HK1173151B - Solid forms of n-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamide - Google Patents

Description

Solid forms of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide

Cross Reference to Related Applications

This application claims benefit of U.S. provisional application serial No. US61/254,614 filed on day 10, 23 of 2009.

Technical Field

The present invention relates to solid state forms, e.g., crystalline forms, of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide, which are modulators of cystic fibrosis transmembrane conductance regulator ("CFTR"). The invention also relates to pharmaceutical compositions comprising crystalline forms of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide and methods of using the same.

Background

ATP cassette transporters (ATP cassette transporters) are a family of membrane transporter proteins that regulate the transport of a wide range of pharmacological agents, potentially toxic drugs and exogenous chemicals, as well as anions. They are homologous membrane proteins that bind and use cellular Adenosine Triphosphate (ATP) to achieve their specific activity. It has been found that some of these transporters are multi-drug resistant proteins (e.g., MDR1-P glycoprotein, or multi-drug resistant protein MRP1) that protect malignant cancer cells from chemotherapeutic agents. To date, 48 such transporters have been identified, classified into families 7 based on their sequence identity and function.

One member of the ATP cassette transporter family commonly associated with disease is the cAMP/ATP-mediated anion channel, CFTR. CFTR is expressed in a variety of cell types, including absorptive and secretory epithelial cells, where it regulates anion flow through the membrane, and regulates the activity of other ion channels and proteins. In epithelial cells, the normal function of CFTR is critical to maintain electrolyte transport throughout the body, including respiratory and digestive tissues. CFTR is composed of approximately 1480 amino acids that encode a protein composed of tandem repeats of transmembrane domains, each containing 6 transmembrane helices and a1 nucleotide binding domain. The 2 transmembrane domains are linked by a large, polar, regulatory (R) -domain with multiple phosphorylation sites that regulate channel activity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced (see Gregory, R.J. et al (1990) Nature 347: 382-Strata 386; Rich, D.P. et al (1990) Nature 347: 358-Strata 362), Riordan, J.R. et al (1989) Science 245: 1066-1073). Defects in this gene cause mutations in CFTR, leading to cystic fibrosis ("CF"), the most common fatal genetic disease of the human body. In the united states, cystic fibrosis affects about one in 2,500 infants. In the general U.S. population, up to 1 million people carry a single copy of such a defective gene, but without significant adverse effects. In contrast, individuals with 2 copies of genes associated with CF can develop debilitating and fatal effects of CF, including chronic lung disease.

In patients with cystic fibrosis, mutations in the endogenously expressed CFTR in the respiratory epithelium result in reduced anion secretion from the apical membrane, imbalanceing ion and fluid transport. The resulting decrease in anion transport leads to an increase in lung mucus accumulation in CF patients with microbial infections that ultimately lead to death. In addition to respiratory disease, CF patients typically have gastrointestinal problems and pancreatic insufficiency, which if left untreated, can lead to death. Furthermore, most men with cystic fibrosis are unable to give birth, while women with cystic fibrosis have reduced fertility. In contrast to the serious consequences of 2 copies of the CF associated gene, individuals carrying a single copy of the CF associated gene showed increased resistance to cholera and diarrhea-probably accounting for the relatively high frequency of the CF gene in this population.

Sequence analysis of the CFTR gene of the CF chromosome revealed numerous disease-causing mutations (Cutting, G.R. et al (1990) Nature 346: 366-. To date, more than 1000 disease-causing mutations in the CF gene have been identified (http:// www.genet.sickkids.on.ca/cftr/app). The most common mutation is a deletion of phenylalanine at position 508 of the CFTR amino acid sequence, commonly referred to as af 508-CFTR. This mutation occurs in about 70% of cystic fibrosis cases and is associated with severe disease.

Deletion of residue 508 in Δ F508-CFTR prevents the nascent protein from folding correctly. This results in the mutant protein not being able to exit the ER and reach the plasma membrane. As a result, the number of channels present in the membrane is much smaller than that observed in cells expressing wild-type CFTR. In addition to impaired trafficking, this mutation leads to defective channel gating. This adds up to the reduced number of channels in the membrane and the defect in gating leading to reduced anion transport through the epithelium, leading to defective ion and liquid transport (Quinton, P.M, (1990), FASEB J.4: 2709-. However, studies have shown that a reduced amount of af 508-CFTR in the membrane is functional, although less than wild-type CFTR. (Dolmans et al (1991), Nature Lond.354: 526-. In addition to Δ F508-CFTR, R117H-CFTR and G551D-CFTR, other disease-causing CFTR mutations that result in defects in trafficking, synthesis and/or channel gating can be up-or down-regulated to alter anion secretion and alter disease progression and/or severity.

Although CFTR transports a variety of molecules in addition to anions, it is clear that this role (transport of anions, chloride and bicarbonate) represents one of the important mechanisms of transport of ions and water across epithelial cells. Other elements include epithelial Na + channels, ENaC, Na⁺/2Cl^-/K⁺Cotransporter, Na⁺-K⁺ATP-ase Pump and basolateral Membrane K⁺Channels, which are responsible for the uptake of chloride ions into the cell.

These elements work together to select from among cells by their selectionSelective expression and localization enable targeted transport across epithelial cells. Through ENaC and CFTR present on the apical membrane with Na expressed on the basolateral surface of the cell⁺-K⁺ATP-ase Pump and Cl^-The concerted activity between the channels, the absorption of chloride ions takes place. The secondary active transport of chloride ions from the luminal side leads to intracellular chloride ion accumulation, which can then passively pass through Cl^-The ion channel leaves the cell, resulting in vector transport. Na on the outer surface of the substrate⁺/2Cl^-/K⁺Cotransporter, Na⁺-K⁺ATP-ase Pump and basolateral Membrane K⁺The arrangement of CFTR on the channel and luminal side coordinates the secretion of chloride ions by CFTR on the luminal side. Since water may never actively transport itself, it flows across the epithelium relying on a slight trans-epithelial osmotic gradient created by the bulk flux of sodium and chloride ions.

It is postulated that defects in bicarbonate transport due to mutations in CFTR result in defects in some secretory functions. See, for example, "cytotoxic fibrosis: immunological fibrosis and mucoviscidiosis," Paul M.Quinton, Lancet 2008; 372:415-417.

Mutations in CFTR associated with moderate CFTR dysfunction are also evident in patients with diseases that share some disease manifestations with CF but do not meet the diagnostic criteria for CF. They include congenital bilateral vasectomy, idiopathic chronic pancreatitis, chronic bronchitis, and chronic rhinosinusitis (rhinosinusitis). Other diseases in which mutant CFTR is considered to be a risk factor along with modifier genes or environmental factors include primary sclerosing cholangitis, allergic bronchopulmonary aspergillosis, and asthma.

Smoking, hypoxia and environmental factors that induce hypoxic signaling have also been shown to impair CFTR function and may cause some forms of respiratory disease, such as chronic bronchitis. Diseases that may result from defective CFTR function but do not meet the diagnostic criteria for CF are characterized as CFTR-associated diseases.

In addition to cystic fibrosis, modulation of CFTR activity may be beneficial in other diseases not directly a result of CFTR mutation, such as CFTR-mediated secretory diseases and other protein folding diseases. CFTR regulates chloride and bicarbonate flow through many cellular epithelia to control fluid movement, protein solubilization, mucus viscosity, and enzyme activity. Defects in CFTR can lead to blockage of the airways or ducts in many organs, including the liver and pancreas. Enhancers are compounds that enhance the gating activity of CFTR present in the cell membrane. Any disease involving mucus thickening, impaired fluid regulation, impaired mucus clearance, or blocked conduits leading to inflammation and tissue destruction may be candidates for enhancers.

These diseases include, but are not limited to, Chronic Obstructive Pulmonary Disease (COPD), asthma, smoking-induced COPD, chronic bronchitis, rhinosinusitis, constipation, dry eye, sjogren's syndrome, gastroesophageal reflux disease, gallstones, rectal prolapse, and inflammatory bowel disease. COPD is characterized by progressive and non-fully reversible airflow limitation. Airflow limitation is due to mucus hypersecretion, emphysema, and bronchiolitis. Activators of mutant or wild-type CFTR provide a potential treatment for mucus hypersecretion and impaired mucociliary clearance common in COPD. Specifically, increasing the secretion of anions across the CFTR promotes fluid transport into airway surface liquids to hydrate mucus and optimize periciliary fluid viscosity. This results in enhanced mucociliary clearance and a reduction in symptoms associated with COPD. Furthermore, by preventing progressive infection and inflammation (due to improved airway clearance), CFTR modulators may prevent or slow down the destruction of airway parenchyma and reduce or reverse the increase in the number and size of mucus secreting cells that are the basis for mucus hypersecretion in airway disease characterized by emphysema. Dry eye disease is characterized by reduced tear production and abnormal tear film lipid, protein and mucin properties. There are many causes of dry eye, some of which include age, Lasik eye surgery, arthritis, medications, chemical/thermal burns, allergies, and diseases such as cystic fibrosis and sjogren's syndrome. Increased anion secretion by CFTR can increase fluid transport from the corneal endothelial cells and secretory glands surrounding the eye to enhance corneal hydration. This helps to alleviate symptoms associated with dry eye. Sjogren's syndrome is an autoimmune disease in which the immune system attacks the systemic fluid-producing glands, including the eye, mouth, skin, respiratory tissues, liver, vagina and intestine. Symptoms include dry eyes, mouth and vagina, and lung disease. The disease is also associated with rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, and polymyositis/dermatomyositis. Protein trafficking defects are thought to cause the disease, thereby limiting treatment options. Modulators of CFTR activity may hydrate the various organs affected by the disease and help alleviate the associated symptoms. Individuals with cystic fibrosis have repeated episodes of ileus and high incidences of rectal prolapse, gallstones, gastroesophageal reflux disease, GI malignancies, and inflammatory bowel disease, suggesting that CFTR function may play an important role in preventing such diseases.

As described above, it is believed that the deletion of residue 508 in Δ F508-CFTR prevents the nascent protein from folding correctly, resulting in the inability of the mutant protein to exit the ER and be transported to the plasma membrane. As a result, insufficient amounts of mature protein are present in the plasma membrane and chloride transport within epithelial tissues is significantly reduced. In fact, this cellular phenomenon of ER deficiency, which processes CFTR via the ER mechanism, has been shown to be the underlying basis not only for CF disease, but also for a wide range of other independent and genetic diseases. Two pathways leading to a failure of the ER mechanism are degradation by loss of coupling to ER export of proteins (coupling), or by ER accumulation of these defective/misfolded proteins [ Aridror M et al, Nature Med.,5(7), pp 745-751 (1999); shasty, B.S. et al, neurochem.International,43, pp 1-7 (2003); rutishauser, J, et al, Swiss Med Wkly,132, pp 211-; morello, JP et al, TIPS,21, pp.466-469 (2000); bross P. et al, Human mut.,14, pp.186-198(1999) ]. The diseases associated with ER dysfunction of the first category are cystic fibrosis (caused by misfolded. DELTA.F 508-CFTR as described above), hereditary emphysema (caused by a 1-antitrypsin; non Piz variants), hereditary hemochromatosis, coagulation-fibrinolysis defects (coaggregation-fibrinolysis defects), such as protein C deficiency, hereditary angioedema type 1, lipid processing defects (processive defects), such as familial hypercholesterolemia, chylomicronemia type 1, betalipoproteinemia-free, lysosomal storage diseases, such as I-cell disease/pseudobulbar disease (pseudohurler), mucopolysaccharidoses (caused by processing enzymes), morbid/Tay-Sachs disease (caused by beta-hexosaminidase), Nakh-Najler syndrome (Crigjler II-UDP-type II-Sachs) (caused by beta-hexosaminidase) Transferase-induced), polyendocrindocrinopathy/hyperinsulinemia, diabetes (induced by insulin receptors), Laron dwarfism (Laron dwarfism) (induced by growth hormone receptors), myeloperoxidase deficiency, primary hypoparathyroidism (induced by pre-parathyroid prohormone), melanoma (induced by tyrosinase). Diseases associated with the latter group of ER dysfunctions are glyceryosis CDG1 type (hypoglycoprotein syndrome), hereditary emphysema (caused by alpha 1-antitrypsin (PiZ variant)), congenital hyperthyroidism, osteogenesis imperfecta (caused by procollagen type I, II, IV), hereditary hypofibrinogenemia (caused by fibrinogen), ACT deficiency (caused by alpha 1-antichymotrypsin), Diabetes Insipidus (DI), neurogenic (neuropysal) DI (caused by vasopressin hormone/V2-receptor), renal DI (caused by aquaporin-II), Charcot-Marie-Tooth Toothoth syndrome (caused by peripheral myelin protein 22), Palmer-Meyer's disease, neurodegenerative diseases, such as Alzheimer's disease (caused by APP beta and senescent proteins), Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, pick's disease, several polyglutamine (polyglutamine) neurological disorders, such as Huntington's disease, spinocerebellar ataxia type I (spinocerebellar ataxia), spinobulbar muscular atrophy, dentatorubral pallidoluysical hypothalamic atrophy, and myotonic dystrophy, and spongiform encephalopathies, such as Creutzfeldt-Jakobdigera (caused by prion protein processing defects), Fabry's disease (caused by lysosomal α -galactosidase A), Straussler-Scheinker syndrome (caused by Prp processing defects), infertility, pancreatitis, pancreatic insufficiency, osteoporosis, osteopenia, Gorham syndrome, chloride channel disease (chloridion channelopathathies), congenital myotonic muscle spasms (Thomson and Becker types), Debart syndrome type III, Dehykura syndrome, shockpaxile syndrome (Trexia), Epilepsy, startle syndrome, lysosomal storage diseases, Angelman syndrome, Primary Ciliary Dyskinesia (PCD), PCD with side-to-side transposition (also known as catagen syndrome), PCD without side-to-side transposition and ciliary dysplasia (ciliary atlas), and liver disease.

Other diseases associated with mutations in CFTR include male sterility due to congenital bilateral absence of the vas deferens (CBAVD), mild pulmonary disease, idiopathic pancreatitis, and allergic bronchopulmonary aspergillosis (ABPA). See "CFTR-opathies: discrete phenyl assisted with cyclic fibrous regulator formulations," Peer G.Noone and Michael R.Knowles, Respir.Res.2001,2:328-332 (incorporated herein by reference).

In addition to upregulating CFTR activity, the reduction of anion secretion by CFTR modulators may be beneficial for the treatment of secretory diarrhea, where epithelial water transport is significantly increased due to secretagogue-activated chloride transport. The mechanisms include elevation of cAMP and stimulation of CFTR.

Although there are many causes of diarrhea, the major consequences of diarrheal disease caused by excessive chloride ion transport are common, including dehydration, acidosis, impaired development and death. Acute and chronic diarrhea represent a major medical problem in many areas of the world. Diarrhea is an important factor in malnutrition and is the leading cause of death in children under 5 years of age (5,000,000 deaths per year).

Secretory diarrhea is also a dangerous condition in patients with acquired immunodeficiency syndrome (AIDS) and chronic Inflammatory Bowel Disease (IBD). Each year 1600 million travelers from developed to developing countries develop diarrhea, the severity and number of cases of diarrhea varying with the country and region traveled.

Thus, there is a need for potent and selective CFTR enhancers for human CFTR wild-type and mutant forms. These mutant CFTRs include, but are not limited to, Δ F508del, G551D, R117H, 2789+5G- > a.

There is also a need for modulators of CFTR activity and compositions thereof, which can be used to modulate CFTR activity in mammalian cell membranes.

There is a need for methods of treating diseases caused by mutations in CFTR using such modulators of CFTR activity.

There is a need for methods of modulating CFTR activity in an ex vivo cell membrane of a mammal.

Furthermore, there is a need for stable solid forms of said compounds which are easy to use in pharmaceutical compositions suitable for use as therapeutic agents.

Summary of The Invention

The present invention relates to solid forms of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide (hereinafter "compound I") having the following structure:

a compound I.

Compound I and pharmaceutically acceptable compositions thereof are useful in the treatment or lessening the severity of a variety of diseases, disorders or conditions including, but not limited to, cystic fibrosis, asthma, COPD due to smoking, chronic bronchitis, rhinosinusitis, constipation, pancreatitis, pancreatic insufficiency, male sterility due to congenital bilateral vasectomy (CBAVD), mild lung disease, idiopathic pancreatitis, allergic bronchopulmonary aspergillosis (ABPA), liver disease, hereditary emphysema, hereditary hemochromatosis, coagulation-fibrinolysis defects, e.g., protein C deficiency, hereditary angioedema type 1, lipid processing defects, e.g., familial hypercholesterolemia, chylomicronemia type 1, abeta-lipoproteinemia, lysosomal storage diseases, e.g., I-cell disease/pseudohuler disease, chronic obstructive pulmonary disease, Mucopolysaccharidosis, morhoff/tay-sachosis, creutzfeldt-jakob type II (Crigler-Najjar type II), polyendocrinopathy/hyperinsulinemia (hyperinsulemia), diabetes mellitus, larmenal dwarfism, myeloperoxidase deficiency (myeloperoxidase deficiency), primary hypoparathyroidism, melanoma, glyceranosis CDG1, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency (ACT deficiency), Diabetes Insipidus (DI), neurogenic DI (neuropyemic DI), renal DI (neurogenic DI), summer-horse-graphics syndrome (char-Marie Tooth syndrome), pelizaeus-merzbachiasis (Perlizaeus-merzcher disease), neurodegenerative diseases, such as alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, progressive amyotrophic lateral sclerosis, and amyotrophic lateral sclerosis, Several polyglutamine neurological disorders (polyglutamine neurological disorders), such as Huntington's disease, spinocerebellar ataxia type I, spinobulbar muscular atrophy, dentatorubral pallidoluysian atrophy and myotonic dystrophy, as well as spongiform encephalopathies, such as hereditary Creutzfeldt-Jakob disease (due to processing deficiency), Fabry disease, Straussler-Scheinker syndrome, COPD, xerophthalmia or Sjogren's disease, osteoporosis, osteopenia, bone healing and bone development (including bone repair, bone regeneration, decreased bone resorption and increased bone deposition), Gorham syndrome, chloride channel pathologies, such as myotonia congenital (Thomson and Becker types), Barter syndrome type III, Huntington's disease, panic syndrome (hyperkkxia), epilepsy, epicyclie syndrome, johnsony syndrome, lysosomal storage syndrome, and lysosomal storage syndrome, Angelman syndrome, Primary Ciliary Dyskinesia (PCD), which term is used to refer to a genetic disorder of ciliary structure and/or function, including PCD with left and right transposition (also known as catagmatic syndrome), PCD without left and right transposition and ciliary dysplasia.

In one aspect, compound I is substantially in a crystalline form, referred to as form B, form a-HCl, or form B-HCl, as described and characterized herein.

In another aspect, compound I is in the form of a hydrochloride salt, referred to as form a-HCl or form B-HCl, as described and characterized herein.

In another aspect, compound I is in the form of a hydrochloride salt, referred to as form B-HCl, as described and characterized herein.

The methods described herein can be used to prepare compositions of the invention comprising form B, form a-HCl, form B-HCl, or any combination of these forms.

Brief Description of Drawings

FIG. 1 is an X-ray powder diffraction pattern of an exemplary sample of form A-HCl.

FIG. 2 is a DSC curve of a representative sample of form A-HCl.

FIG. 3 is a graph of the weight of a representative sample of form A-HCl as a function of temperature generated by thermogravimetric analysis.

FIG. 4 is an FTIR spectrum of a representative sample of form A-HCl.

FIG. 5 is a solid phase of a representative sample of form A-HCl¹³C NMR spectrum.

FIG. 6 is a solid phase of a representative sample of form A-HCl¹⁹F NMR spectrum.

Fig. 7A is an X-ray powder diffraction pattern recorded by instrument 1 for a representative sample of form B.

Fig. 7B is an X-ray powder diffraction pattern recorded by instrument 2 for a representative sample of form B.

Figure 8 is a DSC curve for a representative sample of form B.

FIG. 9 is a graph of the weight of a representative sample of form B as a function of temperature generated by thermogravimetric analysis.

Fig. 10 is an FTIR spectrum of a representative sample of form B.

FIG. 11 is a solid phase of a representative sample of form B¹³C NMR spectrum.

FIG. 12 is a solid phase of a representative sample of form B¹⁹F NMR spectrum.

FIG. 13 is an X-ray powder diffraction pattern of a representative sample of form B-HCl.

FIG. 14 is a DSC curve of a representative sample of form B-HCl.

FIG. 15 is a plot of sample weight as a function of temperature generated by thermogravimetric analysis of a representative sample of form B-HCl.

FIG. 16 is an FTIR spectrum of a representative sample of form B-HCl.

FIG. 17 is a solid phase of a representative sample of form B-HCl¹³C NMR spectrum.

FIG. 18 is a solid phase of a representative sample of form B-HCl¹⁹F NMR spectrum.

Figure 19 is an illustration of the conformational structure of form B based on single crystal X-ray analysis.

FIG. 20 is an illustration of the conformational structure of form A-HCl based on single crystal X-ray analysis.

FIG. 21 is a molecular packing diagram of form A-HCl based on single crystal X-ray analysis.

FIG. 22 is an illustration of the conformational structure of form B-HCl based on single crystal X-ray analysis.

FIG. 23 is a molecular packing diagram of form B-HCl based on single crystal X-ray analysis.

Detailed Description

Definition of

The following definitions as used herein shall apply unless otherwise stated.

The term "ABC-transporter" as used herein refers to an ABC-transporter protein or a fragment thereof comprising at least one binding domain, wherein said protein or fragment thereof is present in vivo or in vitro. The term "binding domain" as used herein refers to a domain on an ABC-transporter that can bind to a modulator. See, e.g., Hwang, T.C. et al, J.Gen.Physiol. (1998):111(3), 477-90.

The term "CFTR" as used herein means the cystic fibrosis transmembrane conductance regulator or mutant with its modulator activity, including but not limited to Δ F508CFTR, R117H CFTR and G551D CFTR (see for example http:// www.genet.sickkids.on.ca/CFTR/app for CFTR mutations).

The term "modulate" as used herein means to increase or decrease in a measurable amount.

The term "normal CFTR" or "normal CFTR function" as used herein refers to a CFTR similar to the wild type without any damage due to environmental factors such as smoking, pollution or any condition that produces inflammation in the lungs.

The term "reduced CFTR" or "reduced CFTR function" as used herein refers to less than normal CFTR or less than normal CFTR function. The term "crystalline" means a compound or composition in which the structural units are arranged in a fixed geometric pattern or lattice such that the crystalline solid has a rigid, large range of order. The structural units constituting the crystal structure may be atoms, molecules or ions. Crystalline solids exhibit a defined melting point.

The term "substantially crystalline" refers to a solid substance that is predominantly arranged in a fixed geometric pattern or lattice having a rigid, large range of order. For example, a substantially crystalline material has a crystallinity greater than about 85% (e.g., greater than about 90% crystallinity, greater than about 95% crystallinity, or greater than about 99% crystallinity). It should also be noted that the term "substantially crystalline" includes the subject word "crystalline" as defined in the preceding paragraph.

For the purposes of the present invention, the chemical elements are identified in the CAS version of the periodic Table of the elements, in accordance with the 75 th edition of the handbook of chemistry and Physics. In addition, general principles of Organic Chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausaltio: 1999, and "March's Advanced Organic Chemistry", 5 th edition, editions Smith, M.B. and March, J., John Wiley & Sons, New York:2001, the entire contents of which are incorporated herein by reference.

Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

Furthermore, unless otherwise indicated, structures described herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the structure described herein, except that deuterium or tritium is substituted for hydrogen, or¹³C-or¹⁴It is within the scope of the present invention to replace carbon with C-enriched carbon. Such compounds are useful, for example, as analytical tools or probes in biological assays. Such compounds, particularly those containing deuterium atoms, may exhibit modified metabolic properties.

Form A-HCl

In one aspect, the invention features a form of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide characterized by form a-HCl.

In certain embodiments, form a-HCl is characterized by one or more of the following peaks, measured in degrees in an X-ray powder diffraction pattern: a peak from about 6.9 to about 7.3 degrees (e.g., about 7.1 degrees) obtained in X-ray powder diffraction using Cu ka radiation; a peak from about 8.0 to about 8.4 degrees, (e.g., about 8.2 degrees); a peak from about 13.9 to about 14.2 degrees, (e.g., about 14.1 degrees); and a peak from about 21.0 to about 21.4 degrees (e.g., about 21.2 degrees).

In certain embodiments, form a-HCl is characterized by one or more of the following peaks, measured in degrees in an X-ray powder diffraction pattern: a peak from about 6.9 to about 7.3 degrees (e.g., about 7.1 degrees) obtained in X-ray powder diffraction using Cu ka radiation; a peak from about 8.0 to about 8.4 degrees, (e.g., about 8.2 degrees); a peak from about 11.9 to about 12.3 degrees, (e.g., about 12.1 degrees); a peak from about 13.5 to about 13.9 degrees, (e.g., about 13.7 degrees); a peak from about 16.2 to about 16.6 degrees, (e.g., about 16.4 degrees); a peak from about 18.5 to about 18.9 degrees, (e.g., about 18.7 degrees); and a peak from about 21.0 to about 21.4 degrees, (e.g., about 21.2 degrees).

In certain embodiments, form a-HCl is characterized by one or more of the following peaks, measured in degrees in an X-ray powder diffraction pattern: a peak from about 6.9 to about 7.2 degrees (e.g., about 7.1 degrees) obtained in X-ray powder diffraction using Cu ka radiation; a peak from about 8.0 to about 8.4 degrees, (e.g., about 8.2 degrees); a peak from about 13.9 to about 14.3 degrees, (e.g., about 14.1 degrees); a peak from about 14.5 to about 14.9 degrees, (e.g., about 14.7 degrees); a peak from about 16.2 to about 16.6 degrees, (e.g., about 16.4 degrees); a peak from about 18.5 to about 18.9 degrees, (e.g., about 18.7 degrees); 3 peaks from about 21.0 to about 22.2 degrees, (e.g., peaks at about 21.2 degrees, about 21.7, and about 21.9); a peak from about 22.6 to about 23.0 degrees, (e.g., about 22.8 degrees); 2 peaks from about 24 to about 25 degrees, (e.g., about 24.6 degrees and about 25.0 degrees); and 2 peaks from about 35.3 to about 36.0 degrees, (e.g., about 35.6 degrees).

In certain embodiments, form a-HCl is characterized by the X-ray powder diffraction pattern provided in fig. 1.

In certain embodiments, form a-HCl is derived from the solid state¹³One or more of the following peaks in the CNMR spectra, measured in parts per million (ppm), are characterized by a peak from about 163.5 to about 163.9ppm (e.g., about 163.7ppm), a peak from about 137.0 to about 137.4ppm (e.g., about 137.2ppm), and a peak from about 121.3 to about 121.7ppm (e.g., about 121.5 ppm).

In certain embodiments, form a-HCl is derived from the solid state¹³One or more peaks in the C NMR spectrum, measured in parts per million (ppm), from about 175.5 to about 175.9ppm of the peak (e.g.About 175.7ppm), from about 163.5 to about 163.9ppm (e.g., about 163.7ppm), from about 142.4 to about 142.8ppm (e.g., about 142.6ppm), from about 140.6 to about 141.0ppm (e.g., about 140.8ppm), from about 137.0 to about 137.4ppm (e.g., 137.2ppm), from about 131.3 to about 131.7ppm (e.g., about 131.5ppm), and from about 121.3 to about 121.7ppm (e.g., about 121.5 ppm).

In certain embodiments, form A-HCl is solid state as shown in FIG. 5¹³C NMR spectrum characterization.

In certain embodiments, form a-HCl is derived from the solid state¹⁹One or more of the following peaks, measured in parts per million (ppm), in the F NMR spectrum are characterized by a peak from about-56.8 to about-57.2 ppm (e.g., about-57.0 ppm), and a peak from about-60.3 to about-60.7 ppm (e.g., about-60.5 ppm).

In certain embodiments, form A-HCl is solid state as shown in FIG. 6¹⁹F NMR spectrum characterization.

In still other embodiments, form a-HCl is characterized by the FTIR spectrum provided in fig. 4.

Form B

In one aspect, the invention features a form of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide characterized by form B.

In certain embodiments, form B is characterized by one or more of the following peaks, measured in degrees in an X-ray powder diffraction pattern: a peak from about 6.5 to about 6.9 degrees (e.g., about 6.7 degrees) obtained in X-ray powder diffraction using Cu ka radiation; a peak from about 9.8 to about 10.2 degrees, (e.g., about 10.0 degrees); a peak from about 11.0 to about 11.4 degrees, (e.g., about 11.2 degrees); a peak from about 13.2 to about 13.6 degrees, (e.g., about 13.4 degrees); and a peak from about 23.8 to about 24.2 degrees, (e.g., about 24.2 degrees).

In certain embodiments, form B is characterized by one or more peaks: a peak from about 6.5 to about 6.9 degrees (e.g., about 6.7 degrees), a peak from about 9.2 to about 9.6 degrees (e.g., about 9.4), a peak from about 11.0 to about 11.4 degrees (e.g., about 11.2 degrees), a peak from about 13.2 to about 13.6 degrees (e.g., about 13.4 degrees), a peak from about 15.0 to about 15.4 degrees (e.g., about 15.2 degrees), a peak from about 17.0 to about 17.4 degrees (e.g., about 17.2 degrees), a peak from about 17.6 to about 18.0 degrees (e.g., about 17.8 degrees), a peak from about 17.9 to about 18.3 degrees (e.g., about 18.1 degrees), a peak from about 19.0 to about 19.4 degrees (e.g., about 19.2), a peak from about 19.9 to about 20.3 degrees (e.g., about 20.1 degrees), a peak from about 19.0 to about 19.4 degrees (e.2 degrees), a peak from about 21.2 degrees to about 24 degrees (e.g., about 2 degrees), a peak from about 21 degrees (e.2 degrees to about 24 degrees), about 26.2 degrees), from about 27.0 to about 27.4 degrees (e.g., about 27.2 degrees), from about 27.5 to about 27.9 degrees (e.g., about 27.7 degrees), and from about 28.7 to about 29.1 degrees (e.g., about 28.9 degrees).

In certain embodiments, form B is characterized by the X-ray powder diffraction pattern provided in fig. 7.

In certain embodiments, form B is prepared from the solid state¹³One or more of the following peaks in the C NMR spectrum, measured in parts per million (ppm): a peak from about 165.1 to about 165.5ppm (e.g., about 165.3ppm), a peak from about 145.7 to about 146.1ppm (about 145.9ppm), a peak from about 132.7 to about 133.1ppm (e.g., about 132.9ppm), and a peak from about 113.2 to about 113.6ppm (e.g., about 113.4 ppm).

In certain embodiments, form B is prepared from the solid state¹³One or more of the following peaks in the C NMR spectrum, measured in parts per million (ppm): from about 175.1 to about 175.5ppm (e.g., about 175.3ppm), from about 165.1 to about 165.5ppm (e.g., about 165.3ppm), from about 141.2 to about 141.6ppm (e.g., about 141.4ppm), from about 145.7 to about 146.1ppm (e.g., about 145.9ppm), from about 132.7 to about 133.1ppm (e.g., about 132.9ppm), from about 123.3 to about 123.7ppm (e.g., about 123.5ppm), from about (anouy) 126.6 to about 123.7ppm127.0ppm peak (e.g., about 126.8ppm), from about 113.2 to about 113.6ppm peak (e.g., about 113.4ppm), from about 117.2 to about 117.6ppm peak (e.g., about 117.4ppm), from about 58.1 to about 58.5ppm peak (e.g., about 58.3ppm), from about 26.7 to about 27.1ppm peak (e.g., about 26.9ppm) and from about 29.o0 to about 29.4ppm peak (e.g., about 29.2 ppm).

In certain embodiments, form B is solid as shown in fig. 11¹³C NMR spectrum characterization.

In certain embodiments, form B is prepared from the solid state¹⁹One or more of the following peaks in the F NMR spectrum, measured in parts per million (ppm): a peak from about-55.9 to about-56.3 ppm (e.g., about-56.1 ppm), and a peak from about-61.9 to about-62.3 ppm (e.g., about-62.1 ppm).

In certain embodiments, form B is solid as shown in fig. 12¹⁹F NMR spectrum characterization.

In another embodiment, the invention features form B N- (4- (7-azabicyclo [2.2.1]]Crystals of hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide having the monoclinic system, P21/c space group, and the following unit cell parameters:α =90 °, β =101.193 °, and γ =90 °.

In one embodiment, the invention provides form B of N- (4- (7-azabicyclo [2.2.1]]Crystals of hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide having the monoclinic system, P21/c space group, and the following unit cell parameters:

in still other embodiments, form B is characterized by the FTIR spectrum provided in fig. 10.

Form B-HCl

In one aspect, the invention features a form of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide characterized by form B-HCl.

In certain embodiments, form B-HCl is characterized by one or more of the following peaks, measured in degrees in an X-ray powder diffraction pattern: a peak from about 8.1 to about 8.5 degrees (e.g., about 8.3 degrees) obtained using Cu ka radiation in X-ray powder diffraction; a peak from about 8.8 to about 9.2 degrees, (e.g., about 9.0 degrees); a peak from about 12.8 to about 13.2 degrees, (e.g., about 13.0 degrees); a peak from about 17.8 to about 18.2 degrees, (e.g., about 18.0 degrees); and a peak from about 22.8 to about 23.2 degrees, (e.g., about 23.0 degrees).

In certain embodiments, form B-HCl is characterized by one or more of the following peaks, measured in degrees in an X-ray powder diffraction pattern: a peak from about 8.1 to about 8.5 degrees (e.g., about 8.3 degrees) obtained using Cu ka radiation in X-ray powder diffraction; a peak from about 14.6 to about 15.1 degrees, (e.g., about 14.8 degrees); a peak from about 16.5 to about 16.9 degrees, (e.g., about 16.7 degrees); 3 peaks from about 17.6 to about 18.4 degrees, (e.g., about 17.8 degrees, about 18.0 degrees, and about 18.2 degrees); 2 peaks from about 21.4 to about 22.1 degrees, (e.g., about 21.7 degrees and about 22.0 degrees); 2 peaks from about 22.8 to about 23.8 degrees, (e.g., peaks at about 23.0 degrees and about 23.6); 2 peaks from about 24.7 to about 25.4 degrees, (e.g., about 24.9 degrees and about 25.2 degrees); a peak from about 26.9 to about 27.3 degrees, (e.g., about 27.1 degrees); a peak from about 30.9 to about 31.3 degrees, (e.g., about 31.1 degrees); and a peak from about 38.2 to about 38.7 degrees, (e.g., about 38.5 degrees).

In certain embodiments, form B-HCl is characterized by one or more of the following peaks, measured in degrees in an X-ray powder diffraction pattern: a peak from about 8.1 to about 8.5 degrees (e.g., about 8.3 degrees) obtained using Cu ka radiation in X-ray powder diffraction; a peak from about 13.8 to about 14.3 degrees, (e.g., about 14.1 degrees); 2 peaks from about 14.6 to about 15.5 degrees, (e.g., about 14.8 degrees and about 15.2 degrees); a peak from about 16.5 to about 16.9 degrees, (e.g., about 16.7 degrees); 3 peaks from about 17.6 to about 18.4 degrees, (e.g., about 17.8 degrees, about 18.0 degrees, and about 18.2 degrees); 2 peaks from about 19.1 to about 19.7 degrees, (e.g., about 19.3 degrees and about 19.5 degrees); 2 peaks from about 21.4 to about 22.1 degrees, (e.g., about 21.7 degrees and about 22.0 degrees); 2 peaks from about 22.8 to about 23.8 degrees, (e.g., about 23.0 degrees and about 23.6 degrees); 4 peaks from about 24.5 to about 25.9 degrees, (e.g., about 24.7 degrees, about 24.9 degrees, about 25.2 degrees, and about 25.7 degrees); a peak from about 26.9 to about 27.3 degrees, (e.g., about 27.1 degrees); 2 peaks from about 27.7 to about 28.3 degrees, (e.g., about 27.9 degrees and about 28.1 degrees); 2 peaks from about 29.5 to about 30.0 degrees, (e.g., about 29.7 degrees and about 29.8 degrees); 2 peaks from about 29.5 to about 30.0 degrees, (e.g., about 29.7 degrees and about 29.8 degrees); a peak from about 30.9 to about 31.3 degrees, (e.g., about 31.1 degrees); a peak from about 32.1 to about 32.5 degrees, (e.g., about 32.3 degrees); 3 peaks from about 33.2 to about 34.1 degrees, (e.g., about 33.4 degrees, about 33.8 degrees, and about 33.9 degrees); a peak from about 35.0 to about 35.4 degrees, (e.g., about 35.2 degrees); a peak from about 36.0 to about 36.4 degrees, (e.g., about 36.2 degrees); and 3 peaks from about 38.3 to about 40.1 degrees, (e.g., about 38.5 degrees, about 38.6 degrees, and about 39.9 degrees).

In certain embodiments, form B-HCl is characterized by the X-ray powder diffraction pattern provided in fig. 13.

In certain embodiments, form B-HCl is derived from the solid state¹³One or more of the following peaks in the C NMR spectrum, measured in parts per million (ppm): from about 168.0 to about 168.4A peak at ppm (e.g., about 168.2ppm), a peak from about 148.5 to about 148.9ppm (e.g., about 148.7ppm), a peak from about 138.6 to about 139.0ppm (e.g., about 138.8ppm), a peak from about 119.6 to about 120.0ppm (e.g., about 119.8ppm), and a peak from about 23.7 to about 24.1ppm (e.g., about 23.9 ppm).

In certain embodiments, form B-HCl is derived from the solid state¹³One or more of the following peaks in the C NMR spectrum, measured in parts per million (ppm): from about 176.1 to about 176.5ppm of the peak (e.g., about 176.3ppm), from about 168.0 to about 168.4ppm of the peak (e.g., about 168.2ppm), from about 148.5 to about 148.9ppm of the peak (e.g., about 148.7ppm), from about 143.0 to about 143.4ppm of the peak (e.g., about 143.2ppm), from about 138.6 to about 139.0ppm of the peak (e.g., about 138.8ppm), 7 from about 119 to about 134ppm of the peak (e.g., about 131.6ppm, about 129.6ppm, about 129.1ppm, about 126.7ppm, about 125.8ppm, about 122.7ppm, and about 119.8ppm), from about 112.1 to about 112.5ppm of the peak (e.g., about 112.3ppm), from about 68.8 to about 69.2ppm of the peak (e.g., about 69.0ppm), from about 66.7 to about 67.6 ppm, about 9.9 ppm of the peak (e.23.9 ppm), and from about 23.9ppm of the peak (e.g., about 23.3 ppm).

In certain embodiments, form B-HCl is derived from the solid state shown in FIG. 17¹³C NMR spectrum characterization.

In certain embodiments, form B-HCl is derived from the solid state¹⁹One or more of the following peaks in the F NMR spectrum, measured in parts per million (ppm): a peak from about-55.4 to about-55.8 ppm (e.g., about-55.6 ppm), and a peak from about-61.8 to about-62.2 ppm (e.g., about-62.0 ppm).

In certain embodiments, form B-HCl is solid state as shown in FIG. 18¹⁹F NMR spectrum characterization.

In still other embodiments, form B-HCl is characterized by the FTIR spectrum provided in fig. 16.

In one aspect, the invention features a pharmaceutical composition that includes form a-HCl, form B-HCl, or any combination of these forms, and a pharmaceutically acceptable adjuvant or carrier.

In one aspect, the invention features a method of treating a CFTR mediated disease in a human, the method including administering to the human an effective amount of form a-HCl, form B-HCl, or any combination of these forms.

In some embodiments, the method comprises administering an additional therapeutic agent.

In some embodiments, the disease is selected from cystic fibrosis, pancreatitis, sinusitis, hereditary emphysema, hereditary hemochromatosis, coagulation-fibrinolysis defects, e.g., protein C deficiency, hereditary angioedema type 1, lipid processing defects, e.g., familial hypercholesterolemia, chylomicronemia type 1, abeta-lipoproteinemia, lysosomal storage diseases, e.g., I-cell disease/pseudohuler disease, mucopolysaccharidosis, sandhoff/tay-sachs disease, creutzfeldt-jakob syndrome type II, polyendocrinopathy/hyperinsulinemia, diabetes, larch dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, glycanosis CDG1, hereditary emphysema, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, and multiple sclerosis, ACT deficiency, Diabetes Insipidus (DI), neurogenic DI, renal DI, Charcot-Marie-Turkey syndrome, Peyer-Meyer's disease, neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, pick's disease, several polyglutamine neurological disorders, such as Huntington's disease, spinocerebellar ataxia type I, spinobulbar muscular atrophy, dentatorubral pallidoluysian atrophy and myotonic dystrophy, and spongiform encephalopathies, such as hereditary pick-Yaya disease, Fabry disease, Straussler-Scheinker syndrome, COPD, xerophthalmia, pancreatic insufficiency, osteoporosis, osteopenia, Gorham syndrome, chloride channel disease, congenital myotonia (Thomson and Becker types), Batt syndrome type III, Dent disease, panic syndrome, epilepsy, or epilepsy, Shock syndrome, lysosomal storage disease, Angelman syndrome, Primary Ciliary Dyskinesia (PCD), PCD with side-to-side transposition (also known as catagory syndrome), PCD without side-to-side transposition and ciliary dysplasia, and sjogren's disease.

In one embodiment, the present invention provides a method of treating cystic fibrosis in a human comprising administering to the human an effective amount of form A-HCl, form B-HCl, or any combination of these forms.

In one aspect, the invention features a pharmaceutical pack or kit that includes form a-HCl, form B-HCl, or any combination of these forms, and a pharmaceutically acceptable carrier.

Other aspects of the invention

Use, formulation and administration

Pharmaceutically acceptable compositions

In one aspect of the invention pharmaceutically acceptable compositions are provided, wherein these compositions comprise form a-HCl, form B, or form B-HCl as described herein, and optionally comprise a pharmaceutically acceptable carrier, adjuvant, or excipient. In some embodiments, these compositions optionally further comprise one or more additional therapeutic agents.

As noted above, the pharmaceutically acceptable compositions of the present invention also comprise pharmaceutically acceptable carriers, adjuvants or excipients, which, as used herein, include any and all solvents, diluents or other liquid carriers, dispersing or suspending agents, surface drugs, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as appropriate for the particular dosage form desired. Remington's Pharmaceutical Sciences, 16 th edition, e.w. martin (mack publishing co., Easton, Pa.,1980) describe various carriers used in formulating pharmaceutically acceptable compositions and known techniques for preparing them. In addition to any conventional carrier media which is incompatible with the compounds of the present invention, e.g., by causing any undesirable biological effect or by interfering in a deleterious manner with any other component of the pharmaceutically acceptable composition, the use of any conventional carrier media is also contemplated as falling within the scope of the present invention. Some examples of substances that can be used as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block copolymers, lanolin, sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc powder; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition, according to the judgment of the formulator.

Use of compounds and pharmaceutically acceptable compositions

In another aspect, the invention provides a method of treating or lessening the severity of a condition, disease or disorder associated with a mutation in CFTR. In some embodiments, the present invention provides a method of treating a condition, disease or disorder associated with a deficiency in CFTR activity, the method comprising administering to a subject, preferably a mammal, in need thereof a composition comprising form a-HCl, form B-HCl, or any combination of these forms, as described herein.

In some embodiments, the invention provides a method of treating a disease associated with reduced CFTR function due to a mutation in a gene encoding CFTR or environmental factors (e.g., smoking). These include cystic fibrosis, asthma, COPD due to smoking, chronic bronchitis, rhinosinusitis, constipation, pancreatitis, pancreatic insufficiency, male sterility due to congenital bilateral absence of the vas deferens (CBAVD), mild lung disease, idiopathic pancreatitis, allergic bronchopulmonary aspergillosis (ABPA), liver disease, hereditary emphysema, hereditary hemochromatosis, coagulation-fibrinolysis defects, such as protein C deficiency, hereditary angioedema type 1, lipid processing defects, such as familial hypercholesterolemia, chylomicronemia type 1, abeta-lipoproteinemia, lysosomal storage diseases, such as I-cell disease/pseudohuler disease, mucopolysaccharidosis, Hoff disease/Tay-Saccharitis, Creutzfeldt-Naja syndrome type II, polyendocrine disease/hyperinsulinemia, Diabetes, larn dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, glyceryosis CDG type 1, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency, Diabetes Insipidus (DI), neurogenic DI, renal DI, summer-horse-tourette syndrome, peyronie-meiosis, neurodegenerative diseases, such as alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, pick's disease, several polyglutamine neurological disorders, such as huntington's disease, spinocerebellar ataxia type I, spinobulbar muscular atrophy, dentatorubral-pallidoluysian atrophy and myotonic dystrophy, and spongiform encephalopathies, such as hereditary creutzfeldt-jakob disease (due to prion protein processing defects), Fabry disease, Straussler-Scheinker syndrome, COPD, dry eye or sjogren's disease, osteoporosis, osteopenia, bone healing and bone development (including bone repair, bone regeneration, decreased bone resorption and increased bone deposition), Gorham syndrome, chloride channel disorders, such as congenital myotonia (Thomson and Becker types), barter syndrome type III, Dent's disease, startle syndrome, epilepsy, startle syndrome, lysosomal storage diseases, Angelman syndrome, and Primary Ciliary Dyskinesia (PCD), which term is used to refer to genetic disorders of ciliary structure and/or function, including PCD with left and right transposition (also known as latag syndrome), PCD free of left and right transposition and ciliary dysplasia.

In certain embodiments, the method comprises treating or lessening the severity of cystic fibrosis in a patient, said method comprising administering to said patient one of the compositions as defined herein. In certain embodiments, the patient has a mutant form of human CFTR. In other embodiments, the patient has one or more of the following mutations: Δ F508, R117H and G551D mutations of human CFTR. In one embodiment, the method comprises treating or lessening the severity of cystic fibrosis in a patient having the Δ F508 mutation in human CFTR, said method comprising administering to said patient one of the compositions as defined herein. In one embodiment, the method comprises treating or lessening the severity of cystic fibrosis in a patient having the G551D mutation of human CFTR, said method comprising administering to said patient one of the compositions as defined herein. In one embodiment, the method comprises treating or lessening the severity of cystic fibrosis in a patient having the Δ F508 mutation in human CFTR on at least 1 allele, said method comprising administering to said patient one of the compositions as defined herein. In one embodiment, the method comprises treating or lessening the severity of cystic fibrosis in a patient having the af 508 mutation of human CFTR on 2 alleles, said method comprising administering to said patient one of the compositions as defined herein. In one embodiment, the method comprises treating or lessening the severity of cystic fibrosis in a patient having the G551D mutation of human CFTR on at least 1 allele, said method comprising administering to said patient one of the compositions as defined herein. In one embodiment, the method comprises treating or lessening the severity of cystic fibrosis in a patient having the G551D mutation of human CFTR on 2 alleles, said method comprising administering to said patient one of the compositions as defined herein.

In certain embodiments, the method comprises reducing the severity of cystic fibrosis in a patient, said method comprising administering to said patient one of the compositions as defined herein. In certain embodiments, the patient has a mutant form of human CFTR. In other embodiments, the patient has one or more of the following mutations: Δ F508, R117H and G551D mutations of human CFTR. In one embodiment, the method comprises reducing the severity of cystic fibrosis in a patient having a Δ F508 mutation in human CFTR, said method comprising administering to said patient one of the compositions as defined herein. In one embodiment, the method comprises reducing the severity of cystic fibrosis in a patient having the G551D mutation of human CFTR, said method comprising administering to said patient one of the compositions as defined herein. In one embodiment, the method comprises reducing the severity of cystic fibrosis in a patient having a Δ F508 mutation in human CFTR on at least 1 allele, said method comprising administering to said patient one of the compositions as defined herein. In one embodiment, the method comprises reducing the severity of cystic fibrosis in a patient having the af 508 mutation of human CFTR on 2 alleles, said method comprising administering to said patient one of the compositions as defined herein. In one embodiment, the method comprises reducing the severity of cystic fibrosis in a patient having the G551D mutation of human CFTR on at least 1 allele, said method comprising administering to said patient one of the compositions as defined herein. In one embodiment, the method comprises reducing the severity of cystic fibrosis in a patient having the G551D mutation of human CFTR on 2 alleles, said method comprising administering to said patient one of the compositions as defined herein.

In certain aspects, the present invention provides a method of treating or lessening the severity of osteoporosis in a patient, comprising administering compound I as described herein to said patient.

In certain embodiments, a method of treating or lessening the severity of osteoporosis in a patient comprises administering to said patient a pharmaceutical composition as described herein.

In certain aspects, the present invention provides a method of treating or lessening the severity of osteopenia in a patient, comprising administering compound I as described herein to said patient.

In certain embodiments, a method of treating or lessening the severity of osteopenia in a patient comprises administering to said patient a pharmaceutical composition as described herein.

In certain aspects, the present invention provides a method of bone healing and/or bone repair in a patient, the method comprising administering to the patient compound I as described herein.

In certain embodiments, a method of bone healing and/or bone repair in a patient comprises administering to the patient a pharmaceutical composition as described herein.

In certain aspects, the present invention provides a method of reducing bone resorption in a patient, comprising administering to the patient compound I as described herein.

In certain embodiments, a method of reducing bone absorption in a patient comprises administering to the patient a pharmaceutical composition as described herein.

In certain aspects, the present invention provides a method of increasing bone deposition in a patient, comprising administering to the patient compound I as described herein.

In certain embodiments, a method of increasing bone deposition in a patient comprises administering to the patient a pharmaceutical composition as described herein.

In certain aspects, the present invention provides a method of treating or lessening the severity of COPD in a patient comprising administering compound I as described herein to said patient.

In certain embodiments, a method of treating or lessening the severity of COPD in a patient comprises administering to said patient a pharmaceutical composition as described herein.

In certain aspects, the present invention provides a method of treating or lessening the severity of COPD caused by smoking in a patient, comprising administering to said patient compound I as described herein.

In certain embodiments, a method of treating or lessening the severity of COPD caused by smoking in a patient comprises administering to said patient a pharmaceutical composition as described herein.

In certain aspects, the present invention provides a method of treating or lessening the severity of chronic bronchitis in a patient, comprising administering to said patient compound I as described herein.

In certain embodiments, a method of treating or lessening the severity of chronic bronchitis in a patient comprises administering to said patient a pharmaceutical composition as described herein.

In some embodiments, the invention provides a method of treating a disease associated with normal CFTR function. These diseases include Chronic Obstructive Pulmonary Disease (COPD), chronic bronchitis, recurrent bronchitis, acute bronchitis, rhinosinusitis, constipation, pancreatitis, including chronic pancreatitis, recurrent pancreatitis and acute pancreatitis, pancreatic insufficiency, male sterility due to congenital bilateral absence of the vas deferens (CBAVD), mild pulmonary disease, idiopathic pancreatitis, liver disease, hereditary emphysema, gallstones, gastroesophageal reflux disease, gastrointestinal malignancies, inflammatory bowel disease, constipation, diabetes, arthritis, osteoporosis, and osteopenia.

In some embodiments, the invention provides a method of treating a disease associated with normal CFTR function, including hereditary hemochromatosis, coagulation-fibrinolysis deficiencies, e.g., protein C deficiency, hereditary angioedema type 1, lipid processing deficiencies, e.g., familial hypercholesterolemia, chylomicronemia type 1, abetalipoproteinemia, lysosomal storage diseases, e.g., I-cell disease/pseudohulerosis, mucopolysaccharidoses, sandhoff/tay-sachs disease, creutzfeldt-jakob syndrome type II, polyendocrinopathy/hyperinsulinemia, diabetes, larron dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, glycanosis CDG type 1, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, diabetes, chronic hypoproteinemia, chronic myelodysplasia, chronic myelopathy, ACT deficiency, Diabetes Insipidus (DI), neurogenic DI, renal DI, Charcot-Marie-Tourette syndrome, Pepper-Meyer's disease, neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, pick's disease, several polyglutamine neurological disorders, such as Huntington's disease, spinocerebellar ataxia type I, spinobulbar muscular atrophy, dentatorubral pallidoluysian atrophy, and myotonic dystrophy, and spongiform encephalopathies, such as hereditary Creutzfeldt-Jakob disease (caused by defects in prion protein processing), Fabry disease, Straussler-Scheinker syndrome, Gorham syndrome, chloride channel disease, congenital myotonia (Thomson and Becker types), Gorhatt syndrome type III, Dent disease, shock syndrome, epilepsy, startle syndrome, lysosomal storage disease, lysosomal diseases, Angelman syndrome, Primary Ciliary Dyskinesia (PCD), PCD with left and right transposition (also known as catagory syndrome), PCD without left and right transposition and ciliary dysplasia, or sjogren's disease, the method comprising the step of administering to the mammal an effective amount of form a-HCl, form B-HCl or any combination of these forms as described herein.

According to an alternative preferred embodiment, the present invention provides a method of treating cystic fibrosis comprising the step of administering to said mammal a composition comprising form a-HCl, form B-HCl or any combination of these forms as described herein, in an effective amount, comprising the step of administering to said mammal an effective amount of a composition.

According to the present invention, an "effective amount" of form a-HCl, form B-HCl, or any combination of these forms or a pharmaceutically acceptable composition is an amount effective to treat or reduce the severity of one or more diseases, disorders, or conditions as described above.

Form a-HCl, form B-HCl, or any combination of these forms, or a pharmaceutically acceptable composition thereof, can be administered in any amount and by any route of administration effective to treat or reduce the severity of one or more diseases, disorders, or conditions as described above.

In some embodiments, form a-HCl, form B-HCl, or any combination of these forms, or a pharmaceutically acceptable composition thereof, is suitable for treating or lessening the severity of cystic fibrosis in a patient who displays residual CFTR activity in the apical membrane of the respiratory epithelium or non-respiratory epithelium. The presence of residual CFTR activity on the surface of epithelial cells can be readily detected using methods known in the art, such as standard electrophysiological, biochemical or histochemical techniques. These methods use in vivo or ex vivo electrophysiological techniques, sweat or saliva Cl^-Concentration measurements or ex vivo biochemical or histochemical techniques to monitor cell surface density to identify CFTR activity. Using these methods, residual CFTR activity can be readily detected in patients who are heterozygous or homozygous for a variety of different mutations, including patients who are heterozygous or homozygous for the most common mutation, af 508.

In another embodiment, the form a-HCl, form B-HCl, or any combination of these forms, or a pharmaceutically acceptable composition thereof, described herein is suitable for treating or lessening the severity of cystic fibrosis in a patient having residual CFTR activity induced or enhanced with a pharmacological method or gene therapy. These methods increase the amount of CFTR present on the cell surface, thereby inducing a hitherto absent CFTR activity in the patient or increasing the existing level of residual CFTR activity in the patient.

In one embodiment, the forms A-HCl, form B-HCl, or any combination of these forms, or a pharmaceutically acceptable composition thereof, described herein are suitable for treating or lessening the severity of cystic fibrosis in a patient having certain genotypes that exhibit residual CFTR activity, e.g., class III mutations (impaired regulation or gating), class IV mutations (altered conductance), or class V mutations (reduced synthesis) (Lee R.Choo-Kang, Pamela L., Zeitin, Type L, II, III, IV, and V cytological fibrosis transmembrane Regulator Defects and Opportunities of Therapy; Currentopinion in Pulmonary Medicine 6: 521-. Other patient genotypes that exhibit residual CFTR activity include patients that are homozygous for one of these types or heterozygous for any other type of mutation, including a class I mutation, a class II mutation, or a mutation without classification.

In one embodiment, the form a-HCl, form B-HCl, or any combination of these forms, or a pharmaceutically acceptable composition thereof, described herein is suitable for treating or lessening the severity of cystic fibrosis in a patient having certain clinical phenotypes, e.g., a mild to moderate clinical phenotype typically associated with an amount of residual CFTR activity in the apical membrane of epithelial cells. These phenotypes include patients exhibiting pancreatic insufficiency or diagnosed with idiopathic pancreatitis and congenital bilateral loss of the vas deferens or mild lung disease.

The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, the severity of the infection, the particular drug, its mode of administration, and the like. The compounds of the present invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein refers to physically discrete units of a drug suitable for the patient to be treated. However, it will be understood that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific effective dosage level for any particular patient or organism will depend upon a variety of factors including the disease to be treated and the severity of the disease; the activity of the particular compound used; the specific composition used; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the particular compound used; the duration of the treatment; drugs used in combination or concomitantly with the specific compound employed, and other factors well known in the medical arts. The term "patient" as used herein refers to an animal, preferably a mammal, most preferably a human.

The pharmaceutically acceptable compositions of the present invention may be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (e.g., by powders, ointments, drops, or patches), bucally, and as an oral or nasal spray, depending on the severity of the infection to be treated. In some embodiments, the compounds of the present invention may be administered orally or parenterally at dosage levels of from about 0.01mg/kg to about 50mg/kg, preferably from about 0.5mg/kg to about 25mg/kg, of the subject's body weight once or more times daily to achieve the desired therapeutic effect.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents, for example, sterile injectable water or oily suspensions. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Acceptable excipients or solvents that may be used include water, ringer's solution, u.s.p. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids (e.g., oleic acid) may be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

In order to prolong the effect of the compounds of the invention, it is often desirable to slow the absorption of the compounds injected subcutaneously or intramuscularly. This can be achieved by using a liquid suspension of crystalline or amorphous material which is poorly water soluble. The rate of absorption of the compound then depends on its dissolution rate, which in turn may depend on the crystal size and crystalline form. Alternatively, delaying absorption of a parenterally administered compound form may be achieved by dissolving or suspending the compound in an oily vehicle. Injectable depot dosage forms (depot forms) are prepared by forming microencapsule matrices of the compounds in biodegradable polymers, such as polylactide-polyglycolide. Depending on the ratio of compound to polymer and the nature of the particular polymer used, the release rate of the compound can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injections can also be prepared by entrapping the compound in liposomes or microemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of the invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or suppository waxes which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and/or a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) dissolution retarders, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) wetting agents, such as cetyl alcohol and glycerol monostearate, h) absorbents, such as kaolin and bentonite, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees (dragees), capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents, and may also be of a composition: they release one or more active ingredients only or preferably in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

The active compound may also be present in microencapsulated form with one or more of the above-mentioned excipients. Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings, controlled release coatings, and other coatings well known in the pharmaceutical formulating art. In these solid dosage forms, the active compound may be mixed with at least one inert diluent, for example sucrose, lactose or starch. These dosage forms may also, for example, contain, as is common practice, other substances than inert diluents, e.g., tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents, and may also be of a composition: they release one or more active ingredients only or preferably in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymers and waxes.

Dosage forms for topical or transdermal administration of the compounds of the invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives or buffers which may be required. Ophthalmic formulations, ear drops and eye drops are also encompassed within the scope of the present invention. Furthermore, the present invention encompasses the use of transdermal patches, which have the additional advantage of controllably delivering the compound to the body. Such dosage forms may be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers may also be used to increase the flux of the compound through the skin. The rate can be controlled by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

It will also be appreciated that form a-HCl, form B-HCl, or any combination of these forms, or a pharmaceutically acceptable composition thereof, described herein may be used in combination therapy, that is, form a-HCl, form B-HCl, or any combination of these forms, or a pharmaceutically acceptable composition thereof, described herein may be administered simultaneously, prior to, or after one or more additional desired treatments or medical procedures. The particular combination of treatments (treatments or manipulations) used in a combination treatment regimen will take into account the compatibility of the desired therapeutic effect to be achieved with the desired treatment and/or manipulation. It will also be appreciated that the treatments used may achieve the desired effect on the same disease (e.g. the compounds of the invention may be administered simultaneously with another drug used to treat the same disease), or they may achieve different effects (e.g. control of any adverse effects). As used herein, an additional therapeutic agent that treats or prevents a particular disease or condition by normal administration is referred to as "appropriate for the disease or condition being treated.

In one embodiment, the additional agent is selected from the group consisting of mucolytics (mucolytics), bronchodilators, antibiotics, anti-infective agents, anti-inflammatory agents, CFTR modulators or nutritional agents other than the compounds of the invention.

In one embodiment, the additional agent is an antibiotic. Typical antibiotics for use herein include tobramycin, including Tobramycin Inhalation Powder (TIP), azithromycin, aztreonam, aerosol forms including aztreonam, amikacin, including liposomal formulations thereof, ciprofloxacin, including formulations thereof suitable for administration by inhalation, levofloxacin, including aerosol formulations thereof, and combinations of two antibiotics, such as fosfomycin and tobramycin.

In another embodiment, the additional agent is a mucolytic (mucolytics). Exemplary mucolytics for use herein include

In another embodiment, the additional agent is a bronchodilator. Typical bronchodilators include salbutamol, metaproterol sulfate, pirbuterol acetate, salmeterol or tetrabulin sulfate.

In another embodiment, the additional agent is effective to restore lung airway surface liquid. This drug improves the movement of the salt inside and outside the cell, making the mucus in the lung airways more easily hydrated and thus more easily cleared. Typical such drugs include hypertonic saline, denufosol sodium ([ [ (3S,5R) -5- (4-amino-2-oxopyrimidin-1-yl) -3-hydroxyoxolanyl-2-yl ] methoxy-hydroxyphosphoryl ] [ [ [ (2R,3S,4R,5R) -5- (2, 4-dioxopyrimidin-1-yl) -3, 4-dihydroxyoxolanyl-2-yl ] methoxy-hydroxyphosphoryl ] oxy-hydroxyphosphoryl ] hydrogen phosphate) or bronchitol (inhalation formulation of mannitol).

In another embodiment, the additional agent is an anti-inflammatory agent, i.e. an agent that can reduce inflammation in the lungs. Typical such drugs for use herein include ibuprofen, docosahexaenoic acid (DHA), sildenafil, inhaled glutathione, pioglitazone, hydroxychloroquine or simvastatin (simavastatin).

In another embodiment, the additional agent decreases the activity of an epithelial sodium channel blocker (ENaC) (either directly by blocking the channel or indirectly by modulating a protease (e.g., serine protease, channel activating protease) that results in an increase in ENaC activity). Typical such agents include camostat (a trypsin-like protease inhibitor), QAU145, 552-02, GS-9411, INO-4995, Aerolytic and amiloride. Additional agents that reduce the activity of epithelial sodium channel blockers (enacs) can be found, for example, in PCT publication No. WO2009/074575, the entire contents of which are incorporated herein by reference in their entirety.

In other diseases described herein, CFTR modulators, e.g., form B-HCl, and form A-HCl, in combination with agents that reduce ENaC activity are useful in the treatment of Liddle syndrome, inflammatory or allergic conditions, including cystic fibrosis, primary ciliary dyskinesia, chronic bronchitis, chronic obstructive pulmonary disease, asthma, respiratory infections, lung cancer, xerostomia and keratoconjunctivitis sicca (keratojunctionality si re), respiratory infections (acute and chronic; viral and bacterial), and lung cancer.

The combination of CFTR modulators, such as form B, form B-HCl and form a-HCl, with agents that reduce ENaC activity is also useful in the treatment of diseases that block epithelial sodium channel-mediated, including diseases other than respiratory diseases, that are associated with dysregulation of fluid through the epithelium, possibly involving physiological abnormalities of protective surface fluids on its surface, such as xerostomia (dry mouth) or keratoconjunctivitis sicca (dry eye). Furthermore, blocking the epithelial sodium channel in the kidney may be used to promote diuresis and thereby induce a hypotensive effect.

Asthma includes intrinsic (non-allergic) asthma and extrinsic (allergic) asthma, mild asthma, moderate asthma, severe asthma, bronchial asthma, exercise-induced asthma, occupational asthma and asthma induced following bacterial infection. Treatment of asthma is also to be understood as including treatment of subjects below 4 or 5 years of age exhibiting wheezing symptoms and diagnosed or diagnosable as "wheezy infants", established patient categories of major medical concern and patients currently identified as typically being initially or early wheezy (for convenience this particular asthmatic condition is referred to as "wheezy infant syndrome"). Prophylactic efficacy in the treatment of asthma is evidenced by a reduction in the frequency or severity of symptomatic attack, e.g., of acute asthma or bronchoconstrictor attack, improvement in lung function, or improvement in airway hyperreactivity. The efficacy may further be evidenced by a reduced need for other palliative therapies, i.e., therapies used or expected to limit or interrupt the onset of symptoms at the time of onset, such as anti-inflammatory (e.g., corticosteroids) or bronchodilation. In particular, prophylactic benefit in asthma is evident in subjects prone to "morning dipping". "morning dipping" is a recognized asthma syndrome, common to a large percentage of asthmatic patients and characterized by asthma attacks, e.g., a few hours between about 4-6am, i.e., at a time generally substantially distant from any previously administered palliative asthma therapy.

Chronic obstructive pulmonary diseases include chronic bronchitis or dyspnea associated therewith, emphysema, and exacerbation of airway hyperreactivity following other drug therapies, particularly other inhaled drug therapies. In some embodiments, a CFTR modulator, e.g., form B-HCl, and form a-HCl, in combination with an agent that decreases ENaC activity is used to treat bronchitis, regardless of type or origin, including, e.g., acute bronchitis, arachidic bronchitis, catarrhal bronchitis, croupus bronchitis, chronic bronchitis, or tuberculous bronchitis.

In another embodiment, the additional agent is a CFTR modulator other than form B, form B-HCl, and form A-HCl, i.e., an agent that has the effect of modulating CFTR activity. Typical such drugs include astalulan (r) ((r))3- [5- (2-fluorophenyl) -1,2, 4-oxadiazol-3-yl]Benzoic acid), sinapultide, lanokift, dilactat (human recombinant neutrophil elastase inhibitor), coprostane (7- { (2R,4aR,5R,7aR) -2- [ (3S) -1, 1-difluoro-3-methylpentyl]-2-hydroxy-6-oxooctahydrocyclopenta [ b]Pyran-5-yl } heptanoic acid) or (3- (6- (1- (2, 2-difluorobenzo [ d ]][1,3]Dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid. In another embodiment, the additional agent is (3- (6- (1- (2, 2-difluorobenzo [ d ]))][1,3]Dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid.

In another embodiment, the additional pharmaceutical agent is a nutraceutical agent. Typical such drugs include pancrelipase (pancreatic enzyme substitute), including enteric pancreatin capsulesOr Dieto(prior one))、Or glutathione inhalation. In one embodiment, the other nutritional agent is pancrelipase.

In one embodiment, the additional agent is a CFTR modulator of a compound of the invention.

The amount of the other therapeutic agent in the compositions of the present invention will not exceed the amount normally administered in compositions containing the therapeutic agent as the only active agent. Preferably, the amount of the other therapeutic agent in the presently disclosed compositions will be about 50% to 100% of the content in a typical composition containing the therapeutic agent as the sole therapeutic agent.

The form a-HCl, form B-HCl, or any combination of these forms, or a pharmaceutically acceptable composition thereof, described herein may also be incorporated into compositions for coating implantable medical devices, such as prostheses, prosthetic valves, vascular grafts, stents (stents), and catheters. Thus, the present invention, in another aspect, includes a composition for coating an implantable device comprising a compound of the present invention as generally described above and in the classes and subclasses herein, and a carrier suitable for coating said implantable device. In another aspect, the present invention includes an implantable device coated with a composition comprising a compound of the present invention as generally described above and in the classes and subclasses herein, and a carrier suitable for coating the implantable device. General preparation of suitable coatings and coated implantable devices is described in U.S. Pat. nos. 6,099,562, 5,886,026, and 5,304,121. The coating is typically a biocompatible polymeric material such as hydrogel polymers, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate and mixtures thereof. The coating may optionally be further covered with a surface layer of a suitable fluorosilicone, polysaccharide, polyethylene glycol, phospholipid, or combinations thereof to impart controlled release characteristics to the composition.

Another aspect of the invention relates to modulating CFTR activity (e.g., in vitro or in vivo) in a biological sample or patient, comprising administering or contacting the biological sample with form a-HCl, form B-HCl, or any combination of these forms, or a pharmaceutically acceptable composition thereof, as described herein. The term "biological sample" as used herein includes, without limitation, cell cultures and extracts thereof; biopsy material obtained from a mammal or an extract thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.

Modulation of CFTR in a biological sample can be used for a variety of purposes known to those skilled in the art. Examples of such purposes include, but are not limited to, the study of CFTR in biological and pathological phenomena; and comparative evaluation of novel CFTR modulators.

In another embodiment, there is provided a method of modulating the activity of an anion channel in vitro or in vivo, comprising the step of contacting the channel with form a-HCl, form B-HCl, or any combination of these forms, or a pharmaceutically acceptable composition thereof, as described herein. In a preferred embodiment, the anion channel is a chloride channel or a bicarbonate channel. In other preferred embodiments, the anion channel is a chloride channel.

According to an alternative embodiment, the present invention provides a method of increasing the number of functional CFTR in a cell membrane, said method comprising the step of contacting said cell with form a-HCl, form B-HCl or any combination of these forms or a pharmaceutically acceptable composition thereof as described herein.

According to another preferred embodiment, the activity of CFTR is determined by measuring the transmembrane voltage potential. The means for measuring the voltage potential across the membrane in a biological sample may employ any method known in the art, such as optical membrane potentiometry or other electrophysiological methods.

Optical membrane potential assays employ voltage-sensitive FRET sensors as described by Gonzalez and Tsien (see Gonzalez, J.E., and R.Y.Tsien (1995) "Voltage sensing by fluorescence response energy transfer in single cells," Biophys J69 (4):1272-80 and Gonzalez, J.E.and R.Y.Tsien (1997), "Improved indicators of fluorescence response energy transfer" Chem Biol 4(4):269-77) in combination with an instrument for measuring fluorescence change, such as a Voltage/ion Probe Reader (PR) (see Gonzalez, J.E., K.Oadades et al (1999) "Cell analysis and discharge analysis" 439).

These voltage-sensitive assays are based on the membrane-soluble, voltage-sensitive dye DiSBAC₂(3) Change in Fluorescence Resonance Energy Transfer (FRET) with the fluorescent phospholipid CC2-DMPE, which is attached to the outer leaflet of the plasma membrane and serves as a FRET donor. Membrane potential (V)_m) Resulting in a negatively charged DiSBAC₂(3) Redistributed across the plasma membrane, the amount of energy transferred from the CC2-DMPE changed accordingly. The change in fluorescence emission can be made using VIPR^TMII monitoring, VIPR^TMII is an integrated liquid processor and fluorescence detector, designed for 96-or 384-well microtiter plate based cell screening.

In another aspect, the present invention provides a kit for determining the activity of CFTR or a fragment thereof in a biological sample in vitro or in vivo, the kit comprising: (i) a composition comprising form a-HCl, form B-HCl, or any combination of these forms or any of the above embodiments; and (ii) instructions for: a) contacting the composition with the biological sample; and b) determining the activity of said CFTR or fragment thereof. In one embodiment, the kit further comprises instructions for: a) contacting an additional compound with the biological sample; b) determining the activity of the CFTR or fragment thereof in the presence of the additional compound; and c) comparing the activity of CFTR in the presence of the additional compound with the activity of CFTR in the presence of form A-HCl, form B-HCl, or any combination of these forms, as described herein. In one embodiment, the step of comparing the activity of the CFTR or fragment thereof provides a method or measure of determining the density of the CFTR or fragment thereof. In a preferred embodiment, the kit is used to determine the density of CFTR.

In one aspect, the invention includes a process for preparing a compound of formula 9a

Or a pharmaceutically acceptable salt thereof, comprising contacting trans-4-aminocyclohexanol with Boc anhydride to produce a compound of formula a

Contacting a compound of formula A with methanesulfonic acid to produce a compound of formula B

Contacting a compound of formula B with trifluoroacetic acid to produce a compound of formula C

Contacting the compound of formula C with a hydroxide to produce a compound of formula 9 a.

In another embodiment, the method further comprises contacting the compound of formula 9a with hydrochloric acid to produce the compound of formula 9.

In order that the invention described herein may be more fully understood, the following examples are provided. It should be understood that these examples are for illustration only and are not to be construed as limiting the invention in any way.

Examples

Method and material

XRPD (X-ray powder diffraction)

Apparatus 1

X-ray powder diffraction (XRPD) data were recorded at room temperature using a Rigaku/MSC MiniFlex Desktop powder X-ray diffractometer (Rigaku, The Woodlands, TX). X-rays were generated using a Cu tube operating at 30kV and 15mA with a K β suppression filter. The divergence slit was variable with the scattering and receiving slits set at 4.2 degrees and 0.3mm slit, respectively. The scan pattern is a Fixed Time (FT) using 0.02 degree steps and a 2.0 second count time. Calibration of the powder X-ray diffractometer using reference standards: 75% sodalite (Na)₃Al₄Si₄O₁₂Cl) and 25% silicon (Rigaku, Cat # 2100/ALS). A6-sample station (SH-LBSI511-RNDB) with zero background sample racks was used. The powder sample was placed on the recessed area and pressed flat with a glass slide.

Apparatus 2

Alternatively, powder X-ray diffraction measurements were performed at room temperature using a PANALYTICAL's X-pert Pro diffractometer with copper radiation (1.54060A). The incident beam source (the incident beam optical) contained a variable divergence slit to ensure constant illumination length on the sample and on the diffracted light sheet (side). The active length measured in scan mode using a fast linear solid state detector was 2.12 degrees 2 θ. Powder samples were filled on the recessed areas of the zero background silicon mount and rotated for better statistics. A symmetric scan from 4-40 degrees 2 theta was performed with a step size of 0.017 degrees and a step scan time of 15.5 seconds.

Apparatus 3

Alternatively, high resolution data was acquired at room temperature on beamline ID31 (european synchrotron radiation device in gelnobu, france). X-rays were generated by an ultra (ex-) vacuum undulator with 3 11 mm slits. Tong (Chinese character of 'tong')The light is monochromatized by an overcooling twin crystal monochromator (Si (111) crystal). The water-cooled slit defines the size of the incident beam (beam absorbance) on the monochromator, and the size of the monochromatic light transmitted onto the sample, in the range of 0.5 to 2.5mm (horizontal) and 0.1 to 1.5mm (vertical). The wavelength used in the experiment isThe diffractometer consists of a series of (a bank of)9 detectors that scan vertically to measure the intensity of divergence as a function of 2 θ. Each detector is behind the Si (111) analyzer crystal with detector channels spaced approximately 2 ° apart. The diffractometer is capable of producing very accurate high resolution diffractograms with peak widths as low as 0.003 °, and peak positions with an accuracy of the order of 0.0001 °. The powder diffraction data was processed and indexed using materials studio (reflection module). The structure was resolved using a powder resolution module of Materials Studio. The resulting analysis was evaluated for structural viability and subsequently refined using Rietveld refinement method.

The XPRD spectra described in the form B embodiment were recorded using either instrument 1 (fig. 7A) or instrument 2 (fig. 7B) with the settings described above. The XPRD spectra described in the examples of form B-HCl and form A-HCl were recorded using instrument 2 with the settings described above. The crystal system, space group and unit cell parameters of form A-HCl and form B-HCl were determined using instrument 3.

Differential Scanning Calorimetry (DSC)

Differential Scanning Calorimetry (DSC) was performed using a TA DSC Q2000 differential scanning calorimeter (TA Instruments, NewCastle, DE). An indium calibration instrument was used. About 2-3mg of the sample was weighed and added to the sealing disk, which was covered with a lid having a hole. The DSC samples were scanned from 25 ℃ to 315 ℃ at a heating rate of 10 ℃/min. By ThermalAdvantage Q Series^TMData were collected by software and analyzed by the Universal Analysis software (tasinstruments, New Castle, DE).

Thermogravimetric analysis (TGA) method

Thermogravimetric analysis (TGA) data were collected on a TA Q500 thermogravimetric analyzer (TA Instruments, New Castle, DE). From 25 ℃ to 350 ℃, a sample weighing about 3-5mg was scanned at a heating rate of 10 ℃/min. Through Thermal Advantage Q Series^TMData were collected by software and analyzed by Universal Analysis software (TA Instruments, New Castle, DE).

FTIR spectroscopy

FTIR spectra were collected from a Thermo Scientific, Nicolet 6700FT-IR spectrometer with an intelligent orbital sampling compartment (total reflection attachment with multiple bounce-back reduction) and a diamond window at 45 degrees. The software for data collection and analysis was: omnic, 7.4. The acquisition settings were as follows:

a detector: DTGS KBr;

spectroscope: ge on KBr;

source: EverGlo IR;

scanning range: 4000-400 cm-1;

gain: 8.0;

optical speed: 0.6329 cm/sec;

pore diameter: 100, respectively;

the scanning times are as follows: 32, a first step of removing the first layer; and

resolution ratio: 4cm^-1

The powder sample was placed directly on the diamond crystal and pressure was applied to conform the sample surface to the diamond crystal surface. A background spectrum is collected and then a sample spectrum is collected.

Solid state nuclear magnetic spectroscopy

Solid state nuclear magnetic spectroscopy (SS NMR) spectra were obtained on a Bruker 400MHz proton frequency wide aperture chromatograph. Obtaining the proton relaxation longitudinal relaxation time by fitting proton saturation recovery data measured by protons to an exponential function: (¹H T₁). Using these values to set a carbon cross-polarization magic angle spinning experiment (¹³C CPMAS), which is typically set at 1.2x¹H T₁And 1.5x¹H T₁In the meantime. Carbon spectra were obtained using linear amplitude variation (from 50% to 100%) on proton channels and 100kHz TPPM decoupling over a 2 millisecond contact time. Typically the Magic Angle Spinning (MAS) speed is 15.0 kHz. The fluorine spectra were obtained using a direct polarization MAS experiment with proton decoupling. 100kHz TPPM decoupling was used. The cyclic delay is set to ≧ 5 ×¹⁹F T₁. Fluorine longitudinal relaxation time by fitting proton decoupling saturation recovery data from fluorine determination to an exponential function: (¹⁹F T₁). The carbon and fluorine spectra were externally compared using high magnetic field resonance with solid phase adamantane set at 29.5 ppm. Using this method, the carbon spectrum is indirectly referenced to tetramethylsilane at 0ppm and the fluorine spectrum is indirectly referenced to nitromethane at 0 ppm.

Preparation example 7-azabicyclo [2.2.1]Heptane hydrochloride (9).

Preparation of trans-4- (tert-butoxycarbonylamino) cyclohexanol (A), method 1. Sodium carbonate (920.2g, 8.682mol, 2 eq) was added to the reaction vessel followed by water (3.000L, 6 vol) and stirred. Dichloromethane (DCM, 4.000L, 4 vol) was added followed by trans-4-aminocyclohexanol (500.0g, 4.341mol) to give a biphasic reaction mixture which was stirred vigorously at rt. Then Boc₂A solution of O (947.4g, 997.3mL, 4.341mol, 1 eq) in DCM (2 volumes) was added quickly dropwise to the vessel and the resulting reaction mixture was stirred at room temperature overnight. The reaction mixture was then filtered and the filter cake was washed with water (2 × 8 volumes). The product was suction dried until it became a dense cake. The cake was then dried in a vacuum oven at 35 ℃ for 24 hours to give 830g of trans-4- (tert-butoxycarbonylamino) cyclohexanol (A) as a crystalline solid.

Preparation of trans-4- (tert-butoxycarbonylamino) cyclohexanol (A), method 2. Two 50L three-neck round bottom flasks were each equipped with a mechanical stirrer and a thermocouple. The flask was placed in a cooled bucket and each flask was then charged with water (8.87L) and trans-4-aminocyclohexanol (1479 g). After about 10 to 30 minutes, trans-4-aminocyclohexanol was dissolved and potassium carbonate (1774.6g) was added to each flask. After about 10 to 20 minutes, the potassium carbonate was dissolved and DCM (2.96L) was charged to each flask. Boc anhydride (3082.6g) in DCM (1479mL) was then added to each flask at a rate to maintain the temperature at 20 to 30 ℃. An ice/water bath was used to control the exotherm and accelerate the addition, which took about 1 to 2 hours. A suspension formed during the addition and the reaction mixture was warmed to room temperature and stirred overnight until the reaction was complete according to the disappearance of Boc anhydride. Heptane (6L) was then charged to each flask and the mixture was cooled to about 0 to 5 ℃. The solids were collected from each flask by filtration using the same filter. The combined solids were washed with heptane (6L) followed by water (8L). The solids were charged to a suitably sized kettle equipped with a mechanical stirrer. Water (12L) and heptane (6L) were added and the resulting suspension was mechanically stirred for 30 to 60 minutes. The solid was collected by filtration, then the filter was washed with water (8L) and heptane (8L), air dried on the filter for 3 days, then dried under vacuum at 30 to 35 ℃ to constant weight to provide the product as a white solid.

Preparation of trans-4- (tert-butoxycarbonylamino) cyclohexyl methanesulfonate (B), method 1. A12L flask was equipped with a nitrogen purge and mechanical stirrer. Trans-4- (tert-butoxycarbonylamino) cyclohexanol (750g, 3.484mol) was introduced followed by tetrahydrofuran (THF, 6.000L, 8 vol) and the mixture stirred. Triethylamine (370.2g, 509.9mL, 3.658mol, 1.05 eq.) was added and the mixture was cooled to 0 ℃. Methanesulfonyl chloride (419.0g, 283.1mL, 3.658mol, 1.05 eq.) was added carefully dropwise, keeping the temperature of the mixture below 5 ℃. After the addition, the mixture was stirred at 0 ℃ for 3h, then gradually warmed to room temperature (17 ℃) and stirred overnight (about 15 h). The mixture was quenched with water (6 vol) and stirred for 15 min. Ethyl acetate (EtOAc, 9.000L, 12 vol.) was added with constant stirringFor 15 minutes. The stirring was stopped, the mixture was allowed to stand for 10min and the aqueous phase was removed. 1N HCl (6 vol, 4.5L) was added and stirring continued for 15 min. The stirring was stopped and the aqueous phase was removed. Add 10% w/v NaHCO₃(4.5L, 6 vol.) the mixture was stirred for 10 minutes. The stirring was stopped and the aqueous phase was removed. Water (6 vol, 4.5L) was added and the mixture stirred for 10 min. The aqueous layer was removed and the organic layer was polished (polish) filtered and concentrated to 4 volumes. Heptane (5.5 vol, 4L) was added and the mixture was concentrated again to dryness to give 988g of trans-4- (tert-butoxycarbonylamino) cyclohexyl methanesulfonate.

Preparation of trans-4- (tert-butoxycarbonylamino) cyclohexyl methanesulfonate (B), method 2. A three-necked round bottom flask equipped with a mechanical stirrer, addition funnel, nitrogen inlet, thermocouple, and drying tube was placed into a cooled bucket. Trans-4- (tert-butoxycarbonylamino) cyclohexanol (2599g, 12.07mol, 1.0 eq), Tetrahydrofuran (THF) (20.8L) and triethylamine (1466g, 14.49mol, 1.2 eq) were added to the flask. The mixture was cooled with an ice-water bath and stirred. Methanesulfonyl chloride (1466g, 12.80mol, 1.06 eq) was added dropwise over a period of 1 hour through the addition funnel. Once the addition was complete, the cold water bath was removed and the reaction mixture was stirred until TLC indicated the starting material was consumed (about 30 minutes). The reaction mixture was then quenched with aqueous hydrochloric acid (223 mL HCl in 6.7L water) and EtOAc (10.4L). The mixture was stirred at room temperature for about 10 to 20 minutes and then transferred to a separatory funnel. The layers were separated and the aqueous layer was discarded. The organic layer was washed with water (2X 4.5L) and saturated aqueous sodium bicarbonate (1X 4.5L), and stirred with anhydrous magnesium sulfate for 5 to 10 minutes to dry. The mixture was filtered and the filter cake was washed with EtOAc (2X 600 mL). The combined eluate and filtrate were concentrated at 40 ℃ under reduced pressure, leaving a white solid. The solid was added to heptane (3L) and cooled in an ice/methanol cooling bucket. Heptane (5L) was added thereto, and the mixture was stirred at 0 to 5 ℃ for not less than 1 hour. The solid was then collected by filtration, washed with cold heptane (0 to 5 ℃,2 × 1.3L), and dried to constant weight at 40 ℃ under vacuum to provide the title compound. Note that: a jacketed reactor may be used in place of a round bottom flask with a cooling barrel and ice bath. Preparation of trans-4-aminocyclohexyl methanesulfonate (C), method 1. Trans-4- (tert-butoxycarbonylamino) cyclohexyl methanesulfonate (985g, 3.357mol) was introduced into a three-necked 12L flask equipped with a stirrer under nitrogen and a vent. DCM (1.970L, 2 volumes) was added at rt and stirring was started. Trifluoroacetic acid (TFA) (2.844kg, 1.922L, 24.94mol, 2 vol.) was added slowly to the mixture in two portions, 1L each. After the first addition, the mixture was stirred for 30min, followed by a second addition. The mixture was stirred at room temperature overnight (15h) to form a clear solution. 2-methyltetrahydrofuran (4 vol) was then added to the reaction mixture, which was stirred for 1 hour. The mixture was then carefully filtered in a hood and suction dried to yield 1100g of the TFA salt of trans-4-aminocyclohexylmethanesulfonate with excess TFA. Preparation of trans-4-aminocyclohexyl methanesulfonate (C), method 2. A 50L three neck round bottom flask was equipped with a mechanical stirrer, addition funnel and thermocouple and placed into a cooled bucket. Trans-4- (tert-butoxycarbonylamino) cyclohexyl methanesulfonate (3474g, 1.0 eq) and DCM (5.9L) were added to the flask. The resulting suspension was stirred at room temperature for 5 to 10 minutes, then trifluoroacetic acid (TFA, 5.9L) was added slowly through an addition funnel over a period of 2.5 hours to control the exotherm and gas release rate generated. The reaction mixture was stirred at room temperature overnight and then cooled to 15 ℃ to 20 ℃ using an ice water bath. 2-methyltetrahydrofuran (2-MeTHF, 11.8L) was then added via an addition funnel at a rate that maintained the internal temperature below 25 deg.C (about 1.5 hours). The initial 4-5L 2-MeTHF addition was exothermic. The resulting suspension was stirred for 1 hour. The solid was collected by filtration and then washed with 2-MeTHF (2X 2.2L) then dried to constant weight under vacuum at room temperature to provide the title compound as a white solid.

7-azabicyclo [2.2.1]Preparation of heptane hydrochloride (9), method 1. A TFA salt of trans-4-aminocyclohexylmethanesulfonate (200g, 650.9mmol) was introduced into a 3L three-neck flask, followed by addition of water (2.200L, 11 vol.). NaOH (78.11g, 1.953mol, 3 equivalents) was added slowly, the temperature of the reaction mixture was kept below 25 ℃ and the mixture was stirred overnight. DCM (1.4L, 7 vol) was then added, the mixture stirred and the organic layer separated.The aqueous layer was then extracted with more DCM (1.4L, 7 vol.) and the DCM layers were combined. HCl (108.5mL, 12M, 1.3020mol, 2 eq) was then added and the mixture was stirred for 30 minutes and then concentrated to dryness on a rotary evaporator. Acetonitrile (10 vol) was added and the mixture was concentrated. This was repeated 3 times until all traces of water were azeotropically removed to provide 7-azabicyclo [2.2.1]Heptane hydrochloride (9). The crude product was recrystallized from acetonitrile (10 volumes) to provide 7-azabicyclo [2.2.1] as a colorless crystalline solid]Heptane hydrochloride (9).¹H NMR(DMSO-d⁶)ppm 8.02-8.04(d)；7.23-7.31(m)；4.59(s)；3.31(s)；2.51-3.3(m)；1.63-1.75(m)；1.45-1.62(m)。

Note that the crude product can also be distilled at about 95 ℃ to 97 ℃ and further recrystallized to replace the addition of DCM for extraction.

Preparation of 7-azabicyclo [2.2.1] heptane hydrochloride (9), method 2. A 50L three-necked round bottom flask was equipped with a mechanical stirrer, addition funnel and thermocouple and placed into a heating mantle. Trans-4-aminocyclohexyl methanesulfonate trifluoroacetate (3000g, 1 eq.) and water (30L) were added to the flask. Since the addition was slightly exothermic, the mixture was stirred and 50% NaOH (2343g, 29.29mol, 3 equivalents) was added through the addition funnel at a rate to maintain the temperature below 25 ℃.

Once the NaOH addition was complete, the reaction mixture was stirred at room temperature overnight. The product is recovered by fractional distillation at reflux temperature (about 100 ℃) at an initial temperature of 95 to 98 ℃. The pH of each fraction was adjusted to 2 by addition of HCl and concentrated at 55 ℃ under reduced pressure to yield a thick paste. Acetonitrile (ACN 1.5L) was added and the resulting suspension was stirred for 30 minutes and then cooled to 0 to 5 ℃ for 1 hour. The solid was collected by filtration, washed with cold (0 to 5 ℃) ACN (2X 600mL) and dried to constant weight at 50 ℃ under vacuum.

A 22L three-necked round bottom flask was equipped with a mechanical stirrer, thermocouple, and condenser, and placed in a heating mantle. The collected solid (2382g), methanol (4.7L) and 2-MeTHF (4.7L) were added to the flask. The resulting suspension was stirred and heated to reflux (about 65 ℃). The reaction flask was transferred to a cooled bucket and the mixture was stirred. 2-MeTHF (4.7L) was then added over a period of 30 minutes through the addition funnel. The resulting suspension was cooled to 0 to 5 ℃ and stirred at this temperature for 30 minutes. The solid was collected by filtration, washed with cold (0 to 5 ℃)2-MeTHF (2X 600mL), then dried under vacuum at 55 ℃ to constant weight.

A 12L three-necked round bottom flask equipped with a mechanical stirrer, thermocouple, nitrogen inlet and condenser was placed in the heating mantle. The crude product (2079g) and ACN (6.2L) were added to the flask. The resulting suspension was stirred and heated to reflux (about 82 ℃ C.) for 30 minutes. The flask was transferred to a cooled bucket and the suspension was slowly cooled to 0 to 5 ℃ and held at this temperature for 1 hour. The solid was collected by filtration, washed with cold (0 to 5 ℃) ACN (3 × 600mL) and dried to constant weight at 55 ℃ under vacuum to provide the title product.

Example 1A: preparation of 4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (7).

Preparation of diethyl 2- ((2-chloro-5- (trifluoromethyl) phenylamino) methylene) malonate (4). 2-chloro-5- (trifluoromethyl) aniline (2) (200g, 1.023mol), diethyl 2- (ethoxymethylene) malonate (3) (276g, 1.3mol) and toluene (100mL) were mixed under nitrogen in a 1L three-necked round bottom flask equipped with a Diesbi condenser. The solution was heated to 140 ℃ with stirring and the temperature was maintained for 4 hours (h). The reaction mixture was cooled to 70 ℃ and hexane (600mL) was added slowly. The resulting slurry was stirred and warmed to room temperature. The solid was collected by filtration, washed with 10% ethyl acetate in hexane (2x400mL) and then dried under vacuum to afford the product as a white solid diethyl 2- ((2-chloro-5- (trifluoromethyl) phenylamino) methylene) malonate (4).¹HNMR(400MHz，DMSO-d⁶)11.28(d，J=13.0Hz，1H)，8.63(d，J=13.0Hz，1H)，8.10(s，1H)，7.80(d，J=8.3Hz，1H)，7.50(dd，J=1.5，8.4Hz，1H)，4.24(q，J=7.1Hz，2H)，4.17(q，J=7.1Hz，2H)，1.27(m，6H)。

Preparation of ethyl 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylate (5A) method 1. A1L three-necked flask was charged with Dow's heat carrier(200mL, 8mL/g) and degassed at 200 ℃ for 1 hour. The solvent was heated to 260 ℃ and diethyl 2- ((2-chloro-5- (trifluoromethyl) phenylamino) methylene) malonate (4) (25g, 0.07mol) was added in portions over 10 minutes (min). The resulting mixture was stirred at 260 ℃ for 6.5h and the resulting ethanol by-product was removed by distillation. The mixture was slowly cooled to 80 ℃. Hexane (150mL) was added slowly over 30 minutes, followed by another 200mL hexane addition. The slurry was stirred until it reached room temperature. The solid was filtered, washed with hexane (3 × 150mL), then dried under vacuum to provide ethyl 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylate (5A) as a tan solid.¹H NMR(400MHz，DMSO-d⁶)11.91(s，1H)，8.39(s，1H)，8.06(d，J=8.3Hz，1H)，7.81(d，J=8.4Hz，1H)，4.24(q，J=7.1Hz，2H)，1.29(t，J=7.1Hz，3H)。

Preparation of ethyl 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylate (5A) method 2. Compound 4(2000g, 5.468mol) was introduced into the reactor. The dow heat carrier (4.000L) was charged to the reactor and nitrogen purged overnight at room temperature. Then stirred and heated to 260 ℃. The formed EtOH was distilled off. The reaction was monitored and after 5.5 hours was complete, the reaction was essentially complete. The heat was removed and the reaction mixture was cooled to 80 ℃ and heptane (2.000L) was added. The mixture was stirred for 30 minutes. Heptane (6.000L) was added to the stirred mixture and stirring was continued overnight. The solid was filtered off, washed with heptane (4.000L) and dried in a vacuum oven at 50 ℃ to afford compound 6A.

Preparation of ethyl 4-oxo-5- (trifluoromethyl) -1H-quinoline-3-carboxylate (6). A5L three-necked flask was charged with ethyl 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylate (5A) (100g, 0.3mol), ethanol (1250mL, 12.5mL/g), and triethylamine (220mL, 1.6 mol). The vessel was then charged with 10g of 10% Pd/C (50% wet) at 5 ℃. The reaction was stirred vigorously at 5 ℃ for 20 hours under a hydrogen atmosphere, after which the reaction mixture was concentrated to a volume of about 150 mL. The product ethyl 4-oxo-5- (trifluoromethyl) -1H-quinoline-3-carboxylate (6) was used directly in the next step as a slurry with Pd/C.

Preparation of 4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (7). Ethyl 4-oxo-5- (trifluoromethyl) -1H-quinoline-3-carboxylate (6) (58g, 0.2mol, crude Pd/C containing reaction slurry) was suspended in NaOH (814mL of 5M, 4.1mol) in a 1L flask with a reflux condenser, heated at 80 ℃ for 18 hours, then at 100 ℃ for 5 hours. The reaction was heated by filtration through packed celite to remove Pd/C, and the celite was rinsed with 1n naoh. The filtrate was acidified to about pH 1 to give a thick white precipitate. The precipitate was filtered and then rinsed with water and cold acetonitrile. The solid was then dried under vacuum to provide 4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (7) as a white solid.¹H NMR(400.0MHz，DMSO-d⁶)15.26(s，1H)，13.66(s，1H)，8.98(s，1H)，8.13(dd，J=1.6，7.8Hz，1H)，8.06-7.99(m，2H)。

EXAMPLE 1B preparation of 4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (7) in a substituted manner And (4) preparing.

Preparation of 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (5B). Ethyl 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylate (5A) (1200g, 3.754mol) was charged to a reaction vessel, followed by addition of 2-propanol (1.200L) and water (7.200L), and stirring. Sodium hydroxide (600.6g, 7.508mol) and water (1.200L) were mixed and cooled to room temperature. Mixing the obtained mixtureCharged to a reaction vessel, which was then heated to 80 ℃ while stirring for 3.5 hours to produce a dark homogeneous mixture. After a further 1 hour, acetic acid (20% w/v of 9.599L, 31.97mol) was added via a dropping funnel over a period of 45 minutes. The reaction mixture was cooled at a rate of 6 ℃/hour while stirring to 22 ℃. The resulting solid was filtered and washed with water (3L) to yield a wet cake (1436 g). The filtrate was passed through a vacuum drying oven with nitrogen bleedDrying to give 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (5B) as a brown solid (1069 g). 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (5B) was purified by slurrying in 1.5L of methanol and stirring for 6 hours. It was then filtered and dried to provide 968.8g of purified 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (5B).

Preparation of 4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (7). 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (5B) (18.5g, 1.00 eq, limiting reagent) was charged to a reaction vessel and MeOH (118mL, 6.4 vol) was added with stirring under an inert atmosphere. Sodium methoxide (3.53g, 1.00 eq) was added dropwise to the reactor over a period of 10 minutes. The mixture was stirred until all solids were in solution (5-10 minutes). Palladium on carbon (2.7g, 0.03 eq) was then added to the reaction mixture. Potassium formate (10.78g, 2 eq) dissolved in MeOH (67mL, 3.6 vol) was added to the reaction mixture over a period of 30 minutes and stirred at room temperature for about 4.5 hours. The reaction was judged to be complete when 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid was not more than 1.0% relative to 4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (7). When the reaction was complete, the mixture was filtered through a pad of celite (the amount of celite used was approximately 2 times the amount of 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (5B) initially charged to the vessel) to remove solids. The celite cake was washed with MeOH (37mL, 2 volumes). The filtrate was charged to a clean reaction vessel and stirred. Acetic acid (7.22mL, 2 equivalents) was continuously charged to the stirred solution over a period of at least 45 minutes and the resulting slurry was stirred for 5-16 hours. The solid was filtered and the cake was washed with MeOH (56mL, 3 volumes), dried under suction, and then dried in vacuo to afford the product as a white/off-white solid.

Alternatively, the potassium formate reagent may be replaced with hydrogen gas.

Example 2A: 4- (7-azabicyclo [2.2.1]]Preparation of hept-7-yl) -2- (trifluoromethyl) aniline (11A) And (4) preparing.

7- [ 4-Nitro-3- (trifluoromethyl) phenyl]-7-azabicyclo [2.2.1]Preparation of heptane (10A), method 1. A solution of 4-fluoro-1-nitro-2- (trifluoromethyl) benzene (8) (6.0g, 28.69mmol) and triethylamine (8.7g, 12.00mL, 86.07mmol) in acetonitrile (50mL) was added under nitrogen to a solution containing 7-azabicyclo [2.2.1] ring]Heptane hydrochloride (9) (4.6g, 34.43mmol flask. the reaction flask was heated at 80 ℃ under nitrogen for 16 hours. then the reaction mixture was cooled and partitioned between water and dichloromethane. the organic layer was washed with 1M HCl, Na was used, and₂SO₄drying, filtering and concentrating to dryness. Purification by silica gel chromatography (0-10% ethyl acetate in hexanes) afforded 7- [ 4-nitro-3- (trifluoromethyl) phenyl as a yellow solid]-7-azabicyclo [2.2.1]Heptane.¹H NMR(400.0MHz，DMSO-d⁶)8.03(d，J=9.1Hz，1H)，7.31(d，J=2.4Hz，1H)，7.25(dd，J=2.6，9.1Hz，1H)，4.59(s，2H)，1.69-1.67(m，4H)，1.50(d，J=7.0Hz，4H)。

Preparation of 7- [ 4-nitro-3- (trifluoromethyl) phenyl ] -7-azabicyclo [2.2.1] heptane (10A), method 2. 4-fluoro-1-nitro-2- (trifluoromethyl) benzene (8) (901g, 4.309mol) was introduced under nitrogen into a 30L jacketed vessel with stirring together with sodium carbonate (959.1g, 9.049mol) and DMSO (5L, 5.5 vol). 7-azabicyclo [2.2.1] heptane hydrochloride (9) (633.4g, 4.740mol) was then added in portions to the vessel and the temperature gradually increased to 55 ℃. The reaction was monitored by HPLC and was considered complete when substrate <1% AUC. The mixture was then diluted with 10 volumes of EtOAc and washed 3 times with water (5.5 volumes). The organic layer was then concentrated to 4 volumes, cyclohexane was added and it was concentrated to 4 volumes. The process of adding cyclohexane and concentrating the resulting solution to 4 volumes was repeated until all EtOAc was removed, the total volume in the flask being about 4 volumes with cyclohexane. The reaction mixture was heated to 60 ℃ on a rotary evaporator for 30 minutes. The solution was then cooled to room temperature while stirring or spinning for 3 hours. All solids were then crystallized and concentrated to dryness to provide 7- [ 4-nitro-3- (trifluoromethyl) phenyl ] -7-azabicyclo [2.2.1] heptane (10A).

Preparation of 7- [ 4-nitro-3- (trifluoromethyl) phenyl ] -7-azabicyclo [2.2.1] heptane (10A), method 3. Tetrabutylammonium bromide (0.05 eq) and 50 wt% KOH (3.6 eq) were added to 4-fluoro-1-nitro-2- (trifluoromethyl) benzene (8) dissolved in 3 volumes of DCM. 7-azabicyclo [2.2.1] heptane hydrochloride (9) was then added at 0-5 ℃. The reaction was warmed to room temperature. The reaction was monitored by HPLC and considered complete when substrate <1% AUC, the layers were separated. The organic layer was washed with 1M HCl and the aqueous layer was discarded. The organic layer was then washed once with water, once with brine and evaporated. The resulting material was recrystallized from cyclohexane under reflux. The solid was filtered, washed with cyclohexane and dried in a vacuum oven at 45 ℃ under nitrogen to afford 7- [ 4-nitro-3- (trifluoromethyl) phenyl ] -7-azabicyclo [2.2.1] heptane.

4- (7-azabicyclo [2.2.1]]Preparation of hept-7-yl) -2- (trifluoromethyl) aniline (11A). Will be charged with 7- [ 4-nitro-3- (trifluoromethyl) phenyl]-7-azabicyclo [2.2.1]A flask of heptane (10A) (7.07g, 24.70mmol) and 10% Pd/C (0.71g, 6.64mmol) was evacuated and then filled with nitrogen. Ethanol (22mL) was added and the reaction flask was fitted with a hydrogen balloon. After stirring vigorously for 12 hours, the reaction mixture was purged with nitrogen and the Pd/C was removed by filtration. The filtrate was concentrated under reduced pressure to a dark oil which was purified by silica gel chromatography (0-15% ethyl acetate in hexane)Purification to provide 4- (7-azabicyclo [2.2.1] as a purple solid]Hept-7-yl) -2- (trifluoromethyl) aniline (11A).¹H NMR(400.0MHz，DMSO-d⁶)6.95(dd, J =2.3, 8.8Hz, 1H), 6.79(d, J =2.6Hz, 1H), 6.72(d, J =8.8Hz, 1H), 4.89(s, 2H), 4.09(s, 2H), 1.61-1.59(m, 4H) and 1.35(d, J =6.8Hz, 4H).

Example 2B: 4- (7-azabicyclo [2.2.1]]Hept-7-yl) -2- (trifluoromethyl) aniline (11B) hydrochloric acid And (3) preparing salt.

Preparation of 4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) aniline hydrochloride (11B), method 1. Palladium on carbon (150g, 5% w/w) was charged under nitrogen to a Buchi hydrogenator (20L capacity) followed by the addition of 7- [ 4-nitro-3- (trifluoromethyl) phenyl ] -7-azabicyclo [2.2.1] heptane (10A, 1500g), prepared as described above in example 2A (method 2), and 2-methyltetrahydrofuran (10.5L, 7 vol). The hydrogenator was then flushed with hydrogen and the mixture was then continuously charged at a pressure of 0.5bar above atmospheric pressure. The mixture was then stirred by cooling the vessel mantle between a temperature of 18 ℃ and 23 ℃. The vessel was evacuated when hydrogen was no longer consumed and when there was no further exotherm. The vessel was then charged with nitrogen at 0.5bar, evacuated again and then charged with nitrogen at 0.5 bar. When HPLC of a filtered aliquot showed no 7- [ 4-nitro-3- (trifluoromethyl) phenyl ] -7-azabicyclo [2.2.1] heptane (10A) (e.g. ≦ 0.5%), the reaction mixture was transferred under nitrogen atmosphere through a filter funnel to the receiving flask using a celite filter. The celite cake was washed with 2-methyltetrahydrofuran (3L, 2 vol). The eluate and filtrate were charged to a vessel equipped with stirring, temperature control and nitrogen atmosphere. 4M HCl in 1, 4-dioxane (1 volume) was added continuously to the vessel over a period of 1 hour at 20 ℃. The mixture was stirred for at least another 10 hours, filtered, washed with 2-methyltetrahydrofuran (2 vol) and dried to give 1519g of 4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) aniline hydrochloride (11B) as a white crystalline solid.

Alternative solvents may also be substituted in this embodiment. For example, MeOH and/or EtOH can replace 2-MeTHF.

Preparation of 4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) aniline hydrochloride (11B), method 2. Palladium on carbon (5% w/w, 150g) was introduced under nitrogen in a Buchi hydrogenator (20L capacity) followed by the addition of 7- [ 4-nitro-3- (trifluoromethyl) phenyl ] -7-azabicyclo [2.2.1] heptane (1500g) and 2-methyltetrahydrofuran (10.5L, 7 vol). The hydrogenator was then flushed with hydrogen and then continuously charged with the stirred mixture at a pressure of 0.5bar above atmospheric pressure. The temperature of the reaction mixture was maintained at 18 ℃ to 23 ℃ by cooling the vessel mantle. The vessel was evacuated when hydrogen was no longer consumed and when there was no further exotherm. The vessel was then charged with nitrogen, evacuated again and then charged with nitrogen again at 0.5 bar. The reaction was considered complete when an aliquot of HPLC was filtered to show that 7- [ 4-nitro-3- (trifluoromethyl) phenyl ] -7-azabicyclo [2.2.1] heptane could not be assayed (< 0.5%). The reaction mixture was then filtered through celite. The remaining slurry was transferred to the receiving flask through a filter funnel containing a celite filter under nitrogen. The celite cake was washed with 2-methyltetrahydrofuran (3L, 2 vol). The filtrate and eluate were transferred to a vessel equipped with stirring means, temperature control and nitrogen atmosphere. 4M HCl in 1, 4-dioxane (1 volume) was added continuously to the vessel over a period of 1 hour at 20 ℃. The resulting mixture was stirred for a further 10 hours, filtered and washed with 2-methyltetrahydrofuran (2 volumes) and dried to yield 1519g of 7- [ 4-amino-3- (trifluoromethyl) phenyl ] -7-azabicyclo [2.2.1] heptane hydrochloride (11B) as a white crystalline solid.

Alternative solvents may also be substituted in this embodiment. For example, MeOH and/or EtOH can replace 2-MeTHF.

Example 3A: n- (4- (7-Nitrogen)Hetero-bicyclo [2.2.1]Hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo Preparation of 5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide (form A).

Propylphosphonic acid cyclic anhydride (T3P (50% solution in ethyl acetate), 52.68mL, 88.48mmol) and pyridine (5.6g, 5.73mL, 70.78mmol) were added at room temperature to 4-oxo-5- (trifluoromethyl) -1H-quinoline-3-carboxylic acid (7) (9.1g, 35.39mmol) and 4- (7-azabicyclo [2.2.1 mmol)]A solution of hept-7-yl) -2- (trifluoromethyl) aniline (11A) (9.2g, 35.74mmol) in 2-methyltetrahydrofuran (91.00 mL). The reaction flask was heated at 65 ℃ for 10 hours under nitrogen. After cooling to room temperature, the reaction mixture was then diluted with ethyl acetate and saturated Na₂CO₃The solution (50mL) was quenched. The layers were separated and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with water and Na₂SO₄Dried, filtered and concentrated to a tan solid. The crude solid was slurried in a 2: 1 mixture of ethyl acetate and diethyl ether, collected by vacuum filtration and washed twice with an ethyl acetate/diethyl ether mixture to provide the crude product as a pale yellow crystalline powder. The powder was dissolved in warm ethyl acetate and absorbed into celite. Purification by silica gel chromatography (0-50% ethyl acetate in dichloromethane) to afford N- (4- (7-azabicyclo [2.2.1] as a white crystalline solid of solid form a]Hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide (compound I). LC/MS M/z 496.0[ M + H ]]⁺Retention time 1.48 minutes (RP-C18,

10-99%CH₃CN/0.05% TFA over 3 min).¹H NMR(400.0MHz，DMSO-d⁶)13.08(s，1H)，12.16(s，1H)，8.88(s，1H)，8.04(dd，J=2.1，7.4Hz，1H)，7.95-7.88(m，3H)，7.22(dd，2.5，8.9Hz，1H)，7.16(d，J=2.5Hz，1H)，4.33(s，2H)，1.67(d，J=6.9Hz，4H)，1.44(d，J=6.9Hz，4H)。

Example 3B: n- (4- (7-azabicyclo [2.2.1]]Hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo Preparation of 5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide (form A-HCl)

2-Methyltetrahydrofuran (0.57L, 1.0 vol) was charged to a 30L jacketed reaction vessel, followed by 4- (7-azabicyclo [2.2.1] c]Hept-7-yl) -2- (trifluoromethyl) aniline (11B) (791g, 2.67mol) as the hydrochloride and 4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (7) (573g, 2.23mol) and a further 5.2L (9.0 vol) of 2-methyltetrahydrofuran. Stirring was started and T3P in 2-methyltetrahydrofuran (2.84kg, 4.46mol) was added to the reaction mixture over a period of 15 minutes. Pyridine (534.0g, 546.0mL, 6.68mol) was then added dropwise over a period of 30 minutes via an addition funnel. The mixture was warmed to 45 ℃ over a period of about 30 minutes and stirred for 12-15 hours. The mixture was then cooled to room temperature and 2-methyltetrahydrofuran (4 vol, 2.29L) was added followed by water (6.9 vol, 4L) to keep the temperature below 30 ℃. Removing the aqueous layer, and subjecting the organic layer to NaHCO₃The saturated aqueous solution was carefully washed twice. The organic layer was then washed with 10% w/w citric acid (5 vol) and finally with water (7 vol). The mixture was polish filtered and transferred to another drying vessel. Adding N- (4- (7-azabicyclo [2.2.1]]Hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide hydrochloride (form A-HCl) (3.281g, 5.570 mmol). Hcl (g) (10 eq) was added in a bubbling fashion over a period of 2 hours and the mixture was stirred overnight. The resulting suspension was filtered, washed with 2-methyltetrahydrofuran (4 volumes), suction dried and dried in a 60 ℃ oven to provide compound I as form a-HCl. The powder diffractogram of form A-HCl is shown in FIG. 1.

Representative XRPD peaks of form a-HCl are provided below in table 1.

TABLE 1 XPRD peaks for form A-HCl.

2-theta (degree)	Relative Strength (%)
		7.1	44.3
8.2	33.3

2-theta (degree)	Relative Strength (%)
		10.8	1.5
11.7	1.5
		12.1	5.8
13.7	3.3
		14.1	32.1
14.7	16.9
		15.0	5.7
16.1	3.0
		16.4	16.9
16.6	3.7
		16.8	1.9
17.6	0.6
		18.7	12.4
18.9	1.9
		19.7	5.4
19.8	6.9
		20.3	1.5
21.2	100.0
		21.7	10.6
21.9	12.3
		22.2	4.0
22.8	21.9
		23.4	9.8
24.6	34.3
		25.0	17.9
25.2	8.6
		25.9	3.6
26.5	1.5
		26，	7.0
27.5	8.3
		28.0	5.3
28.3	1.6
		28.7	3.5
29.0	4.8
		29.2	7.5
29.8	1.40
		30.1	1.8
31.0	5.4
		31.3	2.6
31.9	1.7
		32.3	4.6
32.4	3.7
		32.8	2.3
33.3	3.9
		34.3	1.8
34.5	3.6
		34.7	8.7
35.3	3.0
		35.6	12.7
35.6	18.9
		36.1	3.2
36.8	3.3

2-theta (degree)	Relative Strength (%)
		37.2	1.7
37.8	3.1
		38.5	2.8
39.1	3.1
		39.7	2.3

The single crystal of compound 1 form A-HCl was determined to have a monoclinic system, P2₁The/c space group, and the following unit cell parameters:α =90 °, β =112.303 °, and γ =90 °.

Representative samples of form A-HCl were also evaluated using microscopy.

The DSC curve for a representative sample of form a-HCl is provided in fig. 2.

The TGA profile of a representative sample of form a-HCl is provided in fig. 3.

A representative sample of form a-HCl produced an FTIR spectrum provided in fig. 4.

Table 2 provides the characteristic FTIR absorption of form a-HCl.

Table 2 FTIR absorption of form a HCl.

Position (cm)-1)	Intensity (% reflection)
		407.6	51.1
422.7	69.7
		445.7	62.6
479.1	51.8
		508.4	55.7
538.1	58.6
		568.4	56.7
586.3	65.7
		614.9	66.8
640.5	55.8
		663.2	44.2
669.3	53.2
		687.2	58.2
752.1	34.4
		796.2	32.5
821.7	40.0
		836.8	62.0
868.2	60.8
		884.7	60.5
900.2	56.1
		940.0	59.0
965.4	56.3
		1052.4	35.1
1065.7	40.0
		1109.5	23.9
1122.8	27.3

Position (cm)-1)	Intensity (% reflection)
		1147.7	38.8
1159.3	42.1
		1212.8	37.9
1237.0	65.1
		1255.3	51.4
1270.6	46.6
		1300.8	47.0
1328.6	45.1
		1434.1	41.6
1452.0	61.6
		1521.8	40.2
1568.2	64.0
		1608.8	55.2
1688.5	54.5
		2256.5	68.0
2880.9	73.2
		3676.3	90.5

Also used is Solid State (SS)¹³C and¹⁹f NMR analyses of representative samples of form A-HCl. The respective NMR spectra are provided in fig. 5 and 6. In that¹³C SS NMR and¹⁹several peaks found in the F SS NMR spectrum are described in tables 3 and 4 below.

TABLE 3 form A-HCl¹³C SS NMR peaks.

Peak numbering	Chemical shift (ppm)
		1	175.7
2	163.7
		3	142.6
4	140.8
		5	137.2
6	131.5
		7	129.0
8	126.0
		9	124.8
10	123.8
		11	121.5
12	117.8
		13	112.4
14	65.7
		15	29.2
16	28.3
		17	26.1

TABLE 4 form A-HCl¹⁹F SS NMR peak.

Peak numbering	Chemical shift (ppm)
		1	-57.0
2	-60.5

Example 4A: n- (4- (7-azabicyclo [2.2.1]]Hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo Preparation of 5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide (form B).

2-Methyltetrahydrofuran (1 vol) was charged to a 30L jacketed reaction vessel, followed by the addition of 4- (7-azabicyclo [2.2.1]]Hept-7-yl) -2- (trifluoromethyl) aniline (11B) hydrochloride (1.2 eq) and 4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (7) (573g, 2.228 mol). The vessel was charged with 2-methyltetrahydrofuran (9 vol) and stirring was started. T3P (2 equiv.) in 2-methyltetrahydrofuran was added to the reaction mixture over a period of 15 minutes. Pyridine (3 equivalents) was added rapidly in a dropwise manner using an addition funnel. The mixture is then added with stirring over a period of about 30 minutesHeat to 45 ℃ and hold the temperature for about 5 hours. The mixture was cooled to room temperature. 2-methyltetrahydrofuran (4 vol) was added followed by slow addition of water (6.9 vol) and the reaction temperature was kept below 30 ℃. Removing the aqueous layer, and subjecting the organic layer to NaHCO₃The saturated aqueous solution was washed twice. The organic layer was then carefully washed with 10% w/w citric acid (5 vol) and water (7 vol), polish filtered and then transferred to another drying vessel. 2-Methyltetrahydrofuran (10 vol) was added and stirring was started. Heptane (10 volumes) was added rapidly in a dropwise manner with stirring. The mixture was stirred for a period of about 12 hours and then vacuum filtered. The solid filter cake was introduced into another vessel. Water (15 vol) was charged to the vessel and the suspension was stirred vigorously for 48 hours and then filtered. The solid cake was washed with water (5 vol) and dried to constant weight at 45 ℃ to give compound I, form B.

The powder diffractograms of compound I, form B are shown in figures 7A and 7B.

Table 5: representative XRPD peaks for form B

TABLE 5 XPRD peaks for form B

2-theta (degree)	Relative Strength (%)
		6.7	36.6
9.4	37.2
		10.0	29.5
11.2	35.3
		13.4	70.6
14.8	18.1
		15.2	88.8
15.4	16.6
		17.2	49.5
17.8	48.0
		18.1	83.8
18.8	13.6
		19.2	47.6
20.1	68.9
		21.2	71.8
22.0	42.6
		22.6	7.6
23.5	18.1
		24.0	100.0
25.0	9.6
		25.9	9.7
26.2	44.8
		26.9	26.3
27.2	86.7
		27.7	37.8
28.2	7.4
		28.9	49.0
29.6	21.3
		30.3	8.6
30.6	5.5
		31.2	19.3
32.3	5.5
		33.7	11.4
34.2	8.9
		34.7	12.4
35.1	8.0
		36.7	6.5
38.1	4.7
		39.3	5.3

DSC curves for representative samples of form B are provided in figure 8.

The TGA profile of a representative sample of form B is provided in figure 9.

FTIR spectra of representative samples of form B are provided in fig. 10.

Table 6 provides characteristic FTIR absorption for form B.

Table 6 FTIR absorption of form B.

Position (cm)-1)	Intensity (% reflection)
		406.1	78.6
435.2	93.3
		450.0	91.0
464.3	87.9
		473.7	84.0
490.7	81.3
		505.1	81.2
531.7	81.5
		565.2	79.3
586.1	80.5
		603.0	75.9
642.5	77.3
		661.9	74.3
682.5	78.0
		726.9	80.7
749.6	68.8
		766.4	81.9
798.7	81.3
		823.2	63.4
842.9	93.6
		876.8	83.5
902.3	92.2
		919.5	85.7
976.3	72.4
		1045.8	71.0
1073.6	76.6
		1109.0	65.5
1119.4	65.2
		1139.6	58.8
1167.6	71.4
		1197.9	92.0
1206.6	90.3
		1227.8	81.9
1253.9	79.2
		1272.8	77.9
1285.0	78.6
		1301.1	74.6
1329.6	83.8
		1349.7	76.6
1435.1	75.5
		1466.6	78.2
1501.3	75.0
		1534.8	68.4
1577.4	82.6
		1620.4	83.5
1657.4	79.7

Position (cm)-1)	Intensity (% reflection)
		2952.5	92.2
2988.6	92.1

Also using the solid state¹³C and¹⁹representative samples of form B were analyzed by F NMR. The respective NMR spectra are provided in fig. 11 and 12. In that¹³C SS NMR and¹⁹several peaks found in the F SS NMR spectrum are described in tables 7 and 8 below.

TABLE 7. of form B¹³C SS NMR peaks.

Peak numbering	Chemical shift (ppm)
		1	175.3
2	165.3
		3	145.9
4	141.4
		5	132.9
6	126.8
		7	123.5
8	117.4
		9	113.4
10	58.3
		11	29.2
12	26.9

TABLE 8. of form B¹⁹F SS NMR peak.

Peak numbering	Chemical shift (ppm)
		1	-56.1
2	-62.1

A single crystal of compound 1 form B was mounted on a Micro Mount loop and centered on a Broker Apex II diffractometer equipped with a sealed copper X-ray tube and Apex II CCD detector. Initially, 3 sets of 40 frames were collected to determine the initial cell. A complete data set consisting of 15 scans and 6084 frames is then obtained. Data acquisition was performed at room temperature. Data were integrated and scaled using Apex II software from Bruker AXS. Integration and scaling results in 6176 reflections, of which 2250 is unique. The structure was resolved by direct method in space group P21/c using SHELXTL software. Refinement was also performed using SHELXTL software using the full matrix least squares method on F2. A total of 392 parameters were used for refinement, resulting in a reflection parameter ratio of 5.74. The final refinement indices are wR2=0.0962 and Rl =0.0682 (reflection wR2=0.0850 and Rl =0.0412 for I >2 σ (I)).

In form B N- (4- (7-azabicyclo [2.2.1]]Single crystals of hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide were determined to have monoclinic crystalsThe P21/c space group, and the following unit cell parameters:α =90 °, β =101.193 °, and γ =90 °.

Example 4B: preparation of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide (form B-HCl)

100mL of 2-methyltetrahydrofuran was charged into a three-necked flask with a nitrogen atmosphere equipped with a stirrer. Compound I, form A-HCl (55g, 0.103mol) was added to the flask, followed by 349mL of 2-methyltetrahydrofuran and stirring was started. 28mL of water was added to the flask, which was warmed to an internal temperature of 60 ℃ and stirred for 48 hours. The flask was cooled to room temperature and stirred for 1 hour. The reaction mixture was filtered under vacuum until the filter cake was dried. The solid filter cake was washed twice with 2-methyltetrahydrofuran (4 volumes). The solid filter cake was kept under vacuum for a period of about 30 minutes and transferred to a dry dish. The filter cake was dried under vacuum at 60 ℃ to give form B-HCl as a white crystalline solid.

The powder diffraction pattern of form B-HCl is shown in FIG. 13.

Representative XRPD peaks of form B-HCl are provided below in table 9.

TABLE 9 XRPD peaks for form B-HCl.

2-theta (degree)	Relative Strength (%)
		8.3	93.7
9.0	8.4
		10.9	0.8

2-theta (degree)	Relative Strength (%)
		11.4	1.4
13.0	4.9
		14.1	19.8
14.8	32.7
		15.2	12.6
16.7	23.8
		17.8	37.8
18.0	90.0
		18.2	28.6
19.3	19.0
		19.5	17.5
19.9	2.7
		20.4	9.4
20.6	6.2
		21.7	41.2
22.0	22.2
		23.0	100.0
23.6	20.5
		23.9	4.0
24.1	3.9
		24.5	9.2
24.7	13.0
		24.9	31.9
25.2	22.6
		25.7	12.6
26.1	3.3
		26.7	4.5
27.1	21.3
		27.9	10.6
28.1	18.7
		28.5	4.3
28.7	5.8
		29.7	11.1
29.8	14.2
		30.1	4.0
30.5	8.2
		31.1	30.2
31.5	9.1
		32.3	11.4
32.8	3.8
		33.1	9.2
33.4	11.3
		33.8	11.1
33.9	10.1
		34.1	5.6
34.6	8.5
		34.9	6.4
35.2	10.8
		36.0	4.1
36.2	13.2
		36.4	4.7
37.2	5.9

2-theta (degree)	Relative Strength (%)
		37.6	3.7
37.9	2.2
		38.2	7.5
38.5	22.3
		38.6	13.8
39.9	10.7

The single crystal of compound 1 form B-HCl was determined to have a monoclinic system, P2₁The/a space group, and the following unit cell parameters:α =90 °, β =90.0554 °, and γ =90 °.

Representative samples of form B-HCl were also evaluated using microscopy.

The DSC curve for a representative sample of form B-HCl is provided in fig. 14.

The TGA profile of a representative sample of form B-HCl is provided in fig. 15.

An FTIR spectrum of a representative sample of form B-HCl is provided in fig. 16.

Table 10 provides the characteristic FTIR absorption of form B-HCl.

TABLE 10 FTIR absorption of form B-HCl.

Also using the solid state¹³C and¹⁹f NMR analyses form B-HCl. The respective NMR spectra are provided in fig. 17 and 18. In that¹³C SS NMR and¹⁹several peaks found in the F SS NMR spectrum are listed in tables 11 and 12.

TABLE 11 of form B-HCl¹³C SS NMR peaks.

Peak numbering	Chemical shift (ppm)
		1	176.3
2	168.2
		3	148.7
4	143.2
		5	138.8
6	131.6
		7	129.6
8	129.1
		9	126.7
10	125.8
		11	122.7
12	119.8
		13	112.3
14	69.0
		15	66.9
16	28.3
		17	23.9

TABLE 12 of form B-HCl¹⁹F SS NMR peak.

Peak numbering	F1(ppm)
		1	-55.6
2	-62.0

Example 4C: n- (4- (7-azabicyclo [2.2.1]]Hept-7-yl) -2- (trifluoromethyl) phenyl) -4- Alternative preparation of oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide (form B-HCl) And (4) preparing.

Form A-HCl (14.638g, 27.52mmol) was charged to a 100mL round bottom flask. EtOH (248.9mL) and water (27.82mL) were added. The white slurry was heated to reflux. A clear solution was obtained at 77 ℃. The reaction was cooled to 45 ℃, stirred for 30 minutes, and then cooled to 20 ℃. The mixture was stirred at 20 ℃ for a further 3 hours. The product was filtered and the cake was washed with EtOH. The solid was blow dried in a vacuum oven at 45 ℃ with nitrogen to afford compound I, form B-HCl as a white solid. XRPD analysis confirmed the identity of the solid as form B-HCl.

Note that other solvent combinations such as MeOH/H₂O and IPA/H₂O or the like may be used in place of EtOH/H described in this example₂And O. Examples of alternative solvent combinations are provided in table 13.

Table 13. other solvents that can be used to prepare form B-HCl.

Note also that in examples 3A, 3B and 4A-4C, EtOAC can be used as a solvent instead of 2-MeTHF.

Assays for detecting and measuring the Δ F508-CFTR enhancing properties of compounds

Transmembrane potential optical method for determining Δ F508-CFTR modulating properties of compounds

The assay uses a fluorescent voltage sensing dye to determine transmembrane potential changes as read-outs of increased functional Δ F508-CFTR in NIH3T3 cells using a fluorescent plate reader (e.g., FLIPRIII, Molecular Devices, Inc.). The driving force for this response is that after pre-treating the cells with compounds and then loading the voltage sensing dye, a chloride ion gradient associated with channel activation is generated by a single liquid addition step.

Identification of enhancer compounds

To identify af 508-CFTR enhancers, a double-addition HTS assay format was developed. The HTS assay uses a fluorescent voltage sensing dye to determine the change in transmembrane potential across FLIPR III as a temperature-calibrated Δ F508CFTRMeasurement of increased Δ F508CFTR gating (conductance) in NIH3T3 cells. The driving force for this response is the Cl associated with channel activation with forskolin in a single liquid addition step using a fluorescent plate reader such as FLIPR III, after pre-treatment of cells with the enhancer compound (or DMSO vehicle control) followed by loading of the redistribution dye^-An ion gradient.

Solutions of

Bath solution # 1: (in mM) NaCl 160, KCl 4.5, CaCl₂ 2,MgCl₂1, HEPES 10, pH7.4, NaOH.

An alternative to bath solution #1 includes a bath solution in which the hydrochloride salt is replaced with gluconate.

Cell culture

NIH3T3 mouse fibroblasts stably expressing AF 508-CFTR were used for optical measurement of transmembrane potential. Cells were maintained at 37 ℃ with 5% CO₂And 175cm in 90% humidity²In Dulbecco's modified Eagle's Medium in culture flasks, the medium was supplemented with 2mM glutamine, 10% fetal bovine serum, 1X NEAA, beta-ME, 1X pen/strep and 25mM HEPES. For all optical assays, cells were seeded at-20,000/well on 384-well matrigel-coated plates, incubated at 37 ℃ for 2 hours, and then at 27 ℃ for 24 hours for enhancer analysis. For calibration assays, cells were incubated with and without compounds at 27 ℃ or 37 ℃ for 16-24 hours. Electrophysiological analysis was used to analyze compounds for Δ F508-CFTR modulating properties.

Ussing Chamber analysis

Ussing laboratory experiments were performed on polarized airway epithelial cells expressing Δ F508-CFTR to further characterize modulators of Δ F508-CFTR identified in the optical assay. non-CF and CF airway epithelium was isolated from bronchial tissue and cultured as described above (Galietta, l.j.v., Lantero, s., Gazzolo, a., Sacco, o., Romano, l., Rossi, g.a.,&Zegarra-Moran, O. (1998) In Vitro cell. Dev. biol.34,478-481) In pre-coating with NIH3T 3-conditioned mediumSnapwell^TMSpread flat on the filter. After 4 days, the top medium was removed and the cells were allowed to grow on an air-liquid interface>14 days, then used. This results in a monolayer of fully differentiated columnar cells that are ciliated and characterized by airway epithelium. non-CF HBEs were isolated from non-smokers who did not have any known lung disease. CF-HBE was isolated from patients homozygous for Δ F508-CFTR.

Will grow inSnapwell^TMHBE on cell culture inserts was immobilized on a USsing chamber (physiology Instruments, Inc., San Diego, Calif.) and Cl was measured outside the substrate to the apical end using a voltage clamp system (Department of Bioengineering, University of Iowa, IA)^-Gradient (I)_SC) Transepithelial resistance and short circuit current in the presence of (c). Briefly, under voltage clamp recording conditions (V)_Holding=0mV), HBE was examined at 37 ℃. The solution on the outside of the substrate contained (in mM) 145NaCl, 0.83K₂HPO₄、3.3KH₂PO₄、1.2MgCl₂、1.2CaCl₂10 glucose, 10HEPES (pH adjusted to 7.35 with NaOH), apical solution containing (in mM) 145 sodium gluconate, 1.2MgCl₂、1.2CaCl₂10 glucose, 10HEPES (pH adjusted to 7.35 with NaOH).

Identification of enhancer compounds

Typical protocols use Cl from the outside of the substrate to the top film^-A concentration gradient. To create this gradient, normal ringer's solution (ringer) was used for the substrate outer membrane. In addition, by replacing the NaCl of the apical membrane with equimolar sodium gluconate (titrated to pH7.4 with NaOH), large size Cl spanning the epithelium is obtained^-A concentration gradient. Forskolin (10 μ M) and all test compounds were added to the top surface of the cell culture insert. The effect of the putative af 508-CFTR enhancer was compared to that of the known enhancer genistein.

2. Patch clamp record

Monitoring of total Cl in AF 508-NIH3T3 cells using a perforated patch recording configuration as previously described^-Electric current (Rae, j., Cooper, k., Gates, p.,&watsky, m. (1991) j. neurosci. methods 37, 15-26). Voltage clamp recordings were performed at 22 ℃ using an Axopatch 200B patch clamp amplifier (AxonInstruments inc., Foster City, CA). The pipette solution contained (in mM) 150N-methyl-D-glucosamine (NMDG) -Cl, 2MgCl₂、2CaCl₂10EGTA, 10HEPES and 240. mu.g/ml amphotericin-B (pH adjusted to 7.35 with HCl). The extracellular medium contains (in mM) 150NMDG-Cl, 2MgCl₂、2CaCl₂10HEPES (pH adjusted to 7.35 with HCl). Pulsing, data acquisition and analysis were performed using a PC equipped with a Digidata1320A/D interface and Clampex 8(Axon Instruments Inc.). To activate af 508-CFTR, 10 μ M forskolin and 20 μ M genistein were added to the bath and the current-voltage dependence was monitored every 30 seconds.

Identification of enhancer compounds

The enhancement of macroscopic Δ F508-CFTR Cl in NIH3T3 cells stably expressing Δ F508-CFTR was also investigated by using a perforated patch recording technique^-Electric current (I)_ΔF508) The ability of the cell to perform. Enhancers identified from the optical assay result in I with similar potency and efficacy observed in the optical assay_ΔF508Is increased in a dose-dependent manner. In all cells tested, the reversal potential before and during the application of the enhancing agent was about-30 mV, which is the calculated E_cl(-28mV)。

Cell culture

NIH3T3 mouse fibroblasts stably expressing AF 508-CFTR were used for whole cell recordings. Cells were maintained at 37 ℃ in 5% CO₂And 175cm in 90% humidity²Dulbecco's modified Eagle's Medium in flasks supplemented with 2mM glutamine, 10% fetal bovine serum, 1X NEAA, beta-ME, 1X pen/strep and 25mM HEPES. To perform whole cell recordingIn addition, 2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslips and used to test the activity of the enhancer after incubation at 27 ℃ for 24-48 hours; and incubated with or without a correction compound at 37 ℃ to determine the activity of the correction agent.

3. Single channel recording

The gating activity of temperature-calibrated Δ F508-CFTR and wt-CFTR expressed in NIH3T3 cells was observed using excised membrane inside-out patches (dalemanns, w., Barbry, p., champgny, g., jalat, s., Dott, k., Dreyer, d., crytal, r.g., Pavirani, a., Lecocq, J-p., Lazdunski, M. (1991) Nature 354, 526-. The removal tube contained (in mM): 150NMDG, 150 aspartic acid, 5CaCl₂、2MgCl₂And 10HEPES (pH adjusted to 7.35 with Tris base). The bath contained (in mM): 150NMDG-Cl, 2MgCl₂5EGTA, 10TES and 14Tris base (pH adjusted to 7.35 with HCl). After excision, wt-and Δ F508-CFTR were activated by the addition of 1mM Mg-ATP, 75nM cAMP-dependent protein kinase catalytic subunit (PKA; Promega Corp. Madison, Wis.) and 10mM NaF to inhibit protein phosphatase, which prevented the current from dropping. The pipette potential was maintained at 80 mV. Channel activity was analyzed from membranes containing ≦ 2 active channels. The maximum number of simultaneous openings determines the number of active channels during the experiment. To determine single channel current amplitude, data recorded from 120 sec Δ F508-CFTR activity was filtered "off-line" at 100Hz and then used to construct an amplitude histogram for all points, which was fitted with a multiple Gaussian function using Bio-Patch analysis software (Bio-Logic Comp. France). Total microscopic Current and open probability (P) were determined from 120 seconds of channel Activity_o). Using Bio-Patch software or according to the relation P_o= I/I (N) determine P_oWhere I = mean current, I = single channel current amplitude, N = number of active channels in the membrane.

Cell culture

NIH3T3 mouse fibroblasts stably expressing AF 508-CFTR were used for patch clamp recordings of incised membranes. Maintain the cells inAt 37 ℃ and 5% CO₂And 175cm in 90% humidity²Dulbecco's modified Eagle's Medium in culture flasks supplemented with 2mM glutamine, 10% fetal bovine serum, 1X NEAA, beta-ME, 1X pen/strep and 25mM HEPES. For single channel recording, 2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslips and used after 24-48 hours of incubation at 27 ℃.

Compound I form a is useful as a modulator of ATP-binding cassette transporters. Determination of EC for Compound I form A₅₀(mum) less than 2.0. mu.M. The efficiency of compound I form a was calculated to be 100% to 25%. It should be noted that 100% efficiency is the maximum response obtained using 4-methyl-2- (5-phenyl-1H-pyrazol-3-yl) phenol.

Claims (25)

1. Hydrochloride form a-HCl of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide, wherein the form a-HCl is characterized by the following peaks in an X-ray powder diffraction pattern obtained with Cu ka radiation: a peak at about 7.1 degrees, a peak at about 8.2 degrees, a peak at about 14.1 degrees, a peak at about 14.7 degrees, a peak at about 16.4 degrees, a peak at about 18.7 degrees, a peak at about 21.2 degrees, a peak at about 21.9 degrees, a peak at about 22.8 degrees, a peak at about 24.6 degrees, a peak at about 25.0 degrees, and a peak at about 35.6 degrees.
2. 2. The hydrochloride salt form A-HCl of claim 1, wherein the form A-HCl is in the solid state¹³The CNMR spectrum is characterized by a peak at about 163.7ppm, a peak at about 137.2ppm, and a peak at about 121.5 ppm.
3. 3. The hydrochloride salt form A-HCl of claim 1, wherein the form A-HCl is in the solid state¹⁹The FNMR spectra were characterized by the following peaks: a peak at about-57.0 ppm and a peak at about-60.5 ppm.
4. 4. The hydrochloride salt form a-HCl of any one of claims 1 to 3, wherein the form a-HCl is characterized by a single crystal that is determined to have a monoclinic system; p2₁A/c space group; and the following unit cell parameters:

   α＝90°；

   β -112.303 °; and

   γ＝90°。
5. 5. a pharmaceutical composition comprising the hydrochloride form A-HCl of any one of claims 1 to 4, and a pharmaceutically acceptable adjuvant or carrier.
6. Form B of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide, wherein form B is characterized by the following table in an X-ray powder diffraction pattern: a peak at about 6.7 degrees, a peak at about 9.4 degrees, a peak at about 11.2 degrees, a peak at about 13.4 degrees, a peak at about 15.2 degrees, a peak at about 17.2 degrees, a peak at about 17.8 degrees, a peak at about 18.1 degrees, a peak at about 19.2 degrees, a peak at about 20.1 degrees, a peak at about 21.2 degrees, a peak at about 22.0 degrees, a peak at about 24.0 degrees, a peak at about 26.2 degrees, a peak at about 27.2 degrees, a peak at about 27.7 degrees, and a peak at about 28.9 degrees; or

   Wherein form B is characterized by a single crystal determined to have a monoclinic system, P21/c space group, and the following unit cell parameters:

   α＝90°；

   β -101.193 °; and

   γ＝90°。
7. 7. form B of claim 6, wherein form B is characterized in X-ray powder diffraction by: a peak at about 6.7 degrees, a peak at about 9.4 degrees, a peak at about 11.2 degrees, a peak at about 13.4 degrees, a peak at about 15.2 degrees, a peak at about 17.2 degrees, a peak at about 17.8 degrees, a peak at about 18.1 degrees, a peak at about 19.2 degrees, a peak at about 20.1 degrees, a peak at about 21.2 degrees, a peak at about 22.0 degrees, a peak at about 24.0 degrees, a peak at about 26.2 degrees, a peak at about 27.2 degrees, a peak at about 27.7 degrees, and a peak at about 28.9 degrees.
8. 8. Form B of claim 6, wherein form B is in the solid state¹³The C NMR spectrum was characterized by a peak at about 165.3ppm, a peak at about 145.9ppm, a peak at about 132.9ppm, and a peak at about 113.4 ppm.
9. 9. Form B of claim 6, wherein form B is in the solid state¹⁹The F NMR spectrum is characterized by a peak at about-56.1 ppm and a peak at about-62.1 ppm.
10. 10. A pharmaceutical composition comprising form B according to any one of claims 6 to 9, and a pharmaceutically acceptable adjuvant or carrier.
11. Hydrochloride form B-HCl of N- (4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide, wherein form B-HCl is characterized by the following peaks in an X-ray powder diffraction pattern: a peak at about 8.3 degrees, a peak at about 14.8 degrees, a peak at about 16.7 degrees, a peak at about 17.8 degrees, a peak at about 18.0 degrees, a peak at about 18.2 degrees, a peak at about 21.7 degrees, a peak at about 22.0 degrees, a peak at about 23.0 degrees, a peak at about 23.6 degrees, a peak at about 24.9 degrees, a peak at about 25.2 degrees, a peak at about 27.1 degrees, a peak at about 31.1 degrees, and a peak at about 38.5 degrees.
12. 12. The hydrochloride salt form B-HCl of claim 11, wherein the form B-HCl is in the solid state¹³The C NMR spectrum was characterized by a peak at about 168.2ppm, a peak at about 148.7ppm, a peak at about 138.8ppm, a peak at about 119.8ppm, and a peak at about 23.9 ppm.
13. 13. The hydrochloride salt form B-HCl of claim 11, wherein the form B-HCl is in the solid state¹⁹The F NMR spectrum is characterized by a peak at about-55.6 ppm, and a peak at about-62.0 ppm.
14. 14. The hydrochloride salt form B-HCl of any one of claims 11-13, wherein the form B-HCl is characterized by a single crystal determined to have a monoclinic system, P2₁The/a space group, and the following unit cell parameters:

    α＝90°；

    β -90.0554 °; and

    γ＝90°。
15. 15. a pharmaceutical composition comprising the hydrochloride form B-HCl of any one of claims 11-14, and a pharmaceutically acceptable adjuvant or carrier.
16. 16. Use of the hydrochloride form a-HCl according to any one of claims 1-4, the form B according to any one of claims 6-9 or the hydrochloride form B-HCl of any one of claims 11-14, or any combination of these forms, in the manufacture of a medicament for treating or lessening the severity of a disease in a patient, wherein the disease is selected from the group consisting of cystic fibrosis, hereditary emphysema, chronic obstructive pulmonary disease, and dry eye disease.
17. 17. Use according to claim 16, wherein the disease is cystic fibrosis.
18. 18. The use of claim 17, wherein the patient has a homozygous Δ F508 mutation.
19. 19. The use of claim 17, wherein the patient has the homozygous G551D mutation.
20. 20. The use of claim 17, wherein the patient has a heterozygous Δ F508 mutation.
21. 21. The use of claim 17, wherein the patient has the hybrid G551D mutation.
22. 22. Use of the hydrochloride form a-HCl according to any one of claims 1-4, the form B according to any one of claims 6-9, or the hydrochloride form B-HCl according to any one of claims 11-14, or any combination of these forms, in the manufacture of a medicament for treating or lessening the severity of a disease in a patient, wherein the disease is cystic fibrosis or dry eye.
23. 23. Use of the hydrochloride form a-HCl according to any one of claims 1-4, the form B according to any one of claims 6-9 or the hydrochloride form B-HCl according to any one of claims 11-14, or any combination of these forms, in the manufacture of a medicament for treating a disease or lessening the severity of a disease in a patient, wherein the disease is associated with normal CFTR function.
24. 24. The use of claim 23, wherein the disease is chronic obstructive pulmonary disease or hereditary emphysema.
25. 25. Use of the hydrochloride form a-HCl according to any one of claims 1-4, the form B according to any one of claims 6-9, or the hydrochloride form B-HCl of any one of claims 11-14 in the preparation of a formulation for modulating CFTR activity in a biological sample.