HK1178145B - Solid forms of (r)-1(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-n-(1-(2,3-dihyderoxypropyl) -6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl) -1h-indol-5-yl) cyclopropanecarboxamide - Google Patents

Description

Solid forms of (R) -1(2, 2-difluorobenzo [ D ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide

Technical Field

The present invention relates to solid state forms, e.g., crystalline and amorphous forms, of (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide, pharmaceutical compositions thereof, and methods of use thereof.

Background

CFTR is a cAMP/ATP-mediated anion channel expressed in a variety of cell types, including absorptive and secretory epithelial cells, where the channel regulates anion flux across membranes as well as the activity of other ion channels and proteins. In epithelial cells, the proper functioning of CFTR is critical to maintain electrolyte transport throughout the body, including in respiratory and digestive tissues. CFTR consists of approximately 1480 amino acids, which encodes a protein consisting of tandem repeats of transmembrane domains, each containing 6 transmembrane helices and a nucleotide binding domain. The two transmembrane domains are connected by a large polar regulatory (R) -domain that has 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) Nature347: 382. sup. + 386; Rich, D.P. et al. (1990) Nature347: 358. sup. + 362), (Riordan, J.R. et al. (1989) Science 245: 1066. sup. + 1073). Defects in this gene cause CFTR mutations, leading to cystic fibrosis ("CF"), the most common fatal genetic disease in humans. In the united states, cystic fibrosis affects one in every approximately two thousand five hundred infants. In the general U.S. population, up to 1000 million people carry a single copy of a defective gene without significant adverse effects. In contrast, individuals with double copies of the CF-associated gene suffer from the debilitating and fatal effects of CF, including chronic lung disease.

In patients with cystic fibrosis, mutations in the endogenous expression of CFTR in the respiratory epithelial cells result in reduced apical anion secretion, resulting in an imbalance in ionic and fluid transport. The resulting reduction in anion transport helps to enhance mucus accumulation and concomitant microbial infection in the lungs, which ultimately leads to death in CF patients. In addition to respiratory diseases, CF patients often suffer from gastrointestinal problems and pancreatic insufficiency, which if not treated in a timely manner, can lead to death. In addition, most men with cystic fibrosis are unable to breed and the fertility rate decreases among women with cystic fibrosis. In contrast to the severe effects of the double-copy CF-associated gene, individuals with a single copy of the CF-associated gene exhibit increased tolerance to cholera and to dehydration due to diarrhea-perhaps explaining the relatively high frequency of the CF gene in the population.

Sequence analysis of the CFTR gene of the CF chromosome revealed a number of causative mutations (Cutting, G.R. et al. (1990) Nature 346: 366-. To date, more than 1000 pathogenic mutations of the CF gene have been identified, as reported by the scientific and medical literature. The most common mutation is a phenylalanine deletion at position 508 of the CFTR amino acid sequence (and commonly referred to as Δ F508-CFTR). This mutation occurs in about 70% of cystic fibrosis cases and is associated with severe disease. Other mutations include R117H and G551D.

Deletion of residue 508 of Δ F508-CFTR prevents proper folding of the nascent protein. This results in the inability of the mutein to leave the ER and pass through the plasma membrane. As a result, the number of channels present on the membrane is much less than that observed in cells expressing wild-type CFTR. In addition to impaired flux, mutations also lead to defects in channel gating. The reduced number of channels on the membrane, together with the gating defects, results in reduced transport of anions across epithelial cells, resulting in defects in ion and fluid transport. (Quinton, P.M. (1990), FASEB J. 4: 2709-. However, studies have shown that the number of af 508-CFTR on the membrane is reduced to functional, although less than wild-type CFTR. (Dalemans et al (1991), Nature Lond. 354: 526-. In addition to af 508-CFTR, other causative mutations of CFTR that cause defects in flux, synthesis, and/or channel gating can be up-or down-regulated to alter anion secretion and modify disease progression and/or severity.

Although CFTR transports a variety of molecules in addition to anions, it is clear that this effect (anion transport) represents one of the important mechanisms for transporting ions and water across epithelial cells. Other elements include epithelial Na⁺Channel, ENaC, Na⁺/2Cl^-/K⁺Cotransporter, Na⁺-K⁺ATPase Pump and base side Membrane K⁺Channels, which are responsible for the uptake of chlorine into cells.

These elements act together to achieve both selective expression within the cell and targeted delivery across epithelial cells via their localization. By ENaC and CFTR present on the apical membrane and Na expressed on the basolateral surface of the cell⁺-K⁺-ATPase Pump and Cl^-The coordinated activity of the channels allows chlorine absorption to occur. Secondary active transport of chlorine from the luminal side results in accumulation of chlorine within the cell, which may then pass through Cl^-The channel passively leaves the cell, resulting in vector transport. Na arranged on the outer surface of the substrate⁺/2Cl^-/K⁺Cotransporter, Na⁺-K⁺ATPase Pump and base side Membrane K⁺Channel and luminal CFTR coordinate chlorineSecreted by the luminal CFTR. Because water itself may never be actively transported, its flow across epithelial cells depends on a slight trans-epithelial osmotic gradient created by the bulk flow of sodium and chloride.

As discussed above, it is believed that the deletion of residue 508 of Δ F508-CFTR prevents proper folding of the nascent protein, resulting in the inability of this mutein to leave the ER and pass through the plasma membrane. As a result, insufficient amounts of mature protein are present on the plasma membrane and chloride transport in epithelial tissues is significantly reduced. Indeed, this cellular phenomenon of defective Endoplasmic Reticulum (ER) processing of ATP-binding cassette (ABC) transporters by the ER machinery has been shown to be the basis not only for CF diseases, but also for a wide range of other related and inherited diseases. Two ways in which the ER machinery may not function are degradation by loss of ER export coupling to the protein, or ER accumulation by these defective/misfolded proteins [ Aridor M, et al, nature ed.,5(7) 745 th page 751 (1999); sharthy, b.s., et al, neurochem.international,43pages 1-7 (2003), Rutishauser, j., et al, Swiss Med Wkly,132page 211-222 (2002), Morello, JP et al, TIPS,21page 466-469 (2000); Bross P., et al., Human mut.,14page 186-198 (1999)]。

U.S. published patent application US20090131492, which publication is incorporated herein by reference in its entirety, discloses (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide as a modulator of CFTR activity and thus for the treatment of CFTR mediated diseases such as cystic fibrosis. However, there is a need for stable solid forms of the compounds which can be readily used in pharmaceutical compositions suitable for use as therapeutic agents.

Summary of The Invention

The present invention relates to solid forms of (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide (hereinafter "compound 1") having the following structure:

compound 1

Compound 1 and pharmaceutically acceptable compositions thereof are useful for treating or lessening the severity of CFTR mediated diseases such as cystic fibrosis. In one aspect, compound 1 exists in substantially crystalline and salt free form, referred to as form a, described and characterized herein. In another aspect, compound 1 is present in an amorphous form as described and characterized herein. The nature of the solid in relation to its efficacy as a medicament may depend on the form of the solid. For example, in a drug substance, changes in solid form can lead to differences in properties such as melting point, dissolution rate, oral absorption, bioavailability, toxicology results, and even clinical trial results.

The processes described herein can be used to prepare compositions of the invention comprising form a or the amorphous form or both of compound 1. The amounts and characteristics of the components used in such a process are as described herein.

Brief description of the drawings

FIG. 1 is an X-ray powder diffraction pattern of Compound 1.

Figure 2 is a Differential Scanning Calorimetry (DSC) trace of compound 1.

Figure 3 is a thermogravimetric analysis (TGA) profile of compound 1.

Figure 4 is an X-ray powder diffraction pattern calculated from a single crystal of compound 1 form a.

Figure 5 is an actual X-ray powder diffraction pattern of compound 1 form a prepared by the slurry technique (2 weeks) using DCM as the solvent.

Figure 6 is a Differential Scanning Calorimetry (DSC) trace of compound 1 form a.

Figure 7 is an actual X-ray powder diffraction pattern of compound 1 form a prepared from acetonitrile by a rapid evaporation method.

Figure 8 is an actual X-ray powder diffraction pattern of compound 1 form a prepared by the anti-solvent method using EtOAc and heptane.

Figure 9 is a conformational diagram of compound 1 form a based on single crystal X-ray analysis.

Figure 10 is a conformational diagram showing the stacking sequence of compound 1 form a.

FIG. 11 is a solid state of Compound 1 form A¹³C NMR spectrum (15.0kHz spin).

FIG. 12 is a solid state of Compound 1 form A¹⁹F NMR spectrum (12.5 kHz spin).

FIG. 13 is an X-ray powder diffraction pattern of an amorphous form of Compound 1 from Rapid evaporation rotovap.

Figure 14 is a Modulated Differential Scanning Calorimetry (MDSC) trace of the amorphous form of compound 1 prepared by the rapid evaporation rotary evaporation method.

Figure 15 is a thermogravimetric analysis (TGA) profile of the amorphous form of compound 1 prepared by rapid evaporation rotary evaporation.

Figure 16 is an X-ray powder diffraction pattern of an amorphous form of compound 1 prepared by spray drying.

Figure 17 is a Modulated Differential Scanning Calorimetry (MDSC) trace of the amorphous form of compound 1 prepared by the spray drying method.

FIG. 18 is a solid state of amorphous form of Compound 1¹³C NMR spectrum (15.0kHz spin).

FIG. 19 is a solid state of amorphous form of Compound 1¹⁹F NMR spectrum (12.5 kHz spin).

Detailed Description

Definition of

The following definitions as used herein shall apply, unless otherwise indicated.

The term "CFTR" as used herein means cystic fibrosis transmembrane transport regulator or a variant thereof capable of modulating activity, including, without limitation, mutations of Δ F508 CFTR and G551D CFTR (see, e.g., http:// www.genet.sickkids.on.ca/CFTR/, for CFTR mutations).

The term "amorphous" as used herein refers to a solid form consisting of a disordered array of molecules and having no discernible lattice.

As used herein, "crystalline" refers to 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 long program. The structural units constituting the crystal structure may be atoms, molecules or ions. The crystalline solid showed a fixed melting point.

The term "modulating" as used herein means increasing or decreasing, e.g., activity, by a measurable amount.

The term "chemically stable" as used herein means that compound 1 in solid form does not decompose to one or more different compounds when subjected to specified conditions, e.g., 40 ℃/75% relative humidity for a specified period of time, e.g., 1 day, 2 days, 3 days, 1 week, 2 weeks, or longer. In some embodiments, less than 25% of compound 1 in solid form decomposes, and in some embodiments, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 3%, less than about 1%, less than about 0.5% of said form of compound 1 decomposes under the conditions specified. In some embodiments, no measurable solid form of compound 1 decomposes.

The term "physically stable" as used herein means that the solid form of compound 1 does not change to one or more different physical forms of compound 1 (e.g., different solid forms as measured by XRPD, DSC, etc.) when subjected to particular conditions, e.g., to 40 ℃/75% relative humidity for a particular period of time, e.g., 1 day, 2 days, 3 days, 1 week, 2 weeks, or longer. In some embodiments, less than 25% of the solid form of compound 1 becomes one or more different physical forms when subjected to specified conditions. In some embodiments, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 3%, less than about 1%, less than about 0.5% of the solid form of compound 1 becomes one or more different physical forms of compound 1 when subjected to specified conditions. In some embodiments, no measurable solid form of compound 1 changes to one or more different physical forms of compound 1.

As used herein, the phrase "substantially amorphous compound 1" is used interchangeably with the phrases "amorphous compound 1", "amorphous compound substantially free of crystalline compound 1", and "substantially amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide". In some embodiments, substantially amorphous compound 1 has less than about 30% crystalline compound 1, such as less than about 25% crystalline compound 1, less than about 20% crystalline compound 1, less than about 15% crystalline compound 1, less than about 10% crystalline compound 1, less than about 5% crystalline compound 1, less than about 2% crystalline compound 1.

As used herein, the phrase "substantially crystalline compound 1 form a" is used interchangeably with "compound 1 form a" and "crystalline compound 1 form a substantially free of amorphous compound 1". In some embodiments, substantially crystalline compound 1 form a has less than about 30% amorphous compound 1 or other solid form, e.g., less than about 30% amorphous compound 1 or other solid form, less than about 25% amorphous compound 1 or other solid form, less than about 20% amorphous compound 1 or other solid form, less than about 15% amorphous compound 1 or other solid form, less than about 10% amorphous compound 1 or other solid form, less than about 5% amorphous compound 1 or other solid form, less than about 2% amorphous compound 1 or other solid form. In some embodiments, substantially crystalline compound 1 form a has less than about 1% amorphous compound 1 or other solid forms.

The term "substantially free" (as in the phrase "substantially free of form X"), when referring to a specified solid form (e.g., an amorphous or crystalline form as described herein) of compound 1, means that less than 20% (by weight) of the specified form or co-form (e.g., a crystalline or amorphous form of compound 1) is present, more preferably less than 10% (by weight) of the specified form is present, more preferably less than 5% (by weight) of the specified form is present, and most preferably less than 1% (by weight) of the specified form is present.

The term "substantially pure," when referring to a specified solid form of compound 1 (e.g., an amorphous or crystalline solid form as described herein), means that the specified solid form contains less than 20% (by weight) of residual components such as alternative polymorphic or isomorphic crystalline forms of compound 1 or co-form. It is preferred that the substantially pure solid form of compound 1 contains less than 10% (by weight) of the alternative polymorphic or isomorphic crystalline form of compound 1, more preferably less than 5% (by weight) of the alternative polymorphic or isomorphic crystalline form of compound 1, and most preferably less than 1% (by weight) of the alternative polymorphic or isomorphic crystalline form of compound 1.

As used herein, "dispersion" refers to a dispersion system in which one substance (the dispersed phase) is distributed as discrete units throughout the second substance (the continuous phase or vehicle). The size of the dispersed phase can vary significantly (e.g., nano-sized colloidal particles, to sizes of multiple microns). Typically, the dispersed phase may be a solid, liquid or gas. In the case of solid dispersions, both the dispersed and continuous phases are solid. In pharmaceutical applications, solid dispersions may comprise a crystalline drug (dispersed phase) in an amorphous polymer (continuous phase) or an amorphous drug (dispersed phase) in an amorphous polymer (continuous phase). In some embodiments, the amorphous solid dispersion includes a polymer that constitutes the dispersed phase and a drug that constitutes the continuous phase. In some embodiments, the dispersion comprises amorphous compound 1 or substantially amorphous compound 1.

The term "solid amorphous dispersion" generally refers to a solid dispersion of two or more components, typically a drug and a polymer, but may contain other components such as surfactants or other pharmaceutically acceptable excipients, wherein compound 1 is amorphous or substantially amorphous (e.g., substantially free of crystalline compound 1), and the physical stability and/or dissolution and/or solubility of the amorphous drug is enhanced by the other components.

The terms "about" and "approximately," when used in conjunction with a dose, amount, or weight percentage of an ingredient in a composition or dosage form, as used herein, means a dose, amount, or weight percentage recognized by one of ordinary skill in the art to provide a pharmacological effect equivalent to that obtained from the specified dose, amount, or weight percentage. In particular, the term "about" or "approximately" means an acceptable error for a particular value, as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1,2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value or range.

The abbreviations "MTBE" and "DCM" represent methyl tert-butyl ether and dichloromethane, respectively.

The abbreviation "XRPD" stands for X-ray powder diffraction.

The abbreviation "DSC" stands for differential scanning calorimetry.

The abbreviation "TGA" stands for thermogravimetric analysis.

Unless otherwise indicated, the structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structures, such as the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Thus, single stereochemical isomers as well as mixtures of enantiomers, diastereomers and geometric (or conformational) isomers of the compounds of the present invention are within the scope of the present invention. All tautomeric forms of compound 1 are included herein. For example, compound 1 may exist as a tautomer, both of which are included herein:

in addition, unless otherwise indicated, structures depicted herein are also meant to include compounds that differ only in the presence of atoms that are enriched in one or more isotopes. For example, wherein one or more hydrogen atoms are replaced by deuterium or tritium, or one or more carbon atoms are enriched with¹³C-or¹⁴Carbon-substituted compounds 1 of C-are within the scope of the present invention. Such compounds are useful, for example, as analytical tools, probes in bioassays, or compounds with improved therapeutic conditions.

In one aspect, the invention features (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide described as crystalline form A.

In another embodiment, form a is characterized by one or more peaks at 19.3 to 19.7 °, 21.5 to 21.9 °, and 16.9 to 17.3 ° in an X-ray powder diffraction obtained using Cu ka radiation. In another embodiment, form a is characterized by one or more peaks at about 19.5, 21.7, and 17.1 °. In another embodiment, form a is further characterized by a peak at 20.2 to 20.6 °. In another embodiment, form a is further characterized by a peak at about 20.4 °. In another embodiment, form a is further characterized by a peak at 18.6 to 19.0 °. In another embodiment, form a is further characterized by a peak at about 18.8 °. In another embodiment, form a is further characterized by a peak at 24.5 to 24.9 °. In another embodiment, form a is further characterized by a peak at about 24.7 °. In another embodiment, form a is further characterized by a peak at 9.8 to 10.2 °. In another embodiment, form a is further characterized by a peak at about 10.0 °. In another embodiment, form a is further characterized by a peak at 4.8 to 5.2 °. In another embodiment, form a is further characterized by a peak at about 5.0 °. In another embodiment, form a is further characterized by a peak at 24.0 to 24.4 °. In another embodiment, form a is further characterized by a peak at about 24.2 °. In another embodiment, form a is further characterized by a peak at 18.3 to 18.7 °. In another embodiment, form a is further characterized by a peak at about 18.5 °.

In another embodiment, form a is characterized by a diffraction pattern substantially similar to that of figure 4. In another embodiment, form a is characterized by a diffraction pattern substantially similar to that of figure 5.

In another aspect, the invention features (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide in the form of a crystal having a monoclinic system, a C2 space group, and the following unit cell sizes: a = 21.0952(16) a, α =90 °, b = 6.6287(5) a, β = 95.867(6) °, c = 17.7917(15) a, and γ =90 °.

In another aspect, the invention features a pharmaceutical composition comprising form a and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprises an additional therapeutic agent. In another embodiment, the additional therapeutic agent is selected from a mucolytic agent, a bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR potentiator, or a nutritional agent.

In another aspect, the invention features a method of making form a that includes slurrying (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide in a solvent for an effective amount of time. In another embodiment, the solvent is ethyl acetate, dichloromethane, MTBE, isopropyl acetate, water/ethanol, water/acetonitrile, water/methanol, or water/isopropanol. In another embodiment, the effective amount of time is from 24 hours to 2 weeks. In another embodiment, the effective amount of time is from 24 hours to 1 week. In another embodiment, the effective amount of time is from 24 hours to 72 hours.

In another aspect, the invention features a method of making form a that includes dissolving (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide in a solvent and evaporating the solvent. In another embodiment, the solvent is acetone, acetonitrile, methanol, or isopropanol.

In another aspect, the invention features a method of preparing form A that includes dissolving (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide in a first solvent, and adding (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) cyclopropanecarboxamide 1H-indol-5-yl) cyclopropanecarboxamide is insoluble in the second solvent. In another embodiment, the first solvent is ethyl acetate, ethanol, isopropanol, or acetone. In another embodiment, the second solvent is heptane or water. In another embodiment, the second solvent is added while stirring the first solvent and the solution of (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide.

In another aspect, the invention features a solid, substantially amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide. In another embodiment, amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide contains less than about 5% crystalline (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl ) Cyclopropanecarboxamide.

In another aspect, the invention features a pharmaceutical composition comprising amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprises an additional therapeutic agent. In another embodiment, the additional therapeutic agent is selected from a mucolytic agent, a bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR potentiator, or a nutritional agent.

In another aspect, the invention features a method of preparing amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide including reacting (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide -yl) cyclopropanecarboxamide is dissolved in a suitable solvent and the solvent is removed by rotary evaporation. In another embodiment, the solvent is methanol.

In another aspect, the invention features a solid dispersion comprising amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide and a polymer. In another embodiment, the polymer is Hydroxypropylmethylcellulose (HPMC). In another embodiment, the polymer is hydroxypropylmethylcellulose acetate succinate (HPMCAS).

In another embodiment, the polymer is present in an amount of from 10wt% to 80 wt%. In another embodiment, the polymer is present in an amount of 30 wt.% to 60 wt.%. In another embodiment, the polymer is present in an amount of about 49.5% by weight.

In another embodiment, (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide is present in an amount of 10% by weight to 80% by weight. In another embodiment, (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide is present in an amount of 30% by weight to 60% by weight. In another embodiment, (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide is present in an amount of about 50% by weight.

In another embodiment, the solid dispersion further comprises a surfactant. In another embodiment, the surfactant is sodium lauryl sulfate. In another embodiment, the surfactant is present in an amount of 0.1 wt% to 5 wt%. In another embodiment, the surfactant is present in an amount of about 0.5% by weight.

In another embodiment, the polymer is hydroxypropylmethylcellulose acetate succinate (HPMCAS) present in an amount of 49.5% by weight, the surfactant is sodium lauryl sulfate present in an amount of 0.5% by weight, and the (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide is present in an amount of 50% by weight.

In another aspect, the invention features a pharmaceutical composition that includes a solid dispersion and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprises an additional therapeutic agent. In another embodiment, the additional therapeutic agent is selected from a mucolytic agent, a bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR potentiator, or a nutritional agent.

In another aspect, the invention features a process for preparing amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide comprising contacting (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indole- 5-yl) cyclopropanecarboxamide spray drying.

In another embodiment, the method comprises mixing (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide and a suitable solvent and then spray drying the mixture to obtain amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -N- (1-hydroxy-2-methylpropan-2 -yl) -1H-indol-5-yl) cyclopropanecarboxamide. In another embodiment, the solvent is an alcohol. In another embodiment, the solvent is methanol.

In another embodiment, the method comprises: a) forming a mixture comprising (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide, a polymer, and a solvent; and b) spray drying the mixture to form a solid dispersion.

In another embodiment, the polymer is hydroxypropylmethylcellulose acetate succinate (HPMCAS). In another embodiment, the polymer is present in an amount from 10% by weight to 80% by weight of the solid dispersion. In another embodiment, the polymer is present in an amount of about 49.5% by weight of the solid dispersion. In another embodiment, the solvent is methanol. In another embodiment, the mixture further comprises a surfactant. In another embodiment, the surfactant is Sodium Lauryl Sulfate (SLS). In another embodiment, the surfactant is present in an amount of 0.1 wt% to 5wt% of the solid dispersion. In another embodiment, the surfactant is present in an amount of about 0.5% by weight of the solid dispersion.

In another embodiment, the polymer is hydroxypropyl methyl cellulose acetate succinate (HPMCAS) present in an amount of about 49.5% by weight of the solid dispersion, the solvent is methanol, and the mixture further comprises sodium lauryl sulfate present in an amount of about 0.5% by weight of the solid dispersion.

In another aspect, the invention features a method of treating a CFTR-mediated disease in a subject, the method including administering to the subject an effective amount of form A, amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide, or amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy- 2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide.

In another embodiment, the CFTR mediated disease is selected from the group consisting of cystic fibrosis, asthma, COPD due to smoking, chronic bronchitis, sinusitis, constipation, pancreatitis, pancreatic insufficiency, male infertility due to congenital bilateral vasectomy (CBAVD), mild pulmonary disease, idiopathic pancreatitis, allergic bronchopulmonary aspergillosis (ABPA), liver disease, hereditary emphysema, hereditary hemochromatosis, fibrinolysis deficiency, protein C deficiency, hereditary angioedema type 1, lipid processing deficiency, familial hypercholesterolemia, chylomicronemia type 1, abetalipoproteinemia (abetalipoproteinemia), lysosomal storage diseases, I-cell diseases/pseudohurler disease (pseudo-Hurler), mucodorosis, Sandhof disease/amamentous dementia (Sandhof/Tay-Sachs), crigler-najal syndrome, constipation, post-bowel syndrome, post-operative syndrome, post operative, Polyendocrindocrinopathy/hyperinsulinemia, diabetes mellitus, larnence dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, glycolytic CDG type 1 (glyconosis CDG type 1), congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency, Diabetes Insipidus (DI), neurogenic (renal) DI, renal DI, charcot-marie-tooth syndrome, peyer-meiosis, neurodegenerative diseases, alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, pick's disease, some polyglutamine neurological disorders, huntington's disease, spinocerebellar type I (spinocerebellar) ataxia, spinobulbar muscular atrophy, dentatorulo-pallidoluysian atrophy, myotonic dystrophy, Spongiform encephalopathy, hereditary Creutzfeldt-Jakob disease (due to prion processing defects), Fabry's disease, Sterler-Schlenk syndrome (Straussler-Scheinkersdrome), COPD, xerophthalmia, Sjogren's disease, osteoporosis, osteopenia, Goohan syndrome, chloride channel disease, myotonia congenita (Tomson and Becker forms), Bart's syndrome type III, Dent disease, panic disorder (hyperekplexia), epilepsy, panic disorder, lysosomal storage disease, Angelman syndrome, Primary Ciliary Dyskinesia (PCD), hereditary diseases of ciliary structure and/or function, PCD with visceral inversion (also known as Catalogen syndrome), PCD without visceral inversion, or ciliary hypoplasia. In another embodiment, the CFTR mediated disease is cystic fibrosis. In another embodiment, the subject has cystic fibrosis transmembrane conductance receptor (CFTR) with a Δ F508 mutation. In another embodiment, the subject has cystic fibrosis transmembrane conductance receptor (CFTR) with a R117H mutation. In another embodiment, the subject has cystic fibrosis transmembrane conductance receptor (CFTR) with a G551D mutation.

In another embodiment, the method comprises administering an additional therapeutic agent. In another embodiment, the therapeutic agent is selected from a mucolytic agent, a bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR potentiator, or a nutritional agent.

In another aspect, the invention features formulations comprising form A, amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide, or amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indole- 5-yl) cyclopropanecarboxamide and kits thereof with instructions for use.

Process for preparing compound 1 form a and amorphous forms

Compound 1 is the starting point and can be prepared in one embodiment by coupling an acid chloride moiety with an amine moiety according to schemes 1-4.

Scheme 1. synthesis of acyl chloride moieties.

Scheme 2. alternative synthesis of acid chloride moieties.

Scheme 3. synthesis of amine moieties.

Scheme 4 formation of Compound 1

Compound 1

Methods of forming Compound 1 form A

In one embodiment, form a is prepared by slurrying compound 1 in a suitable solvent for an effective amount of time. In another embodiment, suitable solvents are ethyl acetate, methylene chloride, MTBE, isopropyl acetate, water/ethanol solutions of various ratios, water/acetonitrile solutions of various ratios, water/methanol solutions of various ratios, or water/isopropanol solutions of various ratios. For example, various ratios of water/ethanol solutions include water/ethanol 1:9(vol/vol), water/ethanol 1:1 (vol/vol), and water/ethanol 9:1 (vol/vol). Various ratios of water/acetonitrile solutions include water/acetonitrile 1:9(vol/vol), water/acetonitrile 1:1 (vol/vol), and water/acetonitrile 9:1 (vol/vol). Various ratios of water/methanol solutions include water/methanol 1:9(vol/vol), water/methanol 1:1 (vol/vol), and water/methanol 9:1 (vol/vol). The water/isopropanol solutions of various ratios include water/isopropanol 1:9(vol/vol), water/isopropanol 1:1 (vol/vol) and water/isopropanol 9:1 (vol/vol).

Typically, about 40 mg of compound 1 is slurried with about 1.5 ml of a suitable solvent (target concentration of 26.7 mg/ml) at room temperature for an effective amount of time. In some embodiments, the effective amount of time is from about 24 hours to about 2 weeks. In some embodiments, the effective amount of time is from about 24 hours to about 1 week. In some embodiments, the effective amount of time is from about 24 hours to about 72 hours. The solid was then collected.

In another embodiment, form a is prepared by dissolving compound 1 in a suitable solvent, followed by evaporation of the solvent. In one embodiment, a suitable solvent is one in which compound 1 has a solubility greater than 20 mg/ml. For example, such solvents include acetonitrile, methanol, ethanol, isopropanol, acetone, and the like.

Typically, compound 1 is dissolved in a suitable solvent, filtered, and then left to evaporate slowly or quickly. An example of slow evaporation is to cover a container, such as a vial, containing a solution of compound 1 with a sealing film having a puncture therein. An example of rapid evaporation is the uncovered placement of a container, such as a vial, containing a solution of compound 1. The solid was then collected.

In another aspect, the invention features a method of making form a that includes dissolving compound 1 in a first solvent and adding a second solvent in which compound 1 has poor solubility (< 1 mg/ml). For example, the first solvent can be a solvent in which compound 1 has a solubility greater than 20 mg/ml, such as ethyl acetate, ethanol, isopropanol, or acetone. The second solvent may be, for example, heptane or water.

Typically, compound 1 is dissolved in a first solvent and filtered to remove any seed crystals. The second solvent was added slowly while stirring. The solid precipitated and was collected by filtration.

Process for producing amorphous Compound 1

Starting from compound 1 or compound 1 form a, amorphous forms of compound 1 can be prepared by rotary evaporation or by spray drying methods.

Compound 1 is dissolved in a suitable solvent such as methanol and the methanol is rotary evaporated leaving a foam, yielding compound 1 in amorphous form. In some embodiments, a warm water bath is used to accelerate evaporation.

Compound 1 amorphous form can also be prepared from compound 1 form a using a spray drying process. Spray drying is a process by which a liquid feed is converted into a dry particulate form. Optionally, secondary drying processes such as fluidized bed drying or vacuum drying may be used to reduce residual solvent to pharmaceutically acceptable levels. Typically, spray drying involves contacting a highly dispersed liquid suspension or solution with a sufficient volume of hot air to evaporate and dry the droplets. The formulation to be spray dried may be any solution, coarse suspension, slurry, colloidal dispersion or paste which can be atomized using the spray drying equipment of choice. Under standard procedures, the formulation is sprayed into a warm filtered air stream which evaporates the solvent and conveys the dried product to a collector (e.g., a cyclone). The spent air is then vented with solvent or alternatively the spent air is sent to a condenser to capture and potentially recover solvent. Commercially available equipment types can be used to perform the spray drying. For example, commercially available spray dryers are manufactured by Buchi Ltd. And Ni (e.g. PSD line spray dryer manufactured by Ni ro) (see US 2004/0105820; US 2003/0144257).

Spray drying typically employs from about 3% to about 30% by weight, for example from about 4% to about 20% by weight, preferably at least about 10% solids loaded material (i.e. drug and excipients). Generally, the upper limit of the solids loading is controlled by the viscosity (e.g., pumpability) of the resulting solution and the solubility of the components in the solution. Generally, the viscosity of the solution can determine the particle size in the resulting powder product.

Techniques and methods for Spray Drying can be found in Perry's Chemical Engineering Handbook, 6 th edition, R.H. Perry, D.W. Green & J.O. Malony, McGraw-Hill book co. (1984), and Marshall "Atomization and spread-Drying" 50, chem. Eng. prog. monogr.series 2 (1954). Typically, spray drying is carried out using the following inlet temperatures: from about 60 ℃ to about 200 ℃, e.g., from about 95 ℃ to about 185 ℃, from about 110 ℃ to about 182 ℃, from about 96 ℃ to about 180 ℃, e.g., about 145 ℃. Spray drying is typically carried out with the following exit temperatures: from about 30 ℃ to about 90 ℃, e.g., from about 40 ℃ to about 80 ℃, from about 45 ℃ to about 80 ℃, e.g., about 75 ℃. The atomization flow rate is typically from about 4 kg/h to about 12 kg/h, for example from about 4.3 kg/h to about 10.5 kg/h, for example about 6 kg/h or about 10.5 kg/h. The feed flow rate is typically from about 3 kg/h to about 10 kg/h, for example from about 3.5 kg/h to about 9.0 kg/h, for example about 8 kg/h or about 7.1 kg/h. The atomization ratio is typically from about 0.3 to 1.7, for example from about 0.5 to 1.5, for example about 0.8 or about 1.5.

Removal of the solvent may require a subsequent drying step, such as tray drying, fluidized bed drying (e.g., from about room temperature to about 100 ℃), vacuum drying, microwave drying, drum drying, or double cone vacuum drying (e.g., from about room temperature to about 200 ℃).

In one embodiment, the solid dispersion is fluid bed dried.

In one approach, the solvent comprises a volatile solvent, such as a solvent having a boiling point less than about 100 ℃. In some embodiments, the solvent comprises a mixture of solvents, such as a mixture of volatile solvents or a mixture of volatile and non-volatile solvents. When mixtures of solvents are used, such mixtures may comprise one or more non-volatile solvents, for example, wherein the non-volatile solvent is present in the mixture at less than about 15%, such as less than about 12%, less than about 10%, less than about 8%, less than about 5%, less than about 3%, or less than about 2%.

Preferred solvents are those wherein Compound 1 has a solubility of at least about 10 mg/ml (e.g., at least about 15 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml or more). More preferred solvents include those wherein compound 1 has a solubility of at least about 20 mg/ml.

Exemplary solvents that can be assayed include acetone, cyclohexane, dichloromethane, N-Dimethylacetamide (DMA), N-Dimethylformamide (DMF), 1, 3-dimethyl-2-imidazolidinone (DMI), dimethyl sulfoxide (DMSO), dioxane, ethyl acetate, diethyl ether, glacial acetic acid (HAc), Methyl Ethyl Ketone (MEK), N-methyl-2-pyrrolidone (NMP), methyl tert-butyl ether (MTBE), Tetrahydrofuran (THF), pentane, acetonitrile, methanol, ethanol, isopropanol, isopropyl acetate, and toluene. Exemplary co-solvents include acetone/DMSO, acetone/DMF, acetone/water, MEK/water, THF/water, dioxane/water. In a two-solvent system, the solvent may be present from about 0.1% to about 99.9%. In some preferred embodiments, water is a co-solvent with acetone, wherein water is present at about 0.1% to about 15%, such as about 9% to about 11%, such as about 10%. In some preferred embodiments, water is a co-solvent with MEK, wherein water is present at about 0.1% to about 15%, such as about 9% to about 11%, for example about 10%. In some embodiments, the solvent solution comprises 3 solvents. For example, acetone and water may be mixed with a third solvent such as DMA, DMF, DMI, DMSO, or HAc. In the case where amorphous compound 1 is a component of a solid amorphous dispersion, the preferred solvent dissolves both compound 1 and the polymer. Suitable solvents include those described above, such as MEK, acetone, water, methanol, and mixtures thereof.

The particle size and drying temperature range can be varied to produce the best solid dispersion. As will be appreciated by the skilled practitioner, small particle size will result in improved solvent removal. Applicants have found, however, that smaller particles can result in particle fluffing, which in some cases may not provide an optimal solid dispersion for downstream processing such as tableting. At higher temperatures, crystallization or chemical degradation of compound 1 may occur. At lower temperatures, a sufficient amount of solvent cannot be removed. The process herein provides for optimal particle size and optimal drying temperature.

Typically, the particle size is such that D10 (μm) is less than about 5, such as less than about 4.5, less than about 4.0, or less than about 3.5, D50(μm) is typically less than about 17, such as less than about 16, less than about 15, less than about 14, less than about 13, and D90 (μm) is typically less than about 175, such as less than about 170, less than about 150, less than about 125, less than about 100, less than about 90, less than about 80, less than about 70, less than about 60, or less than about 50. Typically the spray dried particles have a bulk density of from about 0.08 g/cc to about 0.20g/cc, for example from about 0.10 to about 0.15 g/cc, for example about 0.11 g/cc or about 0.14 g/cc. The tap density of the spray dried particles, typically for 10 taps, is in the range of about 0.08 g/cc to about 0.20g/cc, such as about 0.10 to about 0.15 g/cc, such as about 0.11 g/cc or about 0.14 g/cc; in the range of 0.10 g/cc to about 0.25 g/cc, such as about 0.11 to about 0.21 g/cc, such as about 0.15 g/cc, about 0.19 g/cc, or about 0.21 g/cc for 500 taps; in the range of 0.15 g/cc to about 0.27g/cc, such as about 0.18 to about 0.24 g/cc, such as about 0.18g/cc, about 0.19 g/cc, about 0.20g/cc, or about 0.24 g/cc for 1250 taps; and in the range of 0.15 g/cc to about 0.27g/cc, such as about 0.18 to about 0.24 g/cc, for example about 0.18g/cc, about 0.21 g/cc, about 0.23 g/cc, or about 0.24 g/cc for 2500 taps.

Polymer and method of making same

Solid dispersions comprising amorphous compound 1 and a polymer (or solid carrier) are also included herein. For example, compound 1 is present as a component of a solid amorphous dispersion as an amorphous compound. Such solid amorphous dispersions typically comprise compound 1 and a polymer. Exemplary polymers include cellulosic polymers such as HPMC or HPMCAS and pyrrolidone-containing polymers such as PVP/VA. In some embodiments, such solid amorphous dispersions comprise one or more additional excipients such as surfactants.

In one embodiment, the polymer is capable of being dissolved in an aqueous medium. The solubility of the polymer may be pH independent or pH dependent. The latter includes one or more enteric polymers. The term "enteric polymer" refers to a polymer that is preferentially soluble in the less acidic environment of the intestine relative to the more acidic environment of the stomach, e.g., a polymer that is insoluble in acidic aqueous media but soluble when the pH is above 5-6. Suitable polymers should be chemically and biologically inert. To improve the physical stability of solid dispersions, the glass transition temperature (T) of the polymer_g) Should be as high as possible. For example, preferred polymers have a glass transition temperature that is at least equal to or greater than the glass transition temperature of the drug (i.e., compound 1). Other preferred polymers have a glass transition temperature within about 10 to about 15 ℃ of the drug (i.e., compound 1). Examples of suitable glass transition temperatures for the polymer include at least about 90 ℃, at least about 95 ℃, at least about 100 ℃, at least about 105 ℃, at least about 110 ℃, at least about 115 ℃, at least about 120 ℃, at least about 125 ℃, at least about 130 ℃, at least about 135 ℃, at least about 140 ℃, at least about 145 ℃, at least about 150 ℃, at least about 155 ℃, at least about 160 ℃, at least about 165 ℃, at least about 170 ℃, or at least about 175 ℃ (as measured under dry conditions). Without wishing to be bound by theory, it is believed that the preferential mechanism is to have a higher T_gThe polymers of (a) typically have relatively low molecular mobility at room temperature, which may be a key factor in stabilizing the physical stability of amorphous solid dispersions.

In addition, the hygroscopicity of the polymer should be as low as, for example, less than about 10%. For purposes of comparison in this application, the hygroscopicity of the polymer or composition is characterized at about 60% relative humidity. In some preferred embodimentsIn some embodiments, the polymer has a water uptake of less than about 10%, for example, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, or less than about 2%. Hygroscopicity can also affect the physical stability of solid dispersions. In general, moisture adsorption in the polymer can greatly reduce the T of the polymer and the resulting solid dispersion_gThis will further reduce the physical stability of the solid dispersion described above.

In one embodiment, the polymer is one or more water soluble polymers or partially water soluble polymers. Water-soluble or partially water-soluble polymers include, without limitation, cellulose derivatives such as Hydroxypropylmethylcellulose (HPMC), Hydroxypropylcellulose (HPC), or ethylcellulose; polyvinylpyrrolidone (PVP); polyethylene glycol (PEG); polyvinyl alcohol (PVA); acrylates such as polymethacrylate (e.g., Eudragit E); cyclodextrins (e.g., beta-cyclodextrin) and copolymers and derivatives thereof, including, for example, PVP-VA (polyvinylpyrrolidone-vinyl acetate).

In some embodiments, the polymer is Hydroxypropylmethylcellulose (HPMC), such as HPMC E50, HPMC E15, or HPMC60SH 50).

As discussed herein, the polymer can be a pH-dependent enteric polymer. Such pH-dependent enteric polymers include, without limitation, cellulose derivatives (e.g., Cellulose Acetate Phthalate (CAP)), hydroxypropylmethylcellulose phthalate (HPMCP), hypromellose acetate succinate (HPMCAS), carboxymethylcellulose (CMC), or salts thereof (e.g., sodium salts such as (CMC-Na)); cellulose Acetate Trimellitate (CAT), hydroxypropylcellulose acetate phthalate (HPCAP), hydroxypropylmethylcellulose acetate phthalate (HPMCAP) and methylcellulose acetate phthalate (MCAP) or polymethacrylates (e.g., Eudragit E). In some embodiments, the polymer is hypromellose acetate succinate (HPMCAS). In some embodiments, the polymer is hypromellose acetate succinate HG grade (HPMCAS-HG).

In yet another embodiment, the polymer is a polyvinylpyrrolidone copolymer, such as vinylpyrrolidone/vinyl acetate copolymer (PVP/VA).

In embodiments where compound 1 forms a solid dispersion with a polymer, e.g., with an HPMC, HPMCAS, or PVP/VA polymer, the amount of polymer relative to the total weight of the solid dispersion is in the range of about 0.1% to 99% by weight. The percentages of drug, polymer, and other excipients in the dispersion as described are given as weight percentages unless otherwise specified. The amount of polymer is generally at least about 20%, and preferably at least about 30%, such as at least about 35%, at least about 40%, at least about 45%, or about 50% (e.g., 49.5%). The amount is generally about 99% or less, and preferably about 80% or less, such as about 75% or less, about 70% or less, about 65% or less, about 60% or less, or about 55% or less. In one embodiment, the polymer is present in an amount up to about 50% (and even more specifically, between about 40% and 50%, such as about 49%, about 49.5%, or about 50%) of the total weight of the dispersion. HPMC and HPMCAS are available from ShinEtsu in various grades, for example HPMCAS is available in various grades including AS-LF, AS-MF, AS-HF, AS-LG, AS-MG, AS-HG. Each of these grades varies with the percentage of substitution of acetate and succinate.

In some embodiments, compound 1 and the polymer are present in approximately equal amounts, e.g., each of the polymer and the drug constitutes about half of the weight percent of the dispersion. For example, the polymer is present at about 49.5% and the drug is present at about 50%.

In some embodiments, the combined compound 1 and polymer represent 1% -20% w/w total solids content of the non-solid dispersion prior to spray drying. In some embodiments, the combined compound 1 and polymer represent 5% to 15% w/w total solids content of the non-solid dispersion prior to spray drying. In some embodiments, the combined compound 1 and polymer represent about 11% w/w total solids content of the non-solid dispersion prior to spray drying.

In some embodiments, the dispersion further comprises other minor ingredients, such as surfactants (e.g., SLS). In some embodiments, the surfactant is present at less than about 10% of the dispersion, such as less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, about 1%, or about 0.5%.

In embodiments comprising a polymer, the polymer should be present in an amount effective to stabilize the solid dispersion. Stabilizing includes inhibiting or preventing crystallization of compound 1. Such stabilization would inhibit the conversion of compound 1 from the amorphous to crystalline form. For example, the polymer should prevent at least a portion (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or more) of compound 1 from converting from amorphous to crystalline form. Stabilization can be measured, for example, by measuring the glass transition temperature of the solid dispersion, measuring the relaxation rate of the amorphous material, or by measuring the solubility or bioavailability of compound 1.

Suitable polymers for use in combination with compound 1, e.g., to form a solid dispersion such as an amorphous solid dispersion, should have one or more of the following properties:

the glass transition temperature of the polymer should have a temperature that is not less than about 10-15 c below the glass transition temperature of compound 1. Preferably, the glass transition temperature of the polymer is greater than the glass transition temperature of compound 1 and is typically at least 50 ℃ higher than the desired storage temperature of the pharmaceutical product. For example, at least about 100 ℃, at least about 105 ℃, at least about 110 ℃, at least about 120 ℃, at least about 130 ℃, at least about 140 ℃, at least about 150 ℃, at least about 160 ℃ or greater.

The polymer should be relatively non-hygroscopic. For example, the polymer should adsorb less than about 10% water, such as less than about 9%, less than about 8%, less than about 7%, less than about 6%, or less than about 5%, less than about 4%, or less than about 3% water, when stored under standard conditions. Preferably, the polymer should be substantially free of adsorbed water when stored under standard conditions.

The polymer should have similar or better solubility relative to compound 1 in a solvent suitable for spray drying. In a preferred embodiment, the polymer should be dissolved in one or more of the same solvents or solvent systems as compound 1. It is preferred that the polymer is dissolved in a solvent comprising at least one non-hydroxylic solvent such as dichloromethane, acetone or combinations thereof.

When combined with compound 1, the polymer, e.g., in a solid dispersion or in a liquid dispersion, should increase the solubility of compound 1 in aqueous and physiologically compatible media relative to the solubility of compound 1 in the absence of the polymer, or relative to the solubility of compound 1 when combined with a reference polymer. For example, by reducing the amount of amorphous compound 1 that is converted to crystalline compound 1 from a solid amorphous dispersion or from a liquid suspension, the polymer should increase the solubility of amorphous compound 1.

The polymer should reduce the rate of relaxation of the amorphous material.

The polymer should increase the physical and/or chemical stability of compound 1.

The polymer should improve the manufacturability of compound 1.

The polymer should improve one or more of the handling, administration, or storage properties of compound 1.

The polymer should not adversely interact with other pharmaceutically acceptable components such as excipients.

The suitability of a candidate polymer (or other component) can be tested using the spray drying method (or other methods) described herein to form an amorphous composition. Candidate compositions may be compared in terms of stability, resistance to crystal formation, or other properties, and compared to reference formulations, such as formulations of neat amorphous compound 1 or crystalline compound 1. For example, a candidate composition can be tested to determine when it inhibits solvent-mediated crystallization, or under controlled conditions, at least 50%, 75%, 100%, or 110% conversion at a given time and a reference formulation, or a candidate composition can be tested to determine whether it improves bioavailability or solubility relative to crystalline compound 1.

Surface active agent

The solid dispersion or other composition may comprise a surfactant. The surfactant or surfactant mixture should generally reduce the interfacial tension between the solid dispersion and the aqueous medium. Suitable surfactants or surfactant mixtures may also enhance the water solubility and bioavailability of compound 1 from solid dispersions. Surfactants for use in connection with the present invention include, without limitation, sorbitan fatty acid esters (e.g., Spans), polyoxyethylene sorbitan fatty acid esters (e.g., Tweens), Sodium Lauryl Sulfate (SLS), Sodium Dodecylbenzenesulfonate (SDBS), dioctyl sodium sulfosuccinate (Docusate), sodium Deoxycholate (DOSS), sorbitan monostearate, sorbitan tristearate, cetyl trimethylammonium bromide (HTAB), sodium N-dodecylsarcosinate, sodium oleate, sodium myristate, sodium stearate, sodium palmitate, macrogol laurate (Gelucire 44/14), ethylenediaminetetraacetic acid (EDTA), vitamin E d-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS), lecithin, MW 677-692, monosodium glutamate monohydrate, macrogol caprylate caprite (Labrasol), PEG 8 caprylic/capric glycerides, diethylene glycol monoethyl ether (Transcutol), diethylene glycol monoethyl ether, Solutol HS-15, polyethylene glycol stearate 15 (Solutol HS-15), polyethylene glycol/hydroxystearate, taurocholic acid, propylene glycol block polyether F68, block polyether (Pluronic F108) and block polyether (Pluronic F127) (or any other polyoxyethylene-polyoxypropylene copolymer (Pluronic) or saturated polyglycolized glycerides (Gelucers)). Specific examples of such surfactants that may be used in connection with the present invention include, without limitation, Span 65, Span 25, Tween 20, Capryol 90, Pluronic F108, Sodium Lauryl Sulfate (SLS), vitamin E TPGS, segmented polyethers, and copolymers. SLS is generally preferred.

The amount of surfactant (e.g., SLS) relative to the total weight of the solid dispersion can be between 0.1-15%. Preferably, the amount is from about 0.5% to about 10%, more preferably from about 0.5% to about 5%, for example from about 0.5-4%, from about 0.5-3%, from about 0.5-2%, from about 0.5-1% or about 0.5%.

In certain embodiments, the amount of surfactant relative to the total weight of the solid dispersion is at least about 0.1%, preferably about 0.5%. In these embodiments, the surfactant will be present in an amount of no more than about 15%, and preferably no more than about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%. Embodiments are preferred in which the surfactant is present in an amount of about 0.5% by weight.

Candidate surfactants (or other components) may be tested for applicability in the present invention in a manner similar to that described for the test polymers.

Use, dosage form and administration

Pharmaceutically acceptable compositions

In another aspect of the invention, pharmaceutically acceptable compositions are provided, wherein these compositions comprise compound 1 form a or amorphous compound 1 described herein, and optionally a pharmaceutically acceptable carrier, adjuvant or vehicle. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents.

As described above, the pharmaceutically acceptable compositions of the present invention additionally comprise pharmaceutically acceptable carriers, adjuvants or vehicles, which, as used herein, include any and all solvents, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as appropriate for the particular dosage form desired. Remington's Pharmaceutical Sciences, 6 th edition, e.w. Martin (mack publishing co., Easton, Pa., 1980) discloses various carriers for formulating pharmaceutically acceptable compositions and known techniques for their preparation. Except insofar as any conventional carrier medium is incompatible with the compounds of the invention, such as by producing any undesirable biological effect or interacting in a deleterious manner with any other component of a pharmaceutically acceptable composition, its use is contemplated within the scope of the invention. Some examples of materials that can be used as pharmaceutically acceptable carriers include, without limitation, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene block copolymers, lanolin; sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth powder, malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid, pyrogen-free water, isotonic saline, ringer's solution, ethanol and phosphate buffer, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and coloring agents, mold release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition at the discretion of the formulator.

Use of compounds and pharmaceutically acceptable compositions

In yet another aspect, the invention provides methods of treating a condition, disease, or disorder associated with CFTR. In certain embodiments, the present invention provides methods of treating a condition, disease or disorder associated with a deficiency in CFTR activity, comprising administering to a subject, preferably a mammal, in need thereof a composition comprising a solid state form of compound 1 form a or amorphous compound 1 as described herein.

As used herein, a "CFTR mediated disease" is a disease selected from the group consisting of: cystic fibrosis, asthma, COPD due to smoking, chronic bronchitis, sinusitis, constipation, pancreatitis, pancreatic insufficiency, male infertility due to congenital bilateral vasectomy (CBAVD), mild pulmonary disease, idiopathic pancreatitis, allergic bronchopulmonary aspergillosis (ABPA), liver disease, hereditary emphysema, hereditary hemochromatosis, defects in thrombofibrinolysis, protein C deficiency, hereditary angioedema type 1, defects in lipid processing, familial hypercholesterolemia, chylomicronemia type 1, betalipoproteinemia, lysosomal storage diseases, I-cell disease/pseudoheller disease, mucopolysaccharidosis, Sandhof disease/Mongolia dementia, Crigler-Najjar type II syndrome, polyendocrinosis/hyperinsulinemia, diabetes, Ralun dwarfism, Crohn's disease, and Crohn's disease, Myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, glycolytic CDG type 1, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency, Diabetes Insipidus (DI), neurological DI, renal DI, Charcot-Marie-tooth syndrome, Peyer's disease, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, pick's disease, some polyglutamine neurological disorders, Huntington's disease, spinocerebellar ataxia type I, spinobulbar muscular atrophy, dentatorubral globus Louis atrophy, myotonic dystrophy, spongiform encephalopathy, hereditary Creutzfeldt-Jakob disease (due to prion processing defects), Fabry disease, Sterler-Scheck syndrome, COPD, dry eye disease, sjogren's disease, osteoporosis, osteopenia, gaucher's syndrome, chloride channel disease, myotonia congenita (thomson and becker forms), barter syndrome type III, Dent's disease, panic disorder, epilepsy, panic disorder, lysosomal storage disease, Angelman syndrome, Primary Ciliary Dyskinesia (PCD), inherited diseases of ciliary structure and/or function, PCD not accompanied by visceral inversion (also known as catagory syndrome), PCD accompanied by visceral inversion or ciliary hypoplasia.

In certain embodiments, the present invention provides a method of treating a CFTR mediated disease in a human, comprising the step of administering to said human an effective amount of a composition comprising compound 1 form a or amorphous compound 1 described herein.

According to an alternative preferred embodiment, the present invention provides a method of treating cystic fibrosis in a human, the method comprising the step of administering to the human a composition comprising compound 1 form a or amorphous compound 1 as described herein.

According to the present invention, an "effective amount" of compound 1 form a or amorphous compound 1 or a pharmaceutically acceptable composition thereof is an amount effective to treat or reduce the severity of any of the diseases listed above.

Compound 1 form a or amorphous compound 1 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 of the diseases listed above.

In certain embodiments, compound 1 form a or amorphous compound 1 described herein, or a pharmaceutically acceptable composition thereof, is used to treat or reduce the severity of cystic fibrosis in a patient who exhibits residual CFTR activity in the apical membrane of respiratory and non-respiratory epithelial cells. The presence of residual CFTR activity on epithelial surfaces can be readily detected using methods known in the art, such as standard electrophysiological, biochemical or histochemical techniques. Such methods measure the Cl of sweat or saliva using in vivo or in vitro electrophysiological techniques^-Concentration, or in vitro biochemical or histochemical techniques to monitor cell surface density. Using such methods, patients who are heterozygous or homozygous for a number of different mutations, including the most common mutation Δ F508 in homozygosity or heterozygosity and other mutations such as G551Patients with the D mutation or R117H mutation can readily detect residual CFTR activity.

In one embodiment, Compound 1 form A or amorphous Compound 1, or a pharmaceutically acceptable composition thereof, described herein is used to treat or reduce the severity of cystic fibrosis in a patient who is within the range of certain genotypes exhibiting residual CFTR activity, such as class III mutations (impaired regulation or gating), class IV mutations (altered transport), or class V mutations (reduced synthesis) (Lee R. Choo-Kang, Pamela L., Zeitin, Type I, II, III, IV, and V cystic fibrosis nucleic acid production Regulation Defects and opportunities of Therapy; Current Opinion in pure research Medicine 6: 521-. Other patient genotypes exhibiting residual CFTR activity include patients homozygously binding to one of these types or heterozygous for any other type of mutation, including a class I mutation, a class II mutation, or a mutation lacking classification.

In one embodiment, compound 1 form a or amorphous compound 1 described herein, or a pharmaceutically acceptable composition thereof, is used to treat or reduce the severity of cystic fibrosis in a patient who has cystic fibrosis within a range of certain clinical phenotypes, such as moderate to mild clinical phenotypes typically associated with an amount of residual CFTR activity of the apical membrane of epithelial cells. Such phenotypes include patients presenting with pancreatic insufficiency or diagnosed with idiopathic pancreatitis and congenital bilateral absence of 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 unit dosage forms for ease of administration and uniformity of dosage. The expression "unit dosage form" as used herein refers to a physically discrete unit of medicament suitable for the patient to be treated. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention is 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 disorder being treated and the severity of the disorder, the activity of the specific compound employed, the specific composition employed; the age, weight, general health, sex, and diet of the patient; the number of administrations, the route of administration and rate of excretion of the particular compound employed, the duration of the treatment, the drug employed in combination or concomitantly with the particular compound employed, and like factors well known in the medical arts. The term "patient" or "subject" as used herein means an animal, preferably a mammal, and 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 powder, ointment, or drops), buccally, as an oral or nasal spray, etc., depending on the severity of the infection being treated. In certain embodiments, the compounds of the present invention may be administered orally or parenterally at dosage levels of from about 0.01 mg/kg to about 50 mg/kg, and preferably from about 1 mg/kg to about 25 mg/kg of the subject's body weight, once or more a day, to achieve the desired therapeutic effect.

In certain embodiments, the dose of compound 1 form a or amorphous compound 1 in a unit dosage form is from 100mg to 1000 mg. In another embodiment, the dose of compound 1 form a or amorphous compound 1 is 200 mg to 900 mg. In another embodiment, the dose of compound 1 form a or amorphous compound 1 is 300 mg to 800 mg. In another embodiment, the dose of compound 1 form a or amorphous compound 1 is 400 mg to 700 mg. In another embodiment, the dose of compound 1 form a or amorphous compound 1 is 500 mg to 600 mg.

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

Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by the addition of a sterilizing agent in the form of a sterile solid composition which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

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 a suppository wax which is solid at ambient temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity and releases the active compound.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or the following: a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and silicic acid, b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage 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. Tablets, dragees, capsules, pills and granules of solid dosage forms can be prepared with coatings and shells such as enteric coatings as well as other coatings well known in the art of pharmaceutical formulation. They may optionally contain opacifying agents and may also have a composition that they release the active ingredient(s) only, or preferentially, in certain parts of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymers 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 compounds may also be present in microencapsulated form containing one or more of the above-mentioned excipients. Tablets, dragees, capsules, pills and granules of solid dosage forms can be prepared with coatings and shells such as enteric coatings, controlled release coatings and other coatings well known in the art of pharmaceutical formulation. In such solid dosage forms, the active compound may be mixed with at least one inert diluent, for example sucrose, lactose or starch. Such dosage forms may also contain, as is conventional practice, additional substances other 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 have a composition that they release the active ingredient(s) only, or preferentially, in certain parts of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymers and waxes.

It will also be appreciated that compound 1 form a or amorphous compound 1 described herein, or a pharmaceutically acceptable composition thereof, may be used in combination therapy, that is, compound 1 form a or amorphous compound 1 may be administered simultaneously with, prior to, or following one or more other desired treatments and/or medical procedures. The particular combination of therapies (therapeutic agents or surgery) used in a combination regimen should take into account the compatibility of the therapeutic agent and/or surgery required and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve the desired effect on the same disorder (e.g., the compounds of the present invention may be administered simultaneously with another drug used to treat the same disorder), or they may achieve different effects (e.g., control of any side effects). As used herein, an additional therapeutic agent that is typically administered to treat or prevent a particular disease or condition is considered "appropriate for the disease or condition being treated.

In one embodiment, the additional agent is selected from a mucolytic agent, a bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR modulator other than a compound of the invention, or a nutritional agent.

In one 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 agent is N- (5-hydroxy-2, 4-di-tert-butyl-phenyl) -4-oxo-1H-quinoline-3-carboxamide. In another embodiment, the additional agent is selected from table 1:

TABLE 1

In another embodiment, the additional agent is any combination of the above agents. For example, the composition may comprise the compounds 1,3- (6- (1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) cyclopropanecarboxamido) -3-methylpyridin-2-yl) benzoic acid and N- (5-hydroxy-2, 4-di-tert-butyl-phenyl) -4-oxo-1H-quinoline-3-carboxamide. In another example, a composition can comprise compound 1, N- (5-hydroxy-2, 4-di-tert-butyl-phenyl) -4-oxo-1H-quinoline-3-carboxamide, and any one compound from table 1, i.e., compounds 1-14 in table 1, or any combination thereof.

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

In another embodiment, the additional drug is a mucolytic (mucolyte). Exemplary mucolytics (mucolytics) for use herein include Pulmozyme @.

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

In another embodiment, the additional agent is effective to restore pulmonary airway surface fluid. Such drugs improve the movement of salt into and out of the cells, allowing mucus in the pulmonary airways to become more hydrated and therefore more easily cleared. Exemplary such agents include hypertonic saline, denufosol sodium ([ [ (3S, 5R) -5- (4-amino-2-oxopyrimidin-1-yl) -3-hydroxyoxolan-2-yl) ] methoxy-hydroxyphosphoryl ] [ [ (2R,3S,4R,5R) -5- (2, 4-dioxopyrimidin-1-yl) -3, 4-dihydroxyoxolan-2-yl ] methoxy-hydroxyphosphoryl ] oxy-hydroxyphosphoryl ] hydrogen phosphate) or mannitol (bronchitol) (inhalation dosage form of mannitol).

In another embodiment, the additional agent is an anti-inflammatory agent, i.e., an agent that reduces lung inflammation. Exemplary such agents for use herein include ibuprofen, docosahexaenoic acid (DHA), sildenafil, inhaled glutathione, pioglitazone, hydroxychloroquine or simvastatin.

In another embodiment, the additional agent is a CFTR modulator other than compound 1 form I, i.e., an agent that has the effect of modulating CFTR activity. Exemplary such drugs include ataluren ("PTC 124;" 3- [5- (2-fluorophenyl) -1,2, 4-oxadiazol-3-yl ] benzoic acid), cinanaptide, lanoxietat, dilastat (a recombinant human neutrophil elastase inhibitor), cobiprostone (7- { (2R, 4aR, 5R, 7aR) -2- [ (3S) -1, 1-difluoro-3-methylphenyl ] -2-hydroxy-6-oxooctahydrocyclopenta [ b ] pyran-5-yl } heptanoic acid) and N- (5-hydroxy-2, 4-di-tert-butyl-phenyl) -4-oxo-1H-quinoline-3-carboxamide.

In another embodiment, the additional pharmaceutical agent is a nutraceutical agent. Exemplary nutritional agents include pancreatic lipase (pancreatin substitute), comprising Pancrease, Pancreacarb, Ultrase or Creon, Liprotomase ® A @ T @, or T @ T. In one embodiment, the additional nutrient is pancrelipase.

In another embodiment, the additional agent is a compound selected from the group consisting of: gentamicin, curcumin, cyclophosphamide, 4-phenylbutyrate, meglumine, felodipine, nimodipine, philixin B, genistein, apigenin; cAMP/cGMP modulators such as rolipram, sildenafil, milrinone, tadalafil, amrinone, isoproterenol, salbutamol and salmeterol; deoxyspergualin, HSP 90 inhibitors, HSP 70 inhibitors, proteasome inhibitors such as epoxymycin, lactacystin, and the like.

In another embodiment, the additional agent is a compound disclosed in WO 2004028480, WO 2004110352, WO2005094374, WO 2005120497 or WO 2006101740.

In another embodiment, the additional agent is a benzo (c) quinolizine exhibiting CFTR modulatory activityDerivatives or exhibiting CFTR modulating activityThe benzopyran derivative of (1).

In another embodiment, the additional agent is a compound disclosed in US7202262, US6992096, US20060148864, US20060148863, US20060035943, US20050164973, WO2006110483, WO2006044456, WO2006044682, WO2006044505, WO2006044503, WO2006044502 or WO 2004091502.

In another embodiment, the additional agent is a compound disclosed in WO2004080972, WO2004111014, WO2005035514, WO2005049018, WO2006099256, WO2006127588 or WO 2007044560.

These combinations are useful in the treatment of the diseases described herein, including cystic fibrosis. These combinations are also useful in the kits described herein.

The amount of additional therapeutic agent present in the compositions of the present invention should be no more than that which would normally be administered in a composition comprising the therapeutic agent as the only active drug. Preferably, the amount of additional therapeutic agent in the presently disclosed compositions will be in the range of about 50% -100% of the amount typically present in compositions containing the drug as the sole therapeutically active drug.

Compound 1 form a and amorphous form described herein or pharmaceutically acceptable compositions thereof may also be incorporated into compositions for coating implantable medical devices such as prostheses, prosthetic valves, vascular prostheses, stents, and catheters. Thus, in another aspect, the invention includes compositions for coating implantable devices and in classes and subclasses herein, such compositions comprising compound 1 form a and/or amorphous form described herein or a pharmaceutically acceptable composition thereof and a carrier suitable for coating the implantable device. In yet another aspect, the invention includes an implantable device coated with a composition comprising compound 1 form a and/or amorphous form described herein or a pharmaceutically acceptable composition thereof and a carrier suitable for coating the implantable device. The general preparation of suitable coatings and coated implantable devices is described in U.S. patents 6099562, 5886026, and 5304121. Such coating layers are typically biocompatible polymeric materials such as hydrogel polymers, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate, and mixtures thereof. The coating layer may optionally be further covered with a suitable top coating layer of fluorosilicone rubber, polysaccharides, polyethylene glycol, phospholipids or combinations thereof to impart controlled release characteristics to the composition.

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

Examples

Method and material

Modulated Differential Scanning Calorimetry (MDSC) and Differential Scanning Calorimetry (DSC)

Modulated Differential Scanning Calorimetry (MDSC) was used to test the glass transition temperature of amorphous forms of the compounds and spray dried dispersions. Differential Scanning Calorimetry (DSC) is used to determine the melting point of crystalline materials and to distinguish between different polymorphs. Data were collected using a TA DSC Q2000 differential scanning calorimeter (TA Instruments, New Castle, DE). The instrument was calibrated with indium. About 1-5 mg of the sample was weighed into an aluminum sealed pot crimped with a lid having a hole. Samples were scanned from-20 deg.C to 220 deg.C for MDSC at a heating rate of 2 deg.C/min with adjustments of +/-1 deg.C every 60 seconds. The samples were scanned from 25 ℃ to 220 ℃ at a heating rate of 10 ℃/min for DSC. Data was passed through Thermal Advantage QSeries^TMThe software (version: 2.7.0.380) was collected and analyzed by Universal Analysis software (version: 4.4A, build: 4.4.0.5) (TA Instruments, New Castle, DE).

XRPD (X-ray powder diffraction)

X-ray powder diffraction was used to characterize the physical form of the batches of samples produced to date and to characterize the different polymorphs identified. XRPD data for compounds were collected on a PANALYTIC X' pert Pro Powder X-ray diffraction meter (Almelo, the Netherlands). XRPD patterns were recorded at room temperature with copper radiation (1.54060A). X-ray filter generation was suppressed with Nickel K β (Nickel K β) using a Cu sealed tube at 45 kV, 40 mA. The incident beam consists of a variable divergence slit to ensure a constant emission wavelength on the sample and on the diffracted light side; a fast linear solid-state detector with a working length of 2.12 ° 2 θ measured in scan mode was used. The powder samples were compacted in the indented area of the zero background silicon fixture and scanned for better statistical data. The symmetric scan was measured at 4-40 deg. 2 theta with a step size of 0.017 deg. and a scan step time of 15.5 seconds. The Data collection software is an X' pert Data Collector (version 2.2 e). The Data analysis software was either X 'pert Data Viewer (version 1.2d) or X' pert Highscore (version: 2.2 c).

Thermogravimetric analysis (TGA)

TGA was used to study the presence of residual solvent in the characterized batches of samples and to determine the temperature at which decomposition of the samples occurred. TGA data were collected on a TA Q500 Thermogravimetric Analyzer (Thermogravimetric Analyzer) (TAInstants, New Castle, DE). Samples weighing about 2-5 mg were scanned from 25 deg.C-300 deg.C at a heating rate of 10 deg.C/min. Data were obtained by Thermal Advantage Q Series^TMThe software (version 2.5.0.255) was collected and analyzed by general Analysis (Universal Analysis) software (version 4.4A, build 4.4.0.5) (TA Instruments, New Castle, DE).

Compound 1 form a single crystal structure determination

Diffraction data were obtained on a Bruker Apex II diffractometer equipped with a sealed tube Cu ka source and Apex II CCD detector. The structure was resolved and refined using the SHELX program (Sheldrick, g.m., Acta cryst., (2008) a64, 112-. The structure of the C2 space group was resolved and refined based on intensity statistics and systematic extinction. The absolute configuration was determined using similar diffraction. The accuracy of the Flack parameter to 0.00 (18) indicates that the model represents the correct enantiomer [ (R) ].

Solid state NMR

Solid state NMR was performed on a Bruker-Biospin 400 MHz wide-bore spectrometer equipped with a Bruker-Biospin 4 mm HFX probe. The sample was compacted to 4 mm ZrO₂The rotor was rotated under the condition of Magic Angle Spinning (MAS) at a rotational speed of 12.5 kHz. Proton relaxation time first using H MAS T₁Experimental measurement of saturation recovery relaxation to establish¹³Appropriate recycling delay for the C Cross Polarization (CP) MAS test. The CP contact time for the carbon CPMAS experiment was set at 2 ms. CP proton pulses with linear ramps (from 50% to 100%) were used. Hartmann-Hahn matching was optimized based on an external reference sample (glycine). Fluorine MAS spectra were recorded with proton decoupling. To obtain¹³C and¹⁹f, field strength was about 100 kHz using TPPM15 decoupling sequence.

Viride sodium (bis (2-methoxyethoxy) aluminum hydride [ or NaAlH-₂(OCH₂CH₂OCH₃)₂]65 wgt% solution in toluene) from Aldrich Chemicals.

2, 2-difluoro-1, 3-benzodioxole-5-carboxylic acid was purchased from Saltigo (a affiliate of the Lanxess corporation).

Wherever the title of a compound does not correctly describe the structure of the compound, the result is an alternative title and predominates over the structure.

Synthesis of Compound 1

Acid moieties

Synthesis of (2, 2-difluoro-1, 3-benzodioxol-5-yl) -methanol.

Commercially available 2, 2-difluoro-1, 3-benzodioxole-5-carboxylic acid (1.0 equivalent) was slurried in toluene (10 volumes). Red aluminum (Viride) granules (2 equivalent weight) were added through a feeding funnel at a certain speed to keep the temperature within the range of 15 ℃ to 25 ℃. In the process of adding knotWhile bundled, the temperature was allowed to rise to 40 ℃ for 2 hours, and then 10% (w/w) aqueous NaOH (4.0 equiv) was carefully added via an addition funnel, while maintaining the temperature at 40-50 ℃. After stirring for a further 30 minutes, the layers were separated at 40 ℃. The organic phase was cooled to 20 ℃, then washed with water (2 × 1.5 vol) and dried (Na)₂SO₄) Filtered and concentrated to give crude (2, 2-difluoro-1, 3-benzodioxol-5-yl) -methanol, which is used directly in the next step.

Synthesis of 5-chloromethyl-2, 2-difluoro-1, 3-benzodioxole.

(2, 2-difluoro-1, 3-benzodioxol-5-yl) -methanol (1.0 eq) was dissolved in MTBE (5 vol). DMAP (1 mol%) was added in catalytic amount and SOCl was added via addition funnel₂(1.2 equiv.). SOCl is added at a rate to maintain the temperature in the reactor at 15-25 deg.C₂. The temperature was allowed to rise to 30 ℃ for 1 hour, then cooled to 20 ℃ and water (4 volumes) was added via an addition funnel, while maintaining the temperature below 30 ℃. After stirring for a further 30 minutes, the layers were separated. The organic layer was stirred and 10% (w/v) aqueous NaOH (4.4 vol) was added. After stirring for 15-20 minutes, the layers were separated. Then dried (Na)₂SO₄) The organic phase was filtered and concentrated to give crude 5-chloromethyl-2, 2-difluoro-1, 3-benzodioxole, which was used directly in the next step.

Synthesis of (2, 2-difluoro-1, 3-benzodioxol-5-yl) -acetonitrile.

A solution of 5-chloromethyl-2, 2-difluoro-1, 3-benzodioxole (1 eq) in DMSO (1.25 vol) was added to a slurry of NaCN (1.4 eq) in DMSO (3 vol) while maintainingThe temperature is between 30 and 40 ℃. The mixture was stirred for 1 hour, then water (6 vol) was added followed by MTBE (4 vol). After stirring for 30 minutes, the layers were separated. The aqueous layer was extracted with MTBE (1.8 vol). The combined organic layers were washed with water (1.8 vol.) and dried (Na)₂SO₄) Filtered and concentrated to give the crude (2, 2-difluoro-1, 3-benzodioxol-5-yl) -acetonitrile, which is used directly in the next step.¹H NMR (500 MHz, DMSO) 7.44 (br s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.22(dd, J = 8.2, 1.8 Hz, 1H), 4.07 (s, 2H)。

Synthesis of (2, 2-difluoro-1, 3-benzodioxol-5-yl) -1-ethyl acetate-acetonitrile.

The reactor was purged with nitrogen and 900 mL of toluene was added. The solvent was degassed by nitrogen sparging for not less than 16 hours. Then adding Na into the reactor in sequence₃PO₄(155.7 g, 949.5 mmol), bis (dibenzylideneacetone) palladium (0) (7.28 g, 12.66 mmol). A10% w/w solution of tert-butylphosphine in hexane (51.23 g, 25.32 mmol) was added over 10 minutes at 23 deg.C from a nitrogen purged addition funnel. The mixture was allowed to stir for 50 minutes at which time 5-bromo-2, 2-difluoro-1, 3-benzodioxole (75 g, 316.5 mmol) was added over 1 minute. After stirring for another 50 minutes, ethyl cyanoacetate (71.6 g, 633.0 mmol) was added to the mixture over 5 minutes followed by water (4.5 mL) in one portion. The mixture was heated to 70 ℃ over 40 minutes and analyzed by HPLC every 1-2 hours for percent conversion of reactants to products. After complete conversion is observed (typically 100% conversion after 5-8 hours), the mixture is cooled to 20-25 ℃ and filtered through a pad of celite. The celite pad was rinsed with toluene (2X 450 mL) and the combined organics were concentrated to 300 mL under vacuum at 60-65 ℃. 225mL of DMSO was added to the concentrate and concentrated under vacuum at 70-80 ℃ until active distillation of the solvent ceased. The solution was cooled to 20-25 ℃ and diluted with DMSO in the preparation of step 2900 mL。¹H NMR (500MHz, CDCl₃) 7.16-7.10 (m, 2H), 7.03 (d, J = 8.2 Hz, 1H), 4.63 (s, 1H), 4.19(m, 2H), 1.23 (t, J = 7.1 Hz, 3H)。

Synthesis of (2, 2-difluoro-1, 3-benzodioxol-5-yl) -acetonitrile.

To a DMSO solution of (2, 2-difluoro-1, 3-benzodioxol-5-yl) -1-ethyl acetate-acetonitrile from above was added 3N HCl (617.3 mL, 1.85 mol) over 20 minutes while maintaining the internal temperature<At 40 ℃. The mixture was then heated to 75 ℃ over 1 hour and analyzed by HPLC for% conversion every 1-2 hours. When conversion is observed>At 99% (typically after 5-6 h), the reaction was cooled to 20-25 ℃ and extracted with MTBE (2X 525 mL) with sufficient time to allow complete phase separation during extraction. The combined organic extracts were washed with 5% NaCl (2X 375 mL). The solution was then transferred to an apparatus equipped with a cooled receiver flask suitable for 1.5-2.5 torr vacuum distillation. In that<The solution was concentrated in vacuo at 60 ℃ to remove the solvent. (2, 2-difluoro-1, 3-benzodioxol-5-yl) -acetonitrile was distilled from the resulting oil at 125 ℃ and 130 ℃ (oven temperature) at 1.5-2.0 torr. (2, 2-difluoro-1, 3-benzodioxol-5-yl) -acetonitrile isolated as a clear oil in 66% yield from 5-bromo-2, 2-difluoro-1, 3-benzodioxol (2 steps) and with an HPLC purity of 91.5% AUC (equivalent to 95% w/w content).¹H NMR (500 MHz, DMSO) 7.44(br s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.22 (dd, J = 8.2, 1.8 Hz, 1H), 4.07(s, 2H)。

Synthesis of (2, 2-difluoro-1, 3-benzodioxol-5-yl) -cyclopropanecarbonitrile.

Reacting (2, 2-difluoro-1, 3-benzene)Dioxol-5-yl) -acetonitrile (1.0 equiv.), 50 wt% aqueous KOH (5.0 equiv.), 1-bromo-2-chloroethane (1.5 equiv.), and Oct₄A mixture of NBr (0.02 eq.) was heated at 70 ℃ for 1 hour. The reaction mixture was allowed to cool and then treated with MTBE and water. The organic phase was washed with water and brine, and then the solvent was removed to give (2, 2-difluoro-1, 3-benzodioxol-5-yl) -cyclopropanecarbonitrile.¹H NMR (500 MHz, DMSO) 7.43 (d, J = 8.4 Hz, 1H), 7.40 (d, J = 1.9 Hz, 1H), 7.30 (dd, J = 8.4, 1.9Hz, 1H), 1.75 (m, 2H), 1.53 (m, 2H)。

Synthesis of 1- (2, 2-difluoro-1, 3-benzodioxol-5-yl) -cyclopropanecarboxylic acid.

(2, 2-difluoro-1, 3-benzodioxol-5-yl) -cyclopropanecarbonitrile was hydrolyzed using 6M NaOH (8 eq) in ethanol (5 vol) at 80 ℃ overnight. The mixture was cooled to room temperature and the ethanol was evaporated in vacuo. The residue was treated with water and MTBE, 1M HCl was added and the layers were separated. The MTBE layer was then treated with dicyclohexylamine (0.97 eq.). The slurry was cooled to 0 ℃, filtered and washed with heptane to give the corresponding DCHA salt. The salt was treated with MTBE and 10% citric acid and stirred until all solids were dissolved. The layers were separated and the MTBE layer was washed with water and brine. The solvent was changed to heptane, followed by filtration to give 1- (2, 2-difluoro-1, 3-benzodioxol-5-yl) -cyclopropanecarboxylic acid after drying in a vacuum oven overnight at 50 ℃. ESI-MS M/z theoretical 242.04, found 241.58 (M +1)⁺;¹H NMR (500 MHz,DMSO) 12.40 (s, 1H), 7.40 (d, J = 1.6 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H),7.17 (dd, J = 8.3, 1.7 Hz, 1H), 1.46 (m, 2H), 1.17 (m, 2H)。

Amine moieties

Synthesizing 2-bromo-5-fluoro-4-nitroaniline.

3-fluoro-4-nitroaniline (1.0 eq.) was added to the flask followed by ethyl acetate (10 vol) and stirred to dissolve all solids. N-bromosuccinimide (1.0 eq.) was added in portions to maintain the internal temperature at 22 ℃. At the end of the reaction, the reaction mixture was concentrated in vacuo on a rotary evaporator. The residue was slurried in distilled water (5 volumes) to dissolve and remove the succinimide. (the succinimide may also be removed by a water treatment procedure). The water was decanted and the solid was slurried with 2-propanol (5 volumes) overnight. The resulting slurry was filtered and the wet cake was washed with 2-propanol, accompanied by N₂The stream was dried in a vacuum oven at 50 ℃ overnight until a constant weight was reached. A tan solid was isolated (50% yield, 97.5% AUC). Other impurities were bromo-regioisomer (1.4% AUC) and dibromo-adduct (1.1% AUC). H NMR (500 MHz, DMSO) 8.19 (1H, d, J = 8.1 Hz), 7.06 (br. s, 2H), 6.64 (d, 1H, J = 14.3 Hz).

Synthesis of benzyl hydroxy acetic acid-4-ammonium-2-bromo-5-fluoroaniline toluene sulfonate.

To be at N₂The following were added to the thoroughly dried flask: activated powdered 4A molecular sieve (50 wt% based on 2-bromo-5-fluoro-4-nitroaniline), 2-bromo-5-fluoro-4-nitroaniline (1.0 eq.), zinc perchlorate dihydrate (20 mol%) and toluene (8 vol). The mixture NMT was stirred at room temperature for 30 minutes. Finally, (R) -benzyl glycidyl ether (2.0 equivalents) in toluene (2 volumes) was added in a steady flow. The reaction was heated to 80 ℃ (internal temperature) and stirred for about 7 hours or until the 2-bromo-5-fluoro-4-nitroaniline was<5%AUC。

The reaction was cooled to room temperature and celite (50 wt%) was added followed by ethyl acetate (10 vol). The resulting mixture was filtered to remove celite and molecular sieves and washed with ethyl acetate (2 vol). The filtrate was washed with ammonium chloride solution (4 vol, 20% w/v). The organic layer was washed with sodium bicarbonate solution (4 vol. x 2.5% w/v). The organic layer was concentrated in vacuo on a rotary evaporator. The resulting slurry was dissolved in isopropyl acetate (10 volumes) and the solution was transferred to a Buchi hydrogenator.

5wt% Pt (S)/C (1.5 mol%) and in N was added to the hydrogenator₂Next, the mixture was stirred at 30 ℃ (internal temperature). The reactants are successively N₂And a hydrogen purge. The hydrogenator pressure was adjusted to 1 bar of hydrogen and the mixture was stirred rapidly (>1200 rpm). At the end of the reaction, the catalyst was filtered through a pad of celite and washed with dichloromethane (10 volumes). The filtrate was concentrated in vacuo. Any remaining isopropyl acetate was removed with dichloromethane (2 volumes) and concentrated to dryness on a rotary evaporator.

The resulting residue was dissolved in dichloromethane (10 vol). P-toluenesulfonic acid monohydrate (1.2 eq) was added and stirred overnight. The product was filtered, washed with dichloromethane (2 volumes) and dried under suction. The wet cake was transferred to a drying tray and placed in a vacuum oven with N₂Dried at 45 ℃ under a stream until constant weight is reached. Isolation of 4-ammonio-2-bromo-5-fluoroaniline tosylate as an off-white solid

Chiral purity was determined to be >97% ee.

Synthesis of (3-chloro-3-methylbut-1-ynyl) trimethylsilane.

Propargyl alcohol (1.0 eq) was added to the vessel. Aqueous hydrochloric acid (37%, 3.75 vol) was added and stirring was started. During the dissolution of the solid alcohol, a moderate endotherm (5-6 ℃) was observed. The resulting mixture was stirred overnight (16 hours) and slowly turned to a deep red color. Water (5 volumes) was added to a 30L jacketed vessel and then cooled to 10 ℃. The reaction mixture was slowly transferred to water under vacuum while maintaining the internal temperature of the mixture below 25 ℃. Hexane (3 vol) was added and the resulting mixture was stirred for 0.5 h. These phases were allowed to settle and the aqueous phase was drained (pH <1) and discarded. The organic phase was concentrated in vacuo using a rotary evaporator to give the product as a red oil.

Synthesis of (4- (benzyloxy) -3, 3-dimethylbut-1-ynyl) trimethylsilane.

Method A

All equivalents and volume descriptors in this section are based on 250 g of reactant. Magnesium turnings (69.5 g, 2.86mol, 2.0 equivalents) were charged into a 3L 4-neck reactor and stirred with a magnetic stirrer under nitrogen for 0.5 hours. The reactor was submerged in an ice-water bath. A solution of propargyl chloride (250 g, 1.43 mol, 1.0 eq.) in THF (1.8L, 7.2 vol) was added slowly to the reactor with stirring until an initial temperature rise (-10 ℃ C.) was observed. Use of¹H-NMR spectrum confirmed the formation of Grignard reagent by IPC. Once the exotherm subsided, the remainder of the solution was slowly added, during which the batch temperature was maintained<15 ℃ is prepared. The addition required 3.5 hours. The resulting dark green mixture was decanted into a 2L capped bottle.

All equivalents and volume descriptors in this section are based on 500 g of reactant. A22L reactor was charged with a solution of benzyl chloromethyl ether (95%, 375 g, 2.31 mol, 0.8 eq.) in THF (1.5L, 3 vol). The reactor was cooled with an ice water bath. 2 of the 4 batches of grignard reagent prepared above were combined and then added slowly via the addition funnel to the benzyl chloromethyl ether solution while maintaining the batch temperature below 25 ℃. The addition took 1.5 hours. The reaction mixture was stirred overnight (16 hours).

All equivalents and volume descriptors in this section are based on 1kg of reactants. A15% ammonium chloride solution (1.5 kg in 8.5 kg water, 10 volumes) was prepared in a 30L jacketed reactor. The solution was cooled to 5 ℃. The two grignard reaction mixtures were combined and then transferred to an ammonium chloride solution via a manifold container. An exotherm is observed in this quenching, which proceeds, for example, at a rate that maintains the internal temperature below 25 ℃. Once the transfer was complete, the vessel shell temperature was set to 25 ℃. Hexane (8L, 8 vol) was added and the mixture was stirred for 0.5 h. After allowing these phases to settle, the aqueous phase (pH 9) was drained and discarded. The remaining organic phase was washed with water (2L, 2 vol). The organic phase was concentrated in vacuo using a 22L rotary evaporator to afford the crude product as an orange oil.

Method B

Magnesium turnings (106 g, 4.35 mol, 1.0 eq.) were charged to a 22L reactor and then suspended in THF (760mL, 1 vol). The vessel was cooled with an ice water bath to bring the batch temperature to 2 ℃. A solution of propargyl chloride (760 g, 4.35 mol, 1.0 eq) in THF (4.5L, 6 vol) was slowly added to the reactor. After addition of 100 mL, the addition was stopped and the mixture was stirred until a temperature of 13 ℃ was observed, indicating the onset of grignard reagent formation. Once the exotherm subsided, another 500 mL of propargyl chloride solution was slowly added, while maintaining the batch temperature<At 20 ℃. Use of¹H-NMR spectrum confirmed the formation of Grignard reagent by IPC. Slowly adding the remaining propargyl chloride solution while maintaining the batch temperature<At 20 ℃. Addition took 1.5 hours. The resulting dark green solution was stirred for 0.5 hour. Use of¹H-NMR spectrum confirmed the formation of Grignard reagent by IPC. Pure benzyl chloromethyl ether was added to the reactor addition funnel and then added dropwise to the reactor, while maintaining the batch temperature below 25 ℃. The addition took 1.0 hour. The reaction mixture was stirred overnight. Water treatment and concentration were carried out using the same procedure and relative amounts of materials as in method a to give the product as an orange oil.

Synthesis of 4-benzyloxy-3, 3-dimethyl-but-1-yne.

Methanol (6 volumes) was added to a 30L jacketed reactor and then cooled to 5 ℃. To the direction ofPotassium hydroxide (85%, 1.3 equivalents) was added to the reactor. A temperature rise of 15-20 ℃ was observed when the potassium hydroxide was dissolved. The shell temperature was set to 25 ℃. A solution of 4-benzyloxy-3, 3-dimethyl-1-trimethylsilylbut-1-yne (1.0 eq) in methanol (2 volumes) was added and the resulting mixture was stirred until the reaction was complete as monitored by HPLC. Typical reaction times are 3-4 hours at 25 ℃. The reaction mixture was diluted with water (8 vol) and then stirred for 0.5 h. Hexane (6 vol) was added and the resulting mixture was stirred for 0.5 h. These phases were allowed to settle and the aqueous phase was then drained (pH 10-11) and discarded. The organic phase was washed with a solution of KOH (85%, 0.4 eq) in water (8 vol) followed by water (8 vol). The organic phase was then concentrated using a rotary evaporator to give the title material as an orange-yellow oil. The typical purity of this material is in the 80% range, with a single impurity being present.¹H NMR (400MHz, C₆D₆) 7.28 (d, 2 H, J = 7.4 Hz), 7.18 (t, 2 H, J = 7.2 Hz), 7.10 (d,1H, J = 7.2 Hz), 4.35 (s, 2 H), 3.24 (s, 2 H), 1.91 (s, 1 H), 1.25 (s, 6 H)。

Synthesis of Benzylglycolylated 4-amino-2- (4-benzyloxy-3, 3-dimethylbut-1-ynyl) -5-fluoroaniline.

By dissolving in EtOAc (5 vol.) and saturated NaHCO₃The solid was stirred in solution (5 volumes) until a clear organic layer was obtained, leaving 4-ammonium-2-bromo-5-fluoroaniline tosylate as the free base (freebase). The resulting layers were separated and then saturated NaHCO was used₃The organic layer was washed with brine and concentrated in vacuo to give 4-ammonio-2-bromo-5-fluoroaniline tosylate as an oil.

Then, benzylhydroxyacetated-4-ammonium-2-bromo-5-fluoroaniline tosylate (free base, 1.0 equivalent), Pd (OAc) (4.0 mol%), dppb (6.0 mol%) and powdered K were added to the flask₂CO₃(3.0 equivalent weight) of,and stirred at room temperature with acetonitrile (6 volumes). By using N₂The resulting reaction mixture was degassed for about 30 minutes by bubbling through a vent. 4-benzyloxy-3, 3-dimethylbut-1-yne (1.1 eq) dissolved in acetonitrile (2 volumes) was then added in a fast stream, heated to 80 ℃ and stirred until complete consumption of 4-ammonium-2-bromo-5-fluoroaniline tosylate was achieved. The reaction slurry was cooled to room temperature, filtered through a pad of celite and washed with acetonitrile (2 volumes). The filtrate was concentrated in vacuo and the residue was redissolved in EtOAc (6 vol). NH for organic layers₄The Cl solution (20% w/v, 4 vol) and brine (6 vol) were washed twice. The resulting organic layer was concentrated to give a brown oil and used as such in the next reaction.

Synthesis of N-benzyloxyacetized-5-amino-2- (2-benzyloxy-1, 1-dimethylethyl) -6-fluoroindole.

Crude oil of 4-amino-2- (4-benzyloxy-3, 3-dimethylbut-1-ynyl) -5-fluoroaniline phenylacetated with benzyl glycolate was dissolved in acetonitrile (6 volumes) at room temperature and (MeCN) was added₂PdCl₂(15 mol%). The resulting mixture was used N₂The vent was degassed for about 30 minutes. The reaction mixture was then heated at 80 ℃ under N₂Stir overnight under cover. The reaction mixture was cooled to room temperature, filtered through a pad of celite and the filter cake was washed with acetonitrile (1 volume). The resulting filtrate was concentrated in vacuo and redissolved in EtOAc (5 vol). Deloxane-II THP (5% by weight based on the theoretical yield of N-benzyloxyacetized-5-amino-2- (2-benzyloxy-1, 1-dimethylethyl) -6-fluoroindole) was added and stirred at room temperature overnight. The mixture was then filtered through a pad of silica gel (2.5 inch depth, 6 inch diameter filter) and washed with EtOAc (4 vol). The filtrate was concentrated to a dark brown residue and used as such in the next reaction.

The crude N-benzylhydroxyacetoacetated-5-amino-2- (2-benzyloxy-1, 1-dimethylethyl) -6-fluoroindole was purified again:

crude N-benzyloxyacetized-5-amino-2- (2-benzyloxy-1, 1-dimethylethyl) -6-fluoroindole was dissolved in dichloromethane (-1.5 vol) and filtered through a pad of silica gel, initially using 30% EtOAc/heptane, with the impurities discarded. The silica gel pad was then washed with 50% EtOAc/heptane to isolate N-benzylhydroxyacetated-5-amino-2- (2-benzyloxy-1, 1-dimethylethyl) -6-fluoroindole until a pale color was observed in the filtrate. The filtrate was concentrated in vacuo to give a brown oil which crystallized on standing at room temperature.¹H NMR (400 MHz, DMSO) 7.38-7.34 (m, 4 H), 7.32-7.23 (m, 6H), 7.21 (d, 1 H, J = 12.8 Hz), 6.77 (d, 1H, J = 9.0 Hz), 6.06 (s, 1 H), 5.13(d, 1H, J = 4.9 Hz), 4.54 (s, 2 H), 4.46 (br. s, 2 H), 4.45 (s, 2 H), 4.33(d, 1 H, J = 12.4 Hz), 4.09-4.04 (m, 2 H), 3.63 (d, 1H, J = 9.2 Hz), 3.56 (d,1H, J = 9.2 Hz), 3.49 (dd, 1H, J = 9.8, 4.4 Hz), 3.43 (dd, 1H, J = 9.8, 5.7Hz), 1.40 (s, 6 H)。

Synthesis of Compound 1

Synthesis of benzyl protected Compound 1

1- (2, 2-difluoro-1, 3-benzodioxol-5-yl) -cyclopropanecarboxylic acid (1.3 equivalents) was slurried in toluene (2.5 volumes based on 1- (2, 2-difluoro-1, 3-benzodioxol-5-yl) -cyclopropanecarboxylic acid) and the mixture was heated to 60 ℃. Adding SOCl through an addition funnel₂(1.7 equiv.). The resulting mixture was stirred for 2 hours. Toluene and excess SOCl were evaporated using a rotary evaporator₂. Additional toluene (2.5 volumes based on 1- (2, 2-difluoro-1, 3-benzodioxol-5-yl) -cyclopropanecarboxylic acid) was added and distilled again. The crude acid chloride was dissolved in dichloromethane (2 volumes) and added via an addition funnel to a mixture of N-benzylhydroxyacetoxylate-5-amino-2- (2-benzyloxy-1, 1-dimethylethyl) -6-fluoroindole (1.0 eq) and triethylamine (2.0 eq) in dichloromethane (7 volumes) while maintaining 0-3 deg.C (internal temperature)Temperature). The resulting mixture was stirred at 0 ℃ for 4 hours, then allowed to warm to room temperature for reaction overnight. Distilled water (5 vol) was added to the reaction mixture, the NLT was stirred for 30 minutes and the layers were separated. Successively using 20 wt% of K₂CO₃The organic phase was washed (4 volumes x2) and brine (4 volumes) and concentrated to give crude benzyl protected compound 1 as a thick brown oil which was filtered using a pad of silica gel for further purification.

Filtering by a silica gel pad: crude benzyl-protected compound 1 was dissolved in ethyl acetate (3 vol) in the presence of activated carbon Darco-G (10wt%, based on the theoretical yield of benzyl-protected compound 1) and stirred at room temperature overnight. To the mixture was added heptane (3 volumes) and filtered through a pad of silica gel (2 x weight of crude benzyl-protected compound 1). The silica gel pad was washed with ethyl acetate/heptane (1:1, 6 volumes) or until no color was detected in the filtrate. The filtrate was concentrated in vacuo to afford benzyl protected compound 1 as a viscous brown-red oil, which was used directly in the next step.

And (3) secondary purification: the benzyl protected compound 1 was redissolved in dichloromethane (1 volume based on theoretical yield of benzyl protected compound 1) and loaded onto a silica gel pad (2 x weight of crude benzyl protected compound 1). The silica gel pad was washed with dichloromethane (2 volumes, based on the theoretical yield of benzyl protected compound 1) and the filtrate was discarded. The silica gel pad was washed with 30% ethyl acetate/heptane (5 vol) and the filtrate was concentrated in vacuo to give benzyl protected compound 1 as a viscous orange-red oil and used directly in the next step.

Synthesis of Compound 1

Method A

A20L autoclave was purged 3 times with nitrogen and then charged with palladium on charcoal (Evonik E101 NN/W, 5% Pd,60% wet, 200 g, 0.075 mol, 0.04 eq.). The autoclave was then flushed 3 times with nitrogen. A solution of crude benzyl protected compound 1 (1.3 kg,. about.1.9 mol) in THF (8L, 6 vol) was added to the autoclave via suction. The vessel was capped and then flushed 3 times with nitrogen. With gentle stirring, the vessel was purged 3 times with hydrogen and vented to the atmosphere via dilution with nitrogen. The autoclave was pressurized to 3 bar with hydrogen and the stirring rate was increased to 800 rpm. Rapid hydrogen uptake (dissolution) was observed. Once absorption subsided, the vessel was heated to 50 ℃.

The thermostat is turned off at the end of each working day for safety purposes. The vessel was pressurized with hydrogen to 4 bar and then separated from the hydrogen generator dedicated liquid tank.

After 2 days of reaction, additional Pd/C (60 g, 0.023 mol, 0.01 equiv.) was added to the mixture. This was achieved by flushing 3 times with nitrogen and then adding the catalyst through a solid feed port. The reaction was restarted as before. After 4 days, the reaction was deemed complete according to HPLC and the peaks corresponding not only to the starting material but also to the mono-benzylated intermediate disappeared.

The reaction mixture was filtered through a pad of celite. The vessel and filter cake were washed with THF (2L, 1.5 vol). The celite pad was then wetted with water and the filter cake was properly discarded. The combined filtrate and THF washings were concentrated using a rotary evaporator to give the crude product as a black oil, 1 kg.

The equivalents and volumes in the following purifications were based on 1kg of crude material. The crude black oil was dissolved in 1:1 ethyl acetate-heptane. The mixture was charged to a sinter funnel and a pad of silica gel (1.5 kg, 1.5wt. eq.) that had been saturated with 1:1 ethyl acetate-heptane. The silica gel pad was first rinsed with 1:1 ethyl acetate-heptane (6L, 6 vol) and then with pure ethyl acetate (14L, 14 vol). The eluent was collected in 4 fractions, which were analyzed by HPLC.

The equivalents and volumes in the following purifications were based on 0.6 kg of crude material. Fraction 3 was concentrated by rotary evaporation to give a brown foam (600 g) which was then redissolved in MTBE (1.8L, 3 vol). The dark brown solution was stirred at ambient temperature overnight during which crystallization occurred. Heptane (55 mL, 0.1 volume) was added and the mixture was stirred overnight. The mixture was filtered using a buchner funnel and the filter cake was washed with 3:1 MTBE-heptane (900 mL, 1.5 volumes). The filter cake was air dried for 1 hour and then dried under vacuum at ambient temperature for 16 hours to give 253 g of Vxc-661 as an off-white solid.

The equivalents and volumes used for the following purification were based on 1.4 kg of crude material. Fractions 2 and 3 from the above silica gel filtration and the material from the previous reaction were combined and concentrated to give 1.4 kg of black oil. The mixture was filtered again through silica gel as described above (1.5 kg of silica gel eluting with 3.5L, 2.3 volumes of 1:1 ethyl acetate-heptane and 9L, 6 volumes of pure ethyl acetate) and concentrated to give a brown foamy solid (390 g).

The equivalents and volumes used for the following purification were based on 390 g of crude material. The brown solid was insoluble in MTBE and therefore dissolved in methanol (1.2L, 3 vol). The mixture was distilled to 2 volumes using a 4L morton reactor equipped with a long range distillation head. MTBE (1.2L, 3 vol) was added and the mixture distilled back to 2 vol. Add 2 parts MTBE (1.6L, 4 vol) and distill the mixture back to 2 vol. Add 3 parts MTBE (1.2L, 3 vol) and distill the mixture back to 3 vol. GC analysis of the distillate showed it to contain 6% methanol. The thermostat was set to 48 ℃ (below the boiling temperature of the MTBE-methanol azeotrope, which was 52 ℃). The mixture was allowed to cool to 20 ℃ over 2 hours, during which time relatively rapid crystallization occurred. After stirring the mixture for 2 hours, heptane (20 mL, 0.05 volume) was added and the mixture was stirred overnight (16 hours). The mixture was filtered using a buchner funnel and the filter cake was washed with 3:1 MTBE-heptane (800 mL, 2 volumes). The filter cake was air dried for 1 hour and then dried under vacuum at ambient temperature for 16 hours to give 130 g of compound 1 as an off-white solid.

Method B

Benzyl protected compound 1 was dissolved and washed with THF (3 volumes) to remove any remaining residual solvent. Benzyl protected Compound 1 was redissolved in THF (4 vol) and added to a hydrogenation containing 5wt% Pd/C (2.5 mol%,60% wet, Degussa E5E 101 NN/W)In the device. The internal temperature of the reactants was adjusted to 50 ℃ and N was used successively₂(x5) and hydrogen (x3) flush. The hydrogenator pressure was adjusted to 3 bar hydrogen and stirred rapidly (>1100 rpm) of the mixture. At the end of the reaction, the catalyst was filtered through a pad of celite and washed with THF (1 vol). The filtrate was concentrated in vacuo to give a brown foam residue. The resulting residue was dissolved in MTBE (5 vol) and 0.5N HCl solution (2 vol) and distilled water (1 vol) were added. The mixture was stirred for NLT 30 minutes and the resulting layers were separated. Then using 10wt% of K₂CO₃The organic phase was washed with solution (2 volumes x2) and brine wash. The organic layer was added to a solution containing silica gel (25 wt%), Deloxan-THP II (5wt%,75% wet) and Na₂SO₄Was added to the flask and stirred overnight. The resulting mixture was filtered through a pad of celite and washed with 10% THF/MTBE (3 vol). The filtrate was concentrated in vacuo to give crude compound 1 as a pale brown foam.

Recovering compound 1 from the mother liquor: option a.

Filtering by a silica gel pad: the mother liquor was concentrated in vacuo to give a brown foam, which was dissolved in dichloromethane (2 volumes) and filtered through a pad of silica gel (3 x weight of crude compound 1). The pad was washed with ethyl acetate/heptane (1:1, 13 volumes) and the filtrate was discarded. The pad of silica gel was washed with 10% THF/ethyl acetate (10 volumes) and the filtrate was concentrated in vacuo to give compound 1 as a light brown foam. The remaining compound 1 was isolated according to the above crystallization procedure.

Recovering compound 1 from the mother liquor: and (4) selecting item B.

Silica gel column chromatography: after chromatography on silica gel (50% ethyl acetate/hexane-100% ethyl acetate), the desired compound was isolated as a light brown foam. The remaining compound 1 was isolated according to the above crystallization procedure.

FIG. 1 shows the X-ray powder diffraction pattern of Compound 1. The DSC trace for compound 1 is shown in figure 2. The DSC trace in figure 2 indicates that compound 1 is not a pure solid phase. There was an additional peak at 119 ℃ compared to compound 1 form a (see figure 6). The TGA trace of compound 1 is shown in figure 3.

Compound 1 may also be prepared by one of several synthetic routes disclosed in U.S. published patent application US20090131492, which is incorporated herein by reference.

Synthesis of Compound 1 form A

Pulping process

For EtOAc, MTBE, isopropyl acetate or DCM, about 40 mg of compound 1 was added to the vial along with 1-2 ml of any one or more solvents. The slurry was stirred at room temperature for 24 hours-2 weeks and compound 1 form a was collected (with filter) by centrifuging the suspension. Figure 5 discloses the XRPD pattern of compound 1 form a obtained by this method using DCM as solvent.

For EtOH/water solutions, approximately 40 mg of compound 1 was added to 3 separate vials. In the first vial 1.35 ml of EtOH and 0.15 ml of water were added. In a second vial 0.75 ml of EtOH and 0.75 ml of water were added. In a third vial 0.15 ml of EtOH and 1.35 ml of water were added. All 3 vials were stirred at room temperature for 24 hours. Each suspension was then centrifuged (with filter) to collect compound 1 form a.

For the isopropanol/water solution, approximately 40 mg of compound 1 was added to 3 separate vials. In the first vial 1.35 ml of isopropanol and 0.15 ml of water were added. In a second vial 0.75 ml of isopropanol and 0.75 ml of water were added. In a third vial 0.15 ml of isopropanol and 1.35 ml of water were added. All 3 vials were stirred at room temperature for 24 hours. Each suspension was then centrifuged (with filter) to collect compound 1 form a.

For methanol/water solutions, about 40 mg of compound 1 was added to the vial. 0.5 ml of methanol and 1 ml of water are added and the suspension is stirred at room temperature for 24 hours. The suspension was centrifuged (with filter) to collect compound 1 form a.

For acetonitrile, about 50 mg of compound 1 was added to a vial with 2.0 ml of acetonitrile. The suspension was stirred at room temperature for 24 hours and compound 1 form a was collected by centrifugation (with filter).

For acetonitrile/water solutions, approximately 50 mg of compound 1 was dissolved in 2.5 ml of acetonitrile and after sonication a clear solution was obtained. The solution was filtered and 1 ml was aspirated into the vial. 2.25 ml of water were added to give a turbid suspension. The suspension was stirred at room temperature for 24 hours and compound 1 form a was collected by centrifugation (with filter).

Slow evaporation method

Approximately 55 mg of Compound 1 was dissolved in 0.5 ml of acetone to give a clear solution after sonication. The solution was filtered and 0.2ml was aspirated into the vial. The vial is covered with a sealing membrane having a puncture therein and placed. The recrystallized compound 1 form a was collected by filtration.

Fast evaporation method

For isopropanol, approximately 43 mg of compound 1 was dissolved in 2.1 ml of isopropanol to give a clear solution after sonication. The solution was filtered into a vial and placed without a lid. The recrystallized compound 1 form a was collected by filtration.

For methanol, about 58 mg of compound 1 was dissolved in 0.5 ml of methanol to give a clear solution after sonication. The solution was filtered, 0.2ml was aspirated into a lidded vial and placed. The recrystallized compound 1 form a was collected by filtration.

For acetonitrile, about 51 mg of compound 1 was dissolved in 2.5 ml of acetonitrile to obtain a clear solution after sonication. The solution was filtered, half of the solution was aspirated into a cap-less vial and left to stand. The recrystallized compound 1 form a was collected by filtration. Figure 7 discloses an XRPD pattern of compound 1 form a prepared by this method.

Anti-solvent method

For EtOAc/heptane, approximately 30 mg of compound 1 was dissolved in 1.5 ml of EtOAc to give a clear solution after sonication. The solution was filtered and 2.0 ml heptane was added to the filtered solution while stirring slowly. The solution was stirred for an additional 10 minutes and left to stand. The recrystallized compound 1 form a was collected by filtration. Figure 8 discloses an XRPD pattern of compound 1 form a prepared by this method.

For isopropanol/water, approximately 21 mg of compound 1 was dissolved in 1.0 ml of isopropanol to give a clear solution after sonication. The solution was filtered to give 0.8 ml of solution. 1.8 ml of water was added while stirring slowly. An additional 0.2ml of water was added to give a cloudy suspension. Stirring was stopped for 5 minutes to give a clear solution. The solution was stirred for an additional 2 minutes and left to stand. The recrystallized compound 1 form a was collected by filtration.

For ethanol/water, approximately 40 mg of compound 1 was dissolved in 1.0 ml ethanol to give a clear solution after sonication. The solution was filtered and 1.0 ml of water was added. The solution was stirred at room temperature for 1 day. The recrystallized compound 1 form a was collected by filtration.

For acetone/water, approximately 55 mg of compound 1 was dissolved in 0.5 ml of acetone to give a clear solution after sonication. The solution was filtered and 0.2ml was aspirated into the vial. 1.5 ml of water was added followed by another 0.5 ml of water to give a cloudy suspension. The suspension was stirred at room temperature for 1 day. Compound 1 form a was collected by filtration.

Table 2 below summarizes various techniques for forming compound 1 form a.

Table 2.

Solvent	Recrystallization method	Results of residual solids
			ACN	Fast evaporation	Form A
Methanol	Fast evaporation	Form A
			Ethanol	N/A	N/A
IPA	Fast evaporation	Form A
			Acetone (II)	Slow evaporation	Form A
EtOAc	Slurry material	Form A
			DCM	Slurry material	Form A
MTBE	Slurry material	Form A
			Acetic acid isopropyl ester	Slurry material	Form A
Water/ethanol 1:9	N/A	N/A
			Water/ethanol 1:1	Slurry material	Form A
Water/ethanol 9:1	Slurry material	Form A
			Water/ACN 9:4	Slurry material	Form A
Water/methanol 2:1	Slurry material	Form A
			Water/IPA 1:9	N/A	N/A
Water/IPA 9:1	Slurry material	Form A
			Water/IPA 7:3	Slurry material	Form A
Methanol/water 4:3	Slurry material	Form A
			EtOAc/heptane 3:4	Anti-solvent	Form A
IPA/water 2:5	Anti-solvent	Form A
			Ethanol/water 1:1	Anti-solvent	Form A
Acetone/water 1:10	Anti-solvent	Form A
			Ethanol/water 5:6	Anti-solvent	N/A
Toluene	N/A	N/A
			MEK	N/A	N/A
Water (W)	N/A	N/A

The X-ray diffraction pattern calculated from the single crystal structure of compound 1 form a is shown in figure 4. Table 3 lists the peaks calculated for figure 1.

TABLE 3

The actual X-ray powder diffraction pattern of compound 1 form a is shown in figure 5. Table 4 lists the actual peaks of fig. 5.

TABLE 4

The DSC trace for compound 1 form a is shown in figure 6. The melting point of compound 1 form a occurs at about 172-.

Single crystal data were obtained for compound 1 form a, providing additional details about the crystal structure, including lattice size and packing.

Crystal preparation

Crystals of compound 1 form a were obtained by slow evaporation from a concentrated methanol solution (10 mg/ml). Colorless crystals of compound 1 form a having dimensions of 0.20 × 0.05 × 0.05 mm were selected, cleaned with mineral oil, mounted on MicroMount and collected on a Bruker apex iii diffractometer. Resulting in 3 batches of 40 frames separated in the inverted lattice space, providing the orientation matrix and the initial unit cell parameters. The final cell parameters were obtained and refined based on the complete data set.

Experiment of

Each frame was exposed for 30 seconds using a 0.5 ° step size for 0.83 a resolution, resulting in a diffraction data set of inverted lattice space. Data were collected at room temperature [295 (2) K ]. Integration of intensity (Integration of intensities) and refinement of unit cell parameters were achieved using APEXII software. Observation of the crystals after data collection showed no signs of decomposition.

TABLE 5 Crystal data for Compound 1 form A

C26H27F3N2O6	F(000) = 1088
		Mr = 520.50	Dx = 1.397 Mg m-3
Monoclinic, C2	Cu K α radiation, λ = 1.54178 Å
		Hall symbol C2 y	Unit cell parameters from 3945 reflection
a = 21.0952 (16) Å	θ = 2.5°
		b = 6.6287 (5) Å	μ = 0.97 mm-1
c = 17.7917 (15) Å	T = 295 K
		β = 95.867 (6)°	Prism
V = 2474.8 (3) Å 3	0.20 × 0.05 × 0.05 mm
		Z = 4

Geometry: all esds (except the esd of the dihedral angle between the two l.s. planes) were evaluated using the full covariance matrix. Evaluating distances, angles and twist angles esds separately considering cell esds; the correlation between esds of the unit cell parameters is only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of the unit cell esds was used to evaluate the esds of the l.s. plane.

TABLE 6 data collection parameters for Compound 1 form A crystals

APEX II diffractometer	Rint = 0.027
		Radiation source-fine coke sealing tube	θmax = 67.8°, θmin = 2.5°
Graphite (II)	h = -25→24
		8766 measured reflectance	k = -7→7
3945 independent reflection	l = -19→16
		Having a structure of>3510 reflection of 2 σ (I)

Data collection: apex II; crystal modification of a unit cell: apex II; and (3) data simplification: apex II; procedure for resolving the structure: SHELXS97 (Sheldrick, 1990); procedure for refining the structure: SHELXL97 (Sheldrick, 1997); molecular patterning: mercury (Mercury); software for making plate material: pubLCIF.

Table 7 refinement parameters for compound 1 form a crystals.

To F2Fine trimming of	Hydrogen site location inference from neighboring locations
		Least squares matrix of	H atoms treated with a mixture of independent and constrained refinements
R[F2 > 2σ(F2)] = 0.043	w = 1/[σ2(Fo 2) + (0.0821P)2+ 0.2233P]Wherein P = (F)o 2+ 2Fc 2)/3
		wR(F2) = 0.119	(Δ/σ)max < 0.001
S = 1.05	Δ>max = 0.14 e Å-3
		3945 reflection	Δ>min = -0.13 e Å-3
443 parameter	Extinction correction SHELXL, Fc*=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4
		1 constraint	Extinction coefficient 0.00016 (15)
0 constraint	Absolute Structure Flack H D (1983), Acta Crystal. A39, 876-
		Direct method for keeping position and structure of primary atomic site unchanged	Flack parameter 0.00 (18)
Second order atomic site position difference Fourier map

Fine modification: f on ALL reflection²And (7) fine modification. Weighted R-factor wR and goodness of fit S are based on F²Conventional R-factor R is based on F, F vs. negative F²Is set to zero. F²Threshold expression of>2σ(F²) Are used only for calculating R-factors (gt), etc., and are not relevant for the choice of refined reflections. Based on F²Is statistically about twice as large as F-based, and is even larger based on ALL data.

Conformational pictures of compound 1 form a based on single crystal X-ray analysis are shown in figures 9 and 10. The terminal-OH groups are connected via a hydrogen bonding network to form a tetrameric cluster (tetrameric cluster) with 4 adjacent molecules (fig. 10). The other hydroxyl groups act as hydrogen bond donors, forming hydrogen bonds with the carbonyl groups of adjacent molecules. The crystal structure shows a dense packing of molecules. Compound 1 form a is a monoclinic C2 space group with the following unit cell size: a = 21.0952(16) a, b = 6.6287(5) a, c = 17.7917(15) a, β = 95.867(6) °, γ =90 °.

Solid state of compound 1 form a¹³The C NMR spectrum is shown in FIG. 11. Table 8 provides the chemical shifts of the relevant peaks.

Table 8.

Solid state of compound 1 form a¹⁹The F NMR spectrum is shown in FIG. 12. Peaks with an asterisk mean the spinning sidebands. Table 9 provides the chemical shifts of the relevant peaks.

Table 9.

Synthesis of amorphous form of Compound 1

Rotary evaporation method

Amorphous form of compound 1 was also obtained via rotary evaporation. Compound 1 (ca. 10 g) was dissolved in 180 ml MeOH and rotary evaporated in a bath at 50 ℃ as a foam. DSC (fig. 14) and XRPD (fig. 13) confirmed the amorphous form of compound 1. Figure 15 discloses a TGA trace of the amorphous form of compound 1 prepared by this method.

Spray drying method

9.95 g hydroxypropyl methylcellulose acetate succinate HG grade (HPMCAS-HG) was weighed into a 500 ml beaker together with 50 mg Sodium Lauryl Sulfate (SLS). MeOH (200 ml) was mixed with the solid. The material was stirred for 4 hours. To ensure maximum dissolution, the solution was sonicated for 5 minutes after 2 hours of stirring, then stirring was continued for the remaining 2 hours. The final (fin) HPMCAS suspension remains in solution. However, visual observation after tilting the container confirmed that no tacky part remained on the container wall or stuck to the bottom.

Compound 1 form A (10 g) was poured into a 500 ml beaker and the system was stirred on. The solution was spray dried using the following parameters:

description of the formulation: compound 1 form A/HPMCAS/SLS (50/49.5/0.5)

BUCHI small-sized spray drying instrument

T entrance (anchor point) 145 DEG C

T outlet (origin) 75 deg.C

T outlet (end point) 55 DEG C

Nitrogen pressure 75 psi

100% of aspirator

35 percent of pump

40 mm rotary flowmeter

Filter pressure 65 mbar

Condenser temperature-3 deg.C

Run time 1 hour

About 16 g of amorphous form of compound 1 was recovered (80% yield). The amorphous form of compound 1 was confirmed by XRPD (fig. 16) and DSC (fig. 17).

Amorphous form of Compound 1 in solid form¹³The C NMR spectrum is shown in FIG. 8. Table 10 provides the chemical shifts of the relevant peaks.

Table 10.

Amorphous form of Compound 1 in solid form¹⁹The F NMR spectrum is shown in FIG. 19. Peaks with an asterisk mean the spinning sidebands. Table 11 provides the chemical shifts of the relevant peaks.

Table 11.

Table 12 below lists additional analytical data for compound 1.

Table 12.

Test of

Use of detection and measurement of Δ F508-CFTR correction properties of compounds

Membrane potential optical methods for testing compounds for Δ F508-CFTR modulating properties

Optical Membrane potential assays used Gonzalez and Tsien (see Gonzalez, J.E. and R.Y. Tsien (1995) "Voltage sensing by fluorescence sensitivity energy transfer in cells"Biophys J69(4): 1272-80, and Gonzalez, J.E. and R.Y. Tsien (1997) "Improved indicator of cell membrane potential using fluorescence resonance energy transfer (Improved indicators of cell membrane potential using fluorescence resonance energy transfer)"Chem Biol4(4): 269-77) in combination with an instrument used to measure fluorescence changes, such as a Voltage/Ion Probe Reader (VIPR) (see Gonzalez, j.e., k. objects, et al. (1999) "Cell-based assays and instruments (Cell-based assays and instruments) for screening Ion-channel targets"Drug Discov Today4(9): 431-439)。

These voltage sensitive tests are based on the membrane soluble voltage sensitive dye DiSBAC₂(3) And change in fluorescence resonance energy transfer between the fluorescent phospholipid CC2-DMPE attached to the plasma membrane exit leaflet and acting as a FRET donor. Membrane potential (V)_m) Is caused to negatively charge the DiSBAC₂(3) The amount of redistribution across the plasma membrane and energy transfer from CC2-DMPE varied accordingly. Change in fluorescence emission Using VIPR^TMII monitoring, the latter is an integrated liquid processor and fluorescence detector, is designed in 96-or 384-hole microtiter plate for cell-based screening.

1. Identification of calibration Compounds

To identify small molecules that correct for the transport defects associated with Δ F508-CFTR, a separate addition HTS detection format was developed. Cells were incubated in serum-free medium at 37 ℃ for 16 hours in the presence or absence of test compound (negative control). As a positive control, cells spread in 384-well plates were incubated at 27 ℃ for 16 hours to "temperature correct" Δ F508-CFTR. The cells were then washed 3X with Krebs Ringer solution and loaded with voltage sensitive dye. To activate Δ F508-CFTR, wells were incubated with Cl-free^-The culture medium was supplemented with 10. mu.M forskolin and CFTR potentiator, genistein (20. mu.M). Adding Cl-free solution^-The medium of (a) promotes Cl in response to activation of Δ F508-CFTR^-Flow out and the resulting membrane depolarization optically monitored using FRET-based voltage sensor dyes.

2. Validation of synergist Compounds

To identify potentiators of Δ F508-CFTR, a dual-addition HTS assay format was developed. During the first addition, Cl-free solution was added to each well^-With or without the test compound. After 22 seconds, a second addition of Cl-free containing 2-10. mu.M forskolin^-To activate Δ F508-CFTR. Extracellular Cl after two additions^-Concentration 28 mM, which promotes Cl in response to activation of Δ F508-CFTR^-The mixture flows out of the reactor and flows out,and the resulting membrane depolarization optically monitored using FRET-based voltage sensor dyes.

3. Solutions of

Bath #1 (in mM)	NaCl 160、KCl 4.5、CaCl2 2、MgCl21. HEPES 10, pH 7.4 containing NaOH.
		Chlorine-free baths:	the chloride salt in bath #1 was replaced with gluconate.
CC2-DMPE:	Prepared as 10 mM stock solution in DMSO and stored at-20 ℃.
		DiSBAC2(3):	Prepared as 10 mM stock solution in DMSO and stored at-20 ℃.

4. Cell culture

NIH3T3 mouse fibroblasts stably expressing AF 508-CFTR were used for optical measurement of membrane potential. At 175cm²In a culture flask, at 5% CO₂And 90% humidity, at 37 degrees C in 2 mM glutamine, 10% calf serum, 1X NEAA, β -ME, 1X penicillin/streptomycin and 25 mM HEPES supplemented Dulbecco's modified Eagle's medium for all optical determination, the cells 30000/hole in 384 holes coated with substrate glue plate, and at 27 degrees C for 24 hours for synergist identification before cultured at 37 degrees C for 2 hours.For calibration assays, cells were cultured for 16-24 hours at 27 ℃ or 37 ℃ in the presence and absence of the compound.

Electrophysiological experiment for testing the Δ F508-CFTR modulating properties of a compound

1. Ussing Chamber (Using Chamber) experiment

Eustachian (Ussing Chamber) experiments were performed on polarized epithelial cells expressing AF 508-CFTR to further characterize modulators of AF 508-CFTR identified in the optical experiments. FRT grown on Costar Snapwell cell culture ponds (cellculture inserts)^ΔF508-CFTREpithelial cells were mounted in Ussing chamber (Physiologic Instruments, San Diego, CA) and monolayers were short-circuited continuously using a voltage clamp system (bioengineering, university of iowa, IA and Physiologic Instruments, San Diego, CA). Transepithelial membrane resistance was measured by applying a 2-mV pulse. Under these conditions, FRT epithelial cells demonstrated 4K Ω/cm²Or more resistance. The solution was maintained at 27 ℃ and bubbled with air. Electrode potential shifts and fluid resistance were corrected using a cell-free cell. Under these conditions, the current reflects Cl^-Flow through Δ F508-CFTR expressed in apical membrane. I was obtained using the MP100A-CE interface and AcqKnowledge software (v3.2.6; BIOPAC Systems, Santa Barbara, Calif.) numbers_SC。

2. Confirmation of calibration Compounds

Typical protocols use a substrate outside-to-tip membrane Cl^-A concentration gradient. To establish this gradient, normal ringer's solution was applied to the basolateral membrane, while apical NaCl was replaced with equimolar amounts of sodium gluconate (titrated to pH 7.4 with NaOH) to give large Cl across epithelial cells^-A concentration gradient. All experiments were performed with a complete monolayer. To fully activate Δ F508-CFTR, forskolin (10 μ M) and a PDE inhibitor, IBMX (100 μ M) were applied followed by addition of CFTR potentiator genistein (50 μ M).

Incubation of the stabilization Table at Low temperature, as observed for other cell typesFRT cells that reach Δ F508-CFTR increase the functional density of CFTR in the plasma membrane. To determine the activity of the calibration compound, cells were incubated with 10 μ M test compound for 24 hours at 37 ℃ and then washed 3X before recording. Treatment of cAMP-and Genistein-mediated I in Compound-treated cells_SCNormalized to the 27 ℃ and 37 ℃ controls and expressed as percent activity. Pre-incubation of cells with correction compounds significantly increased cAMP-and genistein-mediated I compared to 37 ℃ controls_SC。

3. Identification of potentiator compounds

Typical protocols use a substrate outside-to-tip membrane Cl^-A concentration gradient. To establish this gradient, normal ringer's solution was applied to the basolateral membrane and permeabilized with nystatin (360 μ g/ml), while apical NaCl was replaced with equimolar amounts of sodium gluconate (titrated to pH 7.4 with NaOH) to give large Cl across epithelial cells^-A concentration gradient. All experiments were performed 30 minutes after nystatin infiltration. Forskolin (10 μ M) and all test compounds were added to both sides of the cell culture pond. The potency of the putative AF 508-CFTR potentiator was compared to that of the known potentiator genistein.

4. Solutions of

Substrate-outside solution (in mM):	NaCl (135)、CaCl2(1.2)、MgCl2 (1.2)、K2HPO4 (2.4)、KHPO4(0.6), N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid (HEPES) (10), and glucose (10). The solution was titrated with NaOH to pH 7.4.
		Apical solution (in mM):	with the solution outside the substrateSimilarly, NaCl was replaced with sodium gluconate (135).

5. Cell culture

Expression of Δ F508-CFTR (FRT)^ΔF508-CFTR) Fisher rat epithelial (FRT) cells of (A) were used in the Uussing chamber experiment for putative Δ F508-CFTR modulators identified from our optical experiments. Cells were cultured in a Costar Snapwell cell culture cell at 37 ℃ and 5% CO₂Next, the cells were cultured in 5% calf serum, 100U/ml penicillin and 100 μ g/ml streptomycin supplemented Coon's modified Ham's F-12 medium for 5 days. Cells were incubated at 27 ℃ for 16-48 hours to correct for Δ F508-CFTR before use to characterize the potentiator activity of the compound. To determine the activity of the calibration compound, cells were incubated for 24 hours at 27 ℃ or 37 ℃ in the presence or absence of the compound.

6. Whole cell recording

Macroscopic AF 508-CFTR Current (I) in NIH3T3 cells stably expressing AF 508-CFTR, corrected for temperature and test Compound, was monitored using a perforated membrane, whole cell recordings_ΔF508). Briefly, I was performed at room temperature using an Axomatch 200B patch clamp amplifier (Axon Instruments Inc., Foster City, Calif.)_ΔF508Voltage clamp record of (1). All recordings were obtained at a sampling frequency of 10 kHz and low pass filtering at 1 kHz. The pipette has a resistance of 5-6 M.OMEGA.when filled with intracellular solution. Calculated Cl at room temperature under these recording conditions^-Reversal potential of (E)_Cl) Was-28 mV. All records have>A sealing resistance of 20 G.OMEGA.<A 15M omega series resistance. Pulse generation, data acquisition and analysis were performed using a PC equipped with a Digidata 1320A/D interface along with Clampex 8 (Axon Instruments). The bath contains<250 μ l of saline was continuously perfused at a rate of 2 ml/min using a gravity driven perfusion system.

7. Confirmation of calibration Compounds

To determine the activity of the calibration compound for increasing the density of functional af 508-CFTR in the plasma membrane, we measured the current density 24-hr after treatment with the calibration compound using the perforated patch recording technique described above. To fully activate the Δ F508-CFTR, 10 μ M forskolin and 20 μ M genistein were added to the cells. Under our recording conditions, the current density after 24 hours of incubation at 27 ℃ was higher than that observed after 24 hours of incubation at 37 ℃. These results are consistent with the known effects of low temperature incubation on the density of Δ F508-CFTR in plasma membranes. To determine the effect of the correction compound on the CFTR current density, cells were incubated with 10 μ M of test compound for 24 hours at 37 ℃ and the current density was compared to the 27 ℃ and 37 ℃ control groups (% activity). Prior to recording, cells were washed 3X with extracellular recording medium to remove any residual test compound. Pre-incubation with 10 μ M of a correction compound significantly increased cAMP-and genistein-dependent currents compared to the 37 ℃ control group.

8. Identification of potentiator compounds

The use of a perforated patch recording technique to also study the enhancement of the Δ F508-CFTR potentiator in macroscopic Δ F508-CFTR Cl in NIH3T3 cells stably expressing Δ F508-CFTR^-Stream (I)_ΔF508) The ability of the cell to perform. The potentiators identified from the optical assay induce I with similar potency and effectiveness as observed in the optical assay_ΔF508Is increased in a dose-dependent manner. In all cells examined, the reversal potential before and during the use of the potentiator was about-30 mV, which is the calculated E_Cl(-28 mV)。

9. Solutions of

Intracellular solution (in mM):	cs-aspartic acid (90), CsCl (50), MgCl2(1) HEPES (10) and 240 μ g/ml amphotericinB (pH adjusted to 7.35 with CsOH).
		Extracellular solution (in mM):	N-methyl-D-glucamine (NMDG) -Cl (150), MgCl2(2) CaCl2 (2), HEPES (10) (pH adjusted to 7.35 with HCl).

10. Cell culture

NIH3T3 mouse fibroblasts stably expressing AF 508-CFTR were used for whole cell recordings. At 175cm²In a culture flask, at 5% CO₂And 90% humidity, at 37 degrees C maintained cells with 2 mM glutamine, 10% calf serum, 1 XNEAA, β -ME, 1X penicillin/streptomycin and 25 mM HEPES supplemented Dulbecco's modified Eagle's medium for recording the whole cells, 2500 + 5000 cells inoculated to poly-L-lysine coated glass cover glass and in the 27 ℃ before the use of 24-48 hours to test the synergist activity, and at 37 degrees C in the presence or absence of correction compounds for measuring the activity of correction agent.

11. Single channel recording

The single-channel activity of stably expressed temperature-corrected Δ F508-CFTR and the activity of the potentiator compound in NIH3T3 cells were observed using excised inside-out patches. Briefly, voltage-clamp recordings of single-channel activity were performed at room temperature using an Axopatch 200B patch-clamp amplifier (Axon Instruments, Inc.). All recordings were obtained at a sampling frequency of 10 kHz and low pass filtering at 400 Hz. When filled with extracellular solution, Patch clamps (Patch piptants) were made from Corning Kovar Sealing #7052 glass (World Precision Instruments, Sarasota, FL) and had a resistance of 5-6M Ω. Δ F508-CFTR was activated after excision by addition of 1 mM Mg-ATP and 75 nM cAMP-dependent protein kinase, catalytic subunit (PKA, Promega Inc. Madison, Wis.). After the channel activity stabilized, the membrane was perfused using a gravity-driven micro-perfusion system. The inflow is placed adjacent to the membrane sheet,resulting in complete exchange of the solution within 1-2 seconds. To maintain Δ F508-CFTR activity during rapid perfusion, non-specific phosphatase inhibitor F was added^-(10 mM NaF) was added to the bath. Under these recording conditions, the channel activity remained constant (up to 60 minutes) throughout the patch recording. The current generated by the positive charge moving from the intracellular solution to the extracellular solution (the anions moving in the opposite direction) is shown as a positive current. The clamping voltage (Vp) was maintained at 80 mV.

Channel activity was analyzed from patches 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 seconds of Δ F508-CFTR activity was "off-line" filtered at 100 Hz and then used to construct all point amplitude histograms fitted with a multiple gaussian function using Bio-batch Analysis software (Bio-logic comp. france). The total microscopic current and open probability (Po) were determined from 120 seconds of channel activity. Po was determined using Bio-Patch software or self-correlation Po = I/I (N), where I = mean current, I = single channel current amplitude, and N = number of active channels in the Patch.

12. Solutions of

Extracellular solution (in mM):	NMDG (150), aspartic acid (150), CaCl2 (5)、MgCl2(2) And HEPES (10) (pH adjusted to 7.35 with Tris base).
		Intracellular solution (in mM):	NMDG-Cl (150)、MgCl2(2) EGTA (5), TES (10) and Tris base (14) (pH adjusted to 7.35 with HCl).

13. Cell culture

NIH3T3 mouse fibroblasts stably expressing AF 508-CFTR were used for ionomeric membrane patch clamp recordings. At 175cm²In a culture flask, at 5% CO₂And 90% humidity, at 37 degrees C maintained cells with 2 mM glutamine, 10% calf serum, 1X NEAA, β -ME, 1X penicillin/streptomycin and 25 mM HEPES supplemented Dulbecco's modified Eagle's medium for single channel recording, 2500 + 5000 cells inoculated to poly-L-lysine coated glass cover glass and in the 27 ℃ culture for 24-48 hours.

Using the procedure described above, the activity of compound 1, EC50s, was measured and is shown in table 13.

Table 13.

。

Claims (49)

1. Solid amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide, wherein the amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide Is a spray-dried dispersion.

2. A pharmaceutical composition comprising amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide of claim 1 and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition of claim 2, further comprising an additional therapeutic agent.

4. The pharmaceutical composition of claim 3, wherein the additional therapeutic agent is selected from the group consisting of mucolytic agents, bronchodilators, antibiotics, anti-infective agents, anti-inflammatory agents, CFTR potentiators, or nutritional agents.

5. A process for preparing amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide according to claim 1, wherein the amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) -yl) cyclopropanecarboxamide is a spray dried dispersion comprising dissolving (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide in a suitable solvent and removing the solvent by rotary evaporation.

6. The process of claim 5, wherein the solvent is methanol.

7. A spray-dried solid dispersion comprising amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide of claim 1 and a polymer.

8. The spray dried solid dispersion of claim 7, wherein the polymer is hydroxypropyl methylcellulose.

9. The spray dried solid dispersion of claim 7, wherein the polymer is hydroxypropyl methylcellulose acetate succinate.

10. The spray dried solid dispersion of any of claims 7-9, wherein the polymer is present in an amount of 10wt% to 80 wt%.

11. The spray dried solid dispersion of any of claims 7-9, wherein the polymer is present in an amount of 30 wt% to 60 wt%.

12. The spray dried solid dispersion of any of claims 7-9, wherein the polymer is present in an amount of 49.5% by weight.

13. The spray dried solid dispersion of any of claims 7-9, wherein (R) -1- (2, 2-difluorobenzo [ d ] [1,3] meta-dioxacyclopenten-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide is present in an amount of 10% by weight to 80% by weight.

14. The spray dried solid dispersion of any of claims 7-9, wherein (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide is present in an amount of 30% by weight to 60% by weight.

15. The spray dried solid dispersion of any of claims 7-9, wherein (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide is present in an amount of 50% by weight.

16. The spray dried solid dispersion of any of claims 7-9, further comprising a surfactant.

17. The spray dried solid dispersion of claim 16, wherein the surfactant is sodium lauryl sulfate.

18. The spray dried solid dispersion of claim 16, wherein the surfactant is present in an amount of 0.1 wt% to 5 wt%.

19. The spray dried solid dispersion of claim 18, wherein said surfactant is present in an amount of 0.5% by weight.

20. The spray dried solid dispersion of claim 16, wherein the polymer is hydroxypropyl methylcellulose acetate succinate present in an amount of 49.5% by weight, the surfactant is sodium dodecyl sulfate present in an amount of 0.5% by weight, and (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide is present in an amount of 50% by weight.

21. A pharmaceutical composition comprising the spray-dried solid dispersion of any one of claims 7-20 and a pharmaceutically acceptable carrier.

22. The pharmaceutical composition of claim 21, further comprising an additional therapeutic agent.

23. The pharmaceutical composition of claim 22, wherein the additional therapeutic agent is selected from a mucolytic agent, a bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR potentiator, or a nutritional agent.

24. A process for preparing amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide comprising reacting (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide Formamide spray drying.

25. The process as claimed in claim 24, which comprises mixing (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide with a suitable solvent and spray-drying the mixture to give amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2- Yl) -1H-indol-5-yl) cyclopropanecarboxamide.

26. The method of claim 25, wherein the solvent is an alcohol.

27. The process of claim 26, wherein the solvent is methanol.

28. The method of any one of claims 24 to 27, comprising

a) Forming a mixture comprising (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide, a polymer, and a solvent; and

b) spray drying the mixture to form a solid dispersion.

29. The method of claim 28, wherein the polymer is hydroxypropyl methylcellulose acetate succinate.

30. The method of claim 28, wherein the polymer is present in an amount of 10% by weight to 80% by weight of the solid dispersion.

31. The method of claim 28, wherein the polymer is present in an amount of 49.5% by weight of the solid dispersion.

32. The process of claim 28, wherein the solvent is methanol.

33. The method of claim 28, wherein the mixture further comprises a surfactant.

34. The method of claim 33 wherein the surfactant is sodium lauryl sulfate.

35. The method of claim 33, wherein the surfactant is present in an amount of 0.1 wt% to 5wt% of the solid dispersion.

36. The method of claim 35, wherein the surfactant is present in an amount of 0.5% by weight of the solid dispersion.

37. The method of claim 28, wherein the polymer is hydroxypropyl methyl cellulose acetate succinate present in an amount of 49.5% by weight of the solid dispersion, the solvent is methanol, and the mixture further comprises sodium lauryl sulfate present in an amount of 0.5% by weight of the solid dispersion.

38. Use of the spray-dried amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide according to claim 1, or the spray-dried solid dispersion according to claim 7, for the preparation of a medicament for the treatment of CFTR-mediated diseases selected from cystic fibrosis, asthma, chronic bronchitis, sinusitis, constipation, pancreatitis, pancreatic insufficiency, male infertility due to congenital bilateral absence of vas deferens, mild pulmonary disease, pulmonary, Idiopathic pancreatitis, allergic bronchopulmonary aspergillosis, liver disease, hereditary emphysema, hereditary hemochromatosis, a deficiency in thrombofibrinolysis, a protein C deficiency, hereditary angioedema type 1, a deficiency in lipid processing, familial hypercholesterolemia, chylomicronemia type 1, abetalipoproteinemia, lysosomal storage diseases, I-cell diseases, mucopolysaccharidosis, Sandhof disease, Meng dementia, Crigler-Najal type II syndrome, polyendocrinosis, diabetes, Ralunghur dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, glycolytic CDG type 1, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency, uremia, Charcot-Marie syndrome, Pape-Meissler's disease, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, pick's disease, polyglutamine neurological disorder, Huntington's disease, spinocerebellar ataxia type I, spinobulbar muscular atrophy, dentatorubral pallidoluysian atrophy, myotonic dystrophy, spongiform encephalopathy, hereditary Creutzfeldt-Jakob disease, Fabry's disease, Stusler-Scheck syndrome, COPD, xerosis, Sjogren's disease, osteoporosis, osteopenia, Gohan's syndrome, chloride channel disease, myotonia congenita, Bart's syndrome type III, Dent's disease, epilepsy, panic disorder, lysosomal storage diseases, Angelman syndrome, primary ciliary dyskinesia, hereditary diseases of ciliary structure and/or function, catagmatic syndrome, or ciliary hypoplasia.

39. The use of claim 38, wherein the CFTR mediated disease is selected from cystic fibrosis, COPD, osteoporosis or emphysema.

40. The use of claim 38, wherein the CFTR mediated disease is cystic fibrosis.

41. The use according to claim 38, wherein said diabetes insipidus is neurological diabetes insipidus or renal diabetes insipidus.

42. The use of claim 38, wherein the primary ciliary dyskinesia is a primary ciliary dyskinesia with visceral inversion or a primary ciliary dyskinesia without visceral inversion.

43. The use according to claim 38, wherein the COPD is a cigarette smoking induced COPD.

44. The use of claim 38, wherein the myotonia congenita is tomukong disease or becker myotonia.

45. The use of claim 38, wherein the I-cell disorder is pseudoheller disease.

46. The use of claim 38, wherein the polyendocrinopathy is hyperinsulinemia.

47. The use of any one of claims 38-46, wherein the medicament comprises an additional therapeutic agent.

48. The use of claim 47, wherein the therapeutic agent is selected from a mucolytic agent, a bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR potentiator, or a nutritional agent.

49. A kit comprising the spray dried amorphous (R) -1- (2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) -N- (1- (2, 3-dihydroxypropyl) -6-fluoro-2- (1-hydroxy-2-methylpropan-2-yl) -1H-indol-5-yl) cyclopropanecarboxamide of claim 1, or the solid dispersion of claim 7, and instructions for use thereof.