HK1173150A - Process for preparing modulators of cystic fibrosis transmembrane conductance regulator - Google Patents

Description

Method for producing modulators of cystic fibrosis transmembrane conductance regulator

Cross Reference to Related Applications

The present application claims priority from provisional application u.s. serial No. 61/254,634 filed on 23/10/2009. The contents of which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to methods for making modulators of cystic fibrosis transmembrane conductance regulator ("CFTR").

Background

Cystic Fibrosis (CF) is a recessive genetic disease that affects about 30,000 children and adults in the united states and about 30,000 children and adults in europe. Despite the treatment of CF, there is no cure.

CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial chloride channel that is responsible for the auxiliary regulation of salt and water absorption and secretion in a variety of tissues. Small molecule drugs called potentiating factors that increase the likelihood of CFTR channel opening represent an effective therapeutic strategy for the treatment of CF.

Specifically, CFTR is a cAMP/ATP-mediated anion channel that is expressed in a variety of cell types, including absorptive and secretory epithelial cells, where it regulates transmembrane anion flow through, 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. sup. 386; Rich, D.P. et al (1990) Nature 347: 358. sup. 362), (Riordan, J.R. et al (1989) Science 245: 1066. sup. 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 CF patients, 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, >1000 disease-causing CF gene mutations 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. (Dalemans et al (1991), Nature Lond.354: 526-. In addition to af 508-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 effect (transport of anions) represents the transport of ions andone element of the important mechanism of water. Other elements include epithelial Na + channels, ENaC, Na +/2Cl-/K + cotransporters, 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 achieve targeted transport across epithelial cells through their selective expression and localization in 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 ion 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 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.

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. Indeed, this cellular phenomenon of ER deficiency in processing ABC transporters via the ER machinery has been shown to be the underlying basis not only for CF diseases, but also for a wide range of other independent and genetic diseases.

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 methods of synthesizing compounds useful as modulators of CFTR.

In one aspect, the invention provides a process for preparing a crystalline form of compound 1:

the method comprises the following steps:

(a) reacting compound 2 with compound 3 in the presence of a coupling agent:

wherein the coupling agent is selected from the group consisting of 2-chloro-1, 3-dimethyl-2-imidazolium tetrafluoroborate, HBTU, HCTU, 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine, HATU, HOBT/EDC and

the compounds of formula 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 (processing deficienes), e.g., familial hypercholesterolemia, chylomicronemia type 1, β -lipoproteinemia, lysosomal storage diseases, e.g., I-cell disease/pseudohule disease, Mucopolysaccharidosis, sandhoff disease/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 CDG 1, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency (actDeficienccy), Diabetes Insipidus (DI), neurogenic DI (neuropyemic DI), renal DI (neurogenic DI), summer-horse-graphics syndrome (char-Marie Toothy syndrome), pelizaeus-merzflue disease (perzaeus-merzseeus disease), neurodegenerative diseases, such as alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, progressive amyotrophic lateral sclerosis, several neuro-nuclear disorders (neuro-neuro disorder), neuro-degenerative diseases, Such as huntington's disease, spinocerebellar ataxia type I, spinal and bulbar 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 disease (chlorodecor), e.g. congenital myotonia (Thomson and Becker type), barter syndrome type III, Dent disease, shock syndrome (hypereky fright syndrome), epilepsy, shock syndrome, lysosomal disease, Angelman syndrome, primary dyskinesia (PCD), primary dyskinesia or primary/ciliary dysfunction, Including PCD with left and right transposition (also known as catagory syndrome), PCD without left and right transposition and ciliary dysplasia (ciliary atlas).

In one aspect, compound 1 is in a crystalline form referred to as form a.

In another aspect, compound 1 is in a crystalline form referred to as form B.

In yet another aspect, compound 1 is in a crystalline form referred to as form a-HCl.

In another aspect, compound 1 is in a crystalline form referred to as form B-HCl.

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

Brief Description of Drawings

Figure 1 is an X-ray powder diffraction pattern of a representative sample of compound 1 form a.

Figure 2 is an FTIR spectrum of a representative sample of compound 1 form a.

Figure 3 is a graphical representation of the conformational structure of compound 1 form a based on single crystal X-ray analysis.

Figure 4 is an X-ray powder diffraction pattern of an exemplary sample of compound 1 form a-HCl.

FIG. 5 is a DSC curve of a representative sample of Compound 1, form A-HCl.

FIG. 6 is a graph generated by thermogravimetric analysis of a representative sample of Compound 1 form A-HCl, showing sample weight as a function of temperature.

FIG. 7 is an FTIR spectrum of a representative sample of Compound 1 form A-HCl.

FIG. 8 is a solid phase of a representative sample of Compound 1 form A-HCl¹³C NMR spectrum.

FIG. 9 is a solid phase of a representative sample of Compound 1 form A-HCl¹⁹F NMR spectrum.

Figure 10A is an X-ray powder diffraction pattern recorded with instrument 1 for a representative sample of compound 1 form B.

Figure 10B is an X-ray powder diffraction pattern recorded with instrument 2 for a representative sample of compound 1 form B.

Figure 11 is a DSC curve for a representative sample of compound 1 form B.

Figure 12 is a plot generated by thermogravimetric analysis of a representative sample of compound 1 form B, showing sample weight as a function of temperature.

Figure 13 is an FTIR spectrum of a representative sample of compound 1 form B.

FIG. 14 is a solid phase of a representative sample of Compound 1 form B¹³C NMR spectrum.

FIG. 15 is a solid phase of a representative sample of Compound 1 form B¹⁹F NMR spectrum.

FIG. 16 is an X-ray powder diffraction pattern of Compound 1 form B-HCl.

FIG. 17 is a DSC curve of a representative sample of Compound 1, form B-HCl.

FIG. 18 is a graph generated by thermogravimetric analysis of a representative sample of Compound 1 form B-HCl, showing sample weight as a function of temperature.

FIG. 19 is an FTIR spectrum of a representative sample of Compound 1 form B-HCl.

FIG. 20 is Compound 1Solid phase of a representative sample of form B-HCl¹³C NMR spectrum.

FIG. 21 is a solid phase of a representative sample of Compound 1 form B-HCl¹⁹F NMR spectrum.

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.

The term "stable" as used herein means that the compounds are substantially unchanged when subjected to their preparation, assay, and preferably their recovery, purification, and use for one or more of the purposes disclosed herein. In certain embodiments, a stable compound or chemically feasible compound is one that is substantially unchanged for at least 1 week when maintained at a temperature of 40 ℃ or less in the absence of moisture or other chemical reaction conditions.

Examples of suitable solvents that may be used in the present invention are, without limitation, water, methanol, Dichloromethane (DCM), acetonitrile, Dimethylformamide (DMF), methyl acetate (MeOAc), ethyl acetate (EtOAc), isopropyl acetate (IPAc), tert-butyl acetate (tert-BuOAc), Isopropanol (IPA), Tetrahydrofuran (THF), Methyl Ethyl Ketone (MEK), tert-butanol, diethyl ether (Et), ethyl acetate (IPAc)₂O), methyl-tert-butyl ether (MTBE), 1, 4-dioxane and N-methylpyrrolidone (NMP).

Examples of suitable coupling agents that may be used in the present invention are, without limitation, 1- (3- (dimethylamino) propyl) -3-ethyl-carbodiimide hydrochloride ((dimethyl-amino) propyl) -3-ethyl-carbodiimideEDCI), 2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium Hexafluorophosphate (HBTU), 1-Hydroxybenzotriazole (HOBT), 2- (1H-7-azabenzotriazol-1-yl) -1,1,3, 3-tetramethyluronium Hexafluorophosphate (HATU), 2-chloro-1, 3-dimethyl-2-imidazolium tetrafluoroborate, 1-H-benzotriazol-1- [ bis (dimethylamino) methylene ] phosphonium tetrafluoroborate]-5-chlorohexafluorophosphate (HCTU), 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine and 2-propanephosphonic anhydride

An example of a suitable base that can be used in the present invention is, without limitation, potassium carbonate (K)₂CO₃) N-methylmorpholine (NMM), triethylamine (Et)₃N; TEA), diisopropyl-ethylamine (i-Pr)₂EtN; DIEA), pyridine, potassium hydroxide (KOH), sodium hydroxide (NaOH), and sodium methoxide (NaOMe; NaOCH (NaOCH)₃)。

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, but with deuterium or tritium in place of hydrogen, or with¹³C-or¹⁴C-instead of carbon, is within the scope of the invention. Such compounds are useful, for example, as analytical tools or probes in biological assays.

Method of the invention

In general, the present invention provides methods for the synthesis of compounds useful as modulators of CFTR.

Preparation of Compound 1

In certain embodiments, the present invention provides a process for preparing a crystalline form of compound 1:

the method comprises the following steps:

(a) reacting compound 2 with compound 3 in the presence of a coupling agent

Wherein the coupling agent is selected from the group consisting of 2-chloro-1, 3-dimethyl-2-imidazolium tetrafluoroborate, HBTU, HCTU, 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine, HATU, HOBT/EDC and

in another aspect of this embodiment, compound 3 can be an HCl salt.

In one aspect of this embodiment, at, e.g., K₂CO₃、Et₃N, N-methylmorpholine (NMM), pyridine or Diisopropylethylamine (DIEA) in the presence of a base.

In another aspect of this embodiment, the coupling of compound 2 and compound 3 is carried out in the presence of pyridine or DIEA.

In another aspect of this embodiment, the coupling of compound 2 and compound 3 is performed in the presence of pyridine.

In another aspect of this embodiment, the coupling of compound 2 and compound 3 is performed in the presence of a solvent. In one aspect, the solvent is a polar aprotic solvent. For example, the solvent is selected from ethyl acetate, isopropyl acetate, tetrahydrofuran, methyl ethyl ketone, N-methyl-2-pyrrolidone, acetonitrile, N-dimethylformamide, or 2-methyltetrahydrofuran. More specifically, the solvent is 2-methyltetrahydrofuran.

In another aspect of this embodiment, the coupling of compound 2 and compound 3 is performed at a reaction temperature maintained between 30 ℃ and 80 ℃. In other aspects, the coupling of compound 2 and the aniline of formula 3 is performed at a reaction temperature maintained at 30 ℃ to 80 ℃ (e.g., between about 40 ℃ and 78 ℃, between about 45 ℃ and 75 ℃, between about 50 ℃ and 70 ℃, between about 62 ℃ and 68 ℃, or about 65 ℃), for example, the coupling of compound 2 and compound 3 is performed at a reaction temperature maintained at about 65 ℃.

In another aspect of this embodiment, compound 1 is solid form a.

In another embodiment, the present invention provides compound 1 prepared by the method described in the previous paragraph.

Preparation of Compound 1 form A

In another embodiment, the present invention provides a process for the preparation of compound 1 having solid form a,

reacting compound 2 with compound 3 in the presence of a coupling agent

Wherein the coupling agent is selected from the group consisting of 2-chloro-1, 3-dimethyl-2-imidazolium tetrafluoroborate, HBTU, HCTU, 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine, HATU, HOBT/EDC and

in certain other aspects of this embodiment, compound 3 can be an HCl salt.

In this embodimentIn one aspect, at, for example, K₂CO₃、Et₃N, N-methylmorpholine (NMM), pyridine or Diisopropylethylamine (DIEA) in the presence of a base.

In another aspect of this embodiment, the reaction of compound 2 and compound 3 is carried out in the presence of pyridine or DIEA.

In another aspect of this embodiment, the coupling of compound 2 and compound 3 is performed in the presence of pyridine.

In another aspect of this embodiment, the coupling of compound 2 and compound 3 is carried out in the presence of a polar aprotic solvent. For example, the polar aprotic solvent is selected from ethyl acetate, isopropyl acetate, tetrahydrofuran, methyl ethyl ketone, N-methyl-2-pyrrolidone, acetonitrile, N-dimethylformamide, or 2-methyltetrahydrofuran. More specifically, the coupling of compound 2 and compound 3 is carried out in the presence of 2-methyltetrahydrofuran.

In another aspect of this embodiment, the coupling of compound 2 and compound 3 is performed at a reaction temperature maintained between 30 ℃ and 80 ℃. In other aspects, the coupling of compound 2 and the aniline of formula 3 is performed at a reaction temperature maintained between 30 ℃ and 80 ℃ (e.g., between about 40 ℃ and 78 ℃, between about 45 ℃ and 75 ℃, between about 50 ℃ and 70 ℃, between about 62 ℃ and 68 ℃, or about 65 ℃). For example, the coupling of compound 2 and compound 3 is carried out at a reaction temperature maintained at about 65 ℃.

In another embodiment, the present invention provides compound 1 form a prepared by the method described in the preceding paragraph.

In one embodiment, the present invention provides a process for preparing compound 1 form a, said process comprisingAnd pyridine, at a temperature of about 65 ℃, compound 2 is reacted with compound 3 in the solvent 2-MeTHF for about 10 hours.

Preparation of Compound 1 form B

In another embodiment, the present invention provides a process for preparing compound 1 having solid form B:

the method comprises the following steps:

(a) reacting compound 2 with the hydrochloride salt of compound 3 (3-HCl) in the presence of a coupling agent selected from the group consisting of 2-chloro-1, 3-dimethyl-2-imidazolium tetrafluoroborate, HBTU, HCTU, 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine, HATU, HOBT/EDC and

in one aspect of this embodiment, at, e.g., K₂CO₃、Et₃N, N-methylmorpholine (NMM), pyridine or DIEA in the presence of a base to effect coupling of Compound 2 and hydrochloride salt 3-HCl.

In certain other aspects of this embodiment, the hydrochloride salt 3-HCl can be compound 3.

In another aspect of this embodiment, the coupling of compound 2 and hydrochloride salt 3-HCl is carried out in the presence of pyridine or DIEA.

In another aspect of this embodiment, the coupling of compound 2 and hydrochloride salt 3-HCl is carried out in the presence of pyridine.

In another aspect of this embodiment, the coupling of compound 2 and hydrochloride salt 3-HCl is carried out in the presence of a solvent such as EtOAc, IPAc, THF, MEK, NMP, acetonitrile, DMF, or 2-methyltetrahydrofuran. More specifically, the coupling of compound 2 and hydrochloride salt 3-HCl was carried out in the presence of 2-methyltetrahydrofuran.

In another aspect of this embodiment, the coupling of compound 2 and hydrochloride salt 3-HCl is carried out at a reaction temperature maintained between 15 ℃ and 70 ℃. In other aspects, the coupling of compound 2 and hydrochloride salt 3-HCl is performed at a reaction temperature maintained at 15 ℃ to 70 ℃ (e.g., between about 20 ℃ and 65 ℃, between about 25 ℃ and 60 ℃, between about 30 ℃ and 55 ℃, between about 35 ℃ and 50 ℃, or about 45 ℃). In a preferred aspect of this embodiment, the coupling of the HCl salts of compound 2 and compound 3 is carried out at a reaction temperature maintained at about 45 ℃.

In another aspect, the coupling reaction is stirred for a duration of from about 1.5 hours to about 10 hours. In other aspects, the coupling reaction is stirred for a duration of about 1.5 hours to about 10 hours (e.g., from about 2 hours to about 7 hours, from about 3 hours to about 6 hours, from about 4 hours to about 5.5 hours, or about 5 hours).

In another embodiment, the present invention provides compound 1 form B prepared by the methods described in the preceding paragraphs.

In one embodiment, the present invention provides a process for preparing compound 1 form B, said process comprisingAnd pyridine, compound 2 is reacted with compound 3-HCl in the solvent 2-MeTHF at a temperature of about 45 deg.C for about 5-6 hours.

Preparation of Compound 1 form A-HCl

In another embodiment, the present invention provides a process for preparing the hydrochloride salt of compound 1 having solid form a-HCl:

the method comprises the following steps:

(a) reacting compound 2 with the hydrochloride salt of compound 3 (3-HCl) in the presence of a coupling agent selected from the group consisting of 2-chloro-1, 3-dimethyl-2-imidazolium tetrafluoroborate, HBTU, HCTU, 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine, HATU, HOBT/EDC and

and

(b) treating a mixture of the products of step (a) with HCl.

In one aspect of this embodiment, at, e.g., K₂CO₃、Et₃N, N-methylmorpholine (NMM), pyridine or Diisopropylethylamine (DIEA) in the presence of a base.

In another aspect of this embodiment, step (a) is carried out in the presence of pyridine or DIEA.

In another aspect of this embodiment, step (a) is carried out in the presence of pyridine.

In another aspect of this embodiment, step (a) is carried out in the presence of a polar aprotic solvent. For example, the polar aprotic solvent is selected from ethyl acetate, isopropyl acetate, tetrahydrofuran, methyl ethyl ketone, N-methyl-2-pyrrolidone, acetonitrile, N-dimethylformamide, or 2-methyltetrahydrofuran. More specifically, the coupling of compound 2 and 3-HCl is carried out in the presence of 2-methyltetrahydrofuran.

In another aspect of this embodiment, step (a) is carried out at a reaction temperature maintained between 10 ℃ and 80 ℃. In another aspect of this embodiment, step (a) is carried out at a reaction temperature maintained between 15 ℃ and 70 ℃. In other aspects, the coupling of the HCl salts of compounds 2 and 3 is performed at a reaction temperature maintained at 15 ℃ to 70 ℃ (e.g., between about 20 ℃ and 65 ℃, between about 25 ℃ and 60 ℃, between about 30 ℃ and 55 ℃, between about 35 ℃ and 50 ℃, or about 45 ℃). For example, the coupling of compound 2 and 3-HCl is carried out at a reaction temperature maintained at about 45 ℃.

In another aspect of this embodiment, the time for step (a) is from about 1.5 hours to about 72 hours. In other aspects, the coupling reaction is stirred for a duration of about 1.5 hours to about 72 hours or more (e.g., from about 2 hours to about 48 hours, from about 3 hours to about 24 hours, from about 5 hours to about 20 hours, or from about 12 hours to about 15 hours).

In another aspect of this embodiment, the coupled product in step (a) is treated with hydrogen chloride (HCl) in step (b). For example, HCl gas is bubbled into a mixture comprising the product of the coupling reaction of step (a) and a polar aprotic solvent, such as 2-methyltetrahydrofuran.

Typically, at least about 1 equivalent of HCl gas, and at most about 50 equivalents of HCl gas, are bubbled into the mixture. More typically, from about 2 equivalents (eq.) to about 20 equivalents (from about 5eq to about 15eq, from about 8eq to about 12eq, or about 10eq) of HCl gas is bubbled into the reaction mixture comprising the coupled product of step (a).

Typically, HCl gas is bubbled into the mixture of the product of step (a) and the solvent, e.g., aprotic solvent, over a period of about 0.5 hours to about 5 hours, more typically over a period of about 0.5 hours to about 5 hours (e.g., from about 0.75 hours to about 3 hours, or about 2 hours).

In another embodiment, the present invention provides compound 1 form a-HCl prepared by the method described in the preceding paragraph.

In one embodiment, the present invention provides a process for preparing compound 1 form a-HCl, comprisingAnd pyridine, compound 2 is reacted with compound 3-HCl in the solvent 2-MeTHF at a temperature of about 45 deg.C for about 12-15 hours, followed by treatment with gaseous HCl.

Preparation of Compound 1 form B-HCl

In another aspect, the present invention provides a process for preparing a hydrochloride salt of compound 1 having solid form B-HCl:

the method comprises the following steps:

(a) mixing hydrochloride salt of compound 1 form a-HCl as described herein with an organic solvent and water to produce a mixture:

and

(b) the mixture is heated.

In one aspect of this embodiment, the organic solvent comprises dimethyl sulfoxide, dimethylformamide, dioxane, hexamethylphosphoric triamide, tetrahydrofuran, EtOAc, IPAc, THF, MEK, NMP, acetonitrile, DMF, EtOH, MeOH, isopropanol, or 2-methyltetrahydrofuran. More specifically, the aprotic solvent comprises 2-methyltetrahydrofuran.

In another aspect, the mixing in step (a) is continued in step (b), and the mixture is maintained at a temperature of from about 30 ℃ to about 80 ℃ (e.g., from about 40 ℃ to about 70 ℃, from about 50 ℃ to about 65 ℃, or about 60 ℃). The method of claims 40-43, wherein the mixture is maintained at a temperature of 30 ℃ to about 80 ℃ for a time of from about 12 hours to about 72 hours.

In another aspect, after heating in step (b), the mixture is filtered to produce a filter cake.

In another aspect, after filtration, the filter cake is washed with an aprotic solvent, such as 2-methyltetrahydrofuran.

In another embodiment, the present invention provides compound 1 form B-HCl prepared by the method described in the preceding paragraph.

In one embodiment, the present invention provides a process for preparing compound 1 form B-HCl, the process comprising heating compound 1 form a-HCl in a mixture of 2-MeTHF and water at a temperature of 60 ℃ for 48 hours; cooling to room temperature and filtering the precipitated product; and drying the product under vacuum at a temperature of 60 ℃.

In another embodiment, the present invention provides a process for the preparation of compound 1 form B-HCl comprising heating compound 1 form a-HCl in a mixture of EtOH and water to reflux temperature; cooling to 20 ℃, and stirring for 3 hours; filtering the precipitated product; the product was dried under vacuum at a temperature of 45 ℃.

Preparation of Compounds 2, 3 and 3-HCl

In another embodiment, the present invention provides a process for preparing compound 2:

the method comprises the following steps:

(a) reaction of Compound 4 with diethyl 2- (ethoxymethylene) malonate 5 to produce ester 6A

And

(b) ester 6A is treated with a source of atomic hydrogen, such as hydrogen or formate (format), in a separate step in the presence of a catalyst and a base to produce compound 2

In one aspect, compound 6A is treated with an atomic hydrogen source, such as hydrogen or formate, in the presence of a catalyst to produce compound 6C

Compound 6C is then treated with a base to produce compound 2.

In another aspect, compound 6A is treated with a base to produce compound 6D

Compound 6D is then treated with a source of atomic hydrogen, such as hydrogen or formate, in the presence of a catalyst to produce compound 2.

In certain aspects, the hydrogen source is hydrogen gas. In other aspects, the hydrogen source is formate.

In certain aspects, the catalyst is a palladium catalyst.

In certain aspects, the base is an aqueous sodium hydroxide solution or an aqueous potassium hydroxide solution. For example, the base is aqueous sodium hydroxide.

In one embodiment, the present invention provides a process for preparing compound 2, said process comprising the steps of:

a) reacting compound 4 with compound 5 in toluene at reflux temperature in a dissei apparatus to yield compound 6B;

b) heating compound 6B in a dow heat carrier at a temperature of about 260 ℃ to produce cyclized product compound 6A;

c) hydrogenating compound 6A in EtOH using Pd/C as a catalyst in the presence of hydrogen and triethylamine to produce compound 6C; and

d) hydrolysis of Compound 6C with 5M NaOH to prepare Compound 2

In one embodiment, the present invention provides a process for preparing compound 2, said process comprising the steps of:

a) reacting compound 4 with compound 5 in toluene at reflux temperature in a dissei apparatus to yield compound 6B;

b) heating compound 6B in a dow heat carrier at a temperature of about 260 ℃ to produce cyclized product compound 6A;

c) hydrolyzing compound 6A in a mixture of isopropanol and water using NaOH to produce compound 6D; and

d) compound 6D was hydrogenated in EtOH using Pd/C as catalyst in the presence of formate, sodium methoxide and methanol; and

e) acidification of the solution with acetic acid to produce Compound 2

In another embodiment, the present invention provides a process for preparing compound 3

The method comprises the following steps:

(a) reacting compound 7, wherein Hal is F, Cl, Br or I, with 7-azabicyclo [2.2.1] heptane 8, or a salt thereof, to produce compound 9

And

(b) the nitro moiety in compound 9 is reduced to provide aniline 3.

In one aspect of this embodiment, step (a) is carried out in a polar aprotic solvent in the presence of a base. For example, the base is a tertiary amine base, such as triethylamine, or diisopropylethylamine, or the like, and a solvent, such as acetonitrile.

In another aspect of this embodiment, step (b) is carried out in an alcohol solvent using hydrogen and a transition metal catalyst. For example, the catalyst comprises a group 9 or group 10 transition metal catalyst derived from Pt, Pd, or Ni. More specifically, the catalyst comprises Pd. The alcohol solvent includes alcohols such as isopropyl alcohol, ethanol, methanol, and the like. For example, the solvent includes ethanol.

In another embodiment, the present invention provides a process for preparing compound 3:

the method comprises the following steps:

(a) reacting compound 7, wherein Hal is F, Cl, Br or I, with the hydrochloride salt of 7-azabicyclo [2.2.1] heptane (8-HCl) to produce compound 9

And

(b) the nitro moiety in compound 9 is reduced to provide compound 3.

In one aspect of this embodiment, step (a) is carried out in the presence of a base, such as an inorganic carbonate salt of sodium carbonate, and a polar aprotic solvent, such as DMSO.

In another aspect of this embodiment, step (b) is carried out in an alcohol solvent using hydrogen and a transition metal catalyst. For example, the catalyst comprises a group 9 or group 10 transition metal catalyst derived from Pt, Pd, or Ni. More specifically, the catalyst comprises Pd. The alcohol solvent includes alcohols such as isopropyl alcohol, ethanol, methanol, and the like. For example, the solvent includes ethanol.

In another embodiment, the present invention provides a process for preparing hydrochloride salt 3-HCl:

the method comprises the following steps:

(a) reacting compound 7, wherein Hal is F, Cl, Br or I, with 7-azabicyclo [2.2.1] heptane hydrochloride 8-HCl to yield compound 9

(b) Reducing the nitro moiety in compound 9 to provide aniline 3;

and

(c) treating the product of step (c) with HCl gas to provide 3-HCl.

In one aspect of this embodiment, step (a) is carried out in the presence of a base, such as an inorganic carbonate salt of sodium carbonate, and a polar aprotic solvent, such as DMSO.

In one aspect of this embodiment, step (b) is carried out in an alcohol solvent using hydrogen and a transition metal catalyst. For example, the catalyst comprises a group 9 or group 10 transition metal catalyst derived from Pt, Pd, or Ni. More specifically, the catalyst comprises Pd. The alcohol solvent includes alcohols such as isopropyl alcohol, ethanol, methanol, and the like. For example, the solvent includes ethanol.

In another embodiment, the invention includes a process for preparing Compound 8, or a pharmaceutically acceptable salt thereof,

the process comprises 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

And

contacting the compound of formula C with a hydroxide to prepare the compound of formula 8.

In another embodiment, the invention includes a process for producing a compound of formula 8-HCl,

the method comprises contacting a compound of formula 8 with hydrochloric acid.

In one embodiment, the present invention provides a process for preparing compound 3, comprising the steps of:

a) reacting compound 7 with compound 8-HCl in acetonitrile in the presence of triethylamine at about 80 ℃ for about 16 hours to prepare compound 9; and

b) hydrogenation of Compound 9 in ethanol in the Presence of Hydrogen Using Pd/C as catalyst

In one embodiment, the present invention provides a process for preparing compound 3, comprising the steps of:

a) reacting compound 7 with compound 8-HCl in DMSO in the presence of sodium carbonate at about 55 ℃ to prepare compound 9; and

b) hydrogenation of Compound 9 in ethanol in the Presence of Hydrogen Using Pd/C as catalyst

In one embodiment, the present invention provides a process for preparing compound 3, comprising the steps of:

a) reacting compound 7 with compound 8-HCl in dichloromethane in the presence of sodium hydroxide and tetrabutylammonium bromide to produce compound 9; and

b) hydrogenation of Compound 9 in ethanol in the Presence of Hydrogen Using Pd/C as catalyst

In one embodiment, the present invention provides a process for preparing compound 3-HCl comprising hydrogenating the hydrochloride salt of compound 9 in 2-MeTHF in the presence of hydrogen using Pd/C as a catalyst.

Other aspects of the invention

In one aspect, the invention features a pharmaceutical composition that includes compound 1 form a, compound 1 form a-HCl, compound 1 form B-HCl, or any combination thereof, 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 compound 1 form a, compound 1 form a-HCl, compound 1 form B-HCl, or any combination thereof.

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

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, larron dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, glyceranosis CDG type 1, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency, Diabetes Insipidus (DI), neurogenic DI, renal DI, charcot-marie-tooth syndrome, peyronie'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, spinal and bulbar muscular atrophy, dentatorubral erythrocytic hypothalamic atrophy and myotonic dystrophy, and spongiform encephalopathies, such as hereditary creutzfeldt-jakob disease (due to), fabry disease, Straussler-Scheinker syndrome, and neurodegenerative diseases, 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 diseases, e.g., congenital myotonia (Thomson and Becker types), barter syndrome type III, Dent's disease, startle syndrome, epilepsy, startle syndrome, lysosomal storage diseases, Angelman syndrome, Primary Ciliary Dyskinesia (PCD), the term used to refer to genetic disorders of ciliary structure and/or function, including PCD with left and right transposition (also known as catagory syndrome), PCD without 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 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 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 1 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 1 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, comprising administering to the patient compound 1 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 1 as described herein.

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

In certain aspects, the invention provides a method of increasing bone deposition in a patient, comprising administering to the patient compound 1 as described herein.

In certain embodiments, a method of increasing bone deposition in a patient, the method comprising 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 1 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 1 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 methods of treating or lessening the severity of chronic bronchitis in a patient comprising administering to said patient compound 1 as described herein.

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

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 compound 1 form a, form a-HCl, form B-HCl, or any combination thereof.

In one aspect, the invention features a pharmaceutical package or kit that includes compound 1 form a, form a-HCl, form B-HCl, or any combination of these forms, and a pharmaceutically acceptable carrier.

In one aspect, the invention features N- (4- (7-azabicyclo [2.2.1]]A crystalline form of hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide having a trigonal system, an R-3 space group, and the following unit cell parameters:α =90 °, β =90 °, and γ =120 °.

In one embodiment, the invention provides N- (4- (7-azabicyclo [2.2.1] in form B]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 °.

Use, formulation and administration

In one aspect of the invention, pharmaceutically acceptable compositions are provided, wherein these compositions comprise form a as described herein, and optionally a pharmaceutically acceptable carrier, adjuvant or vehicle. In certain embodiments, these compositions also optionally comprise one or more additional therapeutic agents.

As noted above, the pharmaceutically acceptable compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, adjuvant, or excipient, which, as used herein, includes any and all solvents, diluents, or other liquid excipients, dispersion or suspension aids, surface agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate to the particular dosage form desired. Remington's Pharmaceutical Sciences, 16 th edition, e.w. martin (Mack Publishing co., Easton, Pa., 1980) discloses a variety of carriers for preparing pharmaceutically acceptable compositions and known techniques for preparing the same. So long as it is not the case that any conventional carrier medium is incompatible with the compounds of the present invention, e.g., by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component of a pharmaceutically acceptable composition, its use is contemplated to be within the scope of the present invention. Some examples of materials 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, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, 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; a diol; 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; a ringer's solution; ethanol and phosphate buffer solutions, as well as other non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the combination, according to the judgment of the formulator.

Use of compounds and pharmaceutically acceptable compositions

In yet another aspect, the invention provides methods of treating or lessening the severity of a condition, disease or disorder in which a mutation in CFTR is implicated. In certain embodiments, the present invention provides methods of treating a condition, disease, or disorder involving a deficiency in CFTR activity, comprising administering to a subject, preferably a mammal, in need thereof a composition comprising compound 1 form a, form a-HCl, form B-HCl, or any combination of these forms.

In some embodiments, the invention provides methods of treating diseases associated with reduced CFTR function due to mutations in the gene encoding CFTR or environmental factors (e.g., smoking). These include cystic fibrosis, chronic bronchitis, recurrent bronchitis, acute bronchitis, male infertility due to congenital bilateral absence of the vas deferens (CBAVD), female infertility due to congenital uterine and vaginal loss (CAUV), Idiopathic Chronic Pancreatitis (ICP), idiopathic recurrent pancreatitis, idiopathic acute pancreatitis, chronic rhinosinusitis, primary sclerosing cholangitis, allergic bronchopulmonary aspergillosis, diabetes, dry eye, constipation, allergic bronchopulmonary aspergillosis (ABPA), bone disease (e.g., osteoporosis), and asthma.

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 disorders, myotonia congenital myotonia (Thomson and Becker types), Batt syndrome type III, Dent's disease, shock syndrome, epilepsy, shock syndrome, lysosomal storage disease, lysosomal storage 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, comprising the step of administering to said mammal an effective amount of a composition comprising compound 1 form a, compound 1 form a-HCl, compound 1 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 an effective amount of a composition comprising compound 1 form a, compound 1 form a-HCl, compound 1 form B-HCl or any combination of these forms as described herein.

According to the present invention, an "effective amount" of compound 1 form a, compound 1 form a-HCl, compound 1 form B-HCl, or any combination of these forms, or a pharmaceutically acceptable composition thereof, is an amount effective to treat or reduce the severity of one or more diseases, disorders, or conditions as described above.

Compound 1 form a, compound 1 form a-HCl, compound 1 form B-HCl, or any combination of these forms, or a pharmaceutically acceptable composition thereof, can be administered using any amount and 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, compound 1 form a-HCl, compound 1 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 heterozygous or homozygous for a variety of different mutants, including patients heterozygous or homozygous for the most common mutation af 508.

In another embodiment, compound 1 form a-HCl, compound 1 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 who has 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 another embodiment, the compound 1 form A, compound 1 form A-HCl, compound 1 form B-HCl, or any combination of these forms or pharmaceutically acceptable compositions thereof described herein are useful for cystic fibrosis or lessening the severity of cystic fibrosis in a patient who has certain genotypes exhibiting residual CFTR activity, e.g., a class III mutation (impaired regulation or gating), a class IV mutation (altered transduction), or a class V mutation (reduced synthesis) (Lee R.Choo-Kang, Pamela L., Zeitin, Type I, II, III, IV, and V cysteine fibrosis cancer production Regulator and delivery Opportunities of Therapy; Current Opinion in medicinal Purpurification 6: 521-529, 2000). 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, compound 1 form a-HCl, compound 1 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 clinical phenotypes, e.g., mild to moderate clinical phenotypes 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 absence 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 aqueous or oleaginous 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 will then depend 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, for example, 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) solution 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 compound 1 form a, compound 1 form a-HCl, compound 1 form B-HCl, or any combination of these forms or pharmaceutically acceptable compositions thereof described herein may be used in combination therapy, that is, form a-HCl, form B-HCl, or any combination of these forms or pharmaceutically acceptable compositions 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, modulators of CFTR, such as those described herein, 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 (keratojunctional sis), respiratory infections (acute and chronic; viral and bacterial), and lung cancer.

CFTR modulators, such as those described herein, in combination with agents that reduce ENaC activity are also useful in the treatment of diseases that block epithelial sodium channel-mediated, including diseases other than respiratory diseases, which 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. It may further be evidenced by a reduced need for other palliative therapies, i.e., therapies used or intended to limit or interrupt the onset of symptoms at the time of onset, such as anti-inflammatory (e.g., corticosteroids) or bronchodilatory therapies. 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, such as those described herein, in combination with an agent that reduces ENaC activity is used to treat bronchitis, regardless of type or origin, including, for example, 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 A, 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 composition of the present invention will not exceed the amount normally administered in a composition containing the therapeutic agent as the only active ingredient. 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.

Compound 1 form A, form A-HCl, form B-HCl, or any combination of these forms, or a pharmaceutically acceptable composition thereof, as described herein, can 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 in a patient, comprising administering to the patient or contacting the biological sample with compound 20 form a, 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 compound 20 form a, 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 compound 20 form a, form a-HCl, form B-HCl or any combination of these forms as described herein, or a pharmaceutically acceptable composition thereof.

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 changes in Fluorescence Resonance Energy Transfer (FRET) between the membrane-soluble, voltage-sensitive dye DiSBAC2(3) and 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 compound 20 form a, form a-HCl, form B-HCl, or any combination thereof or any of the above embodiments; and (ii) instructions for: a) contacting an additional 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 composition 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 CFTR activity in the presence of the additional compound to the density of CFTR in the presence of form A, form A-HCl, form B-HCl, or any combination thereof, as described herein. In a preferred embodiment, the kit is used to determine the density of CFTR.

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. The light is monochromatized by a cold-cooled 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). Powder analysis module using Materials StudioAnd (5) analyzing the structure. 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 compared to tetramethylsilane at 0ppm and the fluorine spectrum is indirectly compared at 0ppmControl nitromethane.

Synthetic examples

Preparation examples: 7-azabicyclo [2.2.1] heptane hydrochloride (8-HCl).

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 stirring. 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 3 hours, 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 and stirring continued for 15 min. The stirring was stopped, the mixture was allowed to stand for 10 minutes 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 30 minutes, followed by a second addition. The mixture was stirred at room temperature overnight (15 hours) 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 (8-HCl), method 1. A TFA salt of trans-4-aminocyclohexylmethanesulfonate (200g, 650.9mmol) was introduced into a three-neck flask, followed by addition of water (2.200L, 11 vol.). NaOH (78.11g, 1.953mol, 3 equivalents) was slowly added, 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, the mixture was stirred for 30 minutes, 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. The crude product was recrystallized from acetonitrile (10 volumes) to provide 7-azabicyclo [2.2.1] as a colorless crystalline solid]Heptane hydrochloride (8-HCl).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 (8-HCl), 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 ℃), initial temperature 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 (2).

Preparation of diethyl 2- ((2-chloro-5- (trifluoromethyl) phenylamino) methylene) malonate (6B). 2-chloro-5- (trifluoromethyl) aniline (4) (200g, 1.023mol), diethyl 2- (ethoxymethylene) malonate (5) (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 and stirred to 140 ℃ and held at this temperature for 4 hours. The reaction mixture was cooled to 70 ℃ and hexane (600mL) was added slowly. The resulting slurry was stirred and cooled to room temperature. The solid was collected by filtration, washed with 10% ethyl acetate in hexanes (2 × 400mL), then dried under vacuum to afford a white solid (350g,94% yield) as the desired precipitated product diethyl 2- ((2-chloro-5- (trifluoromethyl) phenylamino) methylene) malonate (6B).¹H NMR(400MHz，DMSO-d⁶)δ11.28(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 (6A) method 1. A1L three-necked flask was charged with Dow's heat carrier(200mL, 8mL/g), which was degassed at 200 ℃ for 1 hour. The solvent was heated to 260 ℃ and diethyl 2- ((2-chloro-5- (trifluoromethyl) phenylamino) methylene) malonate (6B) (25g, 0.07mol) was added in portions over 10 minutes.The resulting mixture was stirred at 260 ℃ for 6.5 hours (h), 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 (min), 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 (6A) as a tan solid.¹H NMR(400MHz，DMSO-d⁶)δ11.91(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 (6A) method 2. Compound 6B (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 (6C). A5L three-necked flask was charged with ethyl 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylate (6A) (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% by weight) 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 (6C), 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 (2). Ethyl 4-oxo-5- (trifluoromethyl) -1H-quinoline-3-carboxylate (6C) (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, and then further heated 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 (2) as a white solid.¹H NMR(400.0MHz，DMSO-d⁶)δ15.26(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: 4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (2) can be prepared instead.

Preparation of 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (6D). Ethyl 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylate (6B) (1200g, 3.754mol) was charged to a vessel, followed by 2-propanol (1.200L) and water (7.200L), and stirred. NaOH (600.6g, 7.508mol) and water (1.200L) were mixed and cooled to room temperature. The resulting NaOH solution was charged to the reaction vessel. The reaction mixture was heated to 80 ℃ and stirred for 3.5 hours to give a dark homogeneous mixture. After a further 1 hour, acetic acid (9.599L [20% w/v solution) was added via dropping funnel over a period of 45 minutes]31.97 mol). The reaction mixture was cooled to 22 ℃ at a rate of 6 ℃/hour and stirred. 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 as a brown solid. By slurrying in 1.5L of methanolAnd stirred for 6 hours to purify 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid. It was then filtered and dried to provide 968.8g of purified 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (6D).

Preparation of 4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (2). 8-chloro-4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (6D) (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. NaOMe (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. It was then 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 no more than 1.0% relative to 4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid. When the reaction was complete, the mixture was filtered through a pad of celite (37g [ approximately twice the amount of starting material 6D ]) 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 provide the title carboxylic acid 2.

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

Example 2A: 4- (7-azabicyclo [2.2.1]]Process for producing hept-7-yl) -2- (trifluoromethyl) aniline (3) And (4) preparation.

7- [ 4-Nitro group-3- (trifluoromethyl) phenyl]-7-azabicyclo [2.2.1]Preparation of heptane (9), method 1. A solution of 4-fluoro-1-nitro-2- (trifluoromethyl) benzene (7) (6.0g, 28.69mmol) and triethylamine (8.7g, 12.00ml, 86.07mmol) in acetonitrile (50ml) was added to a solution containing 7-azabicyclo [2.2.1] benzene (7)]Heptane hydrochloride (8-HCl) (4.6g, 34.43mmol, obtained under nitrogen) in a flask. The reaction flask was heated at 80 ℃ under nitrogen for 16 hours. The reaction mixture was cooled and then partitioned between water and dichloromethane. The organic layer was washed with 1M HCl and Na₂SO₄Drying, 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 (9).¹H NMR(400.0MHz，DMSO-d⁶)δ8.03(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 (9), method 2. 4-fluoro-1-nitro-2- (trifluoromethyl) benzene (7) (901g, 4.309mol) was introduced into a 30L jacketed vessel under nitrogen with sodium carbonate (959.1g, 9.049mol) and DMSO (5L, 5.5 vol) and stirred. 7-azabicyclo [2.2.1] heptane hydrochloride (8-HCl) (633.4g, 4.740mol) was then added in portions to the vessel and the temperature was gradually increased to 55 ℃. When the reaction was substantially complete, the mixture was diluted with 10 volumes of EtOAc and washed 3 times with water (5.5 volumes) until the DMSO in the aqueous layer disappeared (HPLC). The organic layer was concentrated to 4 volumes and then the solvent was exchanged with cyclohexane until all EtOAc was removed, the total volume in the flask was approximately 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 and stirred or spun for 3 hours. When all the solid crystallized, the solution was concentrated to dryness to provide 7- [ 4-nitro-3- (trifluoromethyl) phenyl ] -7-azabicyclo [2.2.1] heptane (9).

7- [ 4-Nitro-3- (trifluoromethyl) phenyl]-7-azabicyclo [2.2.1]Preparation of heptane (9), method 3. By reacting 4-fluoro-1-nitro-2- (trifluoromethyl) benzene was dissolved in 3 volumes of DCM. Tetrabutylammonium bromide (0.05 eq) and KOH (50 wt%, 3.6 eq) were added. Then adding 7-azabicyclo [2.2.1] at 0-5 deg.C]Heptane hydrochloride (8-HCl). The reaction was warmed to room temperature and monitored by HPLC. Once essentially complete, the layers were separated and the organic layer was washed with 1M HCl. The layers were separated and the aqueous layer was discarded. The organic layer was washed 1 time with water, 1 time with brine and then distilled. The resulting material was recrystallized from cyclohexane under reflux. The solid was filtered, washed with cyclohexane and dried in a vacuum oven at 45 ℃ with N₂Gas purge drying to provide 7- [ 4-nitro-3- (trifluoromethyl) phenyl]-7-azabicyclo [2.2.1]Heptane (9).

4- (7-azabicyclo [2.2.1]]Preparation of hept-7-yl) -2- (trifluoromethyl) aniline (3). Will be charged with 7- [ 4-nitro-3- (trifluoromethyl) phenyl]-7-azabicyclo [2.2.1]A flask of heptane (26) (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 and the residue was purified by silica gel chromatography (0-15% ethyl acetate in hexanes) to provide 4- (7-azabicyclo [2.2.1] as a purple solid]Hept-7-yl) -2- (trifluoromethyl) aniline (3) (5.76g,91% yield).¹H NMR(400.0MHz，DMSO-d⁶) δ 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]]Hydrochloric acid of hept-7-yl) -2- (trifluoromethyl) aniline Salt (3-HCl) And (4) preparing.

Preparation of hydrochloride salt of 4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) aniline (3-HCl), method 1. Palladium on carbon (150g, 5% w/w) was charged to a Buchi hydrogen generator (20L capacity) under nitrogen, followed by the addition of hydrochloride salt of 7- [ 4-nitro-3- (trifluoromethyl) phenyl ] -7-azabicyclo [2.2.1] heptane (9) (1500g) and 2-methyltetrahydrofuran (10.5L, 7 vol). Hydrogen was charged into a closed vessel to a pressure above atmospheric +0.5 bar. Vacuum was applied for about 2 minutes, followed by introduction of hydrogen to a pressure of 0.5 bar. This process was repeated 2 times. The hydrogen was then continuously charged at a pressure above atmospheric +0.5 bar. The mixture was stirred and the temperature was maintained between 18 ℃ and 23 ℃ by cooling the jacketed vessel. Once the reaction no longer consumes hydrogen and no longer exotherms, vacuum was again applied. The vessel was charged with nitrogen at 0.5bar, vacuum was again applied, followed by a second charge of 0.5bar nitrogen. When the reaction was substantially complete, the reaction mixture was transferred to a receiving flask through a filter funnel under nitrogen atmosphere 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 another 10 hours (or overnight), filtered, washed with 2-methyltetrahydrofuran (2 volumes) and dried to give 1519g of 4- (7-azabicyclo [2.2.1] hept-7-yl) -2- (trifluoromethyl) aniline hydrochloride (3-HCl) 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-azabicyclo [2.2.1] as form A (Compound 1, form A)] Hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid And (4) preparing amine.

Propyl phosphonic acid cyclic anhydride (in ethyl acetate)To a 50% solution of 4-oxo-5- (trifluoromethyl) -1H-quinoline-3-carboxylic acid (2) (9.1g, 35.39mmol) and 4- (7-azabicyclo [2.2.1 mmol) at room temperature, 52.68mL, 88.48mmol) and pyridine (5.6g, 5.73mL, 70.78mmol) were added]A solution of hept-7-yl) -2- (trifluoromethyl) aniline (3) (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 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 product was slurried in ethyl acetate/diethyl ether (2: 1), collected by vacuum filtration, and washed twice with ethyl acetate/diethyl ether (2: 1) to provide the crude product as a pale yellow crystalline powder. The powder was dissolved in warm ethyl acetate and absorbed on celite. Purification by silica gel chromatography (0-50% ethyl acetate in dichloromethane) afforded N- (4- (7-azabicyclo [2.2.1] as a white crystalline solid]Form a of hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide (compound 1 form a). LC/MS M/z496.0[ M + H ]]⁺Retention time 1.48 min (RP-C)₁₈，10-99%CH₃CN/0.05% TFA over 3 min).¹H NMR(400.0MHz，DMSO-d⁶)δ13.08(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)。

The powder diffractogram of compound 1 form a is shown in figure 1.

Table 1 provides a list of XRPD peaks representing compound 1 form a.

Table 1: representative XRPD peaks of compound 1 form a.

2-theta (degree)	Relative Strength (%)
		7.9	100.0
9.3	10.8
		11.9	12.8
14.4	35.2
		15.1	12.6
15.8	34.1
		17.0	25.2
17.7	13.8
		19.3	39.4
20.1	20.2
		21.4	14.5
21.8	94.2
		23.4	30.0
23.8	92.0
		25.6	8.9
26.8	6.4
		29.4	8.1
29.7	18.1
		30.1	14.2
31.2	9.9

A representative sample of compound 1 form a gave the FTIR spectrum provided in figure 2.

The conformational diagram of compound 1 form a based on single crystal X-ray analysis is shown in figure 3. Diffraction data were obtained on a Bruker Apex II diffractometer equipped with a sealed tube CuK- α source and Apex II CCD detector. Structure (Sh) resolution and refinement using the SHELX programeldrick, g.m., Acta cryst.a64, pp.112-122 (2008)). The structure is resolved and refined into trigonometric and R-3 space groups based on intensity, statistics, and symmetry. Compound 1, form a, has the following unit cell parameters (cell dimension):α =90 °, β =90 °, and γ =120 °.

Example 3B: as form A-HCl(Compound 1 form A-HCl) N- (4- (7-Nitrogen) Hetero-bicyclo [2.2.1]Hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-bis Preparation of hydroquinoline-3-carboxamides

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 hydrochloride (3-HCl) (791g, 2.674mol) and 4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (2) (573g, 2.2278mol) and a further 5.2L (9.0 vol) of 2-methyltetrahydrofuran. Stirring was started and T3P in 2-methyltetrahydrofuran (2.836kg, 4.456mol) was added to the reaction mixture over a period of 15 minutes. Pyridine (534.0g, 546.0mL, 6.684mol) 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. HPLC analysis showed that 4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid was present in an amount of less than 2%. The mixture was then cooled to room temperature and 2-methyltetrahydrofuran (4 vol, 2.292L) was added followed by water (6.9 vol, 4L) to keep the temperature below 30 ℃. The aqueous layer was removed and the residue was washed,the organic layer was washed with 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]]Seed crystals of form A-HCl of hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide hydrochloride (Compound 1 form A-HCl) (3.281g, 5.570 mmol). Hcl (g) (10 eq) was bubbled in over 2 hours and the mixture was stirred overnight. The resulting suspension was filtered, washed with 2-methyltetrahydrofuran (4 volumes), suction dried and dried in an oven at 60 ℃ to constant weight to provide compound 1, form a-HCl.

The powder diffractogram of compound 1 form a-HCl is shown in fig. 4.

Table 2 provides representative XRPD peaks of compound 1 form a-HCl.

Table 2 representative XRPD peaks of compound 1 form a-HCl.

2-theta (degree)	Relative Strength (%)
		7.1	44.3
8.2	33.3
		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
		26.9	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

2-theta (degree)	Relative Strength (%)
		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
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 °.

Samples of compound 1 form a-HCl were also evaluated using microscopy.

A DSC curve for a sample of compound 1 form a-HCl is provided in fig. 5, and a TGA curve for a representative sample of compound 1 form a-HCl is provided in fig. 6.

The FTIR spectra shown for representative samples of compound 1 form a-HCl are provided in fig. 7.

Also using the solid state¹³C and¹⁹compound 1, form a-HCl, was analyzed by F NMR. The respective NMR spectra are provided in fig. 8 and 9. In that¹³C SSNMR and¹⁹several peaks found in the F SSNMR spectra are described in tables 3 and 4.

Table 3: process for preparing compound 1 form A-HCl¹³C SSNMR peaks.

Peak numbering	F1(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

Peak numbering	F1(ppm)
		14	65.7
15	29.2
		16	28.3
17	26.1

Table 4: process for preparing compound 1 form A-HCl¹⁹F SSNMR peak.

Peak numbering	F1(ppm)
		1	-57.0
2	-60.5

Example 4A: n- (4- (7-azabicyclo [2.2.1] as form B (Compound 1, form B)] Hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid And (4) preparing amine.

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 hydrochloride (3-HCl) (1.2 eq) and 4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxylic acid (2) (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 was then heated to 45 ℃ over a period of about 30 minutes with stirring, and the temperature was maintained 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), polished filtered and 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 1 form B.

The powder diffraction patterns of compound 1 form B recorded using instruments 1 and 2 are shown in fig. 10A and 10B, respectively.

Table 5 includes representative XRPD peaks of compound 1 form B

TABLE 5 representative XRPD peaks for Compound 1 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
26.3	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

2-theta (degree)	Relative Strength (%)
		34.2	8.9
34.7	12.4
		35.1	8.0
36.7	6.5
		38.1	4.7
39.3	5.3

A DSC curve for a sample of compound 1 form B is provided in fig. 11, and a TGA curve for a representative sample of compound 1 form B is provided in fig. 12.

FTIR spectra shown for representative samples of compound 1 form B are provided in fig. 13.

Also using the solid state¹³C and¹⁹compound 1, form B, was analyzed by F NMR. The respective NMR spectra are provided in fig. 14 and 15. In that¹³C SSNMR and¹⁹several peaks found in the F SSNMR spectra are described in tables 6 and 7.

Table 6: of Compound 1 form B¹³C SSNMR peaks.

Peak numbering	F1(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 7: of Compound 1 form B¹⁹F SSNMR peak.

Peak numbering	F1(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 solved by direct methods 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 Compound 1 form B N- (4- (7-azabicyclo [2.2.1]]The single crystal of hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-dihydroquinoline-3-carboxamide was determined to have the monoclinic system, P21/c space group, and the following unit cell parameters:α =90 °, β =101.193 °, and γ =90 °.

Example 4B: as form B-HCl(Compound 1 form B-HCl) N- (4- (7-Nitrogen) Hetero-bicyclo [2.2.1]Hept-7-yl) -2- (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1, 4-bis Preparation of hydroquinoline-3-carboxamides

Method 1

100mL of 2-methyltetrahydrofuran was charged into a three-necked flask with a nitrogen atmosphere equipped with a stirrer. Compound 1 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 constant weight. This gave compound 1, form B-HCl, as a white crystalline solid.

Method 2.

Compound 1, 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 filter cake was washed with EtOH. The solid was dried in a vacuum oven at 45 ℃ with nitrogen blow to afford compound 1 as form B-HCl as a white solid.

Note that other solvent combinations, e.g. MeOH/H₂O and IPA/H₂O or the like may be used instead of EtOH/H described in the present embodiment₂O。

Examples of alternative solvent combinations are provided in table 8.

Table 8. other solvents that can be used to prepare form B-HCl.

The powder diffraction pattern of compound form 1B-HCl is shown in FIG. 16.

Table 9: representative XRPD peaks for Compound 1 form B-HCl

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

2-theta (degree)	Relative Strength (%)
		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
		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, P21/a space group, and the following unit cell parameters:α =90 °, β =90.0554 °, and γ =90 °.

Samples of compound form B-HCl were also evaluated using microscopy.

A DSC curve for compound 1 form B-HCl is provided in fig. 17, and a TGA curve for a representative sample of compound 1 form B-HCl is provided in fig. 18.

The FTIR spectra shown for representative samples of compound 1 form B-HCl are provided in fig. 19.

Also using the solid state¹³C and¹⁹f NMR analysis Compound 1, form B-HCl. The respective NMR spectra are provided in fig. 20 and 21. In that¹³C SSNMR and¹⁹several peaks found in the F SSNMR spectra are described in tables 10 and 11:

table 9: process for preparing compound 1 form B-HCl¹³C SSNMAnd (4) peak R.

Peak numbering	F1(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 10: process for preparing compound 1 form B-HCl¹⁹F SSNMR peak.

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

Note 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 on FLIPR III as a measure of increased gating of af 508CFTR (conductance) in temperature-calibrated af 508CFTRNIH 3T3 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, CaCl₂2,MgCl₂1, HEPES 10, ph7.4, containing NaOH.

An alternative to bath solution #1 includes a bath solution in which the chloride 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. Furthermore, the apical membrane was replaced with equimolar sodium gluconate (titrated to pH7.4 with NaOH) to obtain large size Cl spanning the epithelium^-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.

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 correlation was monitored every 30 seconds.

Identification of enhancer compounds

Also investigated the enhancement of AF 508-CFTR enhancer to increase macroscopic AF 508-CFTRCl in NIH3T3 cells stably expressing AF 508-CFTR 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. For whole cell recording, 2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslips and used to test the activity of the enhancer after 24-48 hours of incubation at 27 ℃; and incubated with or without a correction compound at 37 ℃ to determine the activity of the correction agent.

Single channel recording

Cut membrane inside-out pieces (Dalemans, W., Barbry, P., Champgny, G., Jalat, S., Dott, K., Dreyer, D., Crytal, R.G., Pavirani, A., Lecocq, J-P., Lazdunski, M. (1991) Nature were used as described above354,526-. 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, 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. Cells were maintained at 37 ℃ in 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 1 is useful as a modulator of ATP-binding cassette transporters. Determination of EC for Compound 1 form A₅₀(mum) less than 2.0. mu.M.The efficiency of compound 1 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.

Other embodiments

All publications and patents mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the meaning of a term in any patent or publication incorporated by reference contradicts the meaning of the term used in the present disclosure, the meaning of the term in the present disclosure controls. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments of the present invention. One skilled in the art will readily recognize from such discussion and from the accompanying drawings and claims that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention as defined in the following claims.

Claims (69)

1. A process for preparing a substantially crystalline form of compound 1:

the method comprises the following steps:

(a) reacting compound 2 with compound 3 in the presence of a coupling agent

Wherein the coupling agent is selected from the group consisting of 2-chloro-1, 3-dimethyl-2-imidazolium tetrafluoroborate, HBTU, HCTU, 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine, HATU, HOBT/EDC and

2. the process of claim 1, wherein at e.g. K₂CO₃、Et₃N, N-methylmorpholine (NMM), pyridine or Diisopropylethylaniline (DIEA) in the presence of a base.

3. The process of claim 1 or 2, wherein the coupling of compound 2 and compound 3 is carried out in the presence of pyridine or DIEA.

4. The method of any one of claims 1-3, wherein the coupling of compound 2 and compound 3 is performed in the presence of pyridine.

5. The process of any one of claims 1-4, wherein the coupling of compound 2 and compound 3 is performed in the presence of a polar aprotic solvent.

6. The process according to any one of claims 1 to 5, wherein the polar aprotic solvent is selected from ethyl acetate, isopropyl acetate, tetrahydrofuran, methyl ethyl ketone, N-methyl-2-pyrrolidone, acetonitrile, N-dimethylformamide or 2-methyltetrahydrofuran.

7. The process of any one of claims 1-6, wherein the coupling of compound 2 and compound 3 is performed in the presence of 2-methyltetrahydrofuran.

8. The method of any one of claims 1-7, wherein the coupling of compound 2 and compound 3 is performed at a reaction temperature maintained at 30 ℃ to 80 ℃.

9. The method of any one of claims 1-8, wherein compound 1 is solid form a.

10. The method of any one of claims 1-9, wherein at e.g. K₂CO₃、Et₃N, N-methylmorpholine (NMM), pyridine or Diisopropylethylaniline (DIEA) in the presence of a base.

11. The process of any one of claims 1-10, wherein the coupling of compound 2 and compound 3 is performed in the presence of pyridine or DIEA.

12. The method of any one of claims 1-11, wherein the coupling of compound 2 and compound 3 is performed in the presence of pyridine.

13. The method of any one of claims 1-12, wherein the coupling of compound 2 and compound 3 is performed in the presence of a polar aprotic solvent.

14. The process of any one of claims 1-13, wherein the polar aprotic solvent is selected from ethyl acetate, isopropyl acetate, tetrahydrofuran, methyl ethyl ketone, N-methyl-2-pyrrolidone, acetonitrile, N-dimethylformamide, or 2-methyltetrahydrofuran.

15. The method of any one of claims 1-14, wherein the coupling of compound 2 and compound 3 is performed in the presence of 2-methyltetrahydrofuran.

16. The method of any one of claims 1-15, wherein the coupling of compound 2 and compound 3 is performed at a reaction temperature maintained at 30 ℃ to 80 ℃.

17. A process for preparing compound 2:

the method comprises the following steps:

(a) reaction of Compound 4 with diethyl 2- (ethoxymethylene) malonate 5 to produce ester 6A

And

(b) treatment of ester 6A with base to give Compound 6D

And

(c) treating compound 6D with a source of atomic hydrogen in the presence of a catalyst to produce compound 2

18. The method of claim 17, wherein the atomic hydrogen source comprises hydrogen gas.

19. The method of claim 17 or 18, wherein the atomic hydrogen source comprises formate.

20. The method of any one of claims 17-19, wherein the catalyst comprises a palladium catalyst.

21. The process of any one of claims 17-20, wherein the base comprises aqueous sodium hydroxide or aqueous potassium hydroxide.

22. Process for preparing compound 3

The method comprises the following steps:

(a) reacting compound 7, wherein Hal is F, Cl, Br or I, with 7-azabicyclo [2.2.1] heptane 8, or a salt thereof, to produce compound 9

And

(b) reduction of the nitro moiety in Compound 9 to afford Aniline 3

23. The process of claim 22, wherein step (a) is carried out in a polar aprotic solvent in the presence of a base.

24. The process of claim 22 or 23, wherein the base is triethylamine or diisopropylethylamine and the solvent is acetonitrile.

25. The process of any one of claims 22-24, wherein step (b) is performed in an alcohol solvent using hydrogen and a transition metal catalyst.

26. The process of any of claims 22-25, wherein the catalyst comprises a group 9 or group 10 transition metal selected from Pt, Pd or Ni.

27. The process of any of claims 22-26, wherein the catalyst comprises Pd.

28. The method of any one of claims 22-27, wherein the solvent comprises isopropanol, ethanol, or methanol.

29. The method of any one of claims 22-28, wherein the solvent comprises ethanol.

30. The method of any one of claims 22-29, wherein compound 8 is in the form of a salt, compound 8-HCl:

31. the process of any one of claims 22-30, further comprising contacting compound 3 with HCl to provide a hydrochloride salt 3-HCl:

32. the process of any one of claims 22-31, wherein step (a) is performed in the presence of a base such as an inorganic carbonate salt of sodium carbonate and in the presence of a polar aprotic solvent such as DMSO.

33. The process of any one of claims 22-32, wherein step (b) is performed in an alcoholic solvent using hydrogen and a transition metal catalyst.

34. The process of any of claims 22-33, wherein the catalyst comprises a group 9 or group 10 transition metal catalyst derived from Pt, Pd, or Ni and the solvent comprises isopropanol, ethanol, or methanol.

35. The process of any of claims 22-34, wherein the catalyst comprises Pd and the solvent comprises ethanol.

36. A process for preparing compound 1 having solid form B:

the method comprises the following steps:

(a) reacting compound 2 with the hydrochloride salt of compound 3 (3-HCl) in the presence of a coupling agent selected from the group consisting of 2-chloro-1, 3-dimethyl-2-imidazolium tetrafluoroborate, HBTU, HCTU, 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine, HATU, HOBT/EDC and

37. the method of claim 36, wherein at e.g. K₂CO₃、Et₃N, N-methylmorpholine (NMM), pyridine or DIEA in the presence of a base to effect coupling of Compound 2 and hydrochloride salt 3-HCl.

38. The process of claim 36 or 37, wherein the coupling of compound 2 and hydrochloride salt 3-HCl is carried out in the presence of pyridine or DIEA.

39. The process of any one of claims 36-38, wherein the coupling of compound 2 and hydrochloride salt 3-HCl is carried out in the presence of pyridine.

40. The process of any one of claims 36-39, wherein the coupling of Compound 2 and hydrochloride salt 3-HCl is carried out in the presence of a solvent such as EtOAc, IPAc, THF, MEK, NMP, acetonitrile, DMF or 2-methyltetrahydrofuran.

41. The process of any one of claims 36-40, wherein the coupling of Compound 2 and hydrochloride salt 3-HCl is carried out in the presence of 2-methyltetrahydrofuran.

42. The process of any one of claims 36-41, wherein the coupling of Compound 2 and hydrochloride salt 3-HCl is performed at a reaction temperature maintained at 15 ℃ to 70 ℃.

43. The method of any one of claims 36-42, wherein the coupling reaction is stirred for a duration of from about 1.5 hours to about 10 hours.

44. A process for preparing the hydrochloride salt of compound 1 having solid form a-HCl:

the method comprises the following steps:

(a) reacting compound 2 with a hydrochloride salt of formula 3 (3-HCl) in the presence of a coupling agent selected from the group consisting of 2-chloro-1, 3-dimethyl-2-imidazolium tetrafluoroborate, HBTU, HCTU, 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine, HATU, HOBT/EDC andand

(b) treating the product mixture of step (a) with HCl.

45. The method of claim 44, wherein at, e.g., K₂CO₃、Et₃N, N-methylmorpholine (NMM), pyridine or Diisopropylethylaniline (DIEA) in the presence of a base.

46. The process of claim 44 or 45, wherein step (a) is carried out in the presence of pyridine or DIEA.

47. The process of any one of claims 44-46, wherein step (a) is carried out in the presence of pyridine.

48. The process of any one of claims 44-47, wherein step (a) is carried out in the presence of a polar aprotic solvent.

49. The process of any one of claims 44-48, wherein the polar aprotic solvent is selected from ethyl acetate, isopropyl acetate, tetrahydrofuran, methyl ethyl ketone, N-methyl-2-pyrrolidone, acetonitrile, N-dimethylformamide, or 2-methyltetrahydrofuran.

50. The process of any one of claims 44-49, wherein the coupling of compound 2 and 3-HCl is carried out in the presence of 2-methyltetrahydrofuran.

51. The method of any one of claims 44-50, wherein step (a) is performed at a reaction temperature maintained between 10 ℃ and 80 ℃.

52. The method of any one of claims 44-51, wherein step (a) is performed at a reaction temperature maintained at 15 ℃ to 70 ℃.

53. The method of any one of claims 44-52, wherein the time of step (a) is from about 1.5 hours to about 72 hours.

54. The process of any one of claims 44-53, wherein HCl gas is used in step (b).

55. The process of any one of claims 44-54, wherein HCl gas is bubbled into a mixture comprising the product of step (a) and a polar aprotic solvent.

56. The process of any one of claims 44-55, wherein the polar aprotic solvent is 2-methyltetrahydrofuran.

57. The process of any one of claims 44-56, wherein HCl gas is bubbled into the mixture of the product of step (a) and the polar aprotic solvent for a period of from about 0.5 hours to about 5 hours.

58. Process for preparing hydrochloride salt of compound 1 having solid form of B-HCl

The method comprises the following steps:

(a) mixing hydrochloride form a-HCl of compound 1 with an organic solvent and water to produce a mixture;

and

(b) the mixture is heated.

59. The process of claim 58, wherein the organic solvent comprises dimethyl sulfoxide, dimethylformamide, dioxane, hexamethylphosphoric triamide, tetrahydrofuran, EtOAc, IPAc, THF, MEK, NMP, acetonitrile, DMF, EtOH, MeOH, isopropanol, or 2-methyltetrahydrofuran.

60. The method of claim 58 or 59, wherein the aprotic solvent comprises 2-methyltetrahydrofuran.

61. The method of any one of claims 58-60, wherein the temperature is maintained from about 30 ℃ to about 80 ℃.

62. The method of any one of claims 58-61, wherein the mixture is maintained at a temperature of about 30 ℃ to about 80 ℃ for a period of from about 12 hours to about 72 hours.

63. The method of any one of claims 58-62, wherein the mixture is filtered to produce a filter cake.

64. The method of any one of claims 58 to 63, wherein the filtration is followed by washing the filter cake with an aprotic solvent.

65. Compound 1 prepared by the process of claim 1

66. Compound 1 form-A prepared by the process of claim 9

67. Compound 1 form-B prepared by the process of claim 36

68. Compound 1 form A-HCl prepared by the process of claim 44

69. Compound 1 form B-HCl prepared by the process of claim 58