Solid forms of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid

TECHNICAL FIELD OF THE INVENTION [001] The present invention relates to solid state forms, for example, cry stalline forms, of 3-(6-( 1 -(2,2-difluorobenzo[d][l,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin2-yl)benzoic acid, pharmaceutical compositions thereof, processes of manufacturing said forms and a use thereof.

[002] The invention also relates to combinations of 3-(6-( 1 -(2,2difluorobenzo[d][l,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methy!pyridin-2-yl)benzoic acid and N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1 H-quinoline-3-carboxamide, wherein the 3-(6-( 1 -(2,2-difluorobenzo[d][ 1,3]dioxol-5-yl)cyclopropanecarboxamido)-3methylpyridin-2-yl)benzoic acid is in a solid form. The present invention further relates to a pharmaceutical composition thereof and a use thereof.

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I

81796851 [003] CFTR is a cAMP/ATP-mediaied anion channel that is expressed in a variety of · cells types, including absorptive and secretory epithelia cells, where it regulates anion flux across the membrane, as well as the activity of other ion channels and proteins. In epithelia cells, normal functioning of CFTR is critical for the maintenance of electrolyte transport throughout the body, including respiratory and digestive tissue. CFTR is composed of approximately 1480 amino acids that encode a protein made up of a tandem repeat of transmembrane domains, each containing six transmembrane helices and a nucleotide .binding domain. The two transmembrane domains are linked by a large, polar, regulatory (R)-doniain with multiple phosphorylation sites that regulate channel activity and cellular trafficking.

[004] The gene encoding CFTR has been identified and sequenced (See Gregory, R. J. cl al. (1990) Nature 347ΰ 82-386; Rich, D. P. ct rd, (1990) Nature 347:358-362), (Riordan, J. R. et al. (1989) Science 245:1066-1073); A defect in this getre causes mutations in CFTR resulting in cystic fibrosis (CF), the most common fatal genetic disease in humans. Cystic fibrosis affects approximately one in every 2,500 infants in the United States. Within the general United States population, up to 10 million people cany a single copy of the defective gene without apparent ill effects. In contrast, individuals with two copies of the CF associated gene suffer from the debilitating and fatal effects of CF, including chronic lung disease.

[005] In patients with cystic fibrosis, mutations in CFTR endogenously expressed in respiratory epithelia leads to reduced apical anion secretion causing an imbalance in ion and fluid transport. The resulting decrease in anion transport contributes to enhanced mucus accumulation

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PCT/IIS2008/085456 in the lung and the accompanying microbial infections that ultimately cause death in CF patients. In addition to respiratory disease, CF patients typically suffer from gastrointestinal problems and pancreatic insufficiency that, if left untreated, results in death. In addition, the majority of males with cystic fibrosis are infertile and fertility is decreased among females with cystic fibrosis. In contrast to the severe effects of two copies of the CF associated gene, individuals with a single copy of the CF associated gene exhibit increased resistance to cholera and to dehydration resulting from diarrhea - perhaps explaining the relatively high frequency of the CF gene within the population.

[006] Sequence analysis of the CFI'R gene of CF chromosomes has revealed a variety of disease causing mutations (Cutting, G. R. et al. (1990) Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem, B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc. Natl. Acad. Sci. USA 87:8447-8451). To date, > 1000 disease causing mutations in the CF gene have been identified (http://www.genetsickkids.on.ca,^,cftr/)· The most prevalent mutation is a deletion of phenylalanine at position 508 of the CFTR amino acid sequence, and is commonly referred to as AF508-CFTR. This mutation occurs in approximately 70% of the cases of cystic fibrosis and is associated with a severe disease.

[007] The deletion of residue 508 in AF508-CFTR prevents the nascent protein from folding correctly. This results in the inability of the mutant protein to exit the ER, and traffic to the plasma membrane. As a result, the number of channels present in the membrane is far less than observed in cells expressing wild-type CFI'R. In addition to impaired trafficking, the mutation results in defective channel gating. Together, the reduced number of channels in the membrane and the defective gating lead to reduced anion transport across epithelia leading to defective ion and fluid transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studies have shown, however, that the reduced numbers of AF508-CFTR in the membrane are functional, albeit less than wild-type CFTR. (Dalemans et al. (1991), Nature Lond. 354:526-528; Denning et al„ supra; Pasyk and Foskett (1995), J. Cell. Biochem. 270: 12347-50). In addition to AF508CFTR, other disease causing mutations in CFTR that result in defective trafficking, synthesis, and/or channel gating could be up- or down-regulated to alter anion secretion and modify disease progression and/or severity.

[008) Although CFTR transports a variety of molecules in addition to anions, it is clear that this role (the transport of anions) represents one element in an important mechanism of transporting ions and water across the epithelium. The other elements include the epithelial Na* channel, ENaC, Na*/2CT/K⁺ co-transporter, Na’-K’-ATPase pump and the basolaleral membrane K⁺ channels, that are responsible for the uptake of chloride into the cell.

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81796851 (009] These elements work together to achieve directional transport across the epithelium via their selective expression and localization within the cell. Chloride absorption takes place by the coordinated activity of ENaC and CE1K present on the apical membrane and the Na⁺-K⁺-ATPase pump and-Cl- channels expressed on the basolateral surface of the celL Secondary active tiansport of chloride from the luminal side leads to the accumulation of intracellular chloride, which can then passively leave the cell via Cl channels, resulting in a vectorial transport. Arrangement of Na⁺/2C17K⁺ co-transporter, Na⁺-K⁺-ATPase pump and the basolateral membrane K⁺ channels on the basolateral surface and CFTR on the luminal side coordinate the secretion of chloride via CFTR on the luminal side. Because water is probably never actively transported itseffi its flow across epithelia depends on tiny transepKhelial osmotic gradients generated by the bulk flow of sodium and chloride.

(0010] As discussed above, it is believed that the deletion of residue 508 in AF508-CFTR prevents the nascent protein from folding conectly, resulting in the inability of this mutant protein to exit the ER, and traffic to the plasma membrane. As a resuffi insufficient amounts of the mature protein are present at the plasma membrane and chloride transport within epithelial tissues is significantly reduced. Intact, this cellular phenomenon of defective ER processing of ABC transporters by the ER machinery, has been shown to be the underlying basis not only for CF disease, but for a wide range of other isolated and inherited diseases. The two ways that the ER machinery can malfunction is either by loss of coupling to ER export of the proteins leading to degradation, or by the ER accumulation of these defective/misfolded proteins [Aridor M, et al., Nature Med., 5(7), pp 745- 751 (1999); Shastiy, B.S., et al., Neurochem. International, 43. pp 1-7 (2003); Rulishauser, J., ei al., Swiss Med Wkly, 132. pp 211-222 (2002); Morello, JP el al., TTPS, 2L PP. 466- 469 (2000); Brass P., et al., Human Mut., 14, pp. 186-198 (1999)].

(0011 ] 3-(6-( 1 -(2,2-Difluorobenzo[d][l 3 ]dioxol-5-y 1) cyclopropanecarboxamido)-3methylpyridin-2-yl)benzoic acid in salt form is disclosed in International PCT Publication WO 2007056341 as a modulator of CFTR activity and thus useful in treating CFTR-mediated diseases such as cystic fibrosis. However, there is a need for stable solid forms of said compound that can be used readily in pharmaceutical compositions suitable for use as therapeutics.

SUMMARY OF THE INVENTION (0012] The present invention relates to solid forms of 3-(6-( 1-(2,2difluorobenzo[d][l ,3]dioxol-5-yI) cyclopropanecarboxamido)-3-methylpyridin-l-yl)benzoic acid (hereinafter “Compound 1”) which has the structure below:

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81796851

<img file="CA2986286C_D0001.tif" />

[0013] In one aspect, Compound 1 is in a substantially crystalline and salt free form referred to as Form I as described and characterized herein. Processes described herein can be 5 used to prepare lhe compositions of this invention comprising Form I. The amounts and the features of the components used in the processes would be as described herein.

[0014] In another aspect, there is provided a combination comprising: 3-(6-(1-(2,2difluorobenzo[d][l,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid; and N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-lH-quinolinc-3-carboxamide, wherein the 3-(6-( 1 -(2,2-difluorobenzo[d][ 1,3]dioxol-5-yl)cyclopropanecarboxamido)-3methylpyridin-2-yl)benzoic acid is in a solid form.

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81796851

BRIEF DESCRIPTION OF THE DRAWINGS (0015] Figure 1 is an X-ray diffraction pattern calculated from a single crystal structure of Compound 1 in Fonn I.

[0016} Figure 2 is an actual X-ray powder diffraction pattern, of Compound 1 in Form L [0017] Figure 3 is an overlay of an X-ray diffraction pattern calculated from a single crystal of Compound 1 in Form I, and an actual X-ray powder diffraction pattern of Compound 1 in Form L [0018] Figure 4 is a differential scanning calorimetry (DSC) trace of Compound 1 in Form I.

[0019] Figure 5 is a conformational picture of Compound I in Form I based on single crystal X-ray analysis.

[0020] Figure 6 is a conformational picture of Compound 1 in Form I based on single crystal X-ray analysis as a dimer formed through the carboxylic acid groups.

[0021] Figure 7 is a conformational picture of Compound 1 in Form I based on single crystal X-ray analysis showing that the molecules are stacked upon each other.

[0022] Figure 8 is conformational picture of Compound 1 in Form I based on single crystal X-ray analysis showing a different view (down a).

[0023] Figure 9 is an¹HNMR. analysis of Compound 1 in Form I in a 50 mg/niL, 0.5 methyl cellulose-polysorbate 80 suspension at T(0).

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81796851 [0024] Figure 10 is an ¹HNMR analysis of Compound 1 in Form I in a 50 mg/mL, 0.5 methyl cellulose-polysorbate 80 suspension stored at room temperature for 24 hours.

[0025] Figure 11 is an ’HNMR analysis of Compound 1 · HC1 standard.

DETAILED DESCRIPTION OF THE INVENTION [0026] Definitions [0027] As used herein, the following definitions shall apply unless otherwise indicated.

I [0028] The term CFTR as used herein means cystic fibrosis transmembrane conductance regulator or a mutation thereof capable of regulator activity, including, but not limited to, ΔΕ508 CFTR and G551D CFTR. (see, e,g., http7/www.genetsickkids.on.ca/cfir/, for CFTR mutations).

[0029] As used herein “crystalline” refers to compounds or compositions where the structural units are arranged in fixed geometric patterns or lattices, so that crystalline solids have rigid long range order. The structural units that constitute the crystal structure can be atoms, molecules, or ions. Crystalline solids show definite melting points.

(0030] The term modulating as used herein means increasing or decreasing, e.g. activity, by a measurable amount ’ [0031] In one aspect, the invention features a fonn of 3-(6-(1-(2,2diiluoroben2o[d][l,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)bei]2oicacid characterized as Form I.

]0032] In another embodiment, Form 1 is characterized by one or more peaks at

15.4 ±02 degrees, 163 ±02 degrees, and 14.5±02 degrees in an X-ray powder diffraction obtained using Cu K alpha radiation..

]0033] In another embodiment, Form I is characterized by one or more peaks at 15.4, 16.3, and 14.5 degrees.

10034] In another embodiment, Form I is further characterized by a peak at l4.8±02 degrees.

[0035] In another embodiment Form I is further characterized by a peak at 14.8 degrees.

(0036] In another embodiment, Form I is further characterized by a peak at 17.8±02 degrees.

(0037] In another embodiment, Form I is further characterized by a peak at 17.8 degrees. .

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I »

81796851 [0038] In another embodiment, Form I is further characterized by a peak at 16.6±0.2 degrees.

[0039] In another embodiment, Form I is further characterized by a peak at 16.6 degrees.

[0040] In another embodiment, Form I is further characterized by a peak at 7.8±0.2 degrees.

[0041] In another embodiment, Form I is further characterized by a peak at 7.8 degrees.

[0042] In another embodiment, Form I is further characterized by a peak at 26.0±0.2 degrees.

[0043] In another embodiment, Form I is further characterized by a peak at 26.0 degrees.

[0044] In another embodiment, Form I is further characterized by a peak at 21,6±0.2 degrees.

[0045] In another embodiment, Form I is further characterized by a peak at 21.6 degrees.

[0046] In another embodiment, Form I is further characterized by a peak at 23.3±0.2 degrees.

[0047] In another embodiment, Form I is further characterized by a peak at 23.3 degrees.

[0048] In some embodiments, Form I is characterized by a calculated diffraction pattern of Figure 1.

[0049] In some embodiments, Form I is characterized by a diffraction pattern of Figure 2.

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81796851 [0050] In one aspect, the invention features a solid crystalline Form I as defined herein having a particle size distribution of D90 of about 82 pm or less.

[0051] In one aspect, the invention features a solid crystalline Form I as defined herein having a particle size distribution of D50 of about 30 pm or less.

[0052] In one aspect, the invention features a crystal form of 3-(6-(1 -(2,2difluorobenzo[d][l,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid having a monoclinic crystal system, a P2|/n space group, and the following unit cell dimensions: a = 4.9626 (7) Â, b = 12.2994 (18) A, c = 33.075 (4) A, a = 90°, β = 93.938 (9)°, and γ = 90°.

[0053] In one aspect, the invention features a pharmaceutical composition comprising a form of Compound I, as described above, and a pharmaceutically acceptable carrier.

[0054] In one aspect, the present invention features a process of preparing Form I comprising suspending or dissolving the HC1 salt of 3-(6-( 1-(2,2-difluorobenzo[d][l,3]dioxol5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid in an appropriate solvent for an effective amount of time.

[0055] In some embodiments, the appropriate solvent is water or 50% methanol/water mixture.

[0056] In some embodiments, the appropriate solvent is water.

[0057] In some embodiments, the effective amount of time is about 2 to 24 hours. In some embodiments, the effective amount of time is about 2 to about 18 hours. In some embodiments, the effective amount of time is about 2 to about 12 hours.

[0058] In some embodiments, the effective amount of time is about 2 to about 6 hours.

[0059] In one aspect, the invention features a combination comprising: 3-(6-(1-(2,2difluorobenzo[d][l,3]dioxol-5-yl) cyclopropanecarboxamido)-3-mcthylpyridin-2-yl)benzoic acid; and N-(5-hydroxy-2,4-ditcrt-butyl-phenyl)-4-oxo-l H-quinoline-3-carboxamide.

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81796851 [0060] In some embodiments, the 3-(6-(1-(2,2-difluorobenzo[d][l,3]dioxol-5-yl) cyclopropanecarboxamido)-3-methyIpyridin-2-yl)benzoic acid is in the Form 1 in the combination.

[0061] In one aspect, the invention features a pharmaceutical composition comprising

3-(6-( 1 -(2,2-difluorobenzo[d][ 1,3 ]dioxol-5-yl) cyclopropanccarboxamido)-3-methylpyridin-2yl)benzoic acid, N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-lII-quinoline-3-carboxamide, and a pharmaceutically acceptable carrier.

[0062] In some embodiments, the 3-(6-(1-(2,2-difluorobenzo[d][l,3]dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid is in the Form I in the composition.

[0063] In some embodiments, the combination is for use for increasing AF508-CFTR chloride transport compared to 3-(6-(1-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid and N-(5-hydroxy-2,4-ditertbutyl-phenyl)-4-oxo-1 H-quinoline-3-carboxamide alone.

[0064] In one aspect, the Form 1 as defined herein is for use for the manufacture of a medicament.

[0065] In some embodiments, the medicament is for use in the treatment of cystic fibrosis in a subject with a AF508-CFTR mutation.

[0066] In some embodiments, the subject is homozygous for the mutation.

[0067] Methods of Preparing Form I.

[0068] In one embodiment, Form I is prepared from dispersing or dissolving a salt form, such as HCL, of 3-(6-( 1-(2,2-difluorobenzo[d] [1,3 ]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid in an appropriate solvent for an effective amount of time. In another embodiment, Form I is prepared from dispersing a salt

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81796851 form, such as HCL, of 3-(6-(1 -(2,2-difluorobenzo[d][l,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid in an appropriate solvent for an effective amount of time. In another embodiment, Form I is formed directly from 3-(6-(1-(2,2difluorobenzo[d][l,3]dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t5 butylbenzoate and an appropriate acid, such as formic acid. In one embodiment, the HC1 salt form of 3-(6-(1-(2,2-difluorobenzo[d]ll,3]dioxol-5-yl) cyclopropanecarboxamido)-3methylpyridin-2-yl)benzoic acid is the starting point and in one embodiment can be prepared by coupling an acid chloride moiety with an amine moiety according to Schemes 1-3.

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<img file="CA2986286C_D0002.tif" />

|0069| Scheme 1. Synthesis of the acid chloride moiety.

1. Reduction

2. NaOH

<img file="CA2986286C_D0003.tif" />

1. SOCk

2. H₂O

<img file="CA2986286C_D0004.tif" />

1. NaCN

2. H,0

<img file="CA2986286C_D0005.tif" />

<img file="CA2986286C_D0006.tif" />

[0070] Scheme 2. Synthesis of the amine moiety.

<img file="CA2986286C_D0007.tif" />

+

I. K₂COj, Pd(dppf)Cl₂

<img file="CA2986286C_D0008.tif" />

<img file="CA2986286C_D0009.tif" />

urea-hydrogen peroxide phthalic anhydride EtOAc, water

<img file="CA2986286C_D0010.tif" />

CO/Bu

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10071] Scheme 3. Formation of an acid salt of3-(6-(l-(2,2-difluorobcnzo[d][l,3]dioxol5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-y])benzoic acid.

<img file="CA2986286C_D0011.tif" />

<img file="CA2986286C_D0012.tif" />

[0072] Using the HC1, for example, salt form of 3-(6-(1-(2,2-difluorobenzo[d][l,3]dioxol5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid as a starting point, Form I can be formed in high yields by dispersing or dissolving the HCI salt form of 3-(6-(1-(2,2difluorobenzo[d][l ,3]dioxol-5-yI) cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid in an appropriate solvent for an effective amount of time. Other salt forms of 3-(6-(1-(2,2difluorobenzo[d][l ,3]dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid may be used such as, for example, other mineral or organic acid forms. The other salt forms result from hydrolysis of the t-butyl ester with the corresponding acid. Other acids/salt forms include nitric, sulfuric, phosphoric, boric, acetic, benzoic, malonic, and the like. The salt form of 3-(6-(1-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2yl)benzoic acid may or may not be soluble depending upon the solvent used, but lack of solubility does not hinder formation of Form I. For example, in one embodiment, the appropriate solvent may be water or an alcohol/water mixture such as 50% methanol/water mixture, even though the HCI salt form of 3-(6-(1-(2,2-difluorobenzo[d][l,3]dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl)bcnzoic acid is only sparingly soluble in water. In one embodiment, the appropriate solvent is water.

10073] The effective amount of time for formation of Form J from the salt form of 3-(6(l-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2yl)benzoic acid can be any time between 2 to 24 hours or greater. Generally, greater than 24 hours is not needed to obtain high yields (~98%), but certain solvents may require greater amounts of time. It is also recognized that the amount of time needed is inversely proportional to the temperature. That is, the higher the temperature the less time needed to affect dissociation of acid to form Form I. When the solvent is water, stirring the dispersion for approximately 24

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81796851 hours at room temperature gives Form I in an approximately 98% yield. If a solution of the salt form of 3-(6-(l-(2^-difluQrobenzo[d][l,3jdioxol-5-yl) cyclopropanecarboxamido)-3methylpyridin-2-yI)benzoic acid is desired for process purposes, an elevated temperature may be used. After stirring the solution for an effective amount of time at the elevated temperature, recrystallization upon cooling yields substantially pure forms of Form I. In one embodiment, substantially pure refers to greater than about 90% purity. In another embodiment, substantially pure refers to greater than about 95% purity. In another embodiment, substantially pure refers to greater than about 98% purity. In another embodiment, substantially pure refers to greater than about 99% purity. The temperature selected depends in part on the solvent used and is well within the capabilities of someone of ordinary skill in the art to determine. In one embodiment, the temperature is between room temperature and about 80 °C. In another embodiment, the temperature is between room temperature and about 40 °C. In another embodiment, the temperature is between about 40 °C and about 60 °C. In another embodiment, the temperature is between about 60 °C and about 80 °C.

[0074] In some embodiments, Form I may be further purified by recrystallization from an organic solvent Examples of organic solvents include, but are not limited to, toluene, cumene, anisole, 1-butanol, isopropylacetate, butyl acetate, isobutyl acetate, methyl t-butyl ether, methyl isobutyl ketone, or 1-propanol/water (at various ratios). Temperature may be used as described above. For example, in one embodiment, Foim I is dissolved in 1-butanol at 75 °C until it is completely dissolved. Cooling down the solution to 10 °C at a rate of 0.2 “C/min yields crystals of Form I which may be isolated by filtration. · [0075] Uses, Formulation and Administration ' [0076] Pharmaceutically acceptable compositions [0077] In another aspect of the present invention, pharmaceutically acceptable compositions are provided, wherein these compositions comprise Compound 1 as described herein, and optionally comprise a pharmaceutically acceptable carrier, adjuvant or vehicle. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents.

[0078] As described above, the pharmaceutically acceptable compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, adjuvant, or vehicle, which, as used herein, includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form

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PCT/US2008/085456 desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various earners used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof. Except insofar as any conventional earner medium is incompatible with the compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylenepolyoxypropylene-block polymers, wool fat, sugars such as lactose, glucose and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; com oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl lauraie; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

(0079] Uses of Compounds and Pharmaceutically Acceptable Compositions

10080] In yet another aspect, the present invention provides a method of treating a condition, disease, or disorder implicated by CFTR. In certain embodiments, the present invention provides a method of treating a condition, disease, or disorder implicated by a deficiency of CFTR activity, the method comprising administering a composition comprising a solid state form of Form I described herein to a subject, preferably a mammal, in need thereof.

(0081] A “CFTR-mediated disease” as used herein is a disease selected from cystic fibrosis, Hereditary emphysema, Hereditary hemochromatosis, Coagulation-Fibrinolysis deficiencies, such as Protein C deficiency, Type 1 hereditary angioedema, Lipid processing -12CA 2986286 2017-11-21

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PCT/US2008/005456 deficiencies, such as Familial hypercholesterolemia, Type 1 chylomicronemia, Abetalipoprotcinemia, Lysosomal storage diseases, such as I-ccll discasc/Pscudo-Hurlcr, Mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II, Polyendocrinopathy/Hyperinsulcmia, Diabetes mcllitus, Laron dwarfism, Myleoperoxidase deficiency, Primary hypoparathyroidism, Melanoma, Glycanosis CDG type 1, Hereditary emphysema. Congenital hyperthyroidism, Osteogenesis imperfecta. Hereditary hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI), Neurophysea) DI, Neprogenic DI, Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis. Progressive supranuclear plasy, Pick’s disease, several polyglutamine neurological disorders asuch as Huntington, Spinocerebullar ataxia type I, Spinal and bulbar muscular atrophy, Dentatorubal pallidoluysian, and Myotonic dystrophy, as well as Spongiform encephalopathies, such as Hereditary Creutzfeldt-Jakob disease, Fabry disease, Straussler-Scheinker syndrome, COPD, dryeye disease, and Sjogren’s disease.

[0082) In certain embodiments, the present invention provides a method of treating a CFTR-mcdiatcd disease in a human comprising the step of administering to said human an effective amount of a composition comprising Form I described herein.

[0083] According to an alternative preferred embodiment, the present invention provides a method of treating cystic fibrosis in a human comprising the step of administering to said human a composition comprising Form I described herein.

[0084] According to the invention an effective amount of Form I or a pharmaceutically acceptable composition thereof is that amount effective for treating or lessening the severity of any of the diseases recited above.

(0085] Form I or a pharmaceutically acceptable composition thereof may be administered using any amount and any route of administration effective for treating or lessening the severity of one or more of the diseases reicted above.

[0086] In certain embodiments, Form 1 described herein or a pharmaceutically acceptable composition thereof is useful for treating or lessening the severity of cystic fibrosis in patients who exhibit residual CFTR activity in the apical membrane of respiratory and non-rcspiratory epithelia. The presence of residual CFTR activity at the epithelial surface can be readily detected using methods known in the art, e.g., standard electrophysiological, biochemical, or histochemical techniques. Such methods identify CFTR activity using in vivo or ex vivo electrophysiological techniques, measurement of sweat or salivary' Cl concentrations, or er vivo

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PCT/US2008/085456 biochemical or histochemical techniques to monitor cell surface density. Using such methods, residual CFTR activity can be readily detected in patients heterozygous or homozygous for a variety of different mutations, including patients homozygous or heterozygous for the most common mutation, ÂF5O8.

|0087] In one embodiment, Form I described herein or a pharmaceutically acceptable composition thereof is useful for treating or lessening the severity of cystic fibrosis in patients within certain genotypes exhibiting residual CFTR activity, e.g., class III mutations (impaired regulation or gating), class IV mutations (altered conductance), or class V mutations (reduced synthesis) (Lee R. Choo-Kang, Pamela L., Zeitlin, Type 1,11, 111, IK, and V cystic fibrosis Tansmembrane Conductance Regulator Defects and Opportunities of Therapy, Cunent Opinion in Pulmonary Medicine 6:521 - 529,2000). Other patient genotypes that exhibit residual CFTR activity include patients homozygous for one of these classes or heterozygous with any other class of mutations, including class I mutations, class II mutations, or a mutation that lacks classification.

|0088} In one embodiment, Form I described herein or a pharmaceutically acceptable composition thereof is useful for treating or lessening the severity of cystic fibrosis in patients within certain clinical phenotypes, e.g., a moderate to mild clinical phenotype that typically correlates with the amount of residual CFTR activity in the apical membrane of epithelia. Such phenotypes include patients exhibiting pancreatic insufficiency or patients diagnosed with idiopathic pancreatitis and congenital bilateral absence of the vas deferens, or mild lung disease.

|0089] The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular agent, its mode of administration, and the like. The compounds of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression dosage unit form as used herein refers to a physically discrete unit of agent appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician witbin the scope of sound medical judgment. The specific effective dose level for any particular patient or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the

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PCT/US2008/085456 specific compound employed, and like factors well known in the medical arts. The term “patient”, as used herein, means an animal, preferably a mammal, and most preferably a human.

10090] The pharmaceutically acceptable compositions of this invention can be administered to humans and other animals orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, as an oral or nasal spray, or the like, depending on the severity of the infection being treated. In certain embodiments, the compounds of the invention may be administered orally or parenterally at dosage levels of about 0.01 mg/kg to about 50 mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.

10091] In certain embodiments, the dosage amount of Form I in the dosage unit form is from 100 mg to 1,000 mg. In another embodiment, the dosage amount of Form I is from 200 mg to 900 mg. In another embodiment, the dosage amount of Form 1 is from 300 mg to 800 mg. In another embodiment, the dosage amount of Form I is from 400 mg to 700 mg. In another embodiment, the dosage amount of Form I is from 500 mg to 600 mg.

[0092] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0093] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0094] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

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PCT/US2008/085456 [0095) Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, c) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the ease of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0096] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingrcdient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polcthylcnc glycols and the like.

[0097] The active compounds can also be in microencapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystallinc cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or

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[0098] It will also be appreciated that Form I described herein or a pharmaceutically acceptable composition thereof can be employed in combination therapies, that is, Form I can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, an inventive compound may be administered concurrently with another agent used to treat the same disorder), or they may achieve different effects (e.g., control of any adverse effects). As used herein, additional therapeutic agents that are normally administered to treat or prevent a particular disease, or condition, are known as appropriate for the disease, or condition, being treated.

|0099[ In one embodiment, the additional agent is selected from a mucolytic agent, bronchodialator, an anti-biotic, an anti-infective agent, an anti-inflammatory agent, a CFTR modulator other than a compound of the present invention, or a nutritional agent.

[001001 In another embodiment, the additional agent is a compound selected from gentamicin, curcumin, cyclophosphamide, 4-phenylbutyrate, miglustat, felodipine, nimodipine, Philoxin B, geniestein, Apigenin, cAMP/cGMP modulators such as rolipram, sildenafil, milrinone, tadalafi 1, amrinone, isoproterenol, albuterol, and almeterol, deoxyspergualin, HSP 90 inhibitors, HSP 70 inhibitors, proteosome inhibitors such as epoxomicin, lactacystin, etc.

[00101 ] In another embodiment, the additional agent is a compound disclosed in WO 2004028480, WO 2004110352, WO 2005094374, WO 2005120497, or WO 2006101740.

[00102) In another embodiment, the additiona agent is a benzo(c)quinolizinium derivative that exhibits CFTR modulation activity or a benzopyran derivative that exhibits CFTR modulation activity.

1001031 In another embodiment, the addditional agent is a compound disclosed in US7202262, US6992096, US20060148864, US20060148863, VS20060035943, US20050164973, W02006110483, W02006044456, W02006044682, W02006044505, W02006044503, W02006044502, or WO2004091502.

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81796851 [00104] In another embodiment the additional agent is a compound disclosed in W02004080972, W02004111014, W02005035514, W02005049018, W0200600242I, W02006099256, W02006.127588, or W02007044560.

[00105] In another embodiment, the an additional agent selected from compounds disclosed in U.S. Patent Application Serial No. 11/165,818, published as U.S. Published Patent Application No. 2006/0074075, filed June 24,2005.

In another embodiment the additional agent is N-(5-hydroxy-2,4-ditert-butyl-phenyl)-

4-oxo-lH-quinoline-3-carboxamide. These combinations arc useful for treating the diseases described herein including cystic fibrosis. These combinations are also useful in the kits described herein.

[00106] The amount of additional therapeutic agent present in the compositions of this invention will be no more than the amount that would normally be administered in a composition comprising that therapeutic agent as the only active agent Preferably the amount of additional therapeutic agent in the presently disclosed compositions will range from about 50% to 100% of the amount normally present in a composition comprising that agent as the only therapeutically active agent [00107] Form I described herein or a pharmaceutically acceptable composition thereof may also be incorporated into compositions for coating an implantable medical device, such as prostheses, artificial valves, vascular grafts, stents and catheters. Accordingly, the present invention, in another aspect, includes a composition for coating an implantable device comprising Form I described herein or a pharmaceutically acceptable composition thereof, and in classes and subclasses herein, and a carrier suitable for coating said implantable device. In still another aspect, the present invention includes an implantable device coated with a composition comprising Form I described herein or a pharmaceutically acceptable composition thereof] and a carrier suitable for coating said implantable device. Suitable coatings and the general preparation of coated implantable devices arc described in US Patents 6,099,562; 5,886,026; and 5,304» 121. The coatings are typically biocompatible polymeric materials such as a hydrogel polymer, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate, and mixtures thereof. The coatings may optionally be further covered by a suitable topcoat of fluorosilicone, polysaccarides, polyethylene glycol, phospholipids or combinations thereof to impart controlled release characteristics in the composition.

[00108] In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.

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EXAMPLES [00109] Methods &amp; Materials (00110] Differential Scanning Calorimetry (DSC) [00111| The Differential scanning calorimetry (DSC) data of Form I were collected using a DSC Q100 V9.6 Build 290 (TA Instruments, New Castle, DE). Temperature was calibrated with indium and heat capacity was calibrated with sapphire. Samples of 3-6 mg were weighed into aluminum pans that were crimped using lids with 1 pin hole. The samples were scanned from 25°C to 350°C at a heating rate of 1.0°C/min and with a nitrogen gas purge of 50 ml/min. Data were collected by Thermal Advantage Q SeriesTM version 2.2.0.248 software and analyzed by Universal Analysis software version 4. ID (TA Instruments, Newcastle, DE). The reported numbers represent single analyses.

(00112] XRPD (X-ray Powder Diffraction)

100113] The X-Ray diffraction (XRD) data of Fonn I were collected on a Bruker D8 DISCOVER powder diffractometer with HI-STAR 2-dimensional detector and a flat graphite monochromator. Cu sealed tube with Ka radiation was used at 40 kV, 35mA. The samples were placed on zero-background silicon wafers at 25°C. For each sample, two data frames were collected at 120 seconds each at 2 different θ2 angles: 8° and 26°. The data were integrated with GADDS software and merged with Dff'FRACT^plusEVA software. Uncertainties for the reported peak positions are ± 0.2 degrees.

(00114] Vitridel (sodium bis(2-methoxyethoxy)aluminum hydride [or NaAIHXOCHîCH/OCHih], 65 wgt% solution in toluene) was purchased from Aldrich Chemicals.

(00115] 2.2 -Difluoro-l,3-bcnzodioxole-5-carboxylic acid was purchased from Saltigo (an affiliate of the Lanxess Corporation).

(00116| Anywhere in the present application where a name of a compound may not correclly describe the structure of the compound, the structure supersedes the name and governs.

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<img file="CA2986286C_D0013.tif" />

<img file="CA2986286C_D0014.tif" />

(00117) Synthesis of 3-(6-(l-(2,2-difluorobenz<>ldlll3ldioxol-5-vl) cvclopropanecarboxamido)-3-methvipvridin-2-vDbenz<ric acid » HCI.

(00118| Acid Chloride Moiety (00119] Synthesis of (2,2-difluoro-l,3-benzodioxol-5-yl)-metlianol.

1. Vitridc (2 equiv) PhCH₃(10vol)

2. 10% aq (w/w) NaOH (4 equiv) • - CO₂H 86 92% yield |00120| Commercially available 2,2-difluoro-l,3-benzodioxole-5-carboxylic acid (1.0 eq) is slurried in toluene (10 vol). Vitride® (2 eq) is added via addition funnel at a rate to maintain the temperature at 15-25 °C. At the end of addition the temperature is increased to 40 °C for 2 h then 10% (w/w) aq. NaOH (4.0 eq) is carefully added via addition funnel maintaining the temperature at 40-50 °C. After stirring for an additional 30 minutes, the layers are allowed to separate at 40 °C. The organic phase is cooled to 20 °C then washed with water (2x1.5 vol), dried (Na₂SO4), filtered, and concentrated to afford crude (2,2-difluoro-l,3-ben2odioxol-5-yl)methanol that is used directly in the next step.

(001211 Synthesis of 5-chloromethyl-2,2-difluoro-l,3-benzodioxole.

1. SOCI, (1.5 equiv) DMAP (0.01 equiv) MTBE (5 vol)

2. water (4 vol)

82-100% yield (00122) (2,2-difluoro-l 3-benzodioxol-5-yl)-methanol (1.0 eq) is dissolved in MTBE (5 vol). A catalytic amount of DMAP (1 mol %) is added and SOCb (1.2 eq) is added via addition funnel. The SOCh is added at a rate to maintain the temperature in the reactor at 15-25 °C. The temperature is increased to 30 °C for 1 hour then cooled to 20 °C then water (4 vol) is added via addition funnel maintaining the temperature at less than 30 °C. After stirring for an additional 30 minutes, the layers are allowed to separate. The organic layer is stirred and 10% (w/v) aq. NaOH (4.4 vol) is added. After stirring for 15 to 20 minutes, the layers are allowed to separate. The organic phase is then dried (Na^SO.»), filtered, and concentrated to afford crude 5-chloromethyl2,2-difluoro-l ,3-benzodioxole that is used directly in the next step.

<img file="CA2986286C_D0015.tif" />

<img file="CA2986286C_D0016.tif" />

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<img file="CA2986286C_D0017.tif" />

<img file="CA2986286C_D0018.tif" />

100123] Synthesis of (2,2-dinuoro-l,3-benzodioxol-5-yl)-acetonitrile.

1. NaCN (1.4 cquiv) DMSO (3 vol) 30-40 degrees C

2. water (6 vol) MTBE (4 vol)

95-100% yield [00124] A solution of 5-chloromethyl-2,2-difluoro-l,3-benzodioxole (I eq) in DMSO (1.25 vol) is added to a slurry of NaCN (1.4 eq) in DMSO (3 vol) maintaining the temperature between 30-40 °C. The mixture is stirred for 1 hour then water (6 vol) is added followed by MTBE (4 vol). After stirring for 30 min, the layers are separated. The aqueous layer is extracted with MTBE (1.8 vol). The combined organic layers arc washed with water (1.8 vol), dried (NaîSOi). filtered, and concentrated to afford crude (2,2-difluoro-l ,3-benzodioxol-5-yl)acctonitrilc (95%) that is used directly in the next step.

[00125] Synthesis of (2,2-difluoro-13-benzodioxol-5-yl)-cyclopropanecarbonitrile.

<img file="CA2986286C_D0019.tif" />

-bromo-2-chloroethane ( 1.5 equiv) 50% K.OH (5.0 equiv) Oct₄NBr (0.02 equiv) degrees C

<img file="CA2986286C_D0020.tif" />

88-100% yield |00126| A mixture of (2,2-difluoro-l,3-benzodioxol-5-yl)-acetonitrile (1.0 eq), 50 wt % aqueous KOH (5.0 eq) I -bromo-2-chloroclhane (1.5 eq), and Oct>NBr (0.02 eq) is heated at 70 °C for 1 h. The reaction mixture is cooled then worked up with MTBE and water. The organic phase is washed with water and brine then the solvent is removed to afford (2,2-difluoro-l,3benzodioxol-5-yl)-cyclopropanecarbonitrile.

[00127] Synthesis of 1-(2,2-difluoro-l,3-benzodioxol-5-y))-cyclopropanecarboxylic acid.

<img file="CA2986286C_D0021.tif" />

1. 6 M NaOH (8 cquiv)

EtOH (5 vol), 80 degrees C

2. MTBE (10 vol) dicyclohexylamine (1 equiv)

3. MTBE (10 vol)

10% aq citric acid (8 vol)

<img file="CA2986286C_D0022.tif" />

69% yield

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PCT/US2008/085456 |00128| (2,2-difluoro-l,3-benzodioxol-5-yl)-cyclopropanccarbonitrilc is hydrolyzed using 6 M NaOH (8 equiv) in ethanol (5 vol) at 80 °C overnight. The mixture is cooled to room temperature and ethanol is evaporated under vacuum. The residue is taken into water and MTBE, 1 M HC1 was added and the layers are separated. The MTBE layer was then treated with dicyclohexylamine (0.97 equiv). The slurry is cooled to 0 °C, filtered and washed with heptane to give the corresponding DCHA salt. The salt is taken into MTBE and 10% citric acid and stined until all solids dissolve. The layers are separated and the MTBE layer was washed with water and brine. Solvent swap to heptane followed by filtration gives 1-(2,2-difluoio-1,3benzodioxol-5-yl)-cyclopropanecarboxylic acid after drying in a vacuum oven at 50 °C overnight.

[001291 Synthesis of 1-(2,2-difluoro-l,3-benzodioxol-5-yl)-cyclopropanecarbonyl chloride.

<img file="CA2986286C_D0023.tif" />

SOCK,

PhCH₃, degrees C

<img file="CA2986286C_D0024.tif" />

10013011-(2,2-difluoro-I.3-bcnzodioxol-5-yl)-cyclopropanecarboxylic acid (1.2 eq) is slurried in toluene (2.5 vol) and the mixture heated to 60 °C. SOCI2 (1.4 eq) is added via addition funnel. The toluene and SOCI2 are distilled from the reaction mixture after 30 minutes. Additional toluene (2.5 vol) is added and distilled again.

[00131] Amine Moiety

100132] Synthesis of tert-butyl-3-(3-methylpyridin-2-yl)benzoate.

<img file="CA2986286C_D0025.tif" />

CO^Bu

1. toluene, 2M K₂CO_}Pd(dppf)Cl₂,80 degrees C

2. aq. MsOH

3. aq.NaOH _Λ

<img file="CA2986286C_D0026.tif" />

100133] 2-Bromo-3-methylpyridine (1.0 eq) is dissolved in toluene (12 vol). K.2CO3 (4.8 eq) is added followed by water (3.5 vol) and the mixture heated to 65 °C under a stream of N₂ for 1 hour. 3-(r-Butoxycarbonyl)phenylboronic acid (1.05 eq) and PdfdppOChCH^Ch (0.015 eq) are then added and the mixture is heated to 80 °C. After 2 hours, the heat is turned off, water is added (3.5 vol) and the layers are allowed to separate. The organic phase is then washed with water (3.5 vol) and extracted with 10% aqueous methanesulfonic acid (2 eq MsOH, 7.7 vol). The aqueous phase is made basic with 50% aqueous NaOH (2 eq) and extracted with EtOAc (8 vol).

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<img file="CA2986286C_D0027.tif" />

<img file="CA2986286C_D0028.tif" />

The organic layer is concentrated to afford crude terz-butyl-3-(3-methylpyridin-2-yl)benzoate (82%) that is used directly in the next step.

[00134] Synthesis of 2-(3-(iert-butoxycarbonyl)phenyl)-3-inethylpyridine-l-oxide.

100135] ierr-Butyl-3-(3-methylpyridin-2-yl)benzoate (1.0 eq) is dissolved in EtOAc (6 vol). Water (0. 3 vol) is added followed by urea-hydrogen peroxide (3 eq). The phthalic anhydride (3 eq) is added portion-wise as a solid to maintain the temperature in the reactor below 45 °C. After completion of phthalic anhydride addition, the mixture is heated to 45 °C. After stirring for an additional 4 hours, the heat is turned off. 10% w/w aqueous Na₂SO? (1.5 eq) is added via addition funnel. After completion of Na₂SOj addition, the mixture is stirred for an additional 30 minutes and the layers separated. The organic layer is stirred and 10% w/w aq. Na₂CCh (2 eq) is added. After stirring for 30 minutes, the layers arc allowed to separate. The organic phase is washed 13% w/v aq NaCI. The organic phase is then filtered and concentrated to afford crude 2-(3-(iert-butoxycarbonyl)phcnyl)-3-mcthylpyridinc-l-oxidc (95%) that is used directly in the next step.

(00136] Synthesis of tert-butyl-3-(6-amino-3-inethylpyridin-2-yl)benzoate.

<img file="CA2986286C_D0029.tif" />

1. Ms₂0, py, MeCN, 70 degrees C

2. ethanolamine

<img file="CA2986286C_D0030.tif" />

[00137) A solution of 2-(3-(/ert-butoxycarbonyI)phenyl)-3-methylpyridine-l-oxide (1 eq) and pyridine (4 eq) in MeCN (8 vol) is heated to 70 °C. A solution of methanesulfonic anhydride (1.5 eq) in MeCN (2 vol) is added over 50 min via addition funnel maintaining the temperature at less than 75 °C. The mixture is stirred for an additional 0.5 hours after complete addition. The mixture is then allowed to cool to ambient. Ethanolamine (10 eq) is added via addition funnel. After stirring for 2 hours, water (6 vol) is added and the mixture is cooled to 10 °C. After stirring for NLT 3 hours, the solid is collected by filtration and washed with water (3 vol), 2:1 MeCN/water (3 vol), and MeCN (2 x 1.5 vol). The solid is dried to constant weight (&lt;1% difference) in a vacuum oven at 50 °C with a slight N₂ bleed to afford /e//-butyl-3-(6-amino-3methylpyridin-2-yl)benzoate as a red-yellow solid (53% yield).

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PCT/US2008/085456 [00138) Synthesis of 3-( 6-(1-(2,2-difluorobenzo|d||l,3|dioxol-5-yl) cyclopropanecarboxamido)-3-inethylpyridin-2-yI)-t-butylbenzoate.

<img file="CA2986286C_D0031.tif" />

<img file="CA2986286C_D0032.tif" />

[00139] The crude acid chloride is dissolved in toluene (2.5 vol based on acid chloride) and added via addition funnel to a mixture of rm-butyl-3-(6-amino-3-methylpyridin-2yl)benzoate (1 eq), dimethylaminopyridine (DMAP, 0.02 eq), and triethylamine (3.0 eq) in toluene (4 vol based on zew-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate). After 2 hours, water (4 vol based on zerr-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate) is added to the reaction mixture. After stirring for 30 minutes, the layers are separated. The organic phase is then filtered and concentrated to afford a thick oil of 3-(6-(l-(2,2-difluorobenzo[d][1.3]dioxol-5yl) cyclopropanecarboxamido)-3-methylpyridin-2-yI)-t-butylben2oate (quantitative crude yield). MeCN (3 vol based on crude product) is added and distilled until crystallization occurs. Water (2 vol based on crude product) is added and the mixture stirred for 2 h. The solid is collected by filtration, washed with 1:1 (by volume) McCN/watcr (2 x 1 vol based on crude product), and partially dried on the filter under vacuum. The solid is dried to constant weight (&lt;1% difference) in a vacuum oven at 60 °C with a slight Nj bleed to afford 3-(641 -(2,2difluorobenzo[d][l,3]dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl)-tbutylbenzoate as a brown solid.

(00140] Syntheisis of 3-( 6-(1-(2,2-difluorobenzo(d](l,3]dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid * HCL salt.

<img file="CA2986286C_D0033.tif" />

6NHCI MeCN 40 degrees C

<img file="CA2986286C_D0034.tif" />

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100141] To a slurry of 3-(6-(1-(2,2-difluorobenzo[d][l,3 ]dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate (1.0 eq) in MeCN (3.0 vol) is added water (0.83 vol) followed by concentrated aqueous HC1 (0.83 vol). The mixture is heated to 45 ± 5 °C. After stirring for 24 to 48 hours the reaction is complete and the mixture is allowed to cool to ambient. Water (1.33 vol) is added and the mixture stirred. The solid is collected by filtration, washed with water (2 x 0.3 vol), and partially dried on the filter undeT vacuum. The solid is dried to constant weight (&lt;]% difference) in a vacuum oven at 60 °C with a slight Nî bleed to afford 3-(6-(1 -(2,2-difluorobenzo[d][1,3]dioxol-5-y 1) cyclopropanecarf)oxamido)-3methylpyridin-2-yl)benzoic acid · HC1 as an off-white solid.

(00142] Synthesis of 3-(6-(1-(2,2-difluorobenzo|d]|13]dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (Form 1).

<img file="CA2986286C_D0035.tif" />

slurry in water

98%

<img file="CA2986286C_D0036.tif" />

Form I

100143] A slurry of 3-(6-(1-(2,2-difluorobenzo[d][l,3]dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid · HC1 (1 eq) in water (10 vol) is stirred at ambient temperature. A sample is taken after stirring for 24 hours. The sample is filtered and the solid washed with water (2 x). The solid sample is submitted for DSC analysis. When DSC analysis indicates complete conversion to Form 1, the solid is collected by filtration, washed with water (2 x 1.0 vol), and partially dried on the filter under vacuum. The solid is dried to constant weight (&lt;1% difference) in a vacuum oven at 60 °C with a slight N2 bleed to afford Form I as an off-white solid (98% yield). *H NMR (400 MHz, DMSO-d6) 9.14 (s, 1H), 7.997.93 (m, 3H), 7.80-7.78 (m, 1H), 7.74-7.72 (m, 1H), 7.60-7.55 (m, 2H), 7.41-7.33 (m, 2H), 2.24 (s, 311). 1.53-1.51 (m, 2H), 1.19-1.17 (m, 2H).

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PCT/US2008/085456 (00144] Synthesis of 3-( 6-(1-(2,2-difluorobenzo[d)|13)dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (Form I) using water and

<img file="CA2986286C_D0037.tif" />

1. H;0,50% NaOH

2. cone HC1

60-90 °C

<img file="CA2986286C_D0038.tif" />

Form I [001451 To a slurry of 3-(6-(1-(2,2-difluorobenzo[d][l ,3]dioxol-5-yl) cyclopropanecarboxamido)-3-mcthylpyridin-2-yl)bcnzoic acid · HC1 (1 eq) in water (10 vol) stirred at ambient temperature is added 50% w/w aq. NaOH (2.5 eq). The mixture is stirred for NLT 15 min or until a homogeneous solution. Concentrated HC1 (4 eq) is added to crystallize Form I. The mixture is heated to 60 °C or 90 °C if needed to reduce the level of the tbutylbenzoate ester. The mixture is heated until HPLC analysis indicates NMT 0.8% (AUC) tbutylbenzoate ester. The mixture is then cooled to ambient and the solid is collected by filtration, washed with water (3 x 3.4 vol), and partially dried on the filter under vacuum. The solid is dried to constant weight (&lt;1% difference) in a vacuum oven at 60 °C with a slight N? bleed to afford Form I as an off-white solid (97% yield).

100146] Synthesis of 3-(6-(1-(2,2-difluorobenzo|dj[l,3]dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (Form I) directly from benzoate.

<img file="CA2986286C_D0039.tif" />

<img file="CA2986286C_D0040.tif" />

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PCT/US2008/085456 [00147] A solution of 3-(6-(1-(2,2-difluorobcnzo[d][l,3]dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate (1.0 eq) in formic acid (3.0 vol) is heated to 70 ± 10 °C. The reaction is continued until the reaction is complete (NMT 1.0% AUC 3-(6-(1 -(2,2-difluorobenzo[d][ 1,3]dioxol-5-y I) cyclopropanecarboxamido)-3methylpyridin-2-yl)-t-butylbenzoate) or heating for NMT 8 h. The mixture is allowed to cool to ambient. The solution is added to water (6 vol) heated at 50 °C and the mixture stirred. The mixture is then heated to 70 ± 10 °C until the level of 3-(6-(1-(2,2difluorobenzo[d][l,3]dioxol-5yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate is NMT 0.8% (AUC). The solid is collected by filtration, washed with water (2x3 vol), and partially dried on the filter under vacuum. The solid is dried to constant weight (&lt;l% difference) in a vacuum oven at 60 °C with a slight N2 bleed to afford Compound 1 in Form I as an off-white solid.

[00148] An X-ray diffraction pattern calculated from a single crystal structure of Compound 1 in Form I is shown in Figure 1. Tabic 1 lists the calculated peaks for Figure 1.

[00149] Table 1.

<td> Peak : Rank</td><td></td><td> Relative Intensity</td>

<td> 11</td><td> 14.41</td><td> 48.2</td>

<td> 8</td><td> 14.64</td><td> 58.8</td>

<td> 1</td><td> 15.23</td><td> 100.0</td>

<td> 2</td><td> 16.11</td><td> 94.7</td>

<td> 3</td><td> 17.67</td><td> 81.9</td>

<td> 7</td><td> 19.32</td><td> 61.3</td>

<td> 4</td><td> 21.67</td><td> 76.5</td>

<td> 5</td><td> 23.40</td><td> 68.7</td>

<td> 9</td><td> 23.99</td><td> 50.8</td>

<td> 6</td><td> 26.10</td><td> 67.4</td>

<td> 10</td><td> 28.54</td><td> 50.1</td>

[00150] An actual X-ray powder diffraction pattern of Compound 1 in Form I is shown in Figure 2. Table 2 lists the actual peaks for Figure 2.

100151] Table 2.

<td> Peak Rank</td><td> ' _s : 2$ Angle</td><td> Relative In tensity :/ : .</td>

<td> 7</td><td> 7.83</td><td> 37.7</td>

<td> 3</td><td> 14.51</td><td> 74.9</td>

<td> 4</td><td> 14.78</td><td> 73.5</td>

<td> 1</td><td> 15.39</td><td> 100.0</td>

<td> 2</td><td> 16.26</td><td> 75.6</td>

<td> 6</td><td> 16.62</td><td> 42.6</td>

<td> 5</td><td> 17.81</td><td> 70.9</td>

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<td> Teak Rank</td><td> iirtdOwl.........!</td><td> .&lt;. :^ΓΠβ£Ϊ^*·'*'^α· ··'·</td>

<td> 9</td><td> 21.59</td><td> 36.6</td>

<td> 10</td><td> 23.32</td><td> 34.8</td>

<td> 11</td><td> 24.93</td><td> 26.4</td>

<td> 8</td><td> 25.99</td><td> 36.9</td>

[00152] An overlay of an X-ray diffraction pattern calculated from a single crystal structure of Compound 1 in Form I, and an actual X-ray powder diffraction pattern of Compound I in Form I is shown in Figure 3. The overlay shows good agreement between the calculated and actual peak positions, the difference being only about 0.15 degrees.

[00153] The DSC trace of Compound 1 in Form I is shown in Figure 4. Melting for Compound 1 in Form 1 occurs at about 204 °C.

[00154] Conformational pictures of Compound 1 in Form 1 based on single crystal X-ray analysis are shown in Figures 5-8. Figures 6-8 show hydrogen bonding between carboxylic acid groups of a dimer and the resulting stacking that occurs in the crystal. The crystal structure reveals a dense packing of the molecules. Compound 1 in Form I is monoclinic, P2|/n, with the following unit cell dimensions: a = 4.9626(7) A, b ” 12.299(2) A, c = 33.075 (4) Â, β = 93.938(9)°, V = 2014.0 A³, Z = 4. Density of Compound 1 in Form I calculated from structural data is 1.492 g/cm³ at 100 K.

(00155] *HNMR spectra of Compound 1 are shown in Figures 9-11 (Figures 9 and 10 depict Compound 1 in Form 1 in a 50 mg/mL, 0.5 methyl cellulose-polysorbate 80 suspension, and Figure 11 depicts Compound 1 as an HC1 salt).

[001561 Table 3 below recites additional analytical data for Compound 1.

1001571 Table 3.

<td> Cmpd. \.4fa.</td><td> LC/MS</td><td> LC/RT</td><td>¹ .****</td>

<td> 1</td><td> 453.3</td><td> 1.93</td><td> H NMR (400 MHz, DMSO-d6) 9.14 (s, 1H). 7.99-7.93 (m. 3H), 7.80-7.78 (m, 1H), 7.74-7.72 (m, 1H), 7.60-7.55 (m 2H), 7 41-7 33 (m, 2H), 2 24 (s. 3H), 1.53-1.51 (m. 2H). 1.19-1.17 (m, 2H)</td>

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1001581 ^ss^rs

1001591 Assays for Detecting and Measuring AF508-CFTR Correction Properties of Compounds [00160) Membrane potential optical methods for assaying AF508-CFTR modulation properties of compounds [00161J The optical membrane potential assay utilized voltage-sensitive FRET sensors described by Gonzalez and Tsien (See, Gonzalez, J. E. and R. Y. Tsicn (1995) “Voltage sensing by fluorescence resonance energy transfer in single cells” Biophvs J 69(4): 1272-80, and Gonzalez, J. E. and R Y. Tsien (1997) “Improved indicators of cell membrane potential that use fluorescence resonance energy transfer” Chem Biol 4(4): 269-77) in combination with instrumentation for measuring fluorescence changes such as the Voltagc/Ion Probe Reader (VIPR) (See, Gonzalez, J. E., K. Oades, et al. ( 1999) “Cell-based assays and instrumentation for screening ion-channel targets” Drug Discov Today 4(9): 431 -439).

[00162| These voltage sensitive assays arc based on the change in fluorescence resonant energy transfer (FRET) between the membrane-soluble, voltage-sensitive dye, DiSBAC2(3), and a fluorescent phospholipid, CC2-DMPE. which is attached to the outer leaflet of the plasma membrane and acts as a FRET donor. Changes in membrane potential (V_m) cause the negatively charged DiSBACjO) to redistribute across the plasma membrane and the amount of energy transfer from CC2-DMPE changes accordingly. The changes in fluorescence emission were monitored using VIPR™ II, which is an integrated liquid handler and fluorescent detector designed to conduct cell-based screens in 96- or 384-well microtiter plates.

[0016311. Identification of Correction Compounds |00164| To identify small molecules that correct the trafficking defect associated with AF508-CFTR; a single-addition HTS assay format was developed. The cells were incubated in serum-free medium for 16 hrs at 37 °C in the presence or absence (negative control) of test compound. As a positive control, cells plated in 384-wcll plates were incubated for 16 hrs at 27 °C to “temperature-correct” AF508-CFTR. The cells were subsequently rinsed 3X with Krebs Ringers solution and loaded with the voltage-sensitive dyes. To activate AF508-CFTR, 10 μΜ forskolin and the CFTR potentiator, genistein (20 μΜ), were added along with Cl’-free medium to each well. The addition of CT-free medium promoted Cl’ efflux in response to AF508-CFTR activation and the resulting membrane depolarization was optically monitored using the FRETbased voltage-sensor dyes.

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100165] 2, Identification of Potentiator Compounds

100166] To identify potentiators of AF508-CFTR, a double-addition HTS assay format was developed. During the first addition, a Cl’-free medium with or without test compound was added to each well. After 22 sec, a second addition of Cl'-frec medium containing 2 -10 μΜ forskolin was added to activate AF508-CFTR. The extracellular Cl concentration following both additions was 28 mM, which promoted CV efflux in response to AF508-CFTR activation and the resulting membrane depolarization was optically monitored using the FRET-based voltage-sensor dyes.

(00167] ^-Solutions

<td> Bath Solution #1: (in mM)</td><td> NaCl 160, KC14.5, CaCl₂ 2, MgCb 1, HEPES 10, pH 7.4 with NaOH.</td>

<td> Chloride-free bath solution:</td><td> Chloride salts in Bath Solution #1 arc substituted with gluconate salts.</td>

<td> CC2-DMPE:</td><td> Prepared as a 10 mM stock solution in DMSO and stored at -20° C.</td>

<td> DiSBAC₂(3):</td><td> Prepared as a 10 mM stock in DMSO and stored at -20°C.</td>

(00168] 4. Cell Culture (00169] NIH3T3 mouse fibroblasts stably expressing AF508-CFTR are used for optical measurements of membrane potential. The cells are maintained at 37 °C in 5% CO₂ and 90 % humidity in Dulbecco’s modified Eagle’s medium supplemented with 2 mM glutamine, 10 % fetal bovine serum, 1 X NEAA, β-ΜΕ, 1 X pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For all optical assays, the cells were seeded at 30,000/well in 384-well matrigel-coated plates and cultured for 2 hrs at 37 °C before culturing at 27 °C for 24 hrs for the potentiator assay. For the correction assays, the cells arc cultured at 27 °C or 37 °C with and without compounds for 16 - 24 hours.

[00170] Electrophysiological Assays for assaying AF508-CFTR modulation properties of compounds

100171] 1. Using Chamber Assay (00172] Using chamber experiments were performed on polarized epithelial cells

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PCT/IIS2008/085456 expressing AF508-CFTR to further characterize the AF5O8-CFTR modulators identified in the optica) assays, frj⁴*’⁵⁰^⁰™ epithelial cells grown on Costar Snapwell cell culture inserts were mounted in an Ussing chamber (Physiologic Instruments, Inc., San Diego, CA), and the monolayers were continuously short-circuited using a Voltage-clamp System (Department of Bioengineering. University of Iowa, IA, and, Physiologic Instruments, Inc., San Diego, CA). Transepithelial resistance was measured by applying a 2-mV pulse. Under these conditions, the FRT epithelia demonstrated resistances of 4 Κίλ^; cm² or more. The solutions were maintained at 27 °C and bubbled with air. The electrode offset potential and fluid resistance were corrected using a cell-free insert. Under these conditions, the current reflects the flow of Cl' through AF508-CFTR expressed in the apical membrane. The Isc was digitally acquired using an MP100A-CE interface and AcqKnowledge software (v3.2.6; BIOP AC Systems, Santa Barbara, CA).

[0017312. Identification of Correction Compounds |00174| Typical protocol utilized a basolateral to apical membrane Cl* concentration gradient. To set up this gradient, normal ringer was used on the basolateral membrane, whereas apical NaCl was replaced by equimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give a large CI’ concentration gradient across the epithelium. All experiments were performed with intact monolayers. To fully activate AF508-CFTR, forskolin (10 μΜ) and the PDE inhibitor, IBMX (100 μΜ), were applied followed by the addition of the CFTR potentiator, genistein (50 μΜ).

[00175) As observed in other ceil types, incubation at low temperatures of FRT cells stably expressing AF508-CFTR increases the functional density of CFTR in the plasma membrane. To determine the activity of correction compounds, the cells were incubated with 10 μΜ of the test compound for 24 hours at 37°C and were subsequently washed 3X prior to recording. The cAMP- and genistein-mediated Isc in compound-treated cells was normalized to the 27°C and 37°C controls and expressed as percentage activity. Preincubation of the cells with the correction compound significantly increased the cAMP- and genistein-mediated Isc compared to the 37°C controls.

[0017613. Identification of Potentiator Compounds [00177) Typical protocol utilized a basolateral to apical membrane CF concentration gradient. To set up this gradient, normal ringers was used on the basolateral membrane and was permeabilizcd with nystatin (360 pg/ml), whereas apical NaCl was replaced by equimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give a large CF concentration gradient across the -31CA 2986286 2017-11-21

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PCT/US2008/085456 epithelium. All experiments were performed 30 min after nystatin permeabilization. Forskolin (10 μΜ) and all test compounds were added to both sides of the cell culture inserts. The efficacy of the putative AF508-CFTR potentiators was compared to that of the known potentiator, genistein.

100178) 4. Solutions

<td> Basolateral solution (in mM):</td><td> NaCl (135), CaCl₂ (1.2), MgCI₂ (1.2), K₂HPO₄(2.4), KHPO₄ (0.6), N-2-hydroxyethylpiperazineN’-2-elhanesulfonic acid (HEPES) (10), and dextrose (10). The solution was titrated to pH 7.4 with NaOH.</td>

<td> Apical solution (in mM):</td><td> Same as basolateral solution with NaCl replaced with Na Gluconate (135).</td>

100179] 5. Cell Culture

1001801 Fisher rat epithelial (FRT) cells expressing AF508-CFTR _were used for Ussing chamber experiments for the putative AF508-CFTR modulators identified from our optical assays. The cells were cultured on Costar Snapwell cell culture inserts and cultured for five days at 37 °C and 5% CO₂ in Coon’s modified Ham’s F-J 2 medium supplemented with 5% fetal calf serum, 100 U/ml penicillin, and 100 pg/'ml streptomycin. Prior to use for characterizing the potentiator activity of compounds, the cells were incubated at 27 °C for 16 - 48 hrs to correct for the AF508-CFTR. To determine the activity of corrections compounds, the cells were incubated at 27 °C or 37 °C with and without the compounds for 24 hours.

100181] ά. Whole-cell recordings

The macroscopic AF508-CFTR current (Iafsok) in temperature- and test compoundcorrected NIH3T3 cells stably expressing AF508-CFTR were monitored using the perforatedpatch, whole-cell recording. Briefly, voltage-clamp recordings of Lfsos were performed at room temperature using an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc., Foster City, CA). All recordings were acquired at a sampling frequency of 10 kHz and low-pass filtered at 1 kHz. Pipettes had a resistance of 5 - 6 ΜΩ when filled with the intracellular solution. Under these recording conditions, the calculated reversal potential for Cf (Eci) at room temperature was -28 mV. All recordings had a seal resistance > 20 GO and a series resistance < 15 MO. Pulse generation, data acquisition, and analysis were performed using a PC equipped with a Digidata 1320 A/D interface in conjunction with Clampex 8 (Axon Instruments

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Inc.). The bath contained &lt; 250 μΐ of saline and was continuously perifused at a rate of 2 ml/min using a gravity-driven perfusion system.

[00182] 7. Identification of Correction Compounds [00183] To determine the activity of correction compounds for increasing the density of functional AF508-CFTR in the plasma membrane, we used the above-described perforated-patchrecording techniques to measure the current density following 24-hr treatment with the correction compounds. To fully activate AF508-CFTR, 10 μΜ forskolin and 20 μΜ genistein were added to the cells. Under our recording conditions, the current density following 24-hr incubation at 27°C was higher than that observed following 24-hr incubation at 37 °C. These results are consistent with the known effects of low-temperature incubation on the density of AF508-CFTR in the plasma membrane. To determine the effects of correction compounds on CFTR current density, the cells were incubated with 10 μΜ of the test compound for 24 hours at 37°C and the current density was compared to the 27°C and 37°C controls (% activity). Prior to recording, the cells were washed 3X with extracellular recording medium to remove any remaining test compound. Preincubation with 10 μΜ of correction compounds significantly increased the cAMP- and genistein-dependent current compared to the 37°C controls.

(00184] identification of Potentiator Compounds [00185) The ability of AF508 CFTR potentiators to increase the macroscopic AF508CFTR CF current (Iafsos) in NIH3T3 cells stably expressing AF508-CFTR was also investigated using perforated-patch-recording techniques. The potentiators identified from the optical assays evoked a dose-dependent increase in with similar potency and efficacy observed in the optical assays. In all cells examined, the reversal potential before and during potentiator application was around -30 mV, which is the calculated Eci (-28 mV).

10018619. Solutions

Intracellular solution (in mM): Cs-aspartate (90), CsCl (50), MgCh (1), HEPES (10), and 240 pg/ml amphotericin-B (pH adjusted to 7.35 with CsOH).

Extracellular solution (in mM): V-methyl-D-glucaminc (NMDG)-Cl (150), MgCl₂ (2), CaCl₂ (2), HEPES (10) (pH adjusted to 7.35 with HC1).

100187] 10. Cell Culture

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PCT/US2008/085456 (00188] N1H3T3 mouse fibroblasts stably expressing AF5O8-CFTR are used for wholecell recordings. The cells are maintained at 37 °C in 5% CO2 and 90 % humidity in Dulbecco’s modified Eagle’s medium supplemented with 2 mM glutamine, 10 % fetal bovine serum, 1 X NEAA, β-ΜΕ, 1 X pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For whole-cell recordings, 2,500 - 5,000 cells were seeded on poly-L-lysine-coated glass coverslips and cultured for 24 - 48 hrs at 27 °C before use to test the activity of potentiators; and incubated with or without the correction compound at 37 °C for measuring the activity of correctors.

)00189) /1. Single-channel recordings

100190) The single-channel aetdivities of temperature-corrected AF5O8-CFTR stably expressed in NIH3T3 cells and activities of potentiator compounds were observed using excised inside-out membrane patch. Briefly, voltage-clamp recordings of single-channel activity were performed at room temperature with an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.). All recordings were acquired at a sampling frequency of 10 kHz and low-pass filtered at 400 Hz. Patch pipettes were fabricated from Coming Kovar Scaling #7052 glass (World Precision Instruments, Inc., Sarasota, FL) and had a resistance of 5 - 8 ΜΩ when filled with the extracellular solution. The AF5O8-CFTR was activated after excision, by adding I mM Mg-ATP, and 75 nM of the cAMP-dependent protein kinase, catalytic subunit (PKA; Promega Corp. Madison, WT). After channel activity stabilized, the patch was perifused using a gravity-driven microperiusion system. The inflow was placed adjacent to the patch, resulting in complete solution exchange within 1 - 2 sec. To maintain AF508-CFTR activity during the rapid perifusion, the nonspecific phosphatase inhibitor F ( 10 mM NaF) was added to the bath solution. Under these recording conditions, channel activity remained constant throughout the duration of the patch recording (up to 60 min). Currents produced by positive charge moving from the intrato extracellular solutions (anions moving in the opposite direction) arc shown as positive currents. The pipette potential (V_p) was maintained at 80 mV.

(001911 Channel activity was analyzed from membrane patches containing &lt; 2 active channels. The maximum number of simultaneous openings determined the number of active channels during the course of an experiment. To determine the single-channel current amplitude, the data recorded from 120 sec of AF508-CFTR activity was filtered “off-line” at 100 Hz and then used to construct all-point amplitude histograms that were fitted with multigaussian functions using Bio-Patch Analysis software (Bio-Logic Comp. France). The total microscopic cunent and open probability (P_o) were determined from 120 sec of channel activity. The P_o was determined using the Bio-Patch software or from the relationship P_o = Vi(N), where I = mean

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100192] 12. Solutions

<td> Extracellular solution (in mM):</td><td> NMDG (150), aspartic acid (150), CaCI₂ (5), MgCh (2), and HEPES (10) (pH adjusted to 7.35 with Tris base).</td>

<td> Intracellular solution (in mM):</td><td> NMDG-C1 (150), MgCh (2), EGTA (5), TES (10), and Tris base (14) (pH adjusted to 7,35 with HC1).</td>

100193] 15. Cell Culture (00194] NIH3T3 mouse fibroblasts stably expressing AF508-CFTR are used for excisedmembrane patch-clamp recordings. The cells arc maintained at 37 °C in 5% CO> and 90 % humidity in Dulbecco's modified Eagle’s medium supplemented with 2 mM glutamine, 10 % fetal bovine serum, 1 X NEAA, β-ΜΕ, 1 X pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For single channel recordings, 2,500 - 5,000 cells were seeded on poly-L-lysine-coated glass coverslips and cultured for 24 - 48 hrs at 27 °C before use.

(00195] Using the procedures described above, the activity, i.e., EC50s, of Compound 1 has been measured and is shown in Table 4.

[00196] Table 4.

<td colspan="3"> ICfiQÆC® Bins: ♦++&lt;» 2.0 5.0 &lt;♦ . 1</td>

<td colspan="2"></td><td> t ♦♦ «i 400Λ&lt; ♦♦♦ </td>

<td> Cmpd-No.</td><td> . &gt;”?&lt; &gt; ;.</td><td> .BtonedMexEthcacyi 5 &gt; &lt; ·. x ♦ £ ' *1</td>

<td> 1</td><td> +++</td><td> +++ |</td>

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