic ring formed by two Ri taken together along with the phenyl ring to which they are connected has the structure

[0042] p is 1 or 2, and

[0043] R8 and R9 are independently selected from hydrogen, halogen, and Ci-C₃ alkyl.

[0044] In another embodiment, p is 1 and R₈ and R₉ are independently selected from hydrogen, fluoro and methyl.

[0045] In another embodiment, p is 1 and Rg and R9 are both fluoro.

[0046] In one embodiment, the bi-cyclic ring formed by two Ri taken together along with the phenyl ring to which they are connected is selected from

[0047] I <nx anotherr embodi ¾ment, thσe bi-cyclic ri wng formed by two Ri taken together along with the phenyl ring to which they are connected is

[0048] In one embodiment, R₂ and R₃ are each independently of one another selected from hydrogen; fhioro; hydroxyl; cyano; C₂-C₄ alkenyl; C₂-C₄ alkynyl; an optionally substituted CrC₃ alkyl group wherein substitutions are selected from cyano, hydroxyl, C₃-C₆ cycloalkyl, C₃-C₆ heterocycloalkyl, and halogen; an optionally substituted CrC₄ alkyl group having one methylene unit replaced by an oxygen atom (O) wherein substitutions are selected from cyano, hydroxyl, C₃-C₆ cycloalkyl, C₃-C₆ heterocycloalkyl, and halogen; and a C₃-C₆ cycloalkyl group in which a methylene group in the cyclic moiety may optionally be replaced by -NR₆-, -0-, -S-; provided R₂ and R₃ cannot both be hydrogen. Optionally the cycloalkyl groups and heterocycloalkyl groups of R₂ and R may be substituted by halogen. In one embodiment, the C -C alkyl group and O containing C -C alkyl group may optionally be wholly or partly substituted by fluorine atoms.

[0049] In one embodiment, R₂ and R₃ are each independently of one another selected from hydrogen; hydroxyl; cyano; C₂-C₆ alkenyl; C₂-C₆ alkynyl; an optionally substituted Ci- Ce alkyl group wherein substitutions are selected from cyano, hydroxyl, and halogen; an optionally substituted CrC₆ alkyl group having one methylene unit replaced by an oxygen atom (O) wherein substitutions are selected from cyano, hydroxyl, and halogen; and a C₃-C₆ cycloalkyl group in which a methylene group in the cyclic moiety may optionally be replaced by -NR₆-, -0-, -S-; and provided that R₂ and R cannot both be hydrogen.

[0050] In one embodiment, R₂ and R₃ are each independently of one another selected from hydrogen; hydroxyl; cyano; C₂-C₄ alkenyl; C₂-C₄ alkynyl; an optionally substituted Cr C₃ alkyl group wherein substitutions are selected from cyano, hydroxyl, and halogen; an optionally substituted Ci-C₄ alkyl group having one methylene unit replaced by an oxygen atom (O) wherein substitutions are selected from cyano, hydroxyl, and halogen; and provided that R₂ and R₃ cannot both be hydrogen.

[0051] In one embodiment, R₂ and R are each independently of one another selected from hydrogen; hydroxyl; cyano; methyl, ethyl, n-propyl, i-propyl, CF , CHF₂, CH₂F, CH₂CF₃, -OCH₃, -OCH₂CH₃; an optionally substituted CrC₄ alkyl group having one methylene unit replaced by an oxygen atom (O) wherein substitutions are selected from cyano, hydroxyl, and halogen; and provided that R₂ and R cannot both be hydrogen.

[0052] In one embodiment, R₂ and R are each independently of one another selected from hydrogen; hydroxyl; cyano; methyl, ethyl, n-propyl, i-propyl, CF₃, CHF₂, CH₂F, CH₂CF₃, -OCH₃, -OCH₂ CH_3.

[0053] In one embodiment, one of R₂ and R is methyl. [0054] In one embodiment, R₂ and R₃ are each independently of one another selected from hydroxyl; cyano; C₂-C₄ alkenyl; C₂-C₄ alkynyl; an optionally substituted Q-C3 alkyl group wherein substitutions are selected from cyano, hydroxyl, and halogen; an optionally substituted CrC₄ alkyl group having one methylene unit replaced by an oxygen atom (O) and wherein substitutions are selected from cyano, hydroxyl, and halogen.

[0055] In one embodiment, one of R₂ or R₃ is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolidine, piperdine, tetrahydro-2H-pyran, tetrahydrofuran, oxetane, and oxirane. In another embodiment, one of R₂ or R₃ is selected from -CH₂- cycloalkyl, -CH₂-cycloheteroalkyl, -O-cycloalkyl, and -O-cycloheteroalkyl group wherein the cycloalkyl and heterocycloalkyl groups can be selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolidine, piperdine, tetrahydro-2H-pyran, tetrahydrofuran, oxetane, oxirane.

[0056] In one embodiment, R₂ and R are both independently selected from the group

Ci-C₃ alkyl optionally substituted by fluorine.

[0057] In one embodiment, R₂ and R₃ are both methyl.

[0058] In one embodiment, each R₄ and R5 are independently selected from halogen, cyano, hydroxyl, NR₆R7, optionally substituted Q-C3 alkyl wherein substitutions are selected from halogen, cyano, and hydroxyl, optionally substituted C C₄ alkyl having one methylene unit replaced by an oxygen (O) atom, wherein substitutions are selected from cyano, hydroxyl, and halogen;

[0059] In one embodiment, each R₄ and R5 are independently selected from F, CI, Br, hydroxyl, optionally substituted Q-C3 alkyl wherein substitutions are selected from halogen, cyano, and hydroxyl, optionally substituted CrC₄ alkyl having one methylene unit replaced by an oxygen (O) atom, wherein substitutions are selected from cyano, hydroxyl, and halogen;

[0060] In one embodiment, each R₄ and R5 are independently selected from F, CI, Br, hydroxyl, CrC₃ alkyl optionally substituted by F, CI, Br, cyano, and hydroxyl, CrC₄ alkyl having one methylene unit replaced by an oxygen (O) atom, wherein optional substitutions are selected from F, CI, Br, cyano, and hydroxyl;

[0061] In one embodiment, each R₄ and R5 are independently selected from F, CI, Br, hydroxyl, methyl, ethyl, n-propyl, i-propyl, CF₃, CHF₂, CH₂F, CH₂CF₃, methoxy, ethoxy, n- propoxy, i-propoxy, n-butoxy, ethoxy methyl, methoxy ethyl, methoxy methyl, -OCF₃, - OCHF₂, -OCH₂F, -OCH₂CF₃, -CH₂OCF₃.

[0062] In one embodiment, n is 1 and R₄ is methyl. [0063] In one embodiment, there is an R₄ substitutent on the carbon of the pyridine ring adjacent to Cy.

[0064] In one embodiment, the carbon of the pyridine ring adjacent to Cy is substituted by methyl.

[0065] R₆ and R₇ are independently selected from hydrogen and Q-C3 alkyl;

[0066] In one embodiment, m is selected from 0, 1, 2 and 3. In one embodiment, m is selected from 0, 1 and 2. In another embodiment, m is selected from 0 and 1. In one embodiment, m is 0. In one embodiment, m is 1. In one embodiment, m is 2.

[0067] In one embodiment, n is selected from 0, 1 and 2. In another embodiment, n is selected from 0 and 1. In one embodiment, n is 0. In one embodiment, n is 1.

[0068] In one embodiment, o is selected from 0, 1, 2 and 3. In another embodiment, o is selected from 0, 1 and 2. In a further embodiment, o is selected from 0 and 1. In one embodiment, o is 0. In one embodiment, o is 1.

[0069] In one embodiment, the compound of formula 1 is selected from the compounds of Table 1.

Table 1:

carboxylic acid

methylpropanamide

0 methylpropanamide

[0070] When a bond to a substituent is shown to cross a bond connecting two atoms in a ring, then such substituent may be bonded to any atom in the ring. When a substituent is listed without indicating the atom via which such substituent is bonded to the rest of the compound of a given formula, then such substituent may be bonded via any atom in such substituent. Combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds.

[0071] The compounds described herein may have asymmetric centers. Compounds of the present invention containing an asymmetrically substituted atom may be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials. Many geometric isomers of olefins, C=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and may be isolated as a mixture of isomers or as separated isomeric forms. All chiral, diastereomeric, racemic, and geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. All tautomers of shown or described compounds are also considered to be part of the present invention.

[0072] It is to be understood that isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of the invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis. Furthermore, the structures and other compounds and moieties discussed in this application also include all tautomers thereof. Alkenes can include either the E- or Z-geometry, where appropriate.

[0073] 2. Representative Compounds

[0074] Examples 1-42 and Table 1 list representative compounds of Formula 1. The synthetic methods that can be used to prepare each compound are detailed in Examples 1-42, with reference to the synthetic schemes depicted before Example 1, and reference to intermediates described in Example 43. Supporting mass spectrometry data and/or proton NMR data for each compound is also included in Examples 1-42. CFTR modulator activity was determined by the assays described in Example 44 and Example 45. EC₅₀ values were obtained. In the Corrector assay with the CFBE 41o- cells (CFBE) transiently transfected with YFP (see Example 44), compounds of Examples 1-9, 14, and 16-25 had an EC₅₀ of < 5 uM. Compounds of Examples 2, 5, 7, 8, 9, 14, 16-21, 23, 25 had an EC₅₀ < luM.

Compounds of Examples 7, 8, 9, 16, 18, 20, 21, and 23-25 had an EC₅₀ < 0.500.uM. In the Corrector assay with Fisher rat thyroid (FRT) cells stably expressing human AF508-CFTR and yellow fluorescent protein (YFP) (see Example 45), compounds of Examples 7, 8, 10, 11, 14 and 15-42 had an EC₅₀ of < 5 uM. Compounds of Examples 7, 8, 11, 14, 16-19, 21, 24, 25, 28, 30, 32-40, and 42 had an EC₅₀ < luM. Compounds of Examples 7, 8, 14, 24, 33, 34, 37-39, and 42 had an EC₅₀ < 0.500 uM.

C. Definitions

[0075] As used herein, "about" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, "about" will mean up to plus or minus 10% of the particular term.

[0076] The term "acyl" includes compounds and moieties that contain the acetyl radical (CH₃CO-) or a carbonyl group to which a straight o_r branched chain lower alkyl residue is attached.

[0077] The term "alkyl" as used herein refers to a straight or branched chain, saturated hydrocarbon having the indicated number of carbon atoms. For example, (C^Ce) alkyl is meant to include, but is not limited to methyl, ethyl, propyl, isopropyl, butyl, sec- butyl, iert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, and neohexyl. An alkyl group can be unsubstituted or optionally substituted with one or more substituents as described herein.

[0078] The term "alkenyl" as used herein refers to a straight or branched chain unsaturated hydrocarbon having the indicated number of carbon atoms and at least one double bond. Examples of a (C₂-C8) alkenyl group include, but are not limited to, ethylene, propylene, 1-butylene, 2-butylene, isobutylene, sec-butylene, 1-pentene, 2-pentene, isopentene, 1-hexene, 2-hexene, 3-hexene, isohexene, 1-heptene, 2-heptene, 3-heptene, isoheptene, 1-octene, 2-octene, 3-octene, 4-octene, and isooctene. An alkenyl group can be unsubstituted or optionally substituted with one or more substituents as described herein.

[0079] The term "alkynyl" as used herein refers to a straight or branched chain unsaturated hydrocarbon having the indicated number of carbon atoms and at least one triple bond. Examples of a (C₂-C₈) alkynyl group include, but are not limited to, acetylene, propyne, 1-butyne, 2-butyne, 1-pentyne, 2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1- heptyne, 2-heptyne, 3-heptyne, 1-octyne, 2-octyne, 3-octyne, and 4-octyne. An alkynyl group can be unsubstituted or optionally substituted with one or more substituents as described herein.

[0080] The term "alkoxy" as used herein refers to an -O-alkyl group having the indicated number of carbon atoms. For example, a (CrC₆) alkoxy group includes -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O-butyl, -O-sec-butyl, -O-iert-butyl, -O-pentyl, -O- isopentyl, -O-neopentyl, -O-hexyl, -O-isohexyl, and -O-neohexyl.

[0081] The term "aminoalkyl" as used herein, refers to an alkyl group (typically one to six carbon atoms) wherein one or more of the CrC₆ alkyl group' s hydrogen atoms is replaced with an amine of formula -N(R^C)₂, wherein each occurrence of R^c is independently - H or (Ci-C ) alkyl. Examples of aminoalkyl groups include, but are not limited to, -CH₂NH₂, -CH₂CH₂NH₂, -CH₂CH₂CH₂NH₂, -CH₂CH₂CH₂CH₂NH₂, -CH₂CH₂CH₂CH₂CH₂NH₂, - CH₂CH₂CH₂CH₂CH₂CH₂NH₂, -CH₂CH₂CH₂N(CH₃)₂, t-butylaminomethyl,

isopropylaminomethyl, and the like.

[0082] The term "aryl" as used herein refers to a 5- to 14-membered monocyclic, bicyclic, or tricyclic aromatic ring system. Examples of an aryl group include phenyl and naphthyl. An aryl group can be unsubstituted or optionally substituted with one or more substituents as described herein below. Examples of aryl groups include phenyl or aryl heterocycles such as, pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

[0083] As used herein, the term "bioactivity" indicates an effect on one or more cellular or extracellular process (e.g. , via binding, signaling, etc.) which can impact physiological or pathophysiological processes.

[0084] The term "carbonyl" includes compounds and moieties which contain a carbon connected with a double bond to an oxygen atom. Examples of moieties containing a carbonyl include, but are not limited to, aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc. [0085] The term "carboxy" or "carboxyl" means a -COOH group or carboxylic acid.

[0086] "Acidic moiety" as used herein is defined as a carboxylic acid or a carboxylic acid bioisostere. Bioisosteres are substituents or groups with similar physical or chemical properties which produce broadly similar biological properties to a chemical compound. For a review of bioisosteres, see J. Med. Chem, 2011, 54, 2529-2591. Examples of "acidic moiety" include but are not limited to

[0087] "Pharmacophore" is defined as "a set of structural features in a molecule that is recognized at a receptor site and is responsible for that molecule's biological activity" (Gund, Prog. Mol. Subcell. Biol., 5: pp 117-143 (1977)).

[0088] The term "C_m - C_n" means "m" number of carbon atoms to "n" number of carbon atoms. For example, the term "Ci-C " means one to six carbon atoms (C_1; C₂, C₃, C₄, C₅, or C₆). The term "C₂-C₆" includes two to six carbon atoms (C₂, C₃, C₄, C₅, or C₆). The term "C₃-C₆" includes three to six carbon atoms (C₃, C₄, C₅, or C₆).

[0089] The term "cycloalkyl" as used herein refers to a 3- to 14-membered saturated or unsaturated non-aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system. Included in this class are cycloalkyl groups which are fused to a benzene ring.

Representative cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3- cyclohexadienyl, cycloheptyl, cycloheptenyl, 1,3-cycloheptadienyl, 1,4-cycloheptadienyl, - 1,3,5-cycloheptatrienyl, cyclooctyl, cyclooctenyl, 1,3-cyclooctadienyl, 1,4-cyclooctadienyl, - 1,3,5-cyclooctatrienyl, decahydronaphthalene, octahydronaphthalene, hexahydronaphthalene, octahydroindene, hexahydroindene, tetrahydroinden, decahydrobenzocycloheptene, octahydrobenzocycloheptene, hexahydrobenzocycloheptene, tetrahydrobenzocyclopheptene, dodecahydroheptalene, decahydroheptalene, octahydroheptalene, hexahydroheptalene, tetrahydroheptalene, ( 1 s,3s)-bicyclo[ 1.1.0]butane, bicyclo[ 1.1. l]pentane,

bicyclo[2.1.1]hexane, Bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.1.1]heptane, bicyclo[3.2.1]octane, bicyclo[3.3.1]nonane, bicyclo[3.3.2]decane, bicyclo [3.3.]undecane, bicyclo[4.2.2]decane, and bicyclo[4.3.1]decane. A cycloalkyl group can be unsubstituted or optionally substituted with one or more substituents as described herein below.

[0090] The term "halogen" includes fluorine, bromine, chlorine, iodine, etc.

[0091] The term "haloalkyl," as used herein, refers to a CrC₆ alkyl group wherein from one or more of the C -C alkyl group's hydrogen atom is replaced with a halogen atom, which can be the same or different. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, pentachloroethyl, and 1,1,1 -trifluoro-2-bromo-2-chloroethyl.

[0092] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain alkyl, or combinations thereof, consisting of carbon atoms and from one to three heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, and S can be placed at any position of the heteroalkyl group. Examples include -CH₂- CH₂-0-CH₃, -CH₂-CH₂-NH-CH₃, -CH₂-CH₂-N(CH₃)-CH₃, -CH₂-S-CH₂-CH₃, -CH₂-CH₂- S(0)-CH₃, -CH₂-CH₂-S(0)₂-CH₃, and -CH₂-CH=N-OCH₃. Up to two heteroatoms can be consecutive, for example, -CH₂-NH-OCH . When a prefix such as (C₂-C₈) is used to refer to a heteroalkyl group, the number of carbons (2 to 8, in this example) is meant to include the heteroatoms as well. For example, a C₂-heteroalkyl group is meant to include, for example, -CH₂OH (one carbon atom and one heteroatom replacing a carbon atom) and -CH₂SH.

[0093] To further illustrate the definition of a heteroalkyl group, where the heteroatom is oxygen, a heteroalkyl group can be an oxyalkyl group. For instance, (C₂-C5) oxyalkyl is meant to include, for example -CH₂-0-CH₃ (a C₃-oxyalkyl group with two carbon atoms and one oxygen replacing a carbon atom), -CH₂CH₂CH₂CH₂OH, - OCH₂CH₂OCH₂CH₂OH, - OCH₂CH(OH)CH₂OH, and the like.

[0094] The term "heteroaryl" as used herein refers to an aromatic heterocycle ring of

5 to 14 members and having at least one heteroatom selected from nitrogen, oxygen, and sulfur, and containing at least 1 carbon atom, including monocyclic, bicyclic, and tricyclic ring systems. Representative heteroaryls are triazolyl, tetrazolyl, oxadiazolyl, pyridyl, furyl, benzofuranyl, thienyl, benzothienyl, quinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, quinazolinyl, pyrimidyl, azepinyl, oxepinyl, quinoxalinyl and oxazolyl. A heteroaryl group can be unsubstituted or optionally substituted with one or more substituents as described herein below.

[0095] As used herein, the term "heteroatom" is meant to include oxygen (O), nitrogen (N), and sulfur (S).

[0096] As used herein, the term "heterocycle" refers to 3- to 14-membered ring systems which are either saturated, unsaturated, or aromatic, and which contains from 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and wherein the nitrogen and sulfur heteroatoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quaternized, including monocyclic, bicyclic, and tricyclic ring systems. The bicyclic and tricyclic ring systems may encompass a heterocycle or heteroaryl fused to a benzene ring. The heterocycle can be attached via any heteroatom or carbon atom, where chemically acceptable. Heterocycles include heteroaryls as defined above. Representative examples of heterocycles include, but are not limited to, aziridinyl, oxiranyl, thiiranyl, triazolyl, tetrazolyl, azirinyl, diaziridinyl, diazirinyl, oxaziridinyl, azetidinyl, azetidinonyl, oxetanyl, thietanyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, oxazinyl, thiazinyl, diazinyl, dioxanyl, triazinyl, tetrazinyl, imidazolyl, tetrazolyl, pyrrolidinyl, isoxazolyl, furanyl, furazanyl, pyridinyl, oxazolyl, benzoxazolyl, benzisoxazolyl, thiazolyl,

benzthiazolyl, thienyl, pyrazolyl, triazolyl, pyrimidinyl, benzimidazolyl, isoindolyl, indazolyl, benzodiazolyl, benzotriazolyl, benzoxazolyl, benzisoxazolyl, purinyl, indolyl, isoquinolinyl, quinolinyl, and quinazolinyl. A heterocycle group can be unsubstituted or optionally substituted with one or more substituents as described herein below.

[0097] The term "heterocycloalkyl," by itself or in combination with other terms, represents, unless otherwise stated, cyclic versions of "heteroalkyl." Additionally, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of heterocycloalkyl include l-(l,2,5,6-tetrahydropyridyl), 1- piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.

[0098] The term "hydroxyalkyl," as used herein, refers to an alkyl group having the indicated number of carbon atoms wherein one or more of the hydrogen atoms in the alkyl group is replaced with an -OH group. Examples of hydroxyalkyl groups include, but are not limited to, -CH₂OH, -CH₂CH₂OH, -CH₂CH₂CH₂OH, -CH₂CH₂CH₂CH₂OH, - CH₂CH₂CH₂CH₂CH₂OH, -CH₂CH₂CH₂CH₂CH₂CH₂OH, and branched versions thereof.

[0099] The term "hydroxy" or "hydroxyl" includes groups with an -OH or -O^". [00100] As used herein, N-oxide, or amine oxide, refers to a compound derived from a tertiary amine by the attachment of one oxygen atom to the nitrogen atom, R N⁺-0^~. By extension the term includes the analogous derivatives of primary and secondary amines.

[00101] As used herein and unless otherwise indicated, the term "stereoisomer" means one stereoisomer of a compound that is substantially free of other stereoisomers of that compound. For example, a stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. In some embodiments, a stereomerically pure compound comprises greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, for example greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, or greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound.

[00102] As utilized herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals and, more particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered and includes, but is not limited to such sterile liquids as water and oils.

[00103] A "pharmaceutically acceptable salt" or "salt" of a compound of the invention is a product of the disclosed compound that contains an ionic bond, and is typically produced by reacting the disclosed compound with either an acid or a base, suitable for administering to a subject. A pharmaceutically acceptable salt can include, but is not limited to, acid addition salts including hydrochlorides, hydrobromides, phosphates, sulphates, hydrogen sulphates, alkylsulphonates, arylsulphonates, arylalkylsulfonates, acetates, benzoates, citrates, maleates, fumarates, succinates, lactates, and tartrates; alkali metal cations such as Li, Na, and K, alkali earth metal salts such as Mg or Ca, or organic amine salts.

[00104] A "pharmaceutical composition" is a formulation comprising the disclosed compounds or a combination thereof in a form suitable for administration to a subject. A pharmaceutical composition of the invention is preferably formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, oral and parenteral, e.g., intravenous, intradermal, subcutaneous, inhalation, topical, transdermal, transmucosal, and rectal administration.

[00105] The term "substituted, " as used herein, means that any one or more hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency is not exceeded, and that the substitution results in a stable compound. When a substituent is keto (i.e., =0), then 2 hydrogens on the atom are replaced. Ring double bonds, as used herein, are double bonds that are formed between two adjacent ring atoms (e.g. , C=C, C=N, or N=N).

[00106] Substituents for the groups referred to as alkyl, heteroalkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl can be selected from a variety of groups including -OR^d', =0, =NR^d', =N-OR^d', -NR^d'R^d", -SR^d', -halo, - SiR^d'R^d"R^d" ', -OC(0)R^d', -C(0)R^d', -C0₂R^d', -CONR^d'R^d" , -OC(0)NR^d'R^d", - NR^d"C(0)R^d', -NR^d"'C(0)NR^d'R^d", -NR^d"'S0₂NR^d'R^d" , -NR^d"C0₂R^d', -NHC(NH₂)=NH, -NR^a'C(NH₂)=NH, -NHC(NH₂)=NR^d' , -S(0)R^d', -S0₂R^d', -S0₂NR^d'R^d" , -NR^d"S0₂R^d', - CN, and -N0₂, in a number ranging from zero to three, with those groups having zero, one or two substituents being exemplary.

[00107] R^d' , R^d" , and R^d' ' ' each independently refer to hydrogen, unsubstituted (Q-

Cg) alkyl, unsubstituted hetero (C Cg) alkyl, unsubstituted aryl, and aryl substituted with one to three substituents selected from -halo, unsubstituted alkyl, unsubstituted alkoxy, unsubstituted thioalkoxy, and unsubstituted aryl (C C₄) alkyl. When R^d' and R^d" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5- , 6-, or 7-membered ring. For example, -NR^d'R^d" can represent 1-pyrrolidinyl or 4- morpholinyl.

[00108] Typically, an alkyl or heteroalkyl group will have from zero to three substituents, with those groups having two or fewer substituents being exemplary of the present invention. An alkyl or heteroalkyl radical can be unsubstituted or monosubstituted. In some embodiments, an alkyl or heteroalkyl radical will be unsubstituted.

[00109] Exemplary substituents for the alkyl and heteroalkyl radicals include, but are not limited to -OR^d', =0, =NR^d', =N-OR^d', -NR^d'R^d" , -SR^d', -halo, -SiR^d'R^d"R^d" ' , - OC(0)R^d', -C(0)R^d', -C0₂R^d', -CONR^d'R^d", -OC(0)NR^d'R^d" , -NR^d"C(0)R^d', - NR^d"'C(0)NR^d'R^d" , -NR^d ' "S 0₂NR^d ' R^d " , -NR^d"C0₂R^d', -NHC(NH₂)=NH, - NR^a'C(NH₂)=NH, -NHC(NH₂)=NR^d' , -S(0)R^d', -S0₂R^d', -S0₂NR^d'R^d", -NR^d"S0₂R^d', - CN, and -N0₂, where R^d', R^d", and R^d"' are as defined above. Typical substituents can be selected from: -OR^d', =0, -NR^d'R^d" , -halo, -OC(0)R^d', -C0₂R^d', -C(0)NR^d'R^d" , - OC(0)NR^d'R^d" , -NR^d"C(0)R^d', -NR^d"C0₂R^d\ -NR^d"'S0₂NR^d'R^d" , -S0₂R^d', - S0₂NR^d'R^d" , -NR^d"S0₂R^d', -CN, and -N0₂.

[00110] Similarly, substituents for the aryl and heteroaryl groups are varied and selected from: -halo, -OR⁶', -OC(0)R^e', -NR^e'R^e" , -SR^e', -R^e', -CN, -N0₂, -C0₂R^e', - C(0)NR^e'R^e", -C(0)R^e', -OC(0)NR^e'R^e" , -NR^e"C(0)R^e', -NR^e"C0₂R⁶', - NR^e"'C(0)NR^e'R^e", -NR^e"'S0₂NR^e'R^e", -NHC(NH₂)=NH, -NR^e'C(NH₂)=NH, -NH- C(NH₂)=NR^e', -S(0)R^e', -S0₂R^e', -S0₂NR^e'R^e", -NR^e"S0₂R⁶', -N₃, -CH(Ph)₂,

perfluoroalkoxy, and perfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system.

[00111] R⁶', R^e" and R⁶' " are independently selected from hydrogen, unsubstituted

(C C₈) alkyl, unsubstituted hetero (C Cs) alkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted aryl (C C₄) alkyl, and unsubstituted aryloxy (C C₄) alkyl. Typically, an aryl or heteroaryl group will have from zero to three substituents, with those groups having two or fewer substituents being exemplary in the present invention. In one embodiment of the invention, an aryl or heteroaryl group will be unsubstituted or monosubstituted. In another embodiment, an aryl or heteroaryl group will be unsubstituted.

[00112] Two of the substituents on adjacent atoms of an aryl or heteroaryl ring in an aryl or heteroaryl group as described herein may optionally be replaced with a substituent of the formula -T-C(0)-(CH₂)_q-U-, wherein T and U are independently -NH-, -0-, -CH₂- or a single bond, and q is an integer of from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -J-(CH₂)_r-K-, wherein J and K are independently -CH₂-, -0-, -NH-, -S-, -S(O)-, - S(0)₂-, -S(0)₂NR '-, or a single bond, and r is an integer of from 1 to 3. One of the single bonds of the new ring so formed may optionally be replaced with a double bond.

Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CH₂)_s-X-(CH₂)_r, where s and t are independently integers of from 0 to 3, and X is -0-, -NR^f'-, -S-, -S(O)-, -S(0)₂-, or - S(0)₂NR^a'-. The substituent R^f' in -NR^f'- and -S(0)₂NR^f'- is selected from hydrogen or unsubstituted (C -C ) alkyl.

[00113] As used herein, a "secondary active agent" is selected from a mucolytic agent, a bronchodialator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR modulator, a nutritional agent, or any agent known to treat CF. [00114] "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[00115] As used herein the term "therapeutically effective amount" generally means the amount necessary to ameliorate at least one symptom of a disorder to be prevented, reduced, or treated as described herein. The phrase "therapeutically effective amount" as it relates to the compounds of the present invention shall mean dosage that provides the specific pharmacological response for which the compound is administered in a significant number of subjects in need of such treatment. It is emphasized that a therapeutically effective amount of a compound that is administered to a particular subject in a particular instance will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art.

[00116] The phrase "therapeutically effective amount" as it relates to the secondary active agent of the present invention shall mean the dosage that provides the specific pharmacological response for which the secondary active agent is administered in a significant number of subjects in need of such treatment.

[00117] The term "biological sample" includes, but is not limited to, samples of blood

(e.g. , serum, plasma, or whole blood), urine, saliva, sweat, breast milk, vaginal secretions, semen, hair follicles, skin, teeth, bones, nails, or other secretions, body fluids, tissues, or cells.

[00118] D. Pharmaceutical Compositions

[00119] The invention encompasses pharmaceutical compositions comprising at least one compound of the invention described herein and at least one pharmaceutically acceptable carrier. Suitable carriers are described in "Remington: The Science and Practice, Twentieth Edition," published by Lippincott Williams & Wilkins, which is incorporated herein by reference. Pharmaceutical compositions according to the invention may also comprise one or more non-inventive compound active agents.

[00120] The pharmaceutical compositions of the invention can comprise novel compounds described herein, the pharmaceutical compositions can comprise known compounds which previously were not known to have CFTR modulatory activity, or a combination thereof.

[00121] 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 secondary active agents or medical procedures. The particular combination of therapies (secondary agents or procedures) to employ in a combination regimen will take into account compatibility of the desired agents and/or procedures and the desired therapeutic effect to be achieved. The therapies employed may achieve a desired effect for the same disorder (for example, a compound of the present invention may be administered concurrently with a secondary agent used to treat the same disorder), or they may achieve different effects (such as control adverse effects).

[00122] In one embodiment the secondary active agent is selected from a mucolytic agent, a bronchodialator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR modulator, a nutritional agent, or any agent known to treat CF.

[00123] In one embodiment the secondary active agent is a GSNOR inhibitor.

[00124] In one embodiment, the secondary active agent is a GSNOR inhibitor disclosed in WO2010/019903, WO2010/019905, WO2010/019909, WO2010/019910, and WO2011/075478.

[00125] In another embodiment, the secondary active agent is a GSNOR inhibitor disclosed in WO2011/100433, WO2011/099978, WO2012/048181, WO2012/083165, WO2012/083171, and WO 2012/170371.

[00126] In another embodiment, the secondary active agent is selected from

gentamicin, curcumin, cyclophosphamide, 4-phenylbutyrate, miglustat, felodipine, nimodipine, Philoxin B, geniestein, Apigenin, cAMP/cGMP modulators such as rolipram, sildenafil, milrinone, tadalafil, aminone, isoproterenol, albuterol, and almeterol,

deoxyspergualin, HSP 90 inhibitors, HSP 70 inhibitors, proteosome inhibitors such as epoxomicin, lactacystin, terfenadine, enalapril, meclofenamic acid, carbaryl, suprofen, urosolic acid, zaprinast, benzo[c]quinolizinium derivatives that exhibit CFTR modulation activity, modulators of abc transporters, benzopyran derivatives that exhibit CFTR modulation activity, etc.

[00127] The compounds of the invention can be utilized in any pharmaceutically acceptable dosage form, including, but not limited to injectable dosage forms, liquid dispersions, gels, aerosols, ointments, creams, lyophilized formulations, dry powders, tablets, capsules, controlled release formulations, fast melt formulations, delayed release

formulations, extended release formulations, pulsatile release formulations, mixed immediate release and controlled release formulations, etc. Specifically, the compounds of the invention described herein can be formulated: (a) for administration selected from the group consisting of oral, pulmonary, intravenous, intra-arterial, intrathecal, intra-articular, rectal, ophthalmic, colonic, parenteral, intracisternal, intravaginal, intraperitoneal, local, buccal, nasal, and topical administration; (b) into a dosage form selected from the group consisting of liquid dispersions, gels, aerosols, ointments, creams, tablets, sachets, and capsules; (c) into a dosage form selected from the group consisting of lyophilized formulations, dry powders, fast melt formulations, controlled release formulations, delayed release formulations, extended release formulations, pulsatile release formulations, and mixed immediate release and controlled release formulations; or (d) any combination thereof.

[00128] For respiratory infections or pulmonary exacerbations of CF, an inhalation formulation can be used to achieve high local concentrations. Formulations suitable for inhalation include dry power or aerosolized or vaporized solutions, dispersions, or suspensions capable of being dispensed by an inhaler or nebulizer into the endobronchial or nasal cavity of infected patients to treat upper and lower respiratory bacterial infections.

[00129] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can comprise one or more of the following components: (1) a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents; (2) antibacterial agents such as benzyl alcohol or methyl parabens; (3) antioxidants such as ascorbic acid or sodium bisulfite; (4) chelating agents such as ethylenediaminetetraacetic acid; (5) buffers such as acetates, citrates, or phosphates; and (5) agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. A parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[00130] Pharmaceutical compositions suitable for injectable use may comprise sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. The pharmaceutical composition should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[00131] The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol or sorbitol, and inorganic salts such as sodium chloride in the composition.

Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[00132] Sterile injectable solutions can be prepared by incorporating the active reagent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating at least one compound of the invention into a sterile vehicle that contains a basic dispersion medium and any other required ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary methods of preparation include vacuum drying and freeze-drying, both of which yield a powder of a compound of the invention plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[00133] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed, for example, in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the compound of the invention can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.

[00134] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, a nebulized liquid, or a dry powder from a suitable device. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active reagents are formulated into ointments, salves, gels, or creams as generally known in the art. The reagents can also be prepared in the form of suppositories (e.g. , with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[00135] In one embodiment, the compounds of the invention are prepared with carriers that will protect against rapid elimination from the body. For example, a controlled release formulation can be used, including implants and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

[00136] Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[00137] Additionally, suspensions of the compounds of the invention may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery.

Optionally, the suspension may also include suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.

[00138] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of the compound of the invention calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the compound of the invention and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active agent for the treatment of individuals.

[00139] Pharmaceutical compositions according to the invention comprising at least one compound of the invention can comprise one or more pharmaceutical excipients.

Examples of such excipients include, but are not limited to binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, effervescent agents, and other excipients. Such excipients are known in the art. Exemplary excipients include: (1) binding agents which include various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel PH101 and Avicel^® PH102, silicified microcrystalline cellulose (ProSolv SMCC™), gum tragacanth and gelatin; (2) filling agents such as various starches, lactose, lactose

monohydrate, and lactose anhydrous; (3) disintegrating agents such as alginic acid, Primogel, corn starch, lightly crosslinked polyvinyl pyrrolidone, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof; (4) lubricants, including agents that act on the flowability of a powder to be compressed, include magnesium stearate, colloidal silicon dioxide, such as Aerosil 200, talc, stearic acid, calcium stearate, and silica gel; (5) glidants such as colloidal silicon dioxide; (6) preservatives, such as potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride; (7) diluents such as pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and/or mixtures of any of the foregoing; examples of diluents include microcrystalline cellulose, such as

Avicel PH101 and Avicel PHI 02; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose DCL21 ; dibasic calcium phosphate such as Emcompress ; mannitol; starch; sorbitol; sucrose; and glucose; (8) sweetening agents, including any natural or artificial sweetener, such as sucrose, saccharin sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame; (9) flavoring agents, such as peppermint, methyl salicylate, orange flavoring, Magnasweet (trademark of MAFCO), bubble gum flavor, fruit flavors, and the like; and (10) effervescent agents, including effervescent couples such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

[00140] The present invention provides pharmaceutical compositions that are useful in treating or lessening the severity of cystic fibrosis in a patient by administering to said patient an effective amount of a compound of the present invention alone or in combination with one or more secondary active agents (e.g. GSNOR inhibitor). [00141] In one embodiment, the secondary active agent is a GSNOR inhibitor disclosed in WO2010/019903, WO2010/019905, WO2010/019909, WO2010/019910, and WO2011/075478.

[00142] In another embodiment, the secondary active agent is a GSNOR inhibitor disclosed in WO2011/100433, WO2011/099978, WO2012/048181, WO2012/083165, WO2012/083171, and WO 2012/170371.

[00143] In one embodiment the secondary active agent is selected from a mucolytic agent, a bronchodialator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR modulator, a nutritional agent, or any agent known to treat CF.

[00144] In another embodiment, the secondary active agent is selected from gentamicin, curcumin, cyclophosphamide, 4-phenylbutyrate, miglustat, felodipine, nimodipine, Philoxin B, geniestein, Apigenin, cAMP/cGMP modulators such as rolipram, sildenafil, milrinone, tadalafil, aminone, isoproterenol, albuterol, and almeterol,

deoxyspergualin, HSP 90 inhibitors, HSP 70 inhibitors, proteosome inhibitors such as epoxomicin, lactacystin, modulators of abc transporters, benzo[c]quinolizinium derivatives, benzopyran derivatives, etc.

[00145] E. Kits Comprising the Compositions of the Invention

[00146] The present invention also encompasses kits comprising the compositions of the invention. Such kits can comprise, for example, (1) at least one compound of the invention; and (2) at least one pharmaceutically acceptable carrier, such as a solvent or solution. Additional kit components can optionally include, for example: (1) any of the pharmaceutically acceptable excipients identified herein, such as stabilizers, buffers, etc., (2) at least one container, vial, or similar apparatus for holding and/or mixing the kit components; and (3) delivery apparatus, such as an inhaler, nebulizer, syringe, etc.

[00147] F. Methods of Preparing Compounds of the Invention

[00148] The compounds of the invention can readily be synthesized using known synthetic methodologies or via a modification of known synthetic methodologies. As would be readily recognized by a skilled artisan, the methodologies described below allow the synthesis of substituted bicyclic aromatic compoundss having a variety of substituents. Exemplary synthetic methods are described in the Examples section below.

[00149] If needed, further purification and separation of enantiomers and

diastereomers can be achieved by routine procedures known in the art. Thus, for example, the separation of enantiomers of a compound can be achieved by the use of chiral HPLC and related chromatographic techniques. Diastereomers can be similarly separated. In some instances, however, diastereomers can simply be separated physically, such as, for example, by controlled precipitation or crystallization.

[00150] The process of the invention, when carried out as prescribed herein, can be conveniently performed at temperatures that are routinely accessible in the art. In one embodiment, the process is performed at a temperature in the range of about 25°C to about 110°C. In another embodiment, the temperature is in the range of about 40°C to about 100°C. In yet another embodiment, the temperature is in the range of about 50°C to about 95°C.

[00151] Synthetic steps that require a base are carried out using any convenient organic or inorganic base. Typically, the base is not nucleophilic. Thus, in one embodiment, the base is selected from carbonates, phosphates, hydroxides, alkoxides, salts of disilazanes, and tertiary amines.

[00152] The process of the invention, when performed as described herein, can be substantially complete after several minutes to after several hours depending upon the nature and quantity of reactants and reaction temperature. The determination of when the reaction is substantially complete can be conveniently evaluated by ordinary techniques known in the art such as, for example, HPLC, LCMS, TLC, and 1H NMR.

[00153] G. Methods of Treatment

[00154] The invention encompasses methods of preventing or treating (e.g. , alleviating one or more symptoms of) cystic fibrosis through use of one or more of the disclosed compounds. The methods comprise administering a therapeutically effective amount of a compound of the invention to a patient in need. The compositions of the invention can also be used for prophylactic therapy. The compositions of the invention can include one or more secondary active agents.

[00155] In one embodiment, the method is a method of treating or lessening the severity of cystic fibrosis in a patient, comprising the step of administering to said patient an effective amount of a compound of the present invention and pharmaceutical compositions comprising such compounds.

[00156] The compound of the invention used in the methods of treatment according to the invention can be: (1) a compound described herein, or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a prodrug thereof, a metabolite thereof; (2) a compound which was known prior to the present invention, but wherein it was not known that the compound is a CFTR modulator, or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a prodrug thereof, a metabolite thereof; or (3) a compound which was known prior to the present invention, and wherein it was known that the compound is a CFTR modulator, but wherein it was not known that the compound is useful for the methods of treatment described herein, or a pharmaceutically acceptable salt, a stereoisomer, a prodrug, a metabolite, (4) a compound of the present invention in combination with one or more secondary agents.

[00157] The methods of the present invention can be compounds of the invention 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 secondary active agents or medical procedures. The particular combination of therapies (secondary agents or procedures) to employ in a combination regimen will take into account compatibility of the desired agents and/or procedures and the desired therapeutic effect to be achieved. The therapies employed may achieve a desired effect for the same disorder (for example, a compound of the present invention may be administered

concurrently with a secondary agent used to treat the same disorder), or they may achieve different effects (such as control adverse effects).

[00158] The patient can be any animal, domestic, livestock, or wild, including, but not limited to cats, dogs, horses, pigs, and cattle, and preferably human patients. As used herein, the terms patient and subject may be used interchangeably.

[00159] As used herein, "treating" describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a compound of the present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder. More specifically, "treating" includes reversing, attenuating, alleviating, minimizing, suppressing, or halting at least one deleterious symptom or effect of a disease (disorder) state, disease progression, disease causative agent (e.g., bacteria or viruses), or other abnormal condition. Treatment is continued as long as symptoms and/or pathology ameliorate.

[00160] In general, the dosage, i.e. , the therapeutically effective amount, ranges from 1 μg/kg to 10 g/kg and often ranges from 10 μg/kg to 1 g/kg or 10 μg/kg to 100 mg/kg body weight of the subject being treated, per day. [00161] H. Uses

[00162] In subjects with cystic fibrosis, modulation may be achieved, for example, by administering one or more of the disclosed compounds that up regulates CFTR function. These compounds may be administered alone or in combination with other agents as described in detail herein.

[00163] The present invention provides a method of treating a subject afflicted with a disorder ameliorated by CFTR modulation. Such a method comprises administering to a subject a therapeutically effective amount of a compound of the present invention alone or in combination with a secondary active agent.

[00164] The disorders can include pulmonary disorders associated with CFTR modulation in the lungs and airways and/or lung infection and/or lung inflammation and/or lung injury (e.g. , pulmonary hypertension, ARDS, asthma, pneumonia, pulmonary fibrosis/interstitial lung diseases, cystic fibrosis, COPD, primary ciliary dyskinesia, chronic bronchitis, respiratory tract infections); cardiovascular disease and heart disease (e.g., hypertension, ischemic coronary syndromes, atherosclerosis, heart failure, right ventricular hypertrophy, pulmonary artery dilation); diseases characterized by angiogenesis (e.g., coronary artery disease); neurological disorders; pancreatic diseases (e.g., pancreatitis, diabetes), inflammatory diseases (e.g. , inflammatory bowel disease (IBD), Crohn's disease, colitis, arthritis and psoriasis); functional gastrointestinal disorders (e.g., irritable bowel syndrome (IBS), gastroesophageal reflux disease (GERD)); disorders of ocular fluid balance (e.g. keratoconjunctivitis sicca, recurrent corneal erosions, corneal edema, glaucoma, retinal detachment and retinal ischemia); disorders of the salivary gland (e.g., xerostomia, salivary gland hypofunction); reproductive disorders (e.g., infertility, amenorrhea); pregnancy disorders (preeclampsia, HELLP syndrome, gestational diabetes mellitus); bone disorders (e.g., osteoporosis); proliferative cell disorders (e.g. , lung carcinoma); disorders where there is risk of thrombosis occurring; disorders where there is risk of restenosis occurring; diseases where there is risk of apoptosis occurring (e.g., heart failure, atherosclerosis, degenerative neurologic disorders, arthritis, and liver injury); and treatment of psoriasis.

[00165] In one embodiment, the disorder is cystic fibrosis. Compounds of the invention are capable of treating and/or slowing the progression of cystic fibrosis. For approximately 90% of patients with CF, death results from progressive respiratory failure associated with impaired mucus clearance and excessive overgrowth of bacteria and fungi in the airways (Gibson et al., 2003, Proesmans et al., 2008). Compounds of the invention may positively modulate CFTR. Compounds of the invention are capable of treating and/or slowing the progression of CF. In this embodiment, appropriate amounts of compounds of the present invention are an amount sufficient to treat and/or slow the progression of CF and can be determined without undue experimentation by preclinical and/or clinical trials.

[00166] The therapeutically effective amount for the treatment of a subject afflicted with a CFTR mediated disorder. For example, for asthma, a therapeutically effective amount is a bronchodilating effective amount; for cystic fibrosis, a therapeutically effective amount is an airway obstruction ameliorating effective amount or an amount effective in lessening the symptoms in the pancreas, GI tract, and/or liver caused by CF; for ARDS, a therapeutically effective amount is a hypoxemia ameliorating effective amount; for heart disease, a therapeutically effective amount is an angina relieving or angiogenesis inducing effective amount; for hypertension, a therapeutically effective amount is a blood pressure reducing effective amount; for ischemic coronary disorders, a therapeutic amount is a blood flow increasing effective amount; for atherosclerosis, a therapeutically effective amount is an endothelial dysfunction reversing effective amount; for glaucoma, a therapeutic amount is an ocular fluid balancing amount; for diseases characterized by angiogenesis, a therapeutically effective amount is an angiogenesis inhibiting effective amount; for disorders where there is risk of thrombosis occurring, a therapeutically effective amount is a thrombosis preventing effective amount; for disorders where there is risk of restenosis occurring, a therapeutically effective amount is a restenosis inhibiting effective amount; for chronic inflammatory diseases, a therapeutically effective amount is an inflammation reducing effective amount; and for disorders where there is risk of apoptosis occurring, a therapeutically effective amount is an apoptosis preventing effective amount.

[00167] I. Uses in an Apparatus

[00168] The compounds of the present invention or a pharmaceutically acceptable salt thereof, or a stereoisomer, prodrug, metabolite, or N-oxide thereof, can be applied to various apparatus in circumstances when the presence of such compounds would be beneficial. Such apparatus can be any device or container, for example, implantable devices in which a compound of the invention can be used to coat a surgical mesh or cardiovascular stent prior to implantation in a patient. The compounds of the invention can also be applied to various apparatus for in vitro assay purposes or for culturing cells.

[00169] The compounds of the present invention or a pharmaceutically acceptable salt thereof, or a stereoisomer, a prodrug, a metabolite, or an N-oxide thereof, can also be used as an agent for the development, isolation or purification of binding partners to compounds of the invention, such as antibodies, natural ligands, and the like. Those skilled in the art can readily determine related uses for the compounds of the present invention.

EXAMPLES

[00170] The following examples are given to illustrate the present invention. It should be understood, however, that the invention is not to be limited to the specific conditions or details described in these examples. Throughout the specification, any and all references to a publicly available document, including a U.S. patent, are specifically incorporated by reference.

[00171] Examples 1-42 list representative novel analogs of Formula 1 useful as modulators of CFTR. Exemplary schemes below illustrate some general methods of making the analogs of Formula 1. Synthetic methods that can be used to prepare each compound are described in Examples 1-42 below. Supporting mass spectrometry data and/or proton NMR data for each compound is also included in Examples 1-42. Synthetic details for

corresponding Intermediates are detailed in Example 43. Schemes 1-4 below illustrate general methods for preparing analogs described herein.

[00172] Scheme 1:

[00173] A detailed procedure for the synthesis of a compound of Scheme 1 is found in

Example 1.

[00174] Scheme 2 Step 1

[00175] A detailed procedure for the synthesis of a compound of Scheme 2

Example 6.

[00176] Scheme 3

Intermediate 10

A detailed procedure for the synthesis of a compound of Scheme 3 is found

[00178] Scheme 4

[00179] A detailed procedure for the synthesis of a compound of Scheme 4 is found in step 2 of Example 33.

[00180] Example 1: 3-(6-(2-(3-chlorophenyl)-2-methylpropanamido)-3- methylpyridin-2-yl)benzoic acid

[00181] Step 1 (Scheme 1):

[00182] 2-(3-chlorophenyl)-2-methylpropanoic acid (200 mg, 1.01 mmol) was dissolved in DCM (5 mL) and a drop of DMF was added, followed by Oxalyl chloride (3 mmol). The solution was stirred for 1 h at room temperature and then concentrated in vacuo, re-dissolved in 5 mL DCM, and re-concentrated in vacuo. Ethyl 3-(6-amino-3-methylpyridin- 2-yl)benzoate (Intermediate 1, 255 mg, 1.01) was then added to the same flask, dissolved in DCM (5 mL), and stirred for 5 min. Et₃N (200 uL) was then added and the solution was stirred from 2-12 hours until the reaction was complete by LCMS. The solution was diluted with 5 mL H20 and the organics were collected and washed with brine, followed by concentration in vacuo. The crude desired was then purified via flash chromatography (0 - 20% EtOAc in Hexanes) to afford (150 mg) desired ethyl 3-(6-(2-(3-chlorophenyl)-2- methylpropanamido)-3-methylpyridin-2-yl)benzoate. LCMS (M+l) 437.38.

[00183] Step 2 (Scheme 1):

[00184] Ethyl 3-(6-(2-(3-chlorophenyl)-2-methylpropanamido)-3-methylpyridin-2- yl)benzoate (150 mg, 0.34 mmol) was dissolved in 1 mL EtOH, and to it was added 2 mL 4N NaOH. The solution was stirred overnight at room temperature, followed by concentration in vacuo, dilution with 2 mL H20, and acidification to pH = 4 with cone. HC1. The resulting solid was collected via filtration, washed x2 H20 (10 mL), washed xl Isopropyl Ether (5 mL) and dried under vacuum to afford 3-(6-(2-(3-chlorophenyl)-2-methylpropanamido)-3- methylpyridin-2-yl)benzoic acid (78 mg, 0.19 mmol). 1H NMR (DMSO- ₆; 300 MHz) d 9.58 (s, 1H), 7.97 (d, J =9Hz, 2H), 7.90 (d, J =9Hz, 1H), 7.69 (d, J =9Hz, 1H), 7.40 (d, J =9Hz, 1H), 7.32 (m, 5H), 2.23 (s, 3H), 1.59 (s, 6H). LCMS (M+l) 409.35. [00185] Example 2: 3-(6-(2-(3,4-dichlorophenyl)-2-methylpropanamido)-3- methylpyridin-2-yl)benzoic acid. Followed the two step procedure shown in Scheme 1, starting from 2-(3,4-dichlorophenyl)-2-methylpropanoic acid. LCMS (M+l) 443.24. H- NMR (DMSO- , 300MHz) d 9.71 (s, IH), 8.00 (m, 3H), 7.75 (d, J =9Hz, 2H), 7.56 (m, 3H), 7.31 (d, J =12Hz, IH), 2.24 (s, 3H), 1.47 (s, 6H). LCMS (M+l) 443.24.

[00186] Example 3: 3-(6-(2-(3-fluorophenyl)-2-methylpropanamido)-3- methylpyridin-2-yl)benzoic acid. Followed the two step procedure shown in Scheme 1, starting from 2-(3-fluorophenyl)-2-methylpropanoic acid. ¹H NMR (DMSO- ₆; 300 MHz) d 9.53 (s, IH), 8.00 (m, 3H), 7.75 (d, J =9Hz, 2H), 7.58 (dd, J =6Hz, 9Hz, IH), 7.40 (dd, J =6Hz, 9Hz, IH), 7.20 (m, 2H), 7.11 (m, IH), 2.24 (s, 3H), 1.59 (s, 6H). LCMS (M+l) 393.34.

[00187] Example 4: 3-(6-(2-(3,4-difluorophenyl)-2-methylpropanamido)-3- methylpyridin-2-yl)benzoic acid. Followed the two step procedure shown in Scheme 1, starting from 2-(3,4-difluorophenyl)-2-methylpropanoic acid. 1H NMR (DMSO- e; 300 MHz) d 9.58 (s, IH), 8.00 (m, 3H), 7.75 (d, J =9Hz, 2H), 7.59 (t, J =9Hz, IH), 7.41 (m, 2H), 7.17 (m, IH), 2.24 (s, 3H), 1.58 (s, 6H). ¹⁹F-NMR (DMSO-d6; 300 MHz) d 141.95 (m, IF), 138.81 (m, IF). LCMS (M+l) 411.36.

[00188] Example 5: 3-(6-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylpyridin-2-yl)benzoic acid. Followed the two step procedure shown in Scheme 1, starting from 2-(4-chlorophenyl)-2-methylpropanoic acid. 1H NMR (DMSO- e; 300 MHz) d 9.58 (s, IH), 8.00 (m, 3H), 7.75 (d, J = 2H), 7.55 (t, J = IH), 7.39 (bs, 4H), 2.24 (s, 3H), 1.58 (s, 6H). LCMS (M+l) 409.09.

[00189] Example 6: 3-(6-(2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)propanamido)- 3-methylpyridin-2-yl)benzoic acid

[00190] Step 1: (Scheme 2):

[00191] A mixture of 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)propanoic acid

(Intermediate 3, 250 mg, crude) and ethyl 3-(6-amino-3-methylpyridin-2-yl)benzoate

(Intermediate 1, 250 mg, 0.978 mmol) in pyridine (5 mL) was added EDCI (313 mg, 1.63 mmol). The mixture was stirred at 15 °C for 18 hours. The mixture was diluted with H₂0 (30 mL), and the aqueous layer was extracted with EtOAc (20 mL x3). The combined organic layer was washed with brine (30 mL), dried over Na₂S0₄, filtered and concentrated to give the residue. The crude product was purified by silica gel column chromatography (PE/EtOAc = 4/1) to afford 267 mg of ethyl 3-(6-(2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)propanamido)- 3-methylpyridin-2-yl)benzoate as a yellow oil.

[00192] Step 2: (Scheme 2):

[00193] A solution of the compound from step 1 (267 mg, 0.571 mmol) in

THF/H₂0/EtOH (2 mL/2 mL/1 mL) was added LiOH H₂0 (120 mg, 2.85 mmol). The mixture was stirred at 15 °C for 18 hours. The mixture was diluted with H₂0 (10 mL) and neutralized with 1M HCl to pH=7. The aqueous layer was extracted with DCM/MeOH (v/v = 10/1, 20 mL x3) and the combined organic layer was washed with brine (30 mL), dried over Na₂S0₄, filtered and concentrated to give the residue. The crude product was purified by prep-HPLC (0.1% HCl as additive) to afford 87 mg (yield: 35%) of Example 6 as a white solid. 1H NMR (DMSO- ₆, 400 MHz): δ 1.40 (3H, d, J = 6.8 Hz), 2.24 (3H, s), 4.06 (1H, q, J = 6.8 Hz), 7.22 (1H, dd, J = 8.4, 1.6 Hz), 7.33 (1H, d, J = 8.4 Hz), 7.41 (1H, d, J = 1.6 Hz), 7.58 (1H, t, J = 1.6 Hz), 7.71 (1H, d, J = 8.4 Hz), 7.74-7.78 (1H, m), 7.98 (2H, d, J = 8.4 Hz), 8.06 (1H, s), 10.70 (1H, brs). MS: 441.0 [M+H]⁺.

[00194] Example 7: 3-(6-(2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2- methylpropanamido)-3-methylpyridin-2-yl)benzoic acid

[00195] Followed the two step procedure shown in Scheme 2 (detailed procedure in

Example 6) starting from 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2-methylpropanoic acid (Intermediate 4) and ethyl 3-(6-amino-3-methylpyridin-2-yl)benzoate (Intermediate 1). 1H NMR (DMSO- , varian 400 MHz): δ 1.60 (6H, s), 2.24 (3H, s), 7.15 (1H, dd, J = 8.4, 1.6 Hz), 7.36 (1H, d, J = 8.4 Hz), 7.44 (1H, d, J = 1.6 Hz), 7.57 (1H, t, J = 1.6 Hz), 7.70-7.75 (2H, m), 7.94-8.02 (3H, m), 9.55 (1H, brs). MS: 455.0 [M+H]⁺.

[00196] Example 8: 5-(6-(2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2- methylpropanamido)-3-methylpyridin-2-yl)thiophene-3-carboxylic acid

[00197] Followed the two step procedure shown in Scheme 2 (detailed procedure in

Example 6) starting from 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2-methylpropanoic acid (Intermediate 4) and methyl 5-(6-amino-3-methylpyridin-2-yl)thiophene-3-carboxylate (Intermediate 5) with modification. In step 1, the reaction was stirred at 60-70 °C for 16 hours. Following workup, the residue was purified by silica gel column (PE/EtOAc, 5/1). [00198] Step 2: To a solution of the above product (100 mg, 0.211 mmol) in

THF/MeOH (4 mL/2 mL) was added 2N aqueous NaOH (4 mL) at 10-15 °C. The mixture was stirred at 10-15 °C for 16 hours. The mixture was acidified with 2N aqueous HCl to pH = 2 and extracted with EtOAc (25 mL x3). The combined organic layer was washed with brine (25 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was lyophilized, then purified by prep-HPLC. 1H NMR (DMSO- _6, 00 MHz): δ 1.61 (6H, s), 2.48 (3H, s), 7.17 (IH, dd, J = 8.4, 2.0 Hz), 7.38 (IH, d, J = 8.4 Hz), 7.46 (IH, d, J = 2.0 Hz), 7.73 (IH, d, J = 8.4 Hz), 7.76 (IH, d, J = 0.8 Hz), 7.88 (IH, d, J = 8.4 Hz), 8.28 (IH, s), 9.38 (IH, brs). MS: 460.9 [M+H]⁺.

[00199] Example 9: 5-(6-(2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2- methylbutanamido)-3-methylpyridin-2-yl)thiophene-3-carboxylic acid

[00200] Followed the two step procedure shown in Scheme 2 (detailed procedure in

Example 6) starting from 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2-methylbutanoic acid (Intermediate 7) and methyl 5-(6-amino-3-methylpyridin-2-yl)thiophene-3-carboxylate (Intermediate 5) with the modifications described in Example 8. 1H NMR (DMSO- e, 400 MHz): δ 0.78 (3H, t, J = 7.2 Hz), 1.56 (3H, s), 1.90-2.00 (IH, m), 2.15-2.25 (IH, m), 2.48 (3H, s), 7.13 (IH, dd, J = 8.4, 1.6 Hz), 7.38 (IH, d, J = 8.4 Hz), 7.42 (IH, d, J = 2.0 Hz), 7.72 (IH, d, J = 8.4 Hz), 7.75 (IH, s), 7.87 (IH, d, J = 8.0 Hz), 8.24 (IH, s), 9.36 (IH, brs). MS : 474.9 [M+H]⁺.

[00201] Example 10: 3-{6-[2-(4-methoxyphenyl)-2-methylpropanamido]-3- methylpyridin-2-yl}benzoic acid

[00202] Followed the two step procedure shown in Scheme 1 (detailed procedure in

Example 1) starting from 2-(4-methoxyphenyl)-2-methylpropanoic acid. 1H-NMR (DMSO- d6; 300 MHz) d 9.16 (s, IH), 7.99 (m, 3H), 7.74 (d, j=9Hz, 2H), 7.55 (t, j=9Hz, IH), 7.30 (d, j=9Hz, 2H), 6.92 (d, j=9Hz, 2H), 3.72 (s, 3H), 2.23 (s, 3H), 1.56 (s, 6H). LCMS 405.00 [M+H]⁺.

[00203] Example 11: 3-(3-methyl-6-{2-methyl-2-[4- (trifluoromethyl)phenyl]propanamido}pyridin-2-yl)benzoic acid

[00204] Followed the two step procedure shown in Scheme 1 (detailed procedure in

Example 1) starting from 2-methyl-2-(4-(trifluoromethyl)phenyl)propanoic acid. H-NMR (DMSO-d6; 300 MHz) d 9.69 (s, 1H), 7.99 (m, 3H), 7.73 (m, 4H), 7.55 (m, 3H), 2.24 (s, 3H), 1.61 (s, 6H). 19F-NMR (DMSO-d6; 300 MHz) d 60.86 (s, 3F). LCMS 443.24 [M+H]⁺.

[00205] Example 12: 3-(3-methyl-6-{2-methyl-2-[3- (trifluoromethyl)phenyl]propanamido}pyridin-2-yl)benzoic acid

[00206] Followed the two step procedure shown in Scheme 1 (detailed procedure in

Example 1) starting from 2-methyl-2-(3-(trifluoromethyl)phenyl)propanoic acid. LCMS 442.99 [M+H]⁺.

[00207] Example 13: 3-{6-[2-(3-methoxyphenyl)-2-methylpropanamido]-3- methylpyridin-2-yl}benzoic acid

[00208] Followed the two step procedure shown in Scheme 1 (detailed procedure in

Example 1) starting from 2-(3-methoxyphenyl)-2-methylpropanoic acid. LCMS 405.38

[M+H]⁺.

[00209] Example 14: 2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-{6-[3- (hydroxymethyl)phenyl]-5-methylpyridin-2-yl}-2-methylpropanamide

[00210] Step 1:

[00211] A mixture of 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2-methylpropanoic acid

(Intermediate 4, 150 mg, 0.615 mmol), HATU (467 mg, 1.23 mmol) and Et₃N (155 mg, 1.54 mmol) in anhydrous DMF (3 mL) was stirred at 15-20 °C for 15 minutes. Then ethyl 3-(6- amino-3-methylpyridin-2-yl)benzoate (Intermediate 1, 157 mg, 0.615 mmol) was added to the mixture and the resulting reaction mixture was stirred at 50 °C for 16 hours. The mixture was diluted with water (20 mL), extracted with EtOAc (20 mL x2). The combined organic layer was washed with IN aqueous HC1 (20 mL), brine (20 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by silica gel column (PE/EtOAc, 6/1) to give compound ethyl 3-(6-(2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2-methylpropanamido)- 3-methylpyridin-2-yl)benzoate (110 mg, yield: 37%) as a yellow oil.

[00212] Step 2:

[00213] To a solution of the above compound (100 mg, 0.210 mmol) in anhydrous

THF (3 mL) was added LiAlH₄ (12 mg, 0.31 mmol) at -20°C. Then the mixture was stirred at -20 °C for 0.5 hour. The mixture was quenched with water (25 mL) and extracted with EtOAc (20 mL x2). The combined organic layer was washed with brine (25 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by prep-TLC (PE/EtOAc, 2/1) and lyophilized to afford the desired product (27 mg, yield: 30%) as a white solid. 1H NMR (DMSO- ): δ 1.58 (6H, s), 2.21 (3H, s), 4.52 (2H, d, J = 5.6 Hz), 5.20 (1H, t, J = 5.6 Hz), 7.10-7.15 (1H, m), 7.25-7.45 (6H, m), 7.69 (1H, d, J = 8.4 Hz), 7.94 (1H, d, J = 8.4 Hz), 9.46 (1H, brs). MS: 441.0 [M+H]⁺.

[00214] Example 15: 3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-3-hydroxy-2- methylpropanamido]-3-methylpyridin-2-yl}benzoic acid

[00215] Step 1 :

[00216] To a mixture of compound 2-(2,2-difluorobenzo[d] [l,3]dioxol-5-yl)-2-methyl-

3-((tetrahydro-2H-pyran-2-yl)oxy)propanoic acid (Intermediate 8, 150 mg, crude), ethyl 3-(6- amino-3-methylpyridin-2-yl)benzoate (Intermediate 1 , 97 mg, 0.38 mmol) and HATU (215 mg, 0.567 mmol) in anhydrous DCM (4 mL) was added Et₃N (76 mg, 0.76 mmol) at 25-30 °C. Then the resulting reaction mixture was stirred at 25-30 °C for 16 hours. The mixture was diluted with DCM (25 mL), then washed with brine (25 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by prep-TLC (PE/EtOAc, 2/1) to give ethyl 3-(6- (2-(2,2-difluorobenzo[d] [l,3]dioxol-5-yl)-2-methyl-3-((tetrahydro-2H-pyran-2- yl)oxy)propanamido)-3-methylpyridin-2-yl)benzoate (65 mg, yield: 30% for 2 steps) as a yellow oil.

[00217] Step 2:

[00218] To a solution of the above product (65 mg, 0.112 mmol) in THF/MeOH (4 mL/2 mL) was added 2N aqueous NaOH (4 mL) at 25-30 °C. The mixture was stirred at 25- 30 °C for 16 hours. The mixture was acidified with 2N aqueous HC1 to pH = 2 and stirred at 25-30 °C for 0.5 hour. The reaction mixture was extracted with EtOAc (25 mL x2). The combined organic layer was washed with brine (25 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by prep-TLC (PE/EtOAc, 1/1) and lyophilized to afford Example 15 (30 mg, yield: 57%) as a white solid. 1H NMR (DMSO- ₆, Bruker Avance 400 MHz): δ 1.48 (3H, s), 2.25 (3H, s), 3.76 (1H, d, J = 9.2 Hz), 4.07 (1H, d, J = 10.0 Hz), 5.78 (1H, brs), 7.12 (1H, dd, J = 8.4, 1.6 Hz), 7.34 (1H, d, J = 8.4 Hz), 7.41 (1H, d, J = 1.6 Hz), 7.54 (1H, t, J = 7.6 Hz), 7.69 (1H, d, J = 7.6 Hz), 7.76 (1H, d, J = 8.4 Hz), 7.96 (1H, d, J = 1.6 Hz), 8.00 (1H, s), 8.04 (1H, d, J = 8.4 Hz), 9.98 (1H, brs). MS: 471.2

[M+H]⁺. [00219] Example 16: 2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-[6-(3- hydroxypiperidin-l-yl)-5-methylpyridin-2-yl]-2-methylpropanamide

[00220] l-(6-amino-3-methylpyridin-2-yl)piperidin-3-ol (Intermediate 9, crude) was dissolved in pyridine (2 mL) and treated with TMSCl over three hours. A solution of 2-(2,2- difluorobenzo[d][l,3]dioxol-5-yl)-2-methylpropanoyl chloride in DMF was added. The resultant solution was stirred over 2 hours. Aqueous work-up with EtOAc and column purification afforded the final desired product Example 16 ( 9 mg). [M+H⁺]: 434. 1H NMR (300 MHz, Methanol-d4) δ 7.64 (d, J = 8.0 Hz, 1H), 7.48 (dq, J = 8.1, 0.7 Hz, 1H), 7.32 (dd, J = 1.7, 0.7 Hz, 1H), 7.28 - 7.08 (m, 4H), 3.77 (dt, J = 9.0, 4.6 Hz, 1H), 3.33 (p, J = 1.7 Hz, 26H CD30D), 3.19 (d, J = 12.6 Hz, 2H), 2.77 - 2.52 (m, 2H), 2.30 - 2.12 (m, 3H), 2.06 - 1.90 (m, 0.5H), 1.90 - 1.73 (m, 0.5 H), 1.66 (s, 6H), 1.47 - 1.26 (m, 1H). MS: 434.0

[M+H⁺].

[00221] Example 17: l-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2- methylpropanamido]-3-methylpyridin-2-yl}piperidine-3-carboxylic acid

[00222] A mixture of N-(6-chloro-5-methylpyridin-2-yl)-2-(2,2- difluorobenzo[d][l,3]dioxol-5-yl)-2-methylpropanamide (Intermediate 10 , 500 mg, 1.36 mmol), 3-piperidinecarboxylic acid ethyl ester (320 mg, 2.04 mmol), Pd₂(dba)₃ (125 mg, 0.136 mmol, 10 mol%), BINAP (127 mg, 0.204 mmol, 15 mol%) and Cs₂C0₃ (1.33 g, 4.08 mmol) in anhydrous toluene (5 mL) was degassed and purged with N₂ for 3 times. The resulting reaction mixture was heated at 90-100 °C under N₂ atmosphere for 16 hours. The mixture was diluted with water (30 mL), extracted with EtOAc (50 mL x2). The combine organic layer was washed with brine (50 mL), dried over anhydrous Na₂S0₄ and

concentrated. The residue was purified by Combi-Flash (20% EtOAc in PE) to give ethyl 1- (6-(2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2-methylpropanamido)-3-methylpyridin-2- yl)piperidine-3-carboxylate (250 mg, yield: 38%) as a yellow oil.

[00223] Step 2:

[00224] To a solution of the above product (80 mg, 0.16 mmol) in THF/MeOH (4 mL/2 mL) was added 2N aqueous NaOH (4 mL) at 25-30 °C. The reaction mixture was stirred at 25-30 °C for 16 hours. The mixture was acidified with 2N aqueous HC1 to pH = 2 and extracted with EtOAc (25 mL x2). The combined organic layer was washed with brine (25 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by prep- TLC (DCM/MeOH, 20/1) and lyophilized to afford the desired product (40 mg, yield: 53%) as a white solid. 1H NMR (DMSO- _6, 400 MHz): δ 1.40-1.50 (2H, m), 1.57 (6H, s), 1.65-1.75 (IH, m), 1.85-1.95 (IH, m), 2.14 (3H, s), 2.40-2.45 (IH, m, overlap with DMSO peak), 2.55- 2.65 (IH, m), 2.75-2.85 (IH, m), 3.15-3.25 (IH, m), 3.35-3.45 (IH, m, overlap with H₂0 peak), 7.15 (IH, d, J = 8.4 Hz), 7.37 (IH, d, J = 8.4 Hz), 7.45-7.50 (2H, m), 7.54 (IH, d, J = 8.0 Hz), 8.90 (IH, brs). MS: 462.2 [M+H]⁺.

[00225] Example 18: 2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-{6-[3- (hydroxymethyl)piperidin-l-yl]-5-methylpyridin-2-yl}-2-methylpropanamide

[00226] To a solution of ethyl l-(6-(2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2- methylpropanamido)-3-methylpyridin-2-yl)piperidine-3-carboxylate (see Example 17 step 1, 150 mg, 0.307 mmol) in anhydrous THF (5 mL) was added LiAlH₄ (15 mg, 0.37 mmol) at 0 °C. The resulting reaction mixture was stirred at 0 °C for 1 hour. The mixture was quenched with water (25 mL) and extracted with EtOAc (30 mL x2). The combined organic layer was washed with brine (25 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by prep-TLC (DCM/MeOH, 40/1) and lyophilized to afford Example 18 (44 mg, yield: 32%) as a white solid. 1H NMR (DMSO- ₆, 400 MHz): δ 0.95-1.10 (IH, m), 1.50-1.55 (IH, m), 1.57 (6H, s), 1.62-1.78 (3H, m), 2.14 (3H, s), 2.35-2.45 (IH, m), 2.55-2.65 (IH, m), 3.18-3.35 (4H, m), 4.48 (IH, t, J = 5.2 Hz), 7.15 (IH, dd, J = 8.4, 1.6 Hz), 7.37 (IH, d, J = 8.4 Hz), 7.40-7.45 (2H, m), 7.53 (IH, d, J = 8.0 Hz), 8.86 (IH, brs). MS: 448.1 [M+H]⁺.

[00227] Example 19: 5-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2- methylpropanamido]-3-methylpyridin-2-yl}thiophene-2-carboxylic acid

[00228] Step 1: (Scheme 3)

[00229] A mixture of N-(6-chloro-5-methylpyridin-2-yl)-2-(2,2- difluorobenzo[d][l,3]dioxol-5-yl)-2-methylpropanamide (Intermediate 10, 200 mg, 0.542 mmol), thiophene-2-carboxylic acid methyl ester-5-boric acid (151 mg, 0.814 mmol), Pd(dppf)Cl₂ (22 mg, 0.027 mmol, 5 mol%) and Na₂C0₃ (172 mg, 1.63 mmol) in

dioxane/H₂0 (2 mL/0.5 mL) was degassed and purged with N₂ for three times. Then the resulting reaction mixture was heated at 80-90 °C for 16 hours under N₂ atmosphere. The mixture was cooled to room temperature and diluted with water (25 mL), then extracted with EtOAc (25 mL x2). The combined organic layer was washed with brine (25 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by silica gel column

(PE/EtOAc, 5/1) to give N-(6-chloro-5-methylpyridin-2-yl)-2-(2,2- difluorobenzo[d][l,3]dioxol-5-yl)-2-methylpropanamide (236 mg, yield: 92%) as a yellow oil. [00230] Step 2: (Scheme 3)

[00231] To a solution of the above compound (236 mg, 0.497 mmol) in THF/MeOH (4 mL/2 mL) was added 2N aqueous NaOH (4 mL) at 25-30 °C. Then the mixture was heated at 25-30 °C for 16 hours. The mixture was acidified with 2N aqueous HC1 to pH = 2 and extracted with EtOAc (25 mL x2). The combined organic layer was washed with brine (25 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by prep-TLC (PE/EtOAc, 1/2 to 1/1), then lyophilization to afford Example 19 (94 mg, yield: 41%) as a white solid. 1H NMR (DMSO- ₆, 400 MHz): δ 1.61 (6H, s), 2.47 (3H, s), 7.16 (1H, dd, J = 8.4, 1.6 Hz), 7.38 (1H, d, J = 8.4 Hz), 7.46 (1H, d, J = 2.0 Hz), 7.53 (1H, d, J = 3.6 Hz), 7.64 (1H, d, J = 4.0 Hz), 7.73 (1H, d, J = 8.4 Hz), 7.91 (1H, d, J = 8.4 Hz), 9.42 (1H, brs). MS: 461.0 [M+H]⁺.

[00232] Example 20: 5-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2- methylpropanamido]-3-methylpyridin-2-yl}-2-fluorobenzoic acid

[00233] Followed the two step procedure shown in Scheme 3 (detailed procedure in example 19) starting from Intermediate 10 and (3-(ethoxycarbonyl)-4-fluorophenyl)boronic acid. 1H NMR (DMSO- _6, 400 MHz): δ 1.60 (6H, s), 2.24 (3H, s), 7.15 (1H, dd, J = 8.4, 1.2 Hz), 7.31 (1H, t, J = 8.8 Hz), 7.36 (1H, d, J = 8.4 Hz), 7.43 (1H, s), 7.60-7.70 (1H, m), 7.72 (1H, d, J = 8.0 Hz), 7.88 (1H, d, J = 8.0 Hz), 7.97 (1H, d, J = 8.0 Hz), 9.55 (1H, brs). MS: 473.0 [M+H]

[00234] Example 21: 4-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2- methylpropanamido]-3-methylpyridin-2-yl}thiophene-2-carboxylic acid

[00235] Followed the two step procedure shown in Scheme 3 (detailed procedure in example 19) starting from Intermediate 10 and (5-(methoxycarbonyl)thiophen-3-yl)boronic acid. 1H NMR (DMSO- _6, Bruker Avance 400 MHz): δ 1.60 (6H, s), 2.37 (3H, s), 7.15 (1H, dd, J = 8.4, 1.6 Hz), 7.37 (1H, d, J = 8.8 Hz), 7.44 (1H, d, J = 1.6 Hz), 7.68 (1H, d, J = 8.4 Hz), 7.85-8.00 (3H, m), 9.44 (1H, brs). MS: 461.0 [M+H]⁺.

[00236] Example 22: 3-{6-[2-cyano-2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2- methylacetamido] -3-methylpyridin-2-yl}benzoic acid

[00237] Step 1:

[00238] To a mixture of ethyl 2-cyano-2-(2,2-difluorobenzo[d][l,3]dioxol-5- yl)propanoate (Intermediate 11, 150 mg, 0.530 mmol) and ethyl 3-(6-amino-3-methylpyridin- 2-yl)benzoate (Intermediate 1, 271 mg, 1.06 mmol) in anhydrous toluene (2 mL) was added Me₃Al (0.53 mL, 1.06 mmol, 2.0 M in toluene) dropwise at 25-30 °C. The mixture was quenched with IN aqueous HCl (25 mL) and extracted with EtOAc (25 mL x2), washed with brine (25 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by prep-TLC (PE/EtOAc, 5/1) to give ethyl 3-(6-(2-cyano-2-(2,2-difluorobenzo[d][l,3]dioxol-5- yl)propanamido)-3-methylpyridin-2-yl)benzoate (28 mg, yield: 11 ) as a yellow oil.

[00239] Step 2:

[00240] Followed step 2 of Scheme 2 (see Example 6, step 2) to give the desired product. ¹H NMR (DMSO- _6, Bruker Avance 400 MHz): δ 2.08 (3H, s), 2.24 (3H, s), 7.25- 7.45 (2H, m), 7.45-7.55 (2H, m), 7.62 (1H, s), 7.76 (1H, d, J = 8.0 Hz), 7.86 (1H, d, J = 8.0 Hz), 7.94 (1H, d, J = 1.2 Hz), 8.03 (1H, s), 10.50 (1H, brs). MS: 465.9 [M+H]⁺.

[00241] Example 23: 3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2- methylpropanamido]-3-methylpyridin-2-yl}-4-fluorobenzoic acid

[00242] Followed the two step procedure shown in Scheme 3 starting from

Intermediate 10 and 3-borono-4-fluorobenzoic acid ethyl ester. 1H NMR (DMSO- 6, Bruker Avance 400 MHz): δ 1.57 (6H, s), 2.07 (3H, s), 7.12 (1H, dd, J = 8.4, 1.6 Hz), 7.25-7.35 (2H, m), 7.40 (1H, d, J = 1.6 Hz), 7.73 (1H, d, J = 8.8 Hz), 7.88 (1H, d, J = 7.2 Hz), 7.95-8.05 (2H, m), 9.60 (1H, brs). MS: 473.0 [M+H]⁺.

[00243] Example 24: 3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2- methylpropanamido]-3-methylpyridin-2-yl}-5-fluorobenzoic acid

[00244] Followed the two step procedure shown in Scheme 3 starting from

Intermediate 10 and ethyl 3-fluoro-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzoate (Intermediate 12). 1H NMR (DMSO- _6, 00 MHz): δ 1.58 (6H, s), 2.24 (3H, s), 7.13 (1H, dd, J = 8.4, 1.6 Hz), 7.34 (1H, d, J = 8.4 Hz), 7.41 (1H, d, J = 2.0 Hz), 7.60-7.70 (2H, m), 7.75 (1H, d, J = 8.4 Hz), 7.85 (1H, s), 7.99 (1H, d, J = 8.4 Hz), 9.59 (1H, brs). MS: 473.2

[M+H]⁺.

[00245] Example 25: 3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2- methylpropanamido]-3-methylpyridin-2-yl}-2-fluorobenzoic acid

[00246] Followed the two step procedure shown in Scheme 3 starting from

Intermediate 10 and 2-fluoro-3-(ethoxycarbonyl)phenylboronic acid. 1H NMR (DMSO- _6, 400 MHz): δ 1.57 (6H, s), 2.06 (3H, s), 7.12 (1H, dd, J = 8.4, 1.6 Hz), 7.31-7.37 (2H, m), 7.40 (1H, d, J = 1.6 Hz), 7.55-7.60 (1H, m), 7.75 (1H, d, J = 8.4 Hz), 7.85-7.90 (1H, m), 8.02 (1H, d, J = 8.4 Hz), 9.60 (1H, brs). MS: 473.0 [M+H]⁺.

[00247] Example 26: 3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2- methylpropanamido]-3-fluoropyridin-2-yl}benzoic acid

[00248] Step 1:

[00249] A mixture of ethyl 3-(6-amino-3-fluoropyridin-2-yl)benzoate (Intermediate

13, 140 mg, 0.538 mmol), 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2-methylpropanoic acid (Intermediate 4, 140 mg, 0.574 mmol) and EDC.HC1 (154 mg, 0.807 mmol) in pyridine (3 mL) was stirred at 60-70 °C for 16 hours. The mixture was concentrated and the residue was diluted with EtOAc (50 mL), then washed with IN aqueous HC1 (50 mL), brine (50 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by prep-TLC (PE/EtOAc, 3/1) to give ethyl 3-(6-(2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2- methylpropanamido)-3-fluoropyridin-2-yl)benzoate (56 mg, yield: 22%) as a yellow oil.

[00250] Step 2:

[00251] Followed Scheme 3 step 2, wherein the reaction was stirred at 15-20 °C for 2 hours. Acidification, workup and purification were same as described for step 2 of Example 19 to give product as a white solid. 1H NMR (DMSO- _6, 400 MHz): δ 1.62 (6H, s), 7.17 (1H, dd, J = 8.4, 2.0 Hz), 7.38 (1H, d, J = 8.4 Hz), 7.46 (1H, d, J = 2.0 Hz), 7.59 (1H, t, J = 8.0 Hz), 7.86 (1H, dd, J = 10.4, 9.2 Hz), 8.01 (1H, d, J = 8.0 Hz), 8.04-8.15 (2H, m), 8.43 (1H, s), 9.82 (1H, brs). MS Found: 459.5 [M+H]⁺.

[00252] Example 27: 2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-[6-(3- hydroxyphenyl)-5-methylpyridin-2-yl]-2-methylpropanamide

[00253] Followed step 1 of Scheme 3 starting from Intermediate 10 and 3- hydroxyphenylboronic acid to give Example 27 as a white solid. 1H NMR (DMSO- _6, Bruker

Avance 400 MHz): δ 1.58 (6H, s), 2.20 (3H, s), 6.70-6.90 (3H, m), 7.14 (1H, dd, J = 8.4, 1.6

Hz), 7.20 (1H, t, J = 8.0 Hz), 7.36 (1H, d, J = 8.4 Hz), 7.43 (1H, d, J = 1.2 Hz), 7.67 (1H, d, J

= 8.4 Hz), 7.93 (1H, d, J = 8.4 Hz), 9.46 (1H, brs), 9.51 (1H, brs). MS: 426.9 [M+H]⁺.

[00254] Example 28: 2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-[6-(3- acetamidophenyl)-5-methylpyridin-2-yl]-2-methylpropanamide

[00255] Followed step 1 of Scheme 3 starting from Intermediate 10 and 3- acetamidophenylboronic acid to give Example 28 as a white solid. 1H NMR (DMSO- _6, Bruker Avance 400 MHz): δ 1.58 (6H, s), 2.03 (3H, s), 2.20 (3H, s), 7.09 (1H, d, J = 1.6 Hz), 7.14 (1H, d, J = 8.4 Hz), 7.30-7.40 (2H, m), 7.43 (1H, s), 7.55-7.60 (1H, m), 7.64 (1H, s), 7.70 (1H, d, J = 8.4 Hz), 7.96 (1H, d, J = 8.4 Hz), 9.49 (1H, brs), 10.02 (1H, brs). MS: 468.0 [M+H]⁺.

[00256] Example 29: 3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2- methylpropanamido]-3-methylpyridin-2-yl}-4-methoxybenzoic acid

[00257] Followed the two step procedure shown in Scheme 3 starting from

Intermediate 10 and (5-(ethoxycarbonyl)-2-methoxyphenyl)boronic acid to give Example 29 as a white solid. 1H NMR (DMSO- _6, Bruker Avance 400 MHz): δ 1.57 (6H, s), 1.98 (3H, s), 3.77 (3H, s), 7.13 (1H, dd, J = 8.4, 2.0 Hz), 7.17 (1H, d, J = 8.8 Hz), 7.34 (1H, d, J = 8.4 Hz), 7.41 (1H, d, J = 2.0 Hz), 7.60-7.70 (2H, m), 7.90-8.00 (2H, m), 9.49 (1H, brs). MS Found: 485.0 [M+H]⁺.

[00258] Example 30: 3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2- methylpropanamido]-3-methylpyridin-2-yl}-5-methoxybenzoic acid

[00259] Followed the two step procedure shown in Scheme 3 starting from

Intermediate 10 and ethyl 3-methoxy-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)benzoate (Intermediate 14). 1H NMR (DMSO- _6, Bruker Avance 400 MHz): δ 1.59 (6H, s), 2.23 (3H, s), 3.81 (3H, s), 7.14 (1H, dd, J = 8.8, 2.0 Hz), 7.21 (1H, s), 7.35 (1H, d, J = 8.4 Hz), 7.42 (1H, d, J = 2.0 Hz), 7.46 (1H, s), 7.58 (1H, s), 7.72 (1H, d, J = 8.4 Hz), 7.97 (1H, d, J = 8.8 Hz), 9.52 (1H, brs). MS: 485.0 [M+H]⁺.

[00260] Example 31: 3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2- methylpropanamido]-3-methylpyridin-2-yl}-2-methoxybenzoic acid

[00261] Followed the two step procedure shown in Scheme 3 starting from

Intermediate 10 and ethyl 2-methoxy-3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)benzoate (Intermediate 15) to give Example 31 as a white solid. ¹H NMR DMSO-d_6, 400 MHz): δ 1.57 (6H, s), 2.22 (3H, s), 3.78 (3H, s), 7.03 (1H, d, J = 8.8 Hz), 7.13 (1H, d, J = 8.4 Hz), 7.34 (1H, d, J = 8.8 Hz), 7.40-7.45 (2H, m), 7.58 (1H, s), 7.65 (1H, d, J = 8.4 Hz), 7.89 (1H, d, J = 8.8 Hz), 9.45 (1H, brs). MS Found: 485.0 [M+H]⁺.

[00262] Example 32: 5-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2- methylpropanamido]-3-methylpyridin-2-yl}-2-methoxybenzoic acid

[00263] Followed the two step procedure shown in Scheme 3 starting from

Intermediate 10 and ethyl 2-methoxy-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)benzoate (Intermediate 16) to give Example 32 as a white solid. 1H NMR DMSO-d_6, 400 MHz): δ 1.58 (6H, s), 2.23 (3H, s), 3.82 (3H, s), 7.05-7.15 (2H, m), 7.34 (1H, d, J = 8.8 Hz), 7.42 (1H, d, J = 1.6 Hz), 7.56 (1H, d, J = 1.6 Hz), 7.65-7.70 (2H, m), 7.90 (1H, d, J = 8.0 Hz), 9.47 (1H, brs). MS Found: 485.0 [M+H]⁺.

[00264] Example 33: methyl N-(3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2- methylpropanamido]-3-methylpyridin-2-yl}phenyl)carbamate

[00265] Step 1:

[00266] Followed Scheme 3, step 1 starting from Intermediate 10 and 3- aminophenylboroinc acid to give N-(6-(3-aminophenyl)-5-methylpyridin-2-yl)-2-(2,2- difluorobenzo[d][l,3]dioxol-5-yl)-2-methylpropanamide as yellow solid.

[00267] Step 2; ( Scheme 4):

To a mixture of N-(6-(3-aminophenyl)-5-methylpyridin-2-yl)-2-(2,2- difluorobenzo[d][l,3]dioxol-5-yl)-2-methylpropanamide (70 mg, 0.16 mmol) and Et₃N (50 mg, 0.49 mmol) in anhydrous DCM (5 mL) was added methyl chloroformate (47 mg, 0.49 mmol) at 15-20 °C, the resulting reaction mixture was stirred at 15-20 °C for 16 hours. The mixture was diluted with DCM (30 mL), washed with water (30 mL x2), brine (30 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by prep-HPLC (0.1% HCl as additive). Most of the CH₃CN was removed by evaporation under reduced pressure, the remaining solvent was removed by lyophilization to afford Example 33 (48 mg, yield: 33%) as a white solid. 1H NMR (DMSO- _6, 400 MHz): δ 1.60 (6H, s), 2.22 (3H, s), 3.66 (3H, s), 7.09 (1H, d, J = 1.6 Hz), 7.16 (1H, dd, J = 8.4, 1.6 Hz), 7.30-7.40 (2H, m), 7.44 (1H, d, J = 1.6 Hz), 7.51 (1H, d, J = 8.0 Hz), 7.55 (1H, s), 7.82 (1H, d, J = 8.4 Hz), 8.00 (1H, d, J = 8.8 Hz), 9.77 (2H, brs). MS: 484.1 [M+H]⁺.

[00268] Example 34: 2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-[6-(3- methanesulfonamidophenyl)-5-methylpyridin-2-yl]-2-methylpropanamide

[00269] Followed Stepl of Scheme 3 starting with Intermediate 10 and 3-

(methylsulfonylamino)phenyl boronic acid.to give the desired compound as a white solid. 1H NMR (DMSO- _, 400 MHz): δ 1.59 (6H, s), 2.20 (3H, s), 2.98 (3H, s), 7.10-7.20 (2H, m), 7.20-7.28 (2H, m), 7.30-7.40 (2H, m), 7.43 (1H, d, J = 1.6 Hz), 7.71 (1H, d, J = 8.4 Hz), 7.96 (1H, d, J = 8.4 Hz), 9.50 (1H, brs), 9.70 (1H, brs). MS: 525.9 [M+Na]⁺.

[00270] Example 35: 2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methyl-N-[5- methyl-6-(3-propanamidophenyl)pyridin-2-yl]propanamide

[00271] Followed Stepl of Scheme 3 starting with Intermediate 10 and N-(3-(4,4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)phenyl)propionamide (Intermediate 17) to give Example 35 as a white solid. 1H NMR (DMSO- _6, 400 MHz): δ 1.06 (3H, t, J = 7.6 Hz), 1.59 (6H, s), 2.21 (3H, s), 2.32 (2H, q, J = 1.2 Hz), 7.08 (1H, d, J = 1.6 Hz), 7.14 (1H, dd, J = 8.4, 1.6 Hz), 7.31 (1H, d, J = 1.6 Hz), 7.35 (1H, d, J = 8.4 Hz), 7.42 (1H, d, J = 1.6 Hz), 7.61 (1H, d, J = 1.6 Hz), 7.65-7.75 (2H, m), 7.95 (1H, d, J = 8.4 Hz), 9.46 (1H, brs), 10.00 (1H, brs). MS: 482.0 [M+H]⁺.

[00272] Example 36: 2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methyl-N-{5- methyl-6-[3-(2-methylpropanamido)phenyl]pyridin-2-yl}propanamide

[00273] Followed Scheme 3, step 1 starting with Intermediate 10 and 3-

(isobutanoylamino)benzene boronic acid to give Example 36 as a white solid. 1H NMR (DMSO- , Bruker Avance 400 MHz): δ 1.08 (6H, d, J = 6.8 Hz), 1.59 (6H, s), 2.21 (3H, s), 2.55-2.65 (1H, m), 7.09 (1H, d, J = 1.6 Hz), 7.15 (1H, dd, J = 8.4, 1.6 Hz), 7.31 (1H, d, J = 8.0 Hz), 7.35 (1H, d, J = 8.4 Hz), 7.43 (1H, d, J = 1.6 Hz), 7.62 (1H, d, J = 8.0 Hz), 7.65-7.75 (2H, m), 7.95 (1H, d, J = 8.4 Hz), 9.46 (1H, brs), 9.91 (1H, brs). MS: 496.2 [M+H]⁺. [00274] Example 37: ethyl N-(3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2- methylpropanamido]-3-methylpyridin-2-yl}phenyl)carbamate

[00275] Followed Scheme 4 using ethyl chloroforaiate to give the desired Example 37 as a white solid. 1H NMR (DMSO- ₆, 400 MHz): δ 1.24 (3H, t, J = 7.2 Hz), 1.60 (6H, s), 2.22 (3H, s), 4.11 (2H, q, J = 1.2 Hz), 7.08 (1H, d, J = 1.2 Hz), 7.16 (1H, dd, J = 8.8, 2.0 Hz), 7.30-7.40 (2H, m), 7.44 (1H, d, J = 2.0 Hz), 7.51 (1H, d, J = 8.0 Hz), 7.56 (1H, s), 7.81 (1H, d, J = 8.8 Hz), 7.99 (1H, d, J = 8.4 Hz), 9.74 (2H, brs). MS Found: 498.1 [M+H]⁺.

[00276] Example 38: 2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-[6-(3-acetamido- 4-fluorophenyl)-5-methylpyridin-2-yl]-2-methylpropanamide

[00277] Followed Scheme 3, step 1 starting with Intermediate 10 and N-(2-fluoro-5-

(4,4,5, 5-tetramethyl-l,3,2-dioxaborolan-2-yl)phenyl)acetamide (Intermediate 18) to give Example 38 as a white solid. 1H NMR (DMSO-d_6, 400 MHz): δ 1.58 (6H, s), 2.08 (3H, s), 2.22 (3H, s), 7.14 (1H, d, J = 8.0 Hz), 7.15-7.30 (2H, m), 7.35 (1H, d, J = 8.4 Hz), 7.43 (1H, s), 7.70 (1H, d, J = 8.4 Hz), 7.90-8.00 (2H, m), 9.50 (1H, brs), 9.81 (1H, brs). MS: 486.0 [M+H]⁺.

[00278] Example 39: propan-2-yl N-(3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5- yl)-2-methylpropanamido]-3-methylpyridin-2-yl}phenyl)carbamate

[00279] Followed Scheme 3, step 1 starting with Intermediate 10 and 3-

[(isopropoxycarbonyl)amino] phenylboronic acid to give Example 39 as a white solid. 1H NMR (DMSO- _, 400 MHz): δ 1.24 (1H, d, J = 6.4 Hz), 1.58 (6H, s), 2.19 (3H, s), 4.80-4.95 (1H, m), 7.03 (1H, d, J = 8.4 Hz), 7.14 (1H, dd, J = 8.4, 1.6 Hz), 7.30 (1H, t, J = 8.0 Hz), 7.36 (1H, d, J = 8.4 Hz), 7.43 (1H, d, J = 1.6 Hz), 7.47 (1H, d, J = 8.0 Hz), 7.52 (1H, s), 7.69 (1H, d, J = 8.4 Hz), 7.95 (1H, d, J = 8.4 Hz), 9.47 (1H, brs), 9.62 (1H, brs). MS: 512.3

[M+H]⁺.

[00280] Example 40: 2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-[6-(3- ethanesulfonamidophenyl)-5-methylpyridin-2-yl]-2-methylpropanamide

[00281] Followed Scheme 3, step 1 starting with Intermediate 10 and N-(3-(4,4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)phenyl)ethanesulfonamide (Intermediate 19) to give

Example 40 as a white solid. 1H NMR (DMSO- _6, 400 MHz): δ 1.18 (3H, t, J = 1.2 Hz), 1.59

(6H, s), 2.19 (3H, s), 3.07 (2H, q, J = 1.2 Hz), 7.10-7.18 (2H, m), 7.20-7.25 (2H, m), 7.30- 7.40 (2H, m), 7.43 (1H, d, J = 1.6 Hz), 7.70 (1H, d, J = 8.4 Hz), 7.95 (1H, d, J = 8.4 Hz), 9.49 (1H, brs), 9.84 (1H, brs). MS: 539.9 [M+Na]⁺.

[00282] Example 41: 2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-(6-{3- [(dimethylcarbamoyl)amino]phenyl}-5-methylpyridin-2-yl)-2-methylpropanamide

[00283] Followed Scheme 3, step 1 starting with Intermediate 10 and 1,1 -dimethyl- 3-

(3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)phenyl)urea (Intermediate 20) to give Example 40 as a white solid. 1H NMR (DMSO- _6, 400 MHz): δ 1.59 (6H, s), 2.21 (3H, s), 2.91 (6H, s), 6.99 (1H, d, J = 7.6 Hz), 7.15 (1H, dd, J = 8.4, 1.6 Hz), 7.26 (1H, t, J = 8.0 Hz), 7.36 (1H, d, J = 8.4 Hz), 7.43 (1H, d, J = 1.6 Hz), 7.49 (1H, d, J = 8.0 Hz), 7.54 (1H, s), 7.69 (1H, d, J = 8.4 Hz), 7.94 (1H, d, J = 8.4 Hz), 8.34 (1H, brs), 9.45 (1H, brs). MS: 497.3

[M+H]⁺.

[00284] Example 42: 5-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2- methylpropanamido]-3-methylpyridin-2-yl}-2-methylbenzoic acid

[00285] Followed the two step procedure shown in Scheme 3 starting with

Intermediate 10 and methyl 2-methyl-5-(4,4,5,5- tetramethyl-l,3,2-dioxaborolan-2- yl)benzoate to give Example 42 as a white solid. 1H NMR (DMSO- _6, 00 MHz): δ 1.59 (6H, s), 2.23 (3H, s), 2.55 (3H, s), 7.14 (1H, dd, J = 8.4, 1.6 Hz), 7.30-7.37 (2H, m), 7.42 (1H, d, J = 1.6 Hz), 7.56 (1H, dd, J = 8.0, 1.6 Hz), 7.71 (1H, d, J = 8.4 Hz), 7.90 (1H, d, J = 1.6 Hz), 7.95 (1H, d, J = 8.4 Hz), 9.49 (1H, brs). MS: 469.0 [M+H]⁺.

[00286] Example 43: Synthesis of Intermediates

[00287] Intermediate 1: ethyl 3-(6-amino-3-methylpyridin-2-yl)benzoate

[00288] Step 1: Synthesis of ethyl 3-(3-methylpyridin-2-yl)benzoate: A mixture of 2- bromo3-methylpyridine (8.00 g, 47.0 mmol) and 3-ethyloxycarbonylphenylboronic acid (10.0 g, 52.0 mmol) in toluene (200 mL) and H₂0 (94 mL) was added K₂C0₃ (25.9 g, 187 mmol). The resulting mixture was degassed three times and back filled with N₂. Then PdCl₂(dppf) (516 mg, 0.705 mmol) was added and the mixture was degassed three times and back filled with N₂. The mixture was heated at 80 °C for 2 hours. The mixture was diluted with EtOAc (200 mL) and filtered. The filtrate was washed with brine (50 mL), dried over anhydrous Na₂S0₄, and concentrated. The crude product was purified by silica gel column chromatography (PE/EtOAc = 3/1) to give 10.6 g (yield: 94%) of ethyl 3-(3-methylpyridin-2- yl)benzoate as a red oil.

[00289] Step 2: Synthesis of 2-(3-(ethoxycarbonyl)phenyl)-3-methylpyridine 1-oxide.

To a solution of compound ethyl 3-(3-methylpyridin-2-yl)benzoate (9.60 g, 39.8 mmol) in DCM (100 mL) was added m-CPBA (85% purity, 16.1 g, 79.7 mmol). The mixture was stirred at 25°C for 18 hours. The mixture was quenched with saturated NaHS0₃ solution (100 mL). The aqueous layer was extracted with DCM (50 mL x3). The combined organic layers was washed with brine (100 mL), dried over anhydrous Na₂S0₄, filtered and concentrated to dryness. The residue was purified by silica gel column chromatography (EtOAc/MeOH= 10/1) to give 7.50 g (yield: 74%) of 2-(3-(ethoxycarbonyl)phenyl)-3-methylpyridine 1-oxide as a white solid. 1H NMR (CDC1₃, 400 MHz): δ 1.38 (3H, t, J = 12 Hz), 2.12 (3H, s), 4.38 (2H, q, J = 12 Hz), 7.15-7.22 (2H, m), 7.53-7.61 (2H, m), 8.04 (1H, s), 8.14 (1H, d, J = 12 Hz), 8.22-8.27 (1H, m).

[00290] Step 3: Synthesis of ethyl 3-(6-amino-3-methylpyridin-2-yl)benzoate. A suspension of 2-(3-(ethoxycarbonyl)phenyl)-3-methylpyridine 1-oxide (7.86 g, 30.6 mmol) and p-TosCl (7.55 g, 39.8 mmol) in CH₃CN (160 mL) was added pyridine (10 mL). The mixture was heated at 75 °C for 8 hours. The mixture was cooled to room temperature and ethanolamine (18.7 g, 306 mmol) was added. The mixture was stirred at 25 °C for 1 hour. Then CH₃CN was removed in vacuo, and the residue was diluted with H₂0 (50 mL). The aqueous layer was extracted with EtOAc (50 mL x5). The combined organic layer was washed with brine (50 mL), dried over anhydrous Na₂S0₄, filtered and concentrated to dryness. The residue was purified by silica gel column chromatography (PE/EtOAc = 4/1) to give 3.80 g (yield: 48%) of ethyl 3-(6-amino-3-methylpyridin-2-yl)benzoate as a yellow solid. Further purification by prep-HPLC gave the desired Intermediate 1.

[00291] Intermediate 2: 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)propanenitrile

[00292] A solution of 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)acetonitrile (400 mg,

1.99 mmol) in anhydrous THF (18 mL) was added NaH (60% in mineral oil, 95 mg, 2.39 mmol) at -70 °C. After addition, the mixture was stirred at this temperature for 30 minutes. A solution of Mel (339 mg, 2.39 mmol) in anhydrous THF (2 mL) was added and the mixture was stirred at 17 °C for 18 hours. The mixture was quenched with ice water (30 mL). The aqueous layer was extracted with EtOAc (20 mL x3) and the combined organic layer was washed with brine (20 mL), dried over Na₂S0₄, filtered and concentrated to give the residue. The crude product was purified by silica gel column chromatography (PE/EtOAc = 3/1) to afford 355 mg (yield: 83%) of the desired compound as a yellow oil. ¹H NMR (CDC1₃, 400 MHz): δ 1.67 (3H, d, J = 7.2 Hz), 3.92 (1H, q, J = 7.2 Hz), 7.07-7.14 (3H, m).

[00293] Intermediate 3: 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)propanoic acid

[00294] To a stirred solution of Intermediate 2 (250 mg, 1.18 mmol) in EtOH (1 mL) was added NaOH (379 mg, 9.48 mmol) and H₂0 (1.6 mL), the resulting mixture was stirred at 80 °C for 18 hours. The mixture was acidified by 1M HC1 to pH=l. The aqueous layer was extracted with DCM/MeOH (v/v = 10/1, 20 mL x3) and the combined organic layer was washed with brine (20 mL), dried over Na₂S0₄, filtered and concentrated to afford 250 mg of the desired compound which was used directly for the next step.

[00295] Intermediate 4: 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2- methylpropanoic acid:

[00296] Step 1: A solution of 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)acetonitrile (400 mg, 1.99 mmol) in anhydrous THF (8 mL) was added LDA (2M in THF, 2.4 mL, 4.78 mmol) at -78 °C. After addition, the mixture was stirred at this temperature for 15 minutes. A solution of Mel (1.13 g, 7.96 mmol) in anhydrous THF (2 mL) was added and the mixture was stirred at 17 °C for 18 hours. Followed workup and purification procedures described in Intermediate 2 to afford 230 mg (yield: 50%) of 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2- methylpropanenitrile as a yellow solid. 1H NMR (CDC1₃, 400 MHz): δ 1.73 (6H, s), 7.07 (1H, d, J = 8.4 Hz), 7.17-7.27 (2H, m).

[00297] Step 2: To a stirred solution of 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2- methylpropanenitrile (230 mg, 1.02 mmol) in EtOH (1 mL) was added NaOH (572 mg, 14.3 mmol) and H₂0 (1.1 mL), the resulting mixture was stirred at 120 °C for 18 hours. The mixture was acidified by 1M HC1 to pH=l. The aqueous layer was extracted with

DCM/MeOH (v/v = 10/1, 20 mL x3) and the combined organic layer was washed with brine (20 mL), dried over Na₂S0₄, filtered and concentrated to afford 200 mg (yield: 80%) of Intermediate 4 as a yellow solid. 1H NMR (CDC1₃, 400 MHz): δ 1.60 (6H, s), 7.02 (1H, d, J = 8.4 Hz), 7.08-7.16 (2H, m).

[00298] Intermediate 5: methyl 5-(6-amino-3-methylpyridin-2-yl)thiophene-3- carboxylate

[00299] Intermediate 5 was synthesized using standard coupling conditions starting from 6-chloro-5-methylpyridin-2-amine and methyl 5-(4,4,5,5-tetramethyl- 1,3,2- dioxaborolan-2-yl)thiophene-3-carboxylate, Pd(dppf)Cl₂ was used as a catalatlyst in a mixture of dioxane and water.

[00300] Intermediate 6: 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2- methylbutanenitrile

[00301] To a solution of 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)propanenitrile

(Intermediate 2, 200 mg, 0.944 mmol) in anhydrous THF (2 mL) was added LDA (2.4 mL, 2.36 mmol, 1.0 M in THF) dropwise at -78 °C. Then the mixture was stirred at -78 °C for 30 minutes. Iodoethane (589 mg, 3.77 mmol) was added to the mixture at -78 °C. Then the resulting reaction mixture was stirred at 10-15 °C for 16 hours. The mixture was quenched with saturated aqueous NH₄C1 (25 mL) and extracted with EtOAc (25 mL x2). The combined organic layer was washed with brine (25 mL), dried over anhydrous Na₂S0₄ and

concentrated. The residue was purified by silica gel column (PE/EtOAc, 10/1) to give Intermediate 5 (130 mg, yield: 58%) as a yellow solid. ¹H NMR (CDC1₃, 400 MHz): δ 0.97 (3H, t, J = 1.6 Hz), 1.70 (3H, s), 1.85-2.10 (2H, m), 7.07 (1H, d, J = 8.4 Hz), 7.13 (1H, d, J = 1.6 Hz), 7.20 (1H, dd, J = 8.4, 2.0 Hz).

[00302] Intermediate 7: 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2- methylbutanoic acid

[00303] Followed the hydrolysis procedure described for Intermediate 3 starting from

2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2-methylbutanenitrile (Intermediate 6). Crude product was taken on without purification.

[00304] Intermediate 8: 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2-methyl-3- ((tetrahydro-2H-pyran-2-yl)oxy)propanoic acid

[00305] Step 1: Synthesis of 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)propanenitrile:

To a solution of 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)acetonitrile (5.00 g, 25.2 mmol) in anhydrous THF (20 mL) was added NaH (1.10 g, 27.9 mmol, 60% dispersion in mineral oil) in small portions at -78 °C and the mixture was stirred at -78 °C for 0.5 hour. Then a solution of iodomethane (3.90 g, 27.9 mmol) in anhydrous THF (2 mL) was added dropwise to the above mixture. After the completion of addition, the reaction mixture was stirred at 20-25° C for 1 hour. The mixture was quenched with water (50 mL), extracted with EtOAc (50 mL x3). The combined organic layer was washed with brine (100 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by silica gel column (PE/EtOAc, 8/1) to give 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)propanenitrile (3.80 g, yield: 73%) as a yellow oil.

[00306] Step 2: Synthesis of 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-3-hydroxy-2- methylpropanenitrile: To a solution of 2-(2,2-difluorobenzo[d][l,3]dioxol-5- yl)propanenitrile (1.50 g, 7.10 mmol) in anhydrous THF (10 mL) was added LDA (8.5 mL, 8.50 mmol, 1 M in THF) dropwise at -78 °C. After the completion of addition, the reaction mixture was stirred at -78 °C for 0.5 hour. Paraformaldehyde (320 mg, 10.6 mmol) was added to the reaction mixture and the resulting reaction mixture was stirred at 20-25 °C for 16 hours. The mixture was quenched with water (50 mL), extracted with EtOAc (50 mL x3). The combined organic layer was washed with brine (100 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by silica gel column (PE/EtOAc, 5/1) to give 2- (2,2-difluorobenzo[d][l,3]dioxol-5-yl)-3-hydroxy-2-methylpropanenitrile (1.00 g, yield: 59%) as a yellow oil.

Step 3: Synthesis of 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2-methyl-3-((tetrahydro-2H- pyran-2-yl)oxy)propanenitrile: To a mixture of the above product (500 mg, 2.07 mmol) and PPTS (50 mg, 0.21 mmol) in anhydrous DCM (3 mL) was added 3,4-dihydro-2H-pyran (350 mg, 4.15 mmol) at 25-30 °C. The resulting reaction mixture was stirred at 25-30°C for 2 hours. The mixture was diluted with DCM (50 mL), washed with water (30 mL), brine (30 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by silica gel column (PE/EtOAc, 5/1) to give the desired (630 mg, yield: 88%, contains some impurity) as a colorless oil. ¹H NMR (CDC1₃, 400 MHz): δ 1.60-1.74 (5H, m), 1.75-1.80 (3H, m), 1.83- 1.89 (1H, m), 3.57-3.59 (1H, m), 3.93 (1H, dd, J = 18.0, 10.0 Hz), 4.01-4.06 (1H, m), 4.66 (1H, dt, J = 16.0, 3.2 Hz), 4.83 (1H, dd, J = 4.8, 3.2 Hz), 7.08 (1H, d, J = 8.4 Hz), 7.22-7.26 (2H, m).

[00307] Step 4: Synthesis of Intermediate 8: 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-

2-methyl-3-((tetrahydro-2H-pyran-2-yl)oxy)propanoic acid: To a solution of the above product (630 mg, 1.94 mmol) in EtOH/H₂0 (2 mL/2 mL) was added NaOH (930 mg, 23.3 mmol) at 25-30 °C. Then the mixture was heated at 90-100 °C for 48 hours. The mixture was acidified with 10% aqueous citric acid to pH = 5, then extracted with EtOAc (30 mL x3). The combined organic layer was washed with brine (50 mL), dried over anhydrous Na₂S0₄ and concentrated to give Intermediate 8 (150 mg, crude) as a yellow oil, which was directly used for the next step without further purification. [00308] Intermediate 9: l-(6-amino-3-methylpyridin-2-yl)piperidin-3-ol

[00309] Step 1: Synthesis of N-(6-(3-hydroxypiperidin-l-yl)-5-methylpyridin-2-yl)-2-

(3-hydroxypiperidine-l-carbonyl)benzamide: Piperidin-3-ol (0.786 g, 11 mmol) was mixed with 2-(6-chloro-5-methylpyridin-2-yl)isoindoline-l,3-dione (284 mg, 1.10 mmol) in NMP (3 mL) in a microwave tube. The resultant solution was heated at 180°C via a microwave over 5h. Aqueous workup and column purification afforded N-(6-(3-hydroxypiperidin-l-yl)-5- methylpyridin-2-yl)-2-(3-hydroxypiperidine- l-carbonyl)benzamide (130 mg).

[00310] Step 2: Synthesis of Intermediate 9: N-(6-(3-hydroxypiperidin-l-yl)-5- methylpyridin-2-yl)-2-(3-hydroxypiperidine-l-carbonyl)benzamide (130 mg) was suspended in 4 mL of cone. HC1 and 2 mL of water, then heated at reflux over 30 min. Removal of all waters afforded a mixture containing l-(6-amino-3-methylpyridin-2-yl)piperidin-3-ol, which was used in next step without further purification.

[00311] Intermediate 10: N-(6-chloro-5-methylpyridin-2-yl)-2-(2,2- difluorobenzo[d][l,3]dioxol-5-yl)-2-methylpropanamide

[00312] To a mixture of 2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-2-methylpropanoic acid (1.00 g, 4.10 mmol) in SOCl₂ (15 mL) was added 2 drops of DMF at 25-30 °C, then the resulting reaction mixture was heated at 60-70 °C for 1 hour. The mixture was concentrated and the residue was dissolved in anhydrous toluene (3 mL) and concentrated for twice to remove the remaining SOCl₂. The crude acyl chloride was dissolved in anhydrous DCM (5 mL) and added to the mixture of 6-chloro-5-methylpyridin-2-amine (582 mg, 4.10 mmol) and Et₃N (828 mg, 8.20 mmol) in anhydrous DCM (10 mL) at 25-30 °C. The resulting reaction mixture was stirred at 25-30 °C for 1 hour. The mixture was diluted with DCM (80 mL), washed with saturated aqueous NaHC0₃ (30 mL), 2N aqueous HC1 (30 mL), brine (50 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by Combi-Flash (10% EtOAc in PE) to give Intermediate 10 (1.00 g, yield: 67%) as a white solid.

[00313] Intermediate 11: ethyl 2-cyano-2-(2,2-difluorobenzo[d][l,3]dioxol-5- yl)propanoate

[00314] Step 1: A mixture of 5-bromo-2,2-difluoro-l,3-benzodioxole (15.0 g, 63.6 mmol), ethyl cyanoacetate (14.4 g, 127 mmol) and Na₃P0₄ (31.2 g, 191 mmol) in toluene (180 mL) was degassed three times and back filled with N₂. Then Pd(dba)₂ (1.46 g, 2.54 mmol) and PtBu (10% w/w in hexane, 10.3 g, 5.08 mmol) were added. H₂0 (0.9 mL) was added and the mixture was degassed three times and back filled with N₂. The mixture was heated at 90 °C for 18 hours. The mixture was diluted with EtOAc (200 mL) and filtered. The filtrate was washed with H₂0 (100 mL) and brine (50 mL), dried over Na₂S0₄, filtered and concentrated to give 17 g of crude ethyl 2-cyano-2-(2,2-difluorobenzo[d][l,3]dioxol-5- yl)acetate as a black oil, which was used directly for the next step.

[00315] Step 2: To a mixture of ethyl 2-cyano-2-(2,2-difluorobenzo[d][l,3]dioxol-5- yl)acetate (600 mg, 2.23 mmol) and K₂C0₃ (925 mg, 6.69 mmol) in acetone (10 mL) was added iodomethane (791 mg, 5.58 mmol) at 25-30 °C. Then the mixture was stirred at 25-30 °C for 3 hours. To the mixture was added water (50 mL), then extracted with EtOAc (50 mL x2). The combined organic layer was washed with brine (50 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by silica gel column (PE/EtOAc, 10/1) to give ethyl 2-cyano-2-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)propanoate (350 mg, yield: 55%) as a yellow oil. 1H NMR (CDC1₃, 400 MHz): δ 1.28 (3H, t, J = 12 Hz), 1.95 (3H, s), 4.15- 4.35 (2H, m), 7.09 (1H, d, J = 8.4 Hz), 7.27 (1H, d, J = 2.0 Hz), 7.31 (1H, dd, J = 8.4, 2.0 Hz).

[00316] Intermediate 12: ethyl 3-fluoro-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan- 2-yl)benzoate

[00317] Step 1: To a solution of 3-bromo-5-fluorobenzoic acid (2.00 g, 9.13 mmol) in absolute EtOH (20 mL) was added cone. H₂S0₄ (2 mL) at 20-25 °C. The resulting reaction mixture was heated under reflux for 16 hours. The mixture was concentrated and the residue was diluted with EtOAc (100 mL). Then the mixture was washed with saturated aqueous NaHC0₃ (50 mL), brine (50 mL), dried over anhydrous Na₂S0₄ and concentrated to give ethyl 3-bromo-5-fluorobenzoate (2.10 g, yield: 91%) as a yellow oil.

[00318] Step 2: A mixture of ethyl 3-bromo-5-fluorobenzoate (1.00 g, 4.04 mmol), bis(pinacolato)diboron (1.50 g, 6.07 mmol), Pd(dppf)Cl₂ (330 mg, 0.404 mmol, 10 mol%) and KOAc (1.20 g, 12.1 mmol) in anhydrous dioxane (10 mL) was degassed and purged with N₂ for 3 times. Then the resulting reaction mixture was heated at 90-100 °C for 16 hours under N₂ atmosphere. The mixture was filtered and the filtrate was concentrated. The residue was purified by Combi-Flash (PE) to give Intermediate 12 (1.10 g, yield: 92%, contains some pinacol) as a yellow solid. 1H NMR (CDC1₃, 400 MHz): δ 1.35 (12H, s), 1.40 (3H, t, J = 12 Hz), 4.39 (2H, q, J = 12 Hz), 7.60-7.70 (1H, m), 7.75-7.85 (1H, m), 8.23 (1H, s). [00319] Intermediate 13: ethyl 3-(6-amino-3-fluoropyridin-2-yl)benzoate

[00320] Step 1 : A boronic acid coupling similar to Scheme 3 step 1 starting with 2- bromo-3-fluoropyridine and (3-(ethoxycarbonyl)phenyl)boronic acid gave the desired ethyl 3-(3-fluoropyridin-2-yl)benzoate.

[00321] Step 2: To a solution of ethyl 3-(3-fluoropyridin-2-yl)benzoate (500 mg, 2.04 mmol) in anhydrous DCM (10 mL) was added 85% mCPBA (1.05 g, 6.12 mmol) at 15-20 °C. Then the resulting reaction mixture was stirred at 15-20 °C for 48 hours. The mixture was diluted with DCM (50 mL), washed with saturated aqueous Na₂S0₃ (50 mL x2), saturated aqueous NaHC0₃ (50 mL), dried over anhydrous Na₂S0₄ and concentrated to give compound 2-(3-(ethoxycarbonyl)phenyl)-3-fluoropyridine 1-oxide (600 mg, crude).

[00322] Step 3: Pyridine (3 mL) was added to a mixture of the above product (400 mg,

1.52 mmol) and TsCl (440 mg, 2.30 mmol) in anhydrous MeCN (5 mL) at 15-20 °C. Then the resulting reaction mixture was heated at 75 °C for 16 hours. After the mixture was cooled to room temperature, ethanol amine (2.40 g, 38.4 mmol) was added and the resulting reaction mixture was stirred at 15-20 °C for 1 hour. The mixture was concentrated and the residue was diluted with water (50 mL), extracted with EtOAc (50 mL x2). The combined organic layer was washed with water (50 mL x2), brine (50 mL), dried over anhydrous Na₂S0₄ and concentrated. The residue was purified by Combi-Flash (EtOAc in PE, 10-50%) to give Intermediate 13 (110 mg, yield: 27% for 2 steps) as a white solid. 1H NMR (DMSO- ₆, 400 MHz): δ 1.34 (3H, t, J = 7.2 Hz), 4.35 (2H, q, J = 7.2 Hz), 6.09 (2H, brs), 6.51 (1H, dd, J = 9.2, 2.8 Hz), 7.46 (1H, dd, J = 10.8, 8.8 Hz), 7.63 (1H, t, J = 8.0 Hz), 8.00 (1H, dd, J = 8.0, 1.6 Hz), 8.10 (1H, dd, J = 8.0, 1.6 Hz), 8.46 (1H, s).

[00323] Intermediate 14: ethyl 3-methoxy-5-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)benzoate

[00324] Step 1: To a solution of 3-bromo-5-methoxybenzoic acid (1.00 g, 4.33 mmol) in absolute EtOH (15 mL) was added SOCl₂ (1 mL) at 15-20 °C. The resulting reaction mixture was heated under reflux for 16 hours, followed by aqueous workup to give ethyl 3- bromo-5-methoxybenzoate.

[00325] Step 2: Followed the procedure described in step 2 of Intermediate 12 to produce the desired Intermediate 14. 1H NMR (CDC1₃, 400 MHz): δ 1.36 (12H, s), 1.40 (3H, t, J = 7.2 Hz), 3.87 (3H, s), 4.38 (2H, q, J = 7.2 Hz), 7.51 (1H, d, J = 2.0 Hz), 7.66 (1H, d, J = 2.0 Hz), 8.06 (1H, s). [00326] Intermediate 15: ethyl 2-methoxy-3-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)benzoate

[00327] Followed the two step procedure described for intermediate 14, starting from

3-bromo-2-methoxybenzoic acid. 1H NMR (CDC1₃, 400 MHz): δ 1.35 (12H, s), 1.39 (3H, t, J = 7.2 Hz), 3.92 (3H, s), 4.36 (2H, q, J = 7.2 Hz), 6.96 (1H, d, J = 8.4 Hz), 7.89 (1H, dd, J = 8.4, 1.6 Hz), 8.19 (1H, d, J = 2.0 Hz).

[00328] Intermediate 16: ethyl 2-methoxy-5-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)benzoate

[00329] Followed the two step procedure described for intermediate 14, starting from

5-bromo-2-methoxybenzoic acid. 1H NMR (CDC1₃, Bruker Avance 400 MHz): δ 1.33 (12H, s), 1.38 (3H, t, J = 12 Hz), 3.92 (3H, s), 4.35 (2H, q, J = 7.2 Hz), 6.95 (1H, d, J = 8.4 Hz), 7.89 (1H, dd, J = 8.4, 1.6 Hz), 8.18 (1H, d, J = 1.6 Hz).

[00330] Intermediate 17: N-(3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)phenyl)propionamide

[00331] Step 1 : A mixture of 3-bromoaniline (2.00 g, 11.6 mmol) and Et₃N (2.35 g,

23.2 mmol) in anhydrous DCM (20 mL) was added propionyl chloride (1.30 g, 14.0 mmol) dropwise at 10-15 °C. Then the mixture was stirred at 10-15 °C for 16 hours. To the mixture was added water (50 mL) and extracted with EtOAc (50 mL x2). The combined organic layer was washed with brine (50 mL), dried over anhydrous Na₂S0₄ and concentrated. The crude product was washed with PE/EtOAc (5 mL x2, 10/1) to give N-(3- bromophenyl)propionamide (2.30 g, yield: 87%) as a yellow solid.

[00332] Step 2: A mixture of N-(3-bromophenyl)propionamide (500 mg, 2.19 mmol), bis(pinacolato)diboron (835 mg, 3.29 mmol), Pd(dppf)Cl₂ (90 mg, 0.11 mmol, 5 mol%) and KOAc (645 mg, 6.57 mmol) in anhydrous dioxane (6 mL) was degassed and purged with N₂ for three times. Then the resulting reaction mixture was heated at 90-100 °C for 16 hours under N₂ atmosphere. Aqueous workup followed by silica gel column (PE/EtOAc, 2/1) gave Intermediate 17 (400 mg, yield: 66%) as an off-white solid. 1H NMR (CDC1_3> Bruker Avance 400 MHz): δ 1.24 (3H, t, J = 1.6 Hz), 1.33 (12H, s), 2.37 (2H, q, J = 7.6 Hz), 7.20 (1H, brs), 7.34 (1H, t, J = 7.6 Hz), 7.53 (1H, d, J = 7.2 Hz), 7.65 (1H, s), 7.90 (1H, d, J = 8.0 Hz). [00333] Intermediate 18: N-(2-fluoro-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)phenyl)acetamide

[00334] Step 1: To a solution of 5-bromo-2-fluoroaniline (2.00 g, 10.5 mmol) in anhydrous pyridine (20 mL) was added acetic anhydride (1.29 g, 12.6 mmol) at 10-15 °C. The reaction mixture was stirred at 10-15 °C for 16 hours. The mixture was concentrated and the residue was diluted with EtOAc (100 mL). The mixture was washed with 2N aqueous HCl (50 mL x2), brine (50 mL), dried over anhydrous Na₂S0₄ and concentrated. The crude product was washed with PE/EtOAc (5 mL x2, 10/1) to give N-(5-bromo-2- fluorophenyl)acetamide.

[00335] Step 2: Followed step 2 of Intermediate 17 to give the desired Intermediate 18 as a yellow solid. ¹H NMR (CDC1₃, 400 MHz): δ 1.31 (12H, s), 2.21 (3H, s), 7.06 (1H, dd, J = 10.8, 8.4 Hz), 7.36 (1H, brs), 7.50 (1H, t, J = 6.4 Hz), 8.58 (1H, d, J = 8.4 Hz).

[00336] Intermediate 19: N-(3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)phenyl)ethanesulfonamide

[00337] Followed the two step procedure described for Intermediate 17, starting with ethanesulfonyl chloride instead of propionyl chloride to give the product as an off-white solid. 1H NMR (CDC1_3, 400 MHz): δ 1.34 (12H, s), 1.37 (3H, t, J = 7.2 Hz), 3.12 (2H, q, J = 7.2 Hz), 6.37 (1H, brs), 7.36 (1H, d, J = 7.6 Hz), 7.40-7.50 (2H, m), 7.61 (1H, d, J = 7.2 Hz).

[00338] Intermediate 20: l,l-dimethyl-3-(3-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)phenyl)urea

[00339] Step 1: To a suspension of dimethylamine hydrochloride (8.24 g, 101 mmol) in anhydrous THF (30 mL) was added DIPEA (26.1 g, 202 mmol) at 10-15 °C. Then a solution of 3-bromophenyl isocyanate (2.00 g, 10.1 mmol) in anhydrous THF (20 mL) was added to the mixture. The resulting reaction mixture was stirred at 10-15 °C for 16 hours. To the mixture was added water (100 mL), then extracted with EtOAc (100 mL x2). The combined organic layer was washed with brine (100 mL), dried over anhydrous Na₂S0₄ and concentrated. The crude product was washed with PE/EtOAc (10 mL x2, 5/1) to give 3-(3- bromophenyl)-l,l-dimethylurea (1.90 g, yield: 77%) as a white solid.

[00340] Step 2: Followed step 2 of Intermediate 17 to give Intermediate 20 as an off- white solid. 1H NMR (CDC1_3, 400 MHz): δ 1.33 (12H, s), 3.02 (6H, s), 6.33 (1H, brs), 7.32 (1H, t, J = 8.0 Hz), 7.46 (1H, d, J = 7.2 Hz), 7.50 (1H, d, J = 2.0 Hz), 7.80-7.85 (1H, m). [00341] Example 44: HTS of Compounds to Identify CFTR Modulators

[00342] The identification of novel pharmacological modulators (potentiators, correctors, and inhibitors) of the CFTR chloride channel was achieved by performing high- throughput screening using a functional assay (Sui, J. et al, Assay Drug Dev. Technol. 2010 Dec; 8(6):656-68).

[00343] YFP is a derivative of the green fluorescent protein (GFP). Its fluorescence is quenched in the presence of chloride at high concentrations. Its sensitivity to anions has been further improved by mutagenesis. For CFTR, a convenient fluorescent probe is the halide- sensitive yellow fluorescent protein (HS-YFP). The different sensitivity of the HS-YFP toward iodide and chloride allows one to perform assays measuring the transport of anions through the plasma membrane as changes in cell fluorescence. For this assay, the cells expressing HS-YFP were equilibrated in a physiological chloride-rich saline solution (e.g., Dulbecco's PBS). During fluorescence reading, cells were exposed to a high concentration of iodide. Iodide influx quenches the cell fluorescence with a rate that depends on the halide permeability of cell membrane, and therefore, on the activity of anion channels or

transporters.

[00344] Correctors: This protocol was designed to selectively screen compound for

F508del-CFTR correctors on a HTS assay platform. Fischer Rat Thyroid (FRT) cells stably expressing YFP and F508del-CFTR and CFBE 41o- cells (CFBE) transiently transfected with YFP and F508del-CFTR were used for high throughput screening. Cells were incubated for 24-hrs in the presence of test compounds. The cells were washed to remove excess compound, stimulated with 20 μΜ forskolin and 3 μΜ of potentiator P3 in DPBS for 1-2 h, and the YFP signal quenching rate by iodide influx was then measured. The rate of fluorescence quenching is proportionally related to the total CFTR activities in the cell membrane. F508del-CFTR correctors accelerate YFP quenching by increasing the number of CFTR molecules in the plasma membrane. Dose response curves and EC₅₀ for each compound were obtained by fitting the data to the Hill's equation (Jinliang Sui and et al, Assay Drug Dev. Technol. 2010 Dec; 8(6):656-68).

[00345] Potentiators: This protocol is designed to selectively screen compound for

F508del CFTR potentiators on a HTS assay platform. Fischer rat thyroid (FRT) cells stably expressing YFP and F508del-CFTR were incubated overnight at 27 °C to induce maturation of F508del-CFTR. Cells were then washed with PBS buffer, treated for one to two hours at room temperature with Forskolin (20 μΜ) plus varying concentrations of GSNOR inhibitor. Potentiator activity was measured as YFP quenching rate by iodide influx. Iodide enters the cells via active CFTR channels in the plasma membrane, and quenches the YFP fluorescence. The rate of fluorescence quenching is proportionally related to the total CFTR activities in the cell membranes. F508del-CFTR potentiator accelerates YFP quenching by increasing overall CFTR activities in the plasma membrane.

[00346] Results: In the Corrector assay with the CFBE 41o- cells (CFBE) transiently transfected with YFP, compounds of Examples 1-9, 14, and 16-25 had an EC₅₀ of < 5 uM. Compounds of Examples 2, 5, 7, 8, 9, 14, 16-21, 23, 25 had an EC₅₀ < luM in this assay. Compounds of Examples 7, 8, 9, 16, 18, 20, 21, and 23-25 had an EC₅₀ < 0.500 uM.

[00347] Example 45: YFP Based Iodide Influx Assay for the measurement of CFTR activity in FRT cells expressing AF508-CFTR

[00348] Novel pharmacological modulators (correctors, potentiators, and inhibitors) of the

CFTR chloride channel can be identified through high-throughput screening of large chemical libraries using a functional assay. YFP is a derivative of the green fluorescent protein (GFP), whereby the fluorescence is quenched in the presence of chloride at high concentrations. For CFTR, a convenient fluorescent probe is the halide-sensitive yellow fluorescent protein (HS-YFP). Fisher rat thyroid (FRT) cells stably expressing human AF508-CFTR and yellow fluorescent protein (YFP) are used to evaluate the CFTR modulator activities.

[00349] Assay Protocol

[00350] Fisher rat thyroid (FRT) cells stably expressing human AF508-CFTR and yellow fluorescent protein (YFP) were used to evaluate the CFTR corrector activity of the N30 compounds. FRT cells were plated in a black walled, clear bottom 96 well plate at a density of 1.0 x 10⁵ cells/well in 100 μΐ_^ F-12K cell culture medium. Plates were then incubated in a tissue culture incubator for 1 hr. at 37° C. During incubation, N30 compounds and a positive control N1785 from 10 mM primary stocks in DMSO, were diluted as following. A 10-point dilution series of the each compound was prepared in a separate 96 well plate by doing 3-fold dilutions in F-12K cell culture medium starting from 40 μΜ as the highest concentration. One hundred microliters of each dilution was then added to 6 wells containing the FRT cells to obtain final drug concentration of 20.00, 6.667, 2.222, 0.741, 0.247, 0.082, 0.027, 0.009, 0.003, and 0.00015 μΜ. Alternatively, alO-point dilution series of the compounds can be prepared in a separate 96 well plate by doing 3-fold dilutions in F-12K cell culture medium starting from 20 μΜ as the highest concentration. One hundred microliters of each dilution was then added to 6 wells containing the FRT cells to obtain final drug concentrations 10.00, 3.33, 1.11, 0.37, 0.12, 0.041, 0.0137, 0.0045, 0.0015, and 0.0005 μΜ. Each plate has internal controls of a single concentration, 3 μΜ of positive control N1785 and also 0.1 % of DMSO as negative control. The cells were incubated for 24 hrs at 37° C. Following incubation, the cells were washed twice with PBS and stimulated with 75 μΕ of 20 μΜ forskolin and 20 μΜ VX770 in PBS for 1-2 h at RT. To each well, 150 Ε of iodide quenching solution was added and the rate of iodide quenching kinetics was measured on a Molecular Devices FlexStation III plate reader. The YFP signal was measured with excitation/emission filters of 500nm/540nm.

[00351] The vehicle control for each plate was 0.1% DMSO. 5-{6-[2-(2,2-difluoro-2H-l,3- benzodioxol-5 -yl)-2-methylpropanamido] -3 -methylpyridin-2-yl } thiophene-3 -carboxylic acid (Example 8) served as the positive control.

[00352] Data Analysis

[00353] Fluorescent data generated for each compound was analyzed using Molecular

Devices SoftMax® Pro GXP software version 5.4.5.000.

[00354] AF508 CFTR corrector activity was quantified as a percentage of the difference in normalized relative fluorescence units (% Δ norm RFU). This value is also defined as the percentage difference between raw RFU at 24.99 sec (time t₀) and raw RFU at 41.16 sec (time ti). The first RFU is measured right before adding iodide solution and the second raw RFU at 16.17 sec after addition of iodide solution (ti - 1₀). The percent difference in normalized RFU for each replicate was calculated as shown in equation 1 below.

% Δ norm RFU = mean{ [[_rawRFU(t₀) - _rawRFU(ti)] / _rawRFU(t₀)] xl00}

Equation 1

[00355] EC₅₀ of each compound was calculated by plotting concentration on x -axis and % Δ norm RFU on y-axis. EC₅₀ was derived from four parametric non-linear regression equation 2 shown below.

max - min

Equation 2

[00356] Where, max and min are maximum and minimum y-axis values for all the concentrations used on the graph. Error bars used for the graph are standard error of mean. [00357] Results

[00358] In the Corrector assay with Fisher rat thyroid (FRT) cells stably expressing human

AF508-CFTR and yellow fluorescent protein (YFP), compounds of Examples 7, 8, 10, 11, 14 and 15-42 had an EC₅₀ of < 5 uM. Compounds of Examples 7, 8, 11, 14, 16-19, 21, 24, 25, 28, 30, 32-40, and 42 had an EC₅₀ < luM. Compounds of Examples 7, 8, 14, 24, 33, 34, 37-39, and 42 had an EC₅₀ < 0.500 uM.

[00359] Example 46: Ussing chamber measurements of CFTR activity in Cystic

Fibrosis (CF, AF508/AF508) primary human airway epithelial (HAE) cell monolayers treated with compounds of the present invention

[00360] CF HAE cells were grown at an air/liquid interface onto porous membrane supports (Snap well, Corning) to perform short-circuit current measurements using Ussing chambers. Cells were thawed from liquid nitrogen storage, washed and plated as passage 1 on 100 mm cell culture plates for expansion in BEGM culture medium (Randell, et al. Primary Epithelial Cell Models for Cystic Fibrosis Research. Methods Mol Biol 2011; 742: 285-310). At 80-90% confluence, cells are trypsinized, washed, suspended in ALI medium (Randell, et al.), and counted twice for accuracy. The total number of cells seeded as passage 2 in ALI medium on each collagen-coated Snap well insert should be between 100K and 250K, as per standard protocol. Each Snapwell was placed in a single well of a 6-well culture plate.

[00361] CF HAE cells are maintained at 37°C, 95% 0₂ in ALI medium at a

liquid/liquid interface until a confluent monolayer is formed, generally 5-8 days after seeding. HAE cultures were then maintained at an air/liquid interface in 2.5 mL (basolateral) ALI medium (changed every 48 hours). The apical surfaces of the cultures were washed with PBS every other day or as needed to remove mucus accumulation. Cells were maintained under these conditions for no less than 21 days before compounds are added and subsequent short-circuit current measurements are obtained.

[00362] CF HAE cells were washed apically with PBS 18-24 h prior to addition of compounds. Stock dilutions of compounds are made in sterile PBS (PBS concentration never exceeded 0.1%). Snapwells were treated in the basolateral compartment with 2.5 mL ALI containing the test compound at the final concentration. To initiate treatment, one 75 μΐ_^ drop of basolateral medium containing compound was placed on the apical surface of the cultures. Cultures were treated for a total of 24 hours before Ussing chamber experiments are performed.

[00363] Ussing Chamber Experiments

[00364] Experiments were performed in a modified Ussing chamber using LabChart

Software (AD Instruments). Chamber temperature was maintained at 37 C (+/-1 C) by a circulating water bath, and agar bridges are equilibrated in 5 mL bilateral potassium chloride (KC1) for 20 minutes prior to the start of the experiment. Pulse measurements were taken every 20 seconds and recorded digitally. Chambers were zeroed with blank Snap well inserts. Reference measurements of cells were made prior to data acquisition. Basal short circuit current was measured in bilateral Krebs-bicarbonate-Ringer buffer (KBR; 140 mM Na⁺, 120 mM CI^", 5.2 mM K⁺, 1.2 mM Ca²⁺' 1.2 mM Mg²⁺' 2.4 mM HP0₄ ²-' 0.4 mM H₂Po4 , 25 mM HCC _, and 5 mM glucose) (Fulcher et al., Novel Human Bronchial Epithelial Cell Lines for Cystic Fibrosis Research. AJP - Lung Cellular and Molecular Physiology 2009; 296 : L82- L91.) for a minimum of 10 minutes, or until a steady KBR/KBR baseline was obtained. KBR is then aspirated from the apical chamber, and replaced with 5 mL of a modified KBR, high K⁺, low CI^" solution (HKLC; 40 mM Na⁺, 100 mM K⁺, 4.5 mM CI^", 120 mM gluconate, 25 mM HC0₃ ^" _, 2.4 mM HP0₄ ²_ 0.4 mM HP0₄ ^" _, 1.1 mM Ca²⁺' 1.2 mM Mg²⁺' and 5.2 mM glucose) (at 37 C). A new baseline current was then obtained and allowed to stabilize for

approximately 10 minutes. Current agonists and inhibitors were added to Ussing chambers at 10 minute intervals or longer depending on the stability or trend of the current. The following sequential protocol was planned for all chambers regardless of pretreatment condition:

Amiloride (100 μΜ, apical)

Forskolin (10 μΜ, bilateral)

Genistein (10 μΜ, apical)

CFTRinhl72 (10 μΜ, apical)

UTP (100 μΜ, apical).

[00365] Results:

[00366] CF HAE cultures treated with 5- { 6-[2-(2,2-difluoro-2H- 1 ,3-benzodioxol-5- yl)-2-methylpropanamido]-3-methylpyridin-2-yl}thiophene-3-carboxylic acid (Example 8) showed a significant increase in forskolin-mediated short circuit current (μΑ/cm ) compared to the negative vehicle control (DMSO, 0.1%, 24 h). The negative control (DMSO) had a short circuit current value of 0.99 +/- *0.26 μΑ/cm as compared to the increased short circuit current value of Example 8: 5.36 +/- *0.29 μΑ/cm (*= standard error values). Forskolin is a compound that is commonly used to raise cAMP levels and thus increase PKA activity; a pathway that activates CFTR. Moreover, the increase observed in forskolin-mediated short circuit current treated with the compound of Example 8 was confirmed to be through activation of CFTR, since HAE cells treated a specific CFTR inhibitor, (CFTRinhl72) displayed a decrease in the forskolin activated CFTR short circuit current. This measurement using Example 8 in CF HAE cells was reproducibly observed within HAE cells derived from multiple CF patients.

[00367] Example 47: Intestinal current measurements (I CM) study of compounds of the present invention in mice rectal biopsies to detect CFTR function

[00368] Following euthanasia, the murine intestine is removed by fine dissection, taking care to cut but not pull the intestine from its mesenteric attachment. Before mounting in the Ussing chamber, the murine intestine is prepared by seromusculature "stripping" to minimize the influence of the intrinsic neuromuscular system. Seromusculature stripping removes the serosa (visceral peritoneum) and the longitudinal/circular muscle layers of the intestinal wall, leaving only the underlying mucosal elements, primarily the epithelium with minor remnants of muscle. The intestinal section is kept cold during dissection either by prechilling the plate or covering the section with ice-cold RPMI 1640. The tissue is then placed in the Ussing chambers, and voltage clamped to allow continuous monitoring of I_sc and transepithelial resistance R_T. Fluid resistance is first measured and then subtracted from the R_T by Acquire and Analyze 2.3 software, San Diego, CA. The mounted tissue is treated with 10 μΜ indomethacin (bilateral exposure) to reduce the contribution of non-CFTR mediated CI^" channels at baseline conditions. Following stabilization of currents (-30 min) the tissue is treated with amiloride (100 μΜ, mucosal exposure) to block Na⁺ absorption for 15 min. The tissue is then stimulated with 10 μΜ forskolin + 100 μΜ IB MX (bilateral exposure) to raise intracellular cAMP and monitored for 20 minutes. Tissues are then stimulated with carbachol (100 μΜ, serosal exposure) to activate basolateral K⁺ channels and augment CFTR-dependent CI^" secretion. In the presence of functional CFTR at the mucosal cell membrane these agonists generate an upward defection of the I_sc mediated by serosal to mucosal CI^" secretion. In the absence of CFTR, a small K⁺ current is stimulated that produces a downward deflection in the I_sc. Following current stabilization, bumetanide (100 μΜ) is added to the serosal compartment to block the Na⁺/K⁺/2C1^" co-transporter. In the presence of CFTR, bumetanide generates a downward deflection in the I_sc, reflecting inhibition of CFTR- dependent CI^" transport. In the absence of CFTR, bumetanide results in an upward deflection of the I_sc, due to inhibition of the K⁺ secretion. Thus, these maneuvers effectively isolate CFTR activity, producing either a large CFTR-dependent CI^" secretory current or a smaller (reverse polarity) K+ current. Moreover, the addition of bumetanide results in the I_sc also moving in opposing directions dependent on the presence or absence of CFTR at the mucosal membrane.

[00369] Example 48: Western Blot

[00370] The degree of CFTR processing within a cell can be assessed by the relative quantity of different CFTR bands detected by western blot. Properly processed wild- type CFTR is highly glycosylated and migrates as a broad, high molecular weight band (-180-200 kDa, "C-band"). A lesser amount of wild-type CFTR is found as an immature form which has not yet achieved complete folding. Immature CFTR migrates more quickly as a core glycosylated form (-150 kDa, "B-band"). The mutated F508del-CFTR does not properly advance through the protein folding and processing pathways within the cell, and it is minimally glycosylated and eventually degraded. Therefore, when analyzed by western blot, the majority of F508del-CFTR protein is found in the B-band form, and little to none is detected as the slower migrating C-band form. If cells or tissues containing the F508del- CFTR mutant protein are treated with a "corrector" compound, then a shift in the amount of protein from the B-band to the C-band will be observed by Western blot.

[00371] Method for performing Western blot for analyzing the relative amounts of

CFTR C-band and B-band: If using cells, add an appropriate number of cells to a tissue culture plate and grow in a cell-type appropriate growth media at 37 °C for 24-48 hours. For example, FRT cells stably expressing F508del-CFTR were maintained at 37°C, 5% C02 in F12 Coon's Modification medium supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 0.5 mg/mL zeocin, and 0.5 mg/mL G418. FRT cells were plated at 4 x 10⁵ cells/well in a 6 well dish and treated 24 hours after plating. Cells were subconfluent (actively growing) at initiation of experiment.

[00372] Cells were treated with growth medium containing a CFTR modulator or a vehicle control compound for a set amount of time, depending on the experiment.

Compounds were prepared as 10 mM stocks in DMSO and were stored at 0°C. Compounds were screened at a final concentration of 1 μΜ for FRT cells or 0.1% DMSO as a vehicle control. Cells were cultured for 24 hours after the addition of test compound and then harvested for analysis by SDS-PAGE. [00373] After 24 hours of treatment with test compound, medium was removed, cells were washed with lx PBS, and lysed in ice cold RIPA lysis buffer supplemented with a protease inhibitor cocktail. Cell lysates were incubated on ice for 30 minutes and centrifuged for 5 minutes at 25,000 g in an Eppendorf 5417R microcentrifuge at 4°C. Samples were either analyzed directly or stored at -70°C until use.

[00374] Protein lysates from the cell or tissue samples were analyzed using SDS-

PAGE (sodium dodecyl sulfate - polyacrylamide gel electrophoresis) followed by Western blot. Protein concentrations were determined using the BCA method, and approximately 10- 30 μg/ total lysate was combined with sample bluffer containing SDS (or similar detergent) and a reducing agent (such as dithiothreitol, DTT). Samples were heated at 60-70°C for 10 minutes. A fixed amount of each sample is loaded for separation on NuPAGE 3-8% Tris- Acetate gels run in lx Novex Tris-Acetate SDS Running Buffer at 170V for 60 minutes.

[00375] After electrophoresis, the proteins were transferred to PVDF membranes for immunoblotting using an apparatus designed for Western blotting and an appropriate transfer buffer. The membranes were then blocked in Tris-buffered saline solution containing 0.1% Tween (TBS-T) and 5% non-fat milk. The membrane was then incubated in fresh blocking buffer supplemented with an antibody specific for CFTR (e.g. UNC CFTR antibody #596) and incubated for either 2 hours at room temperature or at 4°C overnight with rocking. After incubation with the anti-CFTR antibodies, the membrane was washed 3 x 10 min with TBS- T. Following the wash, the membrane was incubated with a secondary detection antibody, which binds to the anti-CFTR antibody, and contains a conjugate such that the antibody complex can produce a signal that can be detected, for example a chemiluminescent signal. The relative amounts of CFTR C-band and B-band are then visualized and analyzed.

[00376] Results: F508del-CFTR B to C maturation via SDS-PAGE was used to assess corrector activity of 5-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]- 3-methylpyridin-2-yl}thiophene-3-carboxylic acid (Example 8) in Fisher Rat Thyroid (FRT, 1 μΜ compound concentration) cells expressing F508del-CFTR. Activity was expressed as % mature CFTR (C band) relative to total CFTR (B+C band) as quantitated via densitometry. The compound of Example 8 demonstrated 51.5 + 3.3 % mature CFTR compared to vehicle 31.1 + 2.3 % mature CFTR. This number is expressed as the average + SEM (n=7). * * * *

[00377] It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present invention without departing from the spirit or scope of the invention.

Claims

What is Claimed is:

1. A compound of formula 1 and pharmaceutically acceptable salts thereof:

Formula 1

wherein

Cy is selected from

A is selected from -COOH, hydroxyl, -CH₂OH, -CH₂CH₂OH, tetrazole, -NHC(0)R_x, - NHC(0)OR_x, -NHC(0)N(CH₃)₂, and -S0₂R_x;

R_x is selected from the group consisting of methyl, ethyl, i-propyl, and n-propyl;

Ri is selected from

halogen, hydroxyl, cyano, NR

optionally substituted CrC₆ alkyl group wherein substituents are selected from cyano, hydroxyl, and halogen,

optionally substituted Ci-C alkyl group having one methylene unit replaced by an oxygen atom, wherein substituents are selected from cyano, hydroxyl, and halogen,

and two Ri groups taken together to form a 4-7 membered saturated, partially saturated,

or aromatic ring with up to 3 ring atoms independently selected from O, NR₆, and

S and wherein the fused ring may optionally be substituted by one or more halogen or Ci-C₃ alkyl;

R₂ and R₃ are each independently of one another are selected from hydrogen, fluoro, hydroxyl, cyano, C₂-C₆ alkenyl, C₂-C₆ alkynyl,

optionally substituted C -C alkyl group, wherein substitutions are selected from cyano,

hydroxyl, C₃-C₆ cycloalkyl, C₃-C₆ heterocycloalkyl, and halogen,

optionally substituted C -C alkyl group having one methylene unit replaced by an oxygen atom, wherein substituents are selected from cyano, hydroxyl, C₃-C₆ cycloalkyl, C₃-C₆ heterocycloalkyl, and halogen,

a C3-C₆ cycloalkyl group, in which a methylene unit in the cyclic moiety may optionally

be replaced by a -NR₆ - group, an oxygen, or a sulphur atom, and optionally the

cycloalkyl groups and heterocycloalkyl groups may be substituted by halogen, and

provided that R₂ and R₃ cannot both be hydrogen;

each R₄ and R5 are independently selected from

halogen, cyano, hydroxyl, NR₆R₇,

optionally substituted Ci-C₆ alkyl wherein substitutions are selected from halogen, cyano,

and hydroxyl, and

optionally substituted C C₆ alkyl having one methylene unit replaced by an oxygen atom

wherein substitutions are selected from cyano, hydroxyl, and halogen;

R₆ and R₇ are independently selected from hydrogen and Q-C4 alkyl;

m is selected from 0, 1, 2, and 3;

n is selected from 0, 1, 2, and 3; and

o is selected from 0, 1, and 2.

2. The compound of claim 1 wherein A is selected from the group consisting of -COOH, hydroxyl, -CH₂OH, -CH₂CH₂OH, and tetrazole.

3. The compound of claim 1 wherein Cy is selected from the group consisting of

4. The compound of claim 1 wherein Cy is selected from

5. The compound of claim 1 wherein Cy is selected from the group consisting of

6. The compound of claim 1 wherein A is -COOH.

7. The compound of claim 1 wherein R is selected from the group consisting of halogen, hydroxyl, cyano, NR₆R7, an optionally substituted C C_? alkyl group, and an optionally substituted C C₄ alkyl group having one methylene unit replaced by an oxygen atom.

8. The compound of claim 1 wherein two R groups are taken together with the phenyl to which they are attached to form a bi-cyclic ring of formula 2

Formula 2 wherein p is 1 or 2, and

R₈ and R₉ are independently selected from hydrogen, halogen, and Q-C3 alkyl.

9. The compound of claim 8 wherein two R groups are taken together with the phenyl to which they f

10. The compound of claim 1 wherein R₂ and R₃ are independently selected form the group consisting of hydroxyl; cyano; C₂-C₄ alkenyl; C₂-C₄ alkynyl; an optionally substituted Q- C₃ alkyl group; an optionally substituted CrC₄ alkyl group having one methylene unit replaced by an oxygen atom (O), and wherein substitutions are selected from cyano, hydroxyl, and halogen.

11. The compound of claim 1 wherein R₂ and R₃ are independently selected from the group consisting of hydrogen; hydroxyl; cyano; methyl, ethyl, n-propyl, i-propyl, CF₃, CHF₂, CH₂F, CH₂CF₃, -OCH₃, and -OCH₂ CH_3.

12. The compound of claim 1 wherein R₂ and R₃ are both methyl.

13. The compound of claim 1 wherein

Cy is selected from the group consisting

A is -COOH;

R₂ and R are both methyl; and

two Ri groups taken together with the phenyl to which they are attached to form a cyclic ring selected from the group consisting of

14. A compound of claim 1 wherein the compound is selected from the group consisting of 3-{6-[2-(3-chlorophenyl)-2-methylpropanamido]-3-methylpyridin-2-yl}benzoic acid; 3-{6-[2-(3,4-dichlorophenyl)-2-methylpropanamido]-3-methylpyridin-2-yl}benzoic acid; 3-{6-[2-(3-fluorophenyl)-2-methylpropanamido]-3-methylpyridin-2-yl}benzoic acid; 3-{6-[2-(3,4-difluorophenyl)-2-methylpropanamido]-3-methylpyridin-2-yl}benzoic acid; 3-{6-[2-(4-chlorophenyl)-2-methylpropanamido]-3-methylpyridin-2-yl}benzoic acid; 3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)propanamido]-3-methylpyridin-2- yljbenzoic acid;

3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-3-methylpyridin- 2-yl}benzoic acid; 5-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-3-methylpyridin- 2-yl}thiophene-3-carboxylic acid;

5-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylbutanamido]-3-methylpyridin-

2- yl}thiophene-3-carboxylic acid;

3- {6-[2-(4-methoxyphenyl)-2-methylpropanamido]-3-methylpyridin-2-yl}benzoic acid; 3-(3-methyl-6-{2-methyl-2-[4-(trifluoromethyl)phenyl]propanamido} pyridine-2- yl)benzoic acid;

3-(3-methyl-6-{2-methyl-2-[3-(trifluoromethyl)phenyl]propanamido} yridine-2- yl)benzoic acid;

3-{6-[2-(3-methoxyphenyl)-2-methylpropanamido]-3-methylpyridin-2-yl}benzoic acid;

2- (2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-{6-[3-(hydroxymethyl)phenyl]-5- methylpyridin-2-yl } -2-methylpropanamide;

3- {6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-3-hydroxy-2-methylpropanamido]-3- methylpyridin-2-yl}benzoic acid;

2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-[6-(3-hydroxypiperidin-l-yl)-5- methylpyridin-2- yl] -2-methylprop anamide ;

1- {6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-3-methylpyridin-

2- yl}piperidine-3-carboxylic acid;

2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-{6-[3-(hydroxymethyl)piperidin-l-yl]-5- methylpyridin-2-yl } -2-methylpropanamide;

5-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-3-methylpyridin- 2-yl}thiophene-2-carboxylic acid;

5-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-3-methylpyridin- 2-yl}-2-fluorobenzoic acid;

4- {6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-3-methylpyridin-

2- yl}thiophene-2-carboxylic acid;

3- {6-[2-cyano-2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylacetamido]-3- methylpyridin-2-yl}benzoic acid;

3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-3-methylpyridin-

2- yl}-4-fluorobenzoic acid;

3- {6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-3-methylpyridin-

2- yl}-5-fluorobenzoic acid;

3- {6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-3-methylpyridin- 2-yl}-2-fluorobenzoic acid; 3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-3-fluoropyridin- 2-yl}benzoic acid;

2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-[6-(3-hydroxyphenyl)-5-methylpyridin-2- yl] -2-methylpropanamide;

2- (2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-[6-(3-acetamidophenyl)-5-methylpyridin-2- yl] -2-methylpropanamide;

3- {6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-3-methylpyridin-

2- yl}-4-methoxybenzoic acid;

3- {6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-3-methylpyridin-

2- yl}-5-methoxybenzoic acid;

3- {6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-3-methylpyridin- 2-yl}-2-methoxybenzoic acid;

5-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-3-methylpyridin- 2-yl}-2-methoxybenzoic acid;

methyl N-(3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-3- methylpyridin-2- yl } phenyl)c arb amate ;

2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-[6-(3-methanesulfonamidophenyl)-5- methylpyridin-2- yl] -2-methylprop anamide ;

2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methyl-N-[5-methyl-6-(3- propanamidophenyl) yridine-2-yl]propanamide;

2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methyl-N-{5-methyl-6-[3-(2- methylpropanamido)phenyl] yridine-2-yl Jpropanamide;

ethyl N-(3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-3- methylpyridin-2- yl } phenyl)c arb amate ;

2- (2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-[6-(3-acetamido-4-fluorophenyl)-5- methylpyridin-2- yl] -2-methylprop anamide ;

propan-2-yl N-(3-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-

3- methylpyridin-2-yl}phenyl)carbamate;

2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-[6-(3-ethanesulfonamidophenyl)-5- methylpyridin-2- yl] -2-methylprop anamide ;

2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-N-(6-{ 3-[(dimethylcarbamoyl)amino]phenyl}- 5-methylpyridin-2-yl)-2-methylpropanamide; and

5-{6-[2-(2,2-difluoro-2H-l,3-benzodioxol-5-yl)-2-methylpropanamido]-3-methylpyridin- 2-yl}-2-methylbenzoic acid.

15. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1 together with a pharmaceutically accepted carrier or excipient.

16. A method of treatment of a disease or condition which comprises administering a

therapeutically effective amount of a compound of Formula 1 as defined in claim 1 to a patient in need thereof.

17. A method of making a compound of Formula 1 as defined in claim 1.