TW201204353A - Formulations 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 - Google Patents

Abstract

The present invention relates to formulations 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 (Compound 1), pharmaceutical packs or kits thereof, and methods of treatment therewith.

Description

201204353 VI. OBJECTS OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to an oral formulation which comprises (and)-1-(2,2-·-fluorobenz[d][] as described herein. l,3]-oxycyclopentanyl)_n_( 1 _ . (2,3-dipropyl)-6-fluoro1-2-(1-pyridyl-2-methylpropan-2-yl ) _1 only - . Bow _5_ - base) propylene carbamide (Compound 1), water and polyethylene glycol (PEG). The oral formulation may additionally comprise a surfactant and additionally comprise a taste masking agent. Furthermore, the present invention relates to a method of treating a CFTR-mediated disease, such as cystic fibrosis, with such a formulation. The present application claims priority to U.S. Provisional Patent Application Serial No. 61/352,512, the entire disclosure of which is incorporated herein by reference. [Prior Art] CFTR is a cAMP/ATP-mediated anion channel that is expressed in a variety of cell types, including absorbing cells and secreting epithelial cells, where Cftr regulates the anion flux across the cell membrane and other ion channels and proteins. active. In epithelial cells, the normal function of CFTR is critical for maintaining electrolyte transport throughout the body, including beer and digestive tissue. The CFTR consists of about -1480 amino acids encoding a protein consisting of tandem repeats each containing a transmembrane domain of a transmembrane helix and a nucleotide binding domain. The two transmembrane domains are joined by a large polar regulatory (R) domain with multiple acidification sites that regulate channel activity and cell migration. The gene encoding CFTR has been identified and sequenced (see Greg〇ry, R. J. et al. (1990) Nature 347: 382-386; Rich, D. Ρ et al. 156806. doc 201204353 (1990) Nature 347:358-362), (Riordan, JR et al. (1989) Science 245: 1066-1073). Defects in this gene cause a sudden change in CFTR, which causes cystic fibrosis ("CF"), the most common fatal genetic disease in humans. In the United States, approximately one in every 2,500 babies is affected by cystic fibrosis. As many as 10 million people in the entire US population carry a single copy of a defective gene with no apparent adverse effects. In contrast, individuals with two copies of the CF-related gene suffer from the debilitating and lethal effects of CF, including chronic lung disease. In patients with cystic fibrosis, mutations in the CFTR endogenously expressed in the respiratory epithelium result in a decrease in apical anion secretion, resulting in ion and fluid transport imbalances. The resulting decrease in anion transport contributes to increased mucus accumulation in the lungs and accompanying microbial infections, ultimately leading to death in CF patients. In addition to respiratory diseases, CF patients often suffer from gastrointestinal problems and pancreatic insufficiency, and pancreatic insufficiency can cause death if left untreated. In addition, most women with cystic fibrosis have fertility and women with cystic fibrosis have reduced fertility. In contrast to the severe effects of two copies of the CF-related gene, individuals carrying a single copy of the CF-associated gene exhibit increased resistance to cholera and dehydration caused by diarrhea - this may explain the relatively high frequency of CF genes within the population. Sequence analysis of the CFTR gene of the CF chromosome reveals a variety of mutations that cause disease (Cutting, GR et al. (1990) Nature 346: 366-369; Dean, Μ. et al. (1990) Cell 61: 863: 870; and Kerem, BS Et al. (1989) Science 245:1073-1080; Kerem, BS# A (1990) Proc. Natl. Acad. Sci. USA 87:8447-8451). To date, more than 1000 disease-causing mutations in the CF gene have been identified as reported in the scientific and medical literature 156806.doc 201204353. The most common mutation is the absence of phenylalanine at position 508 of the CFTR amino acid sequence and is commonly referred to as ΔΡ508-ΡΤΙΙ. This mutation occurs in approximately 70% of cystic fibrosis cases and is associated with severe disease. Other mutations include R117H and G551D. The absence of residue 508 in F508-CFTR prevents the nascent protein from folding correctly. This can result in the mutant protein not leaving the ER and migrating to the plasma membrane. Therefore, the number of channels present in the cell membrane is much smaller than that observed in cells expressing wild-type CFTR. In addition to migration damage, mutations can also cause defective channel gating. A decrease in the number of channels in the cell membrane together with defect gates causes a decrease in anion transport through the epithelium' resulting in defective ion and fluid transport (Quinton, Ρ M. (1990), FASEB J. 4: 2709-2727). However, studies have shown that although less than wild-type CFTR, the decrease in the number of AF508-CFTR in the cell membrane is still functional (Dalemans et al. (1991), Nature Lond. 354: 526-528; Denning et al. Pasyk and Foskett (1995), J. Cell. Biochem. 270: 12347-50). In addition to AF5 08-CFTR, other disease-causing CFTR mutations that cause defect migration, synthesis, and/or channel gating can be up- or down-regulated to alter anion secretion and improve disease progression and/or severity. Although CFTR transports a variety of molecules in addition to anions, it is clear that this effect (transport anion) represents only one element of an important mechanism for transporting ions and water through the epithelium. Other elements include epithelial Na+ channels, ENaC, Na+/2Cr/K+ cotransporters, Na+-K+-ATPase pumps, and basal cell membrane K+ channels, which are responsible for the uptake of gas ions into cells. 156806.doc 201204353 These elements work together to effect directional transport across the epithelium via their selective expression and localization within the cell. The chloride ion absorption is carried out by the ENaC and CFTR and Na+-K+-ATPase pumps present on the apical membrane and the coordinated activity of the Cl_channels expressed on the side surface of the cell. The secondary active transport of gas ions from the luminal side causes intracellular chloride ion accumulation, and then chloride ions can passively leave the cell via the cr channel, causing vector transport. The Na+/2C17K+ cotransporter, the Na+-K+-ATPase pump and the basal membrane K+ channel, and the CFTR on the luminal side on the basal side surface coordinate the secretion of chloride ions via CFTR on the luminal side. Because water may never be actively transported by itself, it flows through the epithelium by virtue of the tiny transepithelial osmotic gradient produced by the overall flow of sodium and chloride ions. As discussed above, the absence of residue 508 in the phoenix AF508-CFTR prevents the nascent protein from folding properly, resulting in the mutated protein being unable to leave the ER and transport to the plasma membrane. Therefore, the amount of mature protein at the plasma membrane is insufficient and the gas ion transport in the epithelial tissue is significantly reduced. In fact, this cellular phenomenon of defective ER processing of the ΑΤΡ-binding cassette (ABC) transporter caused by the endoplasmic reticulum (ER) mechanism has been shown to be not only a potential basis for CF disease, but also for a wide range of other isolation and inheritance. The underlying basis of sexually transmitted diseases. Two ways in which an ER mechanism can fail can be by loss of coupling with the ER outlet of the protein causing the degradation, or by ER accumulation of the defective/misfolded protein [Aridor Μ et al, Nature Med., 5 ( 7), pp. 745-751 (1999); Shastry, BS et al., Neurochem. International, 43, pp. 1-7 (2003); Rutishauser, J. et al., Swiss Med Wkly, 132, 211-2221 (2002); Morello, JP et al., TIPS, 21, pp. 466-469 (2000); Bross P. et al., Human Mut., 14, pp. 186-198, 156806.doc 201204353 (1999)]

International PCT Publication No. 2010054138 (which is incorporated herein by reference in its entirety) discloses that difluoro benzo[d][l,3]dioxycyclopentene-5-yl)- (1_( 2,3_dipropyl)_6_良_2_(1)yl 2,methylpropyl)吲°_5_yl)cyclopropanecarbamide as a modulator of CFTR activity' is therefore effective in the treatment of CFTR-mediated diseases , such as cystic fibrosis, still. Need to be easily prepared and suitable for use as a therapeutic agent (and)-1-(2,2-difluorobenzo[_,3]dioxolane I group) A pharmaceutical composition of 2,3_dipropyl)·6|2-(1_trans-base_2-methylpropan-2-yl)·ιη_·_5 base) cyprodinil. SUMMARY OF THE INVENTION The present invention relates to (8)_1-(2,2-difluorobenzo[d][l'3]dioxolane-5-yl)-N servant (2,3_2) having the following structure Hydroxypropyl said! (1-1,yl- 2,methylpropan-2-yl)_1Η_^_5•yl) Oral formulation of cyproterone:

Compound 1 is indicated for the treatment or mitigation of a variety of CFTR mediated diseases. In one aspect, the invention provides (4) small (2,2-digang, 3) dioxycyclopentene _5_yl)-N servant (2,3-dihydroxypropyl n 156806.doc 201204353 A formulation of (1-hydroxy-2-mercaptopropan-2-yl)-1H-indol-5-yl)cyclopropanoguanamine (Compound 1), water, and poly(ethylene glycol) (PEG).

In another embodiment, the polyethylene glycol is selected from the group consisting of PEG 300, PEG 400, PEG 500, PEG 600, PEG 700, peg 800, PEG 900 or PEG 1000. In another embodiment, the polyethylene glycol is peg 400. In another embodiment, the concentration of Compound 1 is from about 重量. 〇 5% by weight to about 3% by weight. In another embodiment, the concentration of Compound 1 is from about 1% by weight to about 2% by weight. In another embodiment, the concentration of the hydrazine compound is about 0.1% by weight, 0.25% by weight, or 0.50% by weight. In another embodiment, the concentration of polyethylene glycol is from about 40 weight percent to about 60 weight percent. In another embodiment,

weight%.

In another embodiment, a sex agent. In another embodiment,

The step further comprises interfacial activity anionic, cationic 156806.doc 201204353 or nonionic surfactant. In another embodiment, the surfactant is a nonionic surfactant selected from the group consisting of vitamin Ε d-α-tocopherol peg 1000 succinate (vitamin ε TPGS), polysorbate vinegar 20 , polysorbate 40, polysorbate 6 〇, polysorbate 65, poly mountain 4 alcohol vinegar 80, burnt poly (ethylene oxide), poloxamine (p〇i〇xarnine), burnt base Polyglucoside, octylglucoside, mercapto maltoside, fatty alcohol, cetyl alcohol, oleyl alcohol, cocoamine MEA, cocoamine DEA, Solutol interface

活性 Active agents (such as polyethylene glycol stearate 15 (s〇lut〇1 HS 15)) and cocoamine TEA. In another embodiment, the surfactant is vitamin ε TPGS. In another embodiment t, the concentration of the surfactant is from about 5% by weight to about 15% by weight. In another embodiment, the concentration of the surfactant is from about (7) weight percent to about 12 weight percent. In another embodiment, the concentration of the surfactant is about 11% by weight. In another embodiment, the surfactant is about 5% by weight of Vitamin E TPGS. In another embodiment, the concentration of Compound 1 is from about 5% by weight to about 3% by weight, and the concentration of polyethylene glycol is about 40% by weight. /. Up to about 6 〇 weight β /. And the concentration of the surfactant is from about 5% by weight to about 5% by weight. In another embodiment, the concentration of Compound 1 is from about 5% by weight to about 3% by weight, the polyethylene glycol is PEG 400 and the concentration is from about 4% by weight to about 3% by weight and the surfactant is about 5 6% by weight to 15% by weight of vitamin E TPGS. 3〇 g to 50 g. In another embodiment, in another embodiment, the formulation of Compound 1 is about another embodiment, the formulation is from about 35 g to 45 g (156806.doc 201204353, the formulation is about 40 g. In another embodiment, the formulation of Compound 1 comprises the following: Component (9) Compound 1 0.05 PEG 400 19.95 Vitamin ETPGS 4.48 Water 15.52 In another embodiment, the formulation of Compound 1 comprises the following: Component (g) Compound 1 0.10 PEG 400 19.90 Vitamin ETPGS 4.48 Water 15.52 In another embodiment, the formulation of Compound 1 comprises the following: Component (g) Compound 1 0.20 PEG 400 19.80 Vitamin ETPGS 4.48 Water 15.52 In another embodiment, Compound 1 Formulations include the following: Component (g) Compound 1 0.30 PEG 400 19.70 Vitamin ETPGS 4.48 Water 15.52 In another embodiment, the formulation of Compound 1 comprises the following: 156806.doc -10- 201204353 Component (% by weight) Compound 1 0.10-0.80 PEG 400 48-51 Vitamin ETPGS 10-12 Water 37-40 In another embodiment, the formulation of Compound 1 comprises the following: Component weight (% by weight) Compound 1 0.20-0.60 PEG 400 49-50 Vitamin ETPGS 10-12 Water 37-40 In another embodiment, the formulation of Compound 1 comprises the following: Component (% by weight) Compound 1 0.25 PEG 400 49.75 Vitamin ETPGS 11.20 Water 38.80 In another embodiment, the formulation of Compound 1 comprises the following: Component (% by weight) Compound 1 0.50 PEG 400 49.50 Vitamin ETPGS 11.20 Water 38.80 In another embodiment, the invention provides any further comprising a taste masking agent The above formulation. In another embodiment, the invention provides any of the above formulations further comprising other therapeutic agents. In another embodiment, the additional treatment 156806.doc -11 - 201204353 is selected from the group consisting of mucolytic agents, bronchi An expander, an antibiotic, an anti-infective agent, a CFTR modulator or a nutrient other than a compound. In another aspect, the invention provides a method of treating a CFTR-mediated disease, the disease being selected from cystic Fibrosis, asthma, smoke-induced COPD, 'lingile bronchitis, sinusitis, constipation, pancreatitis, pancreatic function, innate, by congenital No vas deferens on both sides (CBAv (7) caused by male infertility, mild lung disease, idiopathic pancreatitis, allergic bronchial and pulmonary sputum disease (ΑΒΡΑ), liver disease, hereditary emphysema, hereditary pigmentation Septicosis coagulation-fibrinolytic deficiency, protein c deficiency, type I hereditary angioedema, insufficient lipid processing, familial hypercholesterolemia, type I chylomicronemia, no beta lipoproteinemia, lysozyme Body accumulation disease, ^ cell disease / pseudo-Hellez disease (pseud〇_Hurler), mucopolysaccharidosis, Sandof's disease (Tand_Sachs), π-type Kegler - Crigler-Najjar type II, endocrine disease/hyperinsulinemia, diabetes, Laron dwarfism, myeloperoxidase deficiency, primary parathyroid dystrophy, black Oncoma, type I glycolysis CDG, congenital thyroid hyperactivity, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency, diabetes insipidus (DI), neurophysiological DI, renal DI, just Gram-Dy's syndrome (Charc〇t_Marie)

Tooth syndrome), PerHzaeus_Merzbacher disease, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive nucleus Itching, Pick's disease, several polyglutaminine neuropathic disorders, Huntington's disease, type I spinal cord I56806.doc J2 201204353 Brain ataxia, spinal medullary muscular dystrophy, dentate nucleus Pale globule hypothalamic atrophy, muscular dystrophy, spongiform encephalopathy, hereditary Creutzfeldt-Jakob disease (caused by prion protein processing defects), Fabry disease, stu Straussler-Scheinker syndrome, COPD, dry eye disease, Sjogren's disease, osteoporosis, osteopenia, Gorham's Syndrome, chloride channel disease, Congenital myotonia (Thoms on and Becker), Bartter's syndrome type III Dent's disease, excessive seizures, epilepsy, excessive startle, lysosomal storage disease, Angelman syndrome, primary ciliary dyskinesia (PCD), hereditary cilia structure and / Or a functional disorder, PCD with visceral retrograde (also known as Kartagener syndrome), PCD with no visceral retrograde or cilia dysplasia. In another embodiment, the invention provides a method of treating a CFTR-mediated disease selected from the group consisting of cystic fibrosis, COPD, emphysema, dry eye or osteoporosis in a subject, the method comprising administering to the individual an effective amount Any of the above compounds 1 formulations. In another embodiment, the CFTR mediated disease is cystic fibrosis. In another embodiment, the method comprises administering another therapeutic agent. In another embodiment, the additional therapeutic agent is selected from the group consisting of a mucolytic agent, a bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR modulator other than Compound 1, or a nutrient. 156806.doc -13- 201204353 In another aspect, the invention provides a pharmaceutical pack or kit comprising a formulation of any of the above Compounds 1 and instructions for use thereof. The methods described herein can be used to prepare the compositions of the present invention. The amounts and characteristics of the components used in such methods will be as described herein. [Embodiment] Definitions As used herein, the following definitions shall apply unless otherwise stated. The term "rCFTR" as used herein means a cystic fibrosis transmembrane conductance regulator or a mutation thereof having modulatory activity, including but not limited to AF508 CFTR and G551D CFTR (for CFTR mutations, see for example hUP:/ /www.genet.sickkids 〇n ca/cftr/). The term "r-regulation" as used herein means to increase or decrease, for example, activity by a measurable amount. Unless otherwise indicated, structures described herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational) forms) of the structure, such as each. The symmetry center of the ruler and the 8 configuration, (2) and (the magic double bond isomer and the (Ζ) and (Ε) configuration isomers. Therefore, the single stereochemical isomers and enantiomers of the compounds of the invention Mixtures, diastereomeric and geometric isomers (or conformational isomers) mixtures are within the scope of the invention. All tautomeric forms of compound 1 are included herein. For example, the compound oxime may exist as a tautomer. This article includes both of them:

156806.doc -14· 201204353 In addition, unless otherwise stated, the structure described herein also means that && differs only in the presence of seven grams of July or multiple isotopically enriched atoms. Compound. For example, 'where - or a plurality of hydrogens, a group of people, or a compound substituted with one or more magnetic atoms by C- or "C-rich enthalpy! In the scope of the present invention; . Such compounds are useful, for example, as analytical tools, as described in biological assays, or as compounds having improved therapeutic modalities.

The term "protecting group" as used herein, abbreviated as P, refers to any chemical group that introduces a molecule to obtain chemical selectivity in a subsequent chemical reaction by chemical modification of the functional group. Non-limiting examples of alcohol protecting groups include ethenyl (tetra), benzamyl (BZ), benzyl (Bn), methoxyethoxycarbamidine (MEM), dimethoxytrityl (DMT), methoxymethyl bond (MOM), decyloxytrityl (MMT), p-methoxymethyl ether (PMB), pentyl (Piv), tetrahydropyranyl (THp) , trityl (τ〇 and trimethyldecyl (TMS). In one embodiment, the protecting group is Bn, the structure is -CH2C6Hy ^ The abbreviation "DCM" means di-methane. The abbreviation "IpA" means Isopropyl alcohol. The abbreviation "DMSO" means dimethyl slate. The abbreviation "MTBE" means thiol second butyl sulphate. The abbreviation "THF" means tetrahydrofuran. The abbreviation "TEA" means triethylamine. For example, Pd ( The abbreviation "dba" in dba) 2 means diphenylaryleneacetone. For example, the abbreviation r dppf" in Pd(dppf)Cl2 means i-bis(diphenylphosphino)ferrocene. Method for preparing compound 1 Compound 1 Is (i?)-l-(2,2-difluorobenzo[d][i,3]dioxocyclopentene_5yl)-N-(l-(2,3-di-propyl) Base) -6-1·2-(ΐ_ 经基·2_曱基丙_2 156806.doc •15· 201204353 基)-1Η-吲哚-5-based Cyclopropanecarboxamide Amides and in one embodiment, the process according to section 1-3 of the acid chloride with an amine is prepared by coupling of the root portion Scheme 1. Preparation of compound 1.

Wherein Compound 2 was prepared according to Scheme 2. 156806.doc 16- 201204353 Scheme 2. Preparation of Compound 2·

EtO^(

Br~CI

NaOH Bu4NBr

Pd(dba)2, /-BU3P ---► Na3P04, toluene, Η20,7〇π

OEt CN

3NHCI, DMSO, , 丨 750C

1. NaOH 2. HC1

Compound 3 was prepared according to Scheme 3. 156806.doc -17- 201204353 Scheme 3. Preparation of compound 3.

NBS

〇zN F

XX

Br NH2

1. ΐ>χ^〇8η -^ 2. H2/PKS)/C 3. TsOH

© H3N F

TsO@

Pd(OAc)2 dppb K2C03 MeCN

2. (MeCN)2PdCl2 MeCN, 80 °C 3. Silicone filtration

156806.doc -18- 201204353 The formulation of Compound 1 can be prepared according to the following scheme. Disperse PEG in a container and add a weighed amount of Compound I to the PEG and mix at high temperature in a water bath until a solution is formed. Stop heating and stir at room temperature until the solution equilibrates to the solution of PEG and Compound 1. Add a weighed amount of surfactant and mix

In one embodiment, the formulation of Compound 1 can be prepared according to the following scheme. 156806.doc -19- 201204353 Disperse PEG 400 in a container Add a weighed amount of Compound 1 to PEG 400 and stir at high temperature in a water bath until the solution is formed to stop heating and stir at room temperature until the solution equilibrates A weighed amount of the aqueous solution of vitamin ETPCS is added to the solution of PEG 400 and Compound 1 and mixed in one embodiment, and the surfactant is added as a previously prepared aqueous solution. In another embodiment, the concentration of the aqueous surfactant solution is from about 20 to 25 weight percent. In another embodiment, the concentration of the aqueous surfactant solution is about 22% by weight. In another embodiment, the surfactant is Vitamin E TPGS and has a concentration of about 22% by weight. In another embodiment, the elevated temperature is from about 30 °C to 50 °C. In another embodiment, the elevated temperature is between about 35 ° C and 45 ° C. In another embodiment, the elevated temperature is about 40 °C. Formulations of Compound 1 can also be prepared by the following scheme. 156806.doc -20- 201204353

Disperse PEG in a container, add a weighed amount of Compound 1 and a surfactant to the PEG and stir at a high temperature in a water bath, until the solution is formed, stop heating and stir at room temperature until the solution is equilibrated. In one embodiment, a formulation of Compound 1 can be prepared according to the following scheme.

156806.doc -21- 201204353 Disperse PEG 400 in a container ♦ Add a weighed amount of Compound 1 and Vitamin E TTGS to PEG 400 and stir at high temperature in a water bath until the solution is formed to stop heating and stir at room temperature Until the solution is equilibrated with water in this set of examples, Compound 1 and a surfactant (eg, Vitamin E TPGS) are dissolved in PEG (eg, PEG 400), followed by dilution. A surfactant in the form of an aqueous solution was previously added, which provides dilution. In another embodiment, the elevated temperature is between about 30 ° C and 50 ° C. In another embodiment, the elevated temperature is between about 35 ° C and 45 ° C. In another embodiment, the elevated temperature is about 40. . . In the canine study, the Compound 1 formulation of the present invention showed excellent in vivo exposure. PEG co-solvent and vitamin E TPGS-induced microcell formation increased the solubility of Compound 1 from 7 micrograms per milligram of solution to several milligrams. Use, Formulation, and Administration of Aqueous Formulations 156806.doc -22- 201204353 In one aspect of the invention, there is provided an aqueous formulation comprising Compound 1, water and polyethylene glycol as described herein, and optionally Other agents (such as taste masking agents and/or flavoring agents) and other pharmaceutically acceptable carriers, adjuvants or vehicles are included. In certain embodiments, the formulations further comprise one or more additional therapeutic agents as appropriate. It will also be appreciated that Compound 1 can exist as a pharmaceutically acceptable derivative or prodrug. According to the present invention, pharmaceutically acceptable derivatives or prodrugs include, but are not limited to, esters, salts of such esters or, upon administration of a patient in need thereof, capable of providing, directly or indirectly, a compound as otherwise described herein or Any other adduct or derivative of its metabolite or residue. 1. Polyethylene glycol Polyethylene glycol (PEG) is a polyether compound and includes polyethylene oxide (PEO) and polyoxyethylene (POE). PEG, PEO and POE refer to oligomers or polymers of ethylene oxide. The three names are chemically synonymous, but historically PEG tends to refer to oligomers and polymers with molecular masses below 20,000 g/mol, PEO refers to polymers with molecular masses above 20,000 g/mol and POE refers to Any molecular mass of polymer. As used herein, PEG refers to any molecular weight polyethylene glycol suitable for use in a pharmaceutical formulation. Such suitable polyethylene glycols are well known in the art; see, for example, http://www.medicinescomplete.com/mc/excipients/current, which is incorporated herein by reference. Exemplary PEGs include low molecular weight PEGs such as PEG 200, PEG 300, PEG 400, and the like. The number after the term "PEG" indicates the average molecular weight of the particular polymer. For example, PEG 400 is a polyethylene glycol 156806.doc -23-201204353 polymer having an average molecular weight of about 400. In one embodiment, the average molecular weight of the PEG is from about 2 Torr to about 6 Torr. In another embodiment, the PEG is PEG 4 (e.g., PEG having a molecular weight of from about 38 angstroms to about 420 g/mol). 2. Surfactant The surfactant reduces the interfacial tension between water and an organic compound such as Compound 1 by adsorbing it at the interface of the water-compound, thereby promoting the wettability and dissolution of the compound 1. The surfactant increases the solubility of Compound 1 via microcell formation and contributes to the stabilization of the aqueous solution. Surfactants are generally divided into four groups: anionic, cationic, nonionic, and zwitterionic (dual charge). In a preferred embodiment, the surfactant is an anionic, cationic or nonionic surfactant. The anionic surfactant may be selected from the group consisting of lauryl sulfate, lauryl sulfonium salt, lauryl ether sulfate, alkyl benzene sulfonate, butyrate, hexanoate, octoate, citrate, Laurate, myristate, palmate, stearate, arachidate, behenate, myristate, palmitate, oleate, linoleate , α_linolenic acid oleate, peanut oleate, eicosapentaenoate, sinamate or docosahexaenoate. The cationic surfactant may be selected from the group consisting of cetyltrimethylammonium bromide, cetylpyridinium pentoxide, polyethoxylated tallow amine, gasified benzalkonium chloride, and benzethonium chloride. The nonionic surfactant may be selected from the group consisting of vitamin strontium derivatives, polysorbate bismuth oxime poly(ethylene oxide), poloxamine, flaming poly 156806.doc -24- 201204353 glycosides, octyl Glucoside, mercapto maltoside, fatty alcohol, cetyl alcohol, oleic acid, cocoamine MEA, cocoamine DEA and cocoamine TEA. In particular, the nonionic surfactant is a vitamin E derivative, vitamin E, a phenolic polyethylene glycol 1000 succinate (vitamin £ TPGS). Nonionic surfactants also include the trade name s〇lut〇12PEG derivatives. By way of example - polyethylene glycol stearate 15 (s〇lut〇l HS 15;) is prepared by reacting 15 moles of ethylene oxide with 1 mole of hydroxystearic acid. The oral formulation of the present invention typically comprises from about 5 to about 15% by weight of an interfacial active agent. In another embodiment, the concentration of the surfactant is from about 1 Torr to about 12% by weight. In another embodiment, the concentration of the surfactant is about n weight 〇/〇. 3. Masking agent and/or flavoring agent As described above, the oral formulation of Compound 1 preferably includes a taste masking agent. These taste masking agents are metal and soil metal chlorides, including gas, lithium, potassium chloride, magnesium chloride and calcium chloride. Preferred is sodium chloride. The taste masking agent is usually included in the suspension in a taste-masking amount, and the amount of the taste-masking agent is usually from about 0.5 to about 2.0% by weight based on the weight of the suspension. For other salts, the equivalent molar amount can be calculated. In the presence or absence of other sweeteners and/or flavoring agents, his taste masking agent includes sugar. When used, the flavoring agent may be selected from the group consisting of synthetic flavor oils and extracts of flavoring and/or natural oils, plant leaves, flowers, fruits, and the like, and combinations thereof. Such oils may include cinnamon oil, wintergreen oil, walnut oil, clove oil, bay oil, large sesame oil, eucalyptus oil, thyme oil, cedar leaf oil, nutmeg oil, sage oil, bitter almond oil and Cinnamon oil. Vanilla, orange peel oil (including lemon, orange, grape, sour pomelo and grapefruit) and fruit flavor (including 156806.doc -25· 201204353 including rhyme, incense i, pear, peach, strawberry, raspberry, #桃, Hazelnuts, stalks, apricots, etc. are also suitable for use as spices. The amount of flavoring can vary depending on a number of factors, including the desired sensory effect. Typically, the flavoring agent will be present in an amount from about 0. 01 to about 1.0% by weight, based on the total weight of the suspension. The +1/3 formulation may also comprise a pharmaceutically acceptable carrier, adjuvant or vehicle suitable for use in the particular dosage form described above for the removal of water, as used herein, #includes any and all solvents, Diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, granules or emulsifiers, preservatives, solid binders, lubricants and the like.

Remington’s Pharmaceutical Sciences, 16th Edition, E w

Manin (MaCk Pubiishing Co., Easton, pa, 198 〇) discloses various carriers for the formulation of pharmaceutically acceptable compositions and known techniques for preparing them. Unless any conventional carrier medium is incompatible with a compound of the invention, such as by any undesirable biological effect or otherwise in a detrimental manner with any other component of a pharmaceutically acceptable composition, Pre-J The use of such conventional carrier media is within the scope of the invention. Some examples of substances that can be advertised as pharmaceuticals, acceptable carriers include, but are not limited to, bismuth: parental agents, alumina, aluminum stearate, lecithin, blood, 匕*, poor white (啫Such as human blood & albumin, a mixture of saturated plant fatty acids, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride), zinc salts, colloidal cerium oxide , 三夂 = A, vinylpyrrolidone, polyacrylate, wax, polyethylene _ polyoxypropylene block polymer, lanolin, sugar (such as lactose, glucose and sucrose); starch, various: jade starch and Potato powder; malt; gelatin; talc; #形剂, such as = 156806.doc -26- 201204353 fat and suppository wax; oil, 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; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution (Ringer, ss 〇luti〇n); and ethanol, and other non-toxic compatible lubrication (##12-sodium sulphate and magnesium stearate), as well as coloring agents, release agents, coating agents, sweeteners, fragrances, preservatives and anti-deuteration agents may also be present in the cylinder compound. Subject to the judgment of the deployer. Use of a Compound and a Pharmaceutically Acceptable Composition In yet another aspect, the present invention provides a method of treating a condition, disease or condition caused by a scale. In certain embodiments, the invention Provided is a method of treating a disease & disease or condition caused by insufficient CFTR activity, the method comprising administering to an individual in need thereof, preferably a mammal, an oral formulation comprising Compound 1 as described herein. The "CFTR-mediated disease" used in the present invention is a disease selected from the group consisting of cystic fibrosis, asthma, smoke-induced COPD, chronic bronchitis, sinusitis, constipation, visceral inflammation, and dysfunction of the viscera. Male infertility, mild lung disease, idiopathic pancreatitis, allergic bronchial and pulmonary sputum disease (ABPA), liver disease, hereditary emphysema, caused by congenital no vas deferens (CBAVD) Hereditary golden pigmentation, coagulation-fibrinolytic deficiency, protein C deficiency, hereditary vascular leeches, lipid processing deficiencies, familial hypercholesterolemia, spastic chylomicronemia , no beta lipoproteinemia, lysosomal storage disease, [cytopathic / pseudo-Helle disease, sticky stagnation, Shandorf's disease / Thai. Sa's disease, „ type Kerry (four), _ 156806.doc -27- 201204353 Najal syndrome, endocrine disease/Gottung's disease, diabetes, Lalong's dwarfism, myeloperoxidase deficiency, primary hypothyroidism, melanoma, Type 1 glycolysis (10), congenital thyroid hyperactivity, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency, diabetes insipidus (9), neurophysiological DI, renal DI, Chuck _ Ma Lidos's syndrome, Pez' disease, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive pruritus, Pico's disease, several poly-proline Neurological disorders, Huntington's disease, (4) Spinal cord ataxia, vertebral medullary muscular atrophy, dentate nucleus, red nucleus, pale cerebral atrophy, muscular dystrophy, spongiform encephalopathy, hereditary CJD (due to 朊Defects in viral protein processing), Fabry disease, Stresler-Sykes Group, C〇PD, dry eye, Hugh's disease, osteoporosis, osteopenia, Gohan's syndrome, gas ion channel disease, congenital myotonia (Thomson and Becker type) m type Bart Syndrome, Dunte's disease, excessive seizures, epilepsy, excessive startle, lysosomal storage disease, Angman's syndrome, primary ciliary dyskinesia (pcD), hereditary ciliary structure and/or functional disorders, accompanied by Visceral retrograde pcD (also known as Cartagen syndrome), visceral retrograde PCD or cilia dysplasia. In certain embodiments, the invention provides a method of treating a cFTR mediated disease in an individual Which comprises the step of administering to an individual an effective amount of an oral formulation comprising Compound 1 as described herein. According to another embodiment, the invention provides a method for treating cystic fibrosis, emphysema, COPD, dry eye disease in an individual Or a method of osteoporosis comprising the steps of an oral formulation 156806.doc • 28· 201204353 comprising the compound described herein. According to the present invention, the "effective amount" of the oral formulation of Compound 1 is an amount effective to treat or alleviate the severity of any of the above diseases. In certain embodiments, the oral formulations of Compound 1 described herein are useful for treating or reducing the severity of cystic fibrosis in individuals exhibiting residual CFTR activity in the apical membrane of the respiratory epithelium and non-respiratory epithelium. The presence of residual CFTR activity at the epithelial surface is readily detectable using methods known in the art, such as standard electrophysiological, biochemical or histochemical techniques. These methods use in vivo or ex vivo electrophysiological techniques, measuring sweat or salivary cr concentrations, or ex vivo biochemical or histochemical techniques for monitoring cell surface density to identify CFTR activity. Using such methods, residual CFTR activity can be readily detected in a variety of patients with different mutant heterotypic or homotypic junctions, including patients most commonly with mutated ΔΡ508 homozygous or heterotypic junctions. In one embodiment, the oral formulation of Compound 1 described herein is useful for treating certain genotypes that exhibit residual CFTR activity (eg, Class III mutations (regulated or gated impaired), Class IV mutations (conductivity changes) ) or cystic fibrosis in patients with V-type mutations (reduced synthesis)) (Lee R. Choo-Kang, Pamela L., Zeitlin, Type /, //, III, IV, and V cystic fibrosis Tans membrane Conductance Regulator Defects and Opportunities of Therapy, Current Opinion in Pulmonary Medicine 6:521-529, 2000). Other patient genotypes exhibiting residual CFTR activity include heterozygous ligation of one of these classes or heterozygous ligation with any other class of mutations (including class I mutations, class II mutations or unclassified 156806.doc -29-201204353 mutations) The patient. In one embodiment, the oral formulations of the compounds described herein are suitable for treating certain clinical phenotypes (eg, moderate to mild clinical phenotypes typically associated with residual CFTR activity in the epithelial apical membrane). Cystic fibrosis or reduce its severity. These phenotypes include patients with pancreatic insufficiency or patients diagnosed with idiopathic pancreatitis and congenital bilateral vas deferens or mild lung disease. The exact amount required will vary from individual to individual, depending on the species, age and general condition of the individual, the severity of the infection, the particular agent, its mode of administration, and the like. The compounds of the present invention are preferably formulated in unit dosage form gamma for ease of administration and to achieve dose uniformity. The expression "unit dosage form" as used herein refers to a physical individual list of agents suitable for the patient being treated. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of the correct medical judgment. The particular effective amount for any particular patient or organism will depend on a number of factors, including the severity of the condition being treated and the severity of the condition; the particular composition of the particular compound used; the age, weight, and general health of the patient Sex and diet, time of administration of the particular compound used, route of administration; similar factors well known in the art in combination with the particular compound employed. As used herein, synonymously refers to animals, preferably and excretion rates; duration of treatment or concurrent use of drugs; and in medicine, the terms "patient" and "individual" mammals and preferably humans. In a second embodiment, the compound i can be administered at a dose of from 1 mg to about 50 mg per kilogram of body weight per day, preferably from about 1 mg to about 25, 156806.doc -30-201204353, one or more times per day, To achieve the desired therapeutic effect. In certain embodiments, the dosage of Compound 1 in a unit dosage form is from about 5 mg to about 2,000 mg. In another embodiment, the dose of Compound 1 is from about 2 mg to about 900 mg. In another embodiment, the combined dose is from about 3 mg to about 800 mg. In another embodiment, the dose of the compound hydrazine is from about 4 mg to about 700 mg. In another embodiment, the dose of Compound i is from about 5 mg to about 600 mg. ◎ It should also be understood that the oral formulation of Compound 1 described herein can be used in combination therapy, ie, the oral formulation of Compound 1 can be administered simultaneously with, prior to, one or more other desired therapeutic agents or medical procedures. Give or pay after it. The combination of therapies (therapeutics or procedures) used in the combination regimen will take into account the compatibility of the desired therapeutic agent and/or procedure and the desired therapeutic effect to be achieved. It will also be appreciated that the therapy used may achieve the desired effect on the same condition (eg, the compound of the invention may be administered concurrently with another active agent for treating the same condition) or it may effect different effects (eg, control of any side effects). . As used herein, other therapeutic agents that are normally administered to treat or prevent a particular disease or condition are referred to as "appropriate for the disease or condition being treated." In one embodiment, the other agent is selected from the group consisting of a mucolytic agent, a bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR modulator or a nutrient other than the compound of the present invention. In one embodiment, the other therapeutic agent is an antibiotic. Examples of effective antibiotics included in this article include tobramycin (including tobramycin inhalation powder (TIP)), azithromycin (azithromycin), and antrexin (four) generation 〇 _ 156806.doc -31- 201204353 ( Including a nebulized form of antrexam), ainikacin (including its liposome formulation), ciprofl〇xacin (including its formulation suitable for administration by inhalation) , levofloxacin (including its nebulized formulation) and a combination of two antibiotics (such as fosfomycin (f〇sf〇mycin) and tobramycin). In another embodiment, the other agent is mUC〇lyte. Exemplary mokettes that are effective herein include PUlm〇Zyme8. In another embodiment, the other agent is a bronchodilator. Exemplary bronchodilators include albuterol, metaproterolol sulfate, pirbuterol acetate, saimeter〇i or tetrabuline. Sulfate). In another embodiment, the other agent is effective to restore fluid on the surface of the lung trachea. These agents modify the movement of cells and extracellular salts, allowing the mucus in the lungs to hydrate more, thus making it easier to remove. Illustrative of such agents include hypertonic saline, denufoss 1 tetras dium ([[(3S,5R)-5-(4-amino-2-yloxy) Pyrimidine-1-yl)_3_yloxycyclopentan-2-yl]methoxy-hydroxyphosphonium][[[(2Κ,3δ,4Ιι,5Κ)_5_ (2,4-di- oxetidine Dihydroxyoxycyclopenta-2-yl]methoxy-hydroxyphosphonium]oxy-hydroxyphosphonium]hydrogen phosphate) or bronchitol (mannitol inhalation formulation). In the examples, the other agents are anti-inflammatory agents, which may also attenuate the inflammation of the lungs. Examples of effective agents herein include ibuprofen, docosahexaenoic acid φΗΑ, sildena. Sildenafil, 156806.doc -32- 201204353 Inhalation of glutathione, pioglitazone, hydroxychloroquine or simavastatin ° In another embodiment, The other agent is a CFTR modulator other than the compound 1, that is, an agent having an action of regulating CFTR activity. Exemplary such agents include atalulu ("PTC124®"; 3-[5_(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid), West Sinapultide, lancovutide, depelestat (human recombinant neutrophil elastase inhibitor), cobiprostone (7-{(2R, 4aR, 5R,7aR)-2-[(3S)-l,l-difluoro-3-methylpentyl]-2-hydroxy-6-oxooxy octahydrocyclopenta[b]pyran-5 -yl}heptanoic acid) and sense (5-hydroxy-2,4-di-t-butyl-phenyl)-4-o-oxy-111-quinoline>-3-cartoamine. In another embodiment, the other agent is a nutrient. Exemplary nutrients include pancreatic lipase (pectinase replacement), including Pancrease®, Pancreacarb®, Ultrase® or Creon®, Liprotomase® (formerly Trizytek®), Aquadeks® or 耽-glycopeptide inhaler. Among other nutrients are pancreatic lipase. In another embodiment, the other agent is a compound selected from the group consisting of gentamicin, curcumin, cyclamate, 4-phenylbutyrate, miglustat, felol Felodipine, nimodipine, Philoxin B, geniestein, apigenin, cAMP/cGMP modulators (such as rolipram, west) Defera, milrinone, tadalafil, amrinone, isoproterenol, salbuta 156806.doc -33· 201204353 and almeterol, go Deoxyspergualin, HSP 90 inhibitor, HSP 70 inhibitor, proteasome inhibitor (such as epoxomicin, lactaCyStin) and the like. In another embodiment, the other agent is a compound disclosed in WO 2004028480, WO 2004110352, WO 2005094374, WO 2005120497 or WO 2006101740. In another embodiment, the other agent is a benzo(c) quinolizolate rust derivative exhibiting CFTR modulating activity or a benzopyran derivative exhibiting CFTR modulating activity. In another embodiment, the other agents are disclosed in US Pat. No. 7,202,262, US Pat. No. 6,992,096, US Pat. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. Publication No Compound. In another embodiment, the other agents are compounds disclosed in WO 2004080972, WO 2004111014, WO 2005035514, WO 2005049018, WO 2006099256, WO 2006127588 or WO 2007044560. These combinations are useful for treating the diseases described herein, including cystic fibrosis. These combinations also apply to the kits described herein. The amount of other therapeutic agent present in the compositions of the present invention will not exceed the amount normally administered in a composition comprising the therapeutic agent as the sole active agent. Preferably, the amount of the additional therapeutic agent in the presently disclosed compositions will be in the range of from about 50% to 100% of the amount normally present in compositions comprising 156806.doc.34 to 201204353 which is the sole therapeutically active agent. In order to more fully understand the invention described herein, the following examples are set forth. It is to be understood that the examples are for illustrative purposes only and should not be construed as limiting the invention in any way. EXAMPLES Methods and Materials Vitride® (hydrogenated bis(2-decyloxyethoxy)aluminum [or NaAlH2(OCH2CH2OCH3)2], 65% by weight hydrazine solution) was purchased from Aldrich Chemicals. 3-Fluoro-4-nitroaniline was purchased from Capot Chemicals. 5-Bromo-2,2-difluoro-1,3-benzodioxolane was purchased from Alfa Aesar. From Saltigo (a subsidiary of Lanxess Corporation), 2,2-difluoro-1,3-benzodioxocyclopentene-5-carboxylic acid was purchased. In the present application, when the name of the compound may not correctly describe the structure of the compound, the structure replaces the name and is subject to the structure. Synthesis of Compound 1 Acid moiety Synthesis of (2,2-difluoro-1,3-benzodioxocyclopenten-5-yl)-1-acetic acid ethyl ester-acetonitrile

〇 carbazole (four) FV°Y^ 〇

Et0A/CN Na3P04, ΡΛ〇ΑΑγΑ〇Β

A stupid, H20, 70 〇C iN reactor was purged with nitrogen and charged with 900 mL of toluene. The solvent is degassed by nitrogen purge for not less than 16 hours. Then successively load the reactor into 156806.doc •35· 201204353

Na3P04 (155.7 g, 949.5 mmol) and bis(dibenzylideneacetone)palladium (0) (7.28 g, 12.66 mmol). At 23. (1: 10% w/w hexane solution of butylphosphine (5丨23 g, 25.32 mmol) was added from the addition funnel of nitrogen purge for 1 min. The mixture was stirred for 5 min, at this time ^ 5-Bromo-2,2-difluoro-1,3-benzodioxolane (75 g, 316.5 mmol) was added in minutes. After stirring for another 50 minutes, the mixture was charged with cyanoacetate B over 5 minutes. Ester (71.6 g, 633.0 mm 〇l), then water (45 mL) was added in portions. The mixture was heated to 70 <t over 40 minutes and the reaction was converted to a percentage of product by HpLC analysis every 1-2 hours. After complete conversion was observed (usually 100% conversion after 5-8 hours), the mixture was cooled to 2 (TC -25 ° C and passed through diatomaceous earth; care. The mashed earth was washed with toluene (2 x 450 mL) and vacuumed Concentrate the combined organics to 300 mL at 60 ° C - 65 ° C. Add 225 mL of DMSO to the concentrate and concentrate in vacuo at 70 ° c _8 (rc until the active distillation of the solvent is stopped. Cool the solution to 2 :. (:-25. (: and diluted to 900 mL with DMSO to prepare for step 2. NMR (500 MHz, CDC13) δ 7.16-7.10 (m, 2 Η), 7.03 (d, 7 = 8.2 Hz, 1H ), 4.63 (s, ih) 4 .19 (m, 2H),1·23 (t, J=7.i Hz, 3H). Synthesis (2,2--gas benzodioxanthene-5-yl)_Budge

3N HC1, '-1

DMSO, 75 0C Add 3 N HC1 to DMSO solution of (2,2-di-I,3-benzodioxol-5-yl)-1-acetic acidacetonitrile in DMSO over 20 min ( ;617; 3 mL, 1.85 mol) while maintaining the internal temperature < 4 (TC. followed by heating the mixture to 75 ° C via i 156806.doc -36 - 201204353 and analyzing the percent conversion by HPLC every 1-2 hours. When a percent conversion was observed > 99% (usually after 5-6 hours), the reaction was cooled to 20 °C - 25 °C and extracted with MTBE (2 x 525 mL) for a sufficient time to allow complete phase separation during extraction. The organic extract was washed with 5% 'NaCl (2 x 375 mL). The solution was then transferred to a device equipped with a cooled receiving flask for 1.5-2.5 Torr vacuum distillation. The solution was concentrated under vacuum at 60 ° C to remove Solvent. Distilled from the obtained oil at 125 ° C - 130 ° C (oven temperature) and 1.5-2.0 Torr (2,2-difluoro-1,3-benzodioxolanpentene-5 -yl)-acetonitrile. Separation of (2,2-difluoro-1,3-benzene) as a clear oil from 5-bromo-2,2-difluoro-1,3-benzodioxolane P-dioxetane-5-yl)-acetonitrile, yield 66% (2 And HPLC purity of 91.5% AUC (corresponding to 95% of w/w assay). 1H NMR (500 MHz, DMSO) δ 7.44 (br s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.22 (dd, /=8.2, 1.8 Hz, 1H), 4.07 (s, 2H). Synthesis of (2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropane Nitrile

〇 χχχ〇Ν

Bu4NBr, 50% w/w NaOH M1BE 50% w/w The NaOH solution was degassed by nitrogen purge for not less than 16 hours. The right amount of MTBE is similarly degassed for several hours. The degassed 1 丁 8 ugly (143 1111 〇, (2,2-difluoro-1,3-benzodioxol-5-yl) was successively charged into a nitrogen purged reactor. - acetonitrile (40.95 g, 207.7 mmol) and tetrabutylammonium bromide (2.25 g, 10.38 mmol). The volume of the mixture was recorded and mixed 156806.doc -37-201204353 was degassed by nitrogen purge for 30 minutes. Degassing is sufficient to restore the mixture to the original volume before degassing. The degassed 50°/〇w/w NaOH (143 mL) was charged to the scrambled mixture at 23.0 ° C for 10 minutes. 1-Bromo-2-ethaneethane (44.7 g, 311.6 mmol) was then charged over 30 minutes. The percent conversion of the reactants was analyzed by HPLC at 1 hour intervals. Stirring was stopped and the phases were separated prior to sampling. The top organic phase was sampled for analysis. When a percent conversion was > 99% (usually after 2.5-3 hours), the reaction mixture was cooled to 10 °C and water was added at a rate to maintain temperature <25 °C. (461 mL) Adjust the temperature to 20 ° C to 25 ° C and separate the phases. Note: The time should be sufficient to allow complete phase separation. The aqueous phase uses MTBE ( 123 m L) The extracted and combined organic phase is washed with 1 N HCl (163 mL) and 5% NaCl (163 mL) and concentrated in vacuo at 40 ° C - 50 ° C (2,2-difluoro-1,3- a solution of benzodioxol-5-yl)-cyclopropanecarbonitrile in hydrazine to 164 mL. The solution was charged with ethanol (256 mL) and vacuumed at 50 ° C -60 ° C. Concentrate to 164 mL. Fill in ethanol (256 mL) and concentrate the mixture to 164 mL in vacuo at 50 ° C - 60 ° C. Cool the resulting mixture to 20 ° C -25 ° C and dilute to 266 mL with ethanol to prepare Used in the next step. 1H NMR (500 MHz, DMSO) δ 7.43 (d, J = 8.4 Hz, 1H), 7.40 (d, / = 1.9 Hz, 1H), 7.30 (dd, J = 8.4, 1.9 Hz, 1H), 1.75 (m, 2H), 1.53 (m, 2H). Synthesis of 1-(2,2-difluoro-1,3-benzodioxocyclopent-5-yl)-cyclopropanecarboxylic acid

6NNaOH

EtOH, 80 °C

156806.doc -38* 201204353 Loading into the ethanol solution of (2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile in the previous step over 20 minutes 6N NaOH (277 mL) and heated to an internal temperature of 77 〇c_78t over 45 minutes. The progress of the reaction was monitored by HPLC after 16 hours. Note: Monitoring (2,2-difluorocyclopentan-5-yl)-cyclopropane carbonitrile with (2,2-difluoroj,% benzodioxane Partial hydrolysis of 'cyclopentene _5_yl)-cyclopropanenitrile yields consumption of both primary guanamines. When the percent conversion was observed > 99% (usually 100% ^ conversion after 16 hours), the reaction mixture was cooled to 25 eC and charged with ethanol (41 mL) and DCM (164 mL). The solution was cooled to 10 ° C and charged with 6 N HCl (290 mL) at a temperature maintained at < 25 ° C. The temperature was raised to 20 ° C - 25 t: after the separation of the phases. The bottom organic phase was collected and the top aqueous phase was back extracted with DCM (164 mL). Note that due to the high concentration of inorganic salts, the aqueous phase is slightly cloudy before and after extraction. The organics were combined and concentrated in vacuo to 164 mL. Toluene (328 mL) was charged and the mixture was concentrated to 164 mL· at 70 C -75 °C. The mixture was cooled to 45 C, charged with MTBE (364 mL) and sparged at 60 ° C for 20 min. The 〇 solution was cooled to 25 C and finely filtered to remove residual inorganic salts. The reactor was washed with MTBE (123 mL) / month and the solid was collected. The combined organics were transferred to a clean reactor for preparation for the next step. Separation of 1-(2,2-difluoro-1,3-benzodioxocyclopentene-5-yl)-cyclopropanecarboxylic acid

The solution of 1-(2,2-difluoro-i,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid in the previous step was reduced to 164 mL and charged with toluene (328 mL). And 156806.doc -39- 201204353 concentrated to 164 mL at 70 ° C -75 ° C. The mixture is then heated to a temperature of from 100 ° C to 105 ° C to obtain a homogeneous solution. After stirring at this temperature for 30 minutes, the solution was cooled to 5 ° C over 2 hours and maintained at 5. (The next 3 hours. The mixture was then filtered and the reactor was washed with cold 1:1 toluene/n-heptane (2×l 23 mL) and the solid was collected. The material was dried in vacuo at 55 C for 17 hr. 1-(2,2-difluoro-; i,3-benzodioxocyclopenten-5-yl)-cyclopropanecarboxylic acid. From (2,2-difluoro.4,3_ stupid and dioxane 1-(2,2-Difluoro-indole-3-peroxydioxol-5_yl)-cyclopropanoic acid was isolated from cyclopentene-5-yl)-acetonitrile in a yield of 79% (yield: 79%) 3 steps, including separation) and HPLc purity was 99.0% 八; (: £81-]^8»2/2 calculated value 242.04, experimental value 241.5 8 (M+l) + ; NMR (500 MHz, DMSO) δ 12.40 (s, 1H), 7.4 〇 (d, /=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). Alternative synthesis of acid moiety (2,2-difluoro-1,3-benzodioxolane-5-yl)-methanol 1. Vitride (2 equivalent)

PhCH3 (10 volumes)

2· 10% (w/w) NaOH aqueous solution (4 equivalents) Yield 86%-92% Commercially available 2,2-difluoro-1,3-benzodioxolane-5-carboxylic acid (1 〇 equivalent) A slurry was formed in toluene (1 〇 volume). Vitride® (2 equivalents) was added via an addition funnel at a rate to maintain a temperature between 15 ° C and 25 t. At the end of the addition, the dish was increased to 40 C for 2 hours, then a 10% (w/w) aqueous solution of Na〇H (4·〇 equivalent) was carefully added via an addition funnel while maintaining the temperature at 156806.doc 201204353 40C 50C Stir for another 3 minutes, at 4 〇. . The layers are separated. Cool the organic phase to 2 ° C. Then wash with water (2 x 1.5 vol), dry (Na2S 〇 4), pass through and concentrate to give a crude (2,2-di-tun-1,3-stac. Cyclopentene-5·yl)·methanol, which was used directly in the next step. Synthesis of 5-gas methyl-2,2-difluoro-1,3-benzodioxolane 1· S0C12 (1.5 equivalents) DMAP (〇.〇1 equivalent) MTBE (5 vol)

❹

2. Water (4 volumes) Yield 82%~100%

Q (2,2-difluoro-i,3-stupoxydioxol-5-yl)methanol equivalent) > glutathion was dissolved in MTBE (5 vol). A catalytic amount of DMAP (1 mol%) was added via an addition funnel and s〇ch (12 equivalents) was added. S〇ci2 was added at a rate to maintain the temperature in the reactor from 15 ° C to 25 ° C. The temperature was raised to 3 Torr <>c, held for 1 hour, then cooled to 2 (rc, then water (4 vol) was added via an addition funnel while maintaining the temperature below 30 ° C. After stirring for an additional 30 minutes, The layers were separated. The organic layer was stirred and a 1% (w/v) aqueous solution of Na〇H (4 4 vol.) was added. After 15 to 20 minutes of mixing, the layers were separated. The organic phase was then dried (Na2S〇4). Concentration gave crude 5-mercapto-2,2-difluoro-i,3-benzodioxolane which was used directly in the next step. Synthesis (2,2-difluoro-1,3 _Benzo-dioxocyclopenten-5-yl)-acetonitrile 1- NaCN (1.4 eq.) DMSO (3 vol)

30-40〇C 2. Water (6 volumes) MTBE (4 volumes)^ Yield 95%-100% F 〇-^ΝίΑ^ΟΝ 156806.doc • 41- 201204353 5-chloroanthryl-2,2- - a solution of fluoro-1,3-benzodioxolane (丨 equivalent) in DMSO (1.25 vol) was added to a slurry of NaCN (14 eq.) in DMSO (3 vol) while maintaining The temperature is between 30 ° C and 40 ° C. The mixture was stirred for 1 hour, followed by successive addition of water (6 volumes) and MTBE (4 volumes). After 30 minutes of disruption, the layers were separated. The aqueous layer was extracted with MTBE (1.8 vol). The combined organic layers were washed with water (~8 vol.), dried (NajO4), filtered and concentrated to give crude (2,2-difluoro-1,3-benzodioxol-5-yl) - Acetonitrile (95%) 'It was used directly in the next step. The remaining steps are the same as described above for the synthesis of the acid moiety. Amine moiety synthesis 2 - desert-5 - gas-4-decyl aniline

EtOAc 50%

The flask was successively charged with 3-fluoro-4-nitroaniline (1.0 eq.) and ethyl acetate (1 vol.) and stirred to dissolve all solids. N-bromobutanimide (1.0 eq.) was added in portions to maintain an internal temperature of 22 〇c. At the end of the reaction, the reaction mixture was concentrated in vacuo on a rotary evaporator. The residue was slurried in steamed water (5 volumes) to dissolve and remove the succinimide (the succinimide was also removed by a water treatment procedure). The water was decanted and the solid formed a slurry in 2-propanol (5 vol.) overnight. The resulting slurry was filtered and the wet cake was washed with 2-propanol' in a vacuum oven. (: Dry under N2 flow overnight until the weight is strange. Separate a tan solid (yield 50 〇 / 〇, 97.5% AUC). Other impurities are brominated positional isomers (1.4% AUC) and dibromo Compound (丨156806.doc • 42· 201204353 AUC). 4 NMR (500 MHz, DMSO) 8.19 (1 H,d,/=8.1 Hz), 7.06 (br·s,2 H),6.64 (d,1) H, «7=14.3 Hz) ° Synthesis of (i?)-l-((4-amino-2-bromo-5-fluorophenyl)amino)_3_(benzyloxy)propan-2-ol P-toluenesulfonate o2n

F 3〇c

Br νη2

1) ΐ>χ^0Βη catalyst Zn(C104)2-2H20 benzene, 80°C

2) H2, Pt (SVC IPAc 3) Ts0H-H20 DCM

OBn The fully dried flask was charged with the following under N2: active powdered 4A molecular sieve (50% by weight, based on 2-bromine-5-fluoro-4-nitroanilide), 2-bromo-5-fluoro- 4-Nitroaniline (1.0 eq.), dehydrated acid (20 mol%) and toluene (8 vol). The mixture was stirred at room temperature for 30 minutes for NMT. Finally, toluene (2 vol) containing (R)-benzylglycidyl ether (2.0 eq.) was added as a steady stream. Heat the reaction to 80 ° C (internal temperature) and mix for about 7 hrs or until 2-bromo-5-fluoro-4-nitroaniline < 5% AUC » Cool the reaction to room temperature and add the vines one after the other Soil (50% by weight) and 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 a solution of ammonium hydride (4 volumes, 2% w/v). The organic layer was washed with sodium bicarbonate solution (4 volumes χ 25% w/v). The organic layer was concentrated in vacuo on a rotary evaporator. The resulting slurry was dissolved in isopropyl acetate (10 vol) and the solution was transferred to a Bucher hydrogenator. 156806.doc • 43- 201204353 The hydride was charged with 5 wt% Pt (S ) / c (1.5 mol %) and the mixture was stirred at 3 Torr < t (internal temperature) N2. The reactants were washed successively with helium and hydrogen. The hydrogenator pressure was adjusted to 1 bar of hydrogen and the mixture was rapidly stirred (> 12 rpm). At the end of the reaction, the catalyst was filtered through a pad of Celite and washed with dichloromethane (10 vol). The filtrate was concentrated in vacuo. Any residual isopropyl acetate was captured with di-methane (2 vol) and concentrated to dryness using a rotary evaporator. The resulting residue was dissolved in dichloro-hexane (10 vol). Add p-toluenesulfonic acid monohydrate (1 - 2 equivalents) and stir overnight. The product was filtered and washed with dichloromethane (2 vol) and suction dried. The wet cake was transferred to a drying tray and placed in a vacuum oven and dried under a stream of N2 at 45 ° C until the weight was constant. Separation of p-toluic acid salt of (pseudo-丨-((4-amino-2-bromo-5-fluorophenyl)amino)_3_(presentyloxy)propan-2-ol as an off-white solid Measured to palm purity >97% ee. Synthesis of (3-a-3-methylbut-1-ynyl)trimethylnonane

TMS

Pure HCI 90%

Propargyl alcohol (1.0 eq. of aqueous hydrochloric acid (37%, 3.75 vol)) was added to the vessel and stirring was started. A moderate endotherm (C6C) was observed during solid alcohol dissolution, and the mixture was aged overnight (16 hours). The mixture slowly turned dark red. Water (5 vol) was charged to a 30 L jacketed vessel, followed by cooling to 10 C vacuum and the reaction mixture was slowly transferred to water maintaining the internal temperature of the mixture below 25. Hexane (3 vol) was added and the resulting mixture was stirred for 5 hours. The phases were allowed to settle and the aqueous phase was drained (ρ Η < 1) and discarded. The organic phase was concentrated in vacuo using a rotary evaporator at 156806.doc • 44 - 201204353 Red oily product β synthesis (4-(benzyloxy)_3,3-dimercapto-1 -ynyl) trimethyl fever

TMS

1. Mg --- 2. BnOCH2Cl

TMS OBn

Method A

All equivalent and volume descriptors in this section are based on 25 〇g of reactant. Magnesium swarf (69.5 g, 2_86 mol, 2.0 eq.) was charged to a 3 L four-necked reactor and stirred with a magnetic stirrer under nitrogen for 0.5 hour. The reactor was immersed in an ice-water bath. A solution of propargyl gas (25 〇g, 1.43 mol, i.o eq.) in Thf (1.8 L, 7.2 vol) was slowly added to the reactor with stirring until an exotherm (about 10. 〇) was observed. Grignard reagent formation was confirmed by IPC using Ih_nmr spectroscopy. After the exotherm subsided, the remaining solution was slowly added while maintaining the material temperature <15C>C. Adding takes about 3.5 hours. The resulting dark green mixture was decanted into a 2 L capped bottle. All of the equivalent and volume descriptors in this enthalpy are based on 5 〇〇 g of reactant. A 22 L reactor was charged with a solution of benzyl methyl methyl ether (95 〇 / 〇, mM, 〇. 8 eq.) in THF (1.5 L, 3 vol). In the ice_water bath, the reaction is cooled. The two batches of Grignard reagents prepared as described above were combined and slowly added to the benzyl chloromethyl-solution via an addition funnel to maintain the material temperature below 25t. Adding takes 1.5 hours to mix the reaction mixture overnight (16 hours). All equivalent and volume descriptors in this file are based on 1 kg of reactant. Prepare 15% gasification solution in water in a 3〇 L jacketed reactor 156806.doc •45- 201204353, 10 volumes). Cool the solution to 5X:. The two batches of the Grignard reaction mixture prepared as described above were combined and then transferred to a vaporized ammonium solution via a header vessel. An exotherm was observed in this quenching, which was carried out at a rate to keep the internal temperature below 25. After the transfer is completed, set the container jacket temperature to 25 < t. Hexane (8 L, 8 vol) was added and the mixture was stirred for 5 hours. After the phases settled, the aqueous phase (pH 9) was discharged and discarded. The remaining organic phase was washed with water (2 L, 2 vol). The organic phase was concentrated in vacuo using a 22 L rotary evaporator to afford crude material.

Method B Magnesium swarf (106 g, 4.35 mol, ίο equivalent) was charged to a 22 L reactor, followed by suspension in THF (76 〇 mL, 丨 volume). Cool the container in an ice-water bath to bring the material to a temperature of 2t:. A solution of propargyl gas (76 〇g, «5 mol' U equivalent) in butyl HF (4.5 L, 6 vol) was slowly added to the reactor. After the addition of 100 mL, the addition was stopped and the mixture was stirred until the 13C exotherm was observed, indicating that the Grignard reagent was started to be produced. After the sputum subsided, slowly add another 500 mL of hydrazine chloride solution, and the temperature of the material (4) was &lt; 2 〇 t:. The Grignard reagent formation was confirmed by lpc using lH_NMR spectroscopy. Slowly add the remaining block of propyl chloride solution to maintain the material temperature <grab. The addition takes about 1.5 hours. The resulting dark green solution was stirred for 5 hours. Gliner reagent formation was confirmed by lpc using 'NMR spectroscopy. Pure benzylmethyl chlorodecyl ether was charged to the reactor addition funnel and then added dropwise to the reactor to maintain the material temperature below 25 t:. Adding need U Xiaoxiao. The reaction mixture was given overnight. The product was treated with the same procedure as in Method A and the mass of the related material was subjected to water-based treatment and concentration to give the product as an orange oil. 156806.doc • 46· 201204353 Synthesis of 4-benzyloxy_3,3·dimethyl τ acetylene

TMS

Yield of KOH MeOH OBn 2 step 88% OBn

The 30 L jacketed reactor was charged with methanol (6 vol) and then cooled to completion. A hydroxide clock (85%, u equivalent) was added to the reaction. A 15 aC 2 〇t: exotherm was observed when the potassium pentoxide was dissolved. The jacket temperature is set to hungry. Add a solution of 4_benzyloxy-3,3-dimethyl dimethyltridecyl succinyl acetylene (1 〇曰) in methanol (2 vol) and stir the resulting mixture until the reaction is complete, such as HPLC Monitored. The typical reaction time at 25 ° C is 34 hours. The reaction mixture was diluted with water (8 vol), then hexanes (yield) and hexanes (6 vols) was added and the mixture was stirred for 5 hours. The phases were allowed to settle' and then the aqueous phase (pH 10-11) was drained and discarded. The organic phase was washed successively with a solution of hydrazine (85 ° / .' 〇 · 4 equivalents) in water (8 vol) and water (8 vol). The organic phase was then concentrated using a rotary evaporator to give the title material as a yellow oil. The typical purity of this material is in the range of 80%, in which a single impurity is mainly present. 1H NMR (400 MHz, C6D6) δ 7.28 (d, 2 Η, J = 7.4 Hz), 7.18 (t, 2 H, J = 7.2 Hz), 7.10 (d, 1H, J=J.2

Hz), 4.35 (s, 2 H), 3.24 (s, 2 H), 1.91 (s, 1 H), 1.25 (s, 6 H) 〇

Synthesis of iV-benzyl alcohol glycolated-5-amino-2-(2-benzyloxy·ι,;[_dimethylethyl)-6-like π-drinking method A synthesis (and)-1 -((4-Amino-2-(4-(benzyloxy)-3,3-dimethylbutan--yne) 156806.doc •47- 201204353 1-yl)-5-fluorophenyl) Amino)-3-(benzyloxy)propan-2-ol

Pd(OAc)r dppb, K2C〇3, Cu 丨, water

Stirring (i〇-Bu((4-amino-2-bromo-5-fluoro)amino)-3-() in dichloromethane (5 vol) and saturated NaHC03 (5 vol) Phenyloxy)propan-2-ol p-toluenesulfonate solid until a clear organic layer is obtained to freely basify. The resulting layers are separated, and the organic layer is washed successively with NaHC03 saturated solution (5 vol) and brine. Concentration gave the amino-amino-2-bromo-5-fluorophenyl)amino)-3-(dumptoxy)propan-2-ol free base as an oil. Acetate (0.01 equivalents), dppb (0.015 equivalents), Cul (0.015 equivalents), and potassium carbonate (3 equivalents) were suspended in acetonitrile (丨 2 vol). After stirring for 5 minutes, 4-phenylhydrazineoxy-3,3-dimercapto--yne was added. "Equivalent" solution in acetonitrile (0.2 vol). The mixture was purged with nitrogen for an hour, followed by the addition of (phantom-1-((4-amino-2-bromo-5-fluorophenyl)amino)-3-(benzyloxy)propan-2-ol free A solution of the base (1 eq.) in acetonitrile (4 vol.). The mixture was then purged with nitrogen for 1 hour and then heated to 80 s. The progress of the reaction was monitored by HPLC and the reaction was usually completed in 3-5 hours. The mixture was washed with acetonitrile (4 volumes), and the combined filtrate was co-evaporated until the dry mixture was sensitive to the next reactor. Λ)-1-((4-Amino-2-(4-(phenylhydroxy)-3,3-didecylbut-1-yne-I group)_5_acrozyl)amino)- The 3-acetonitrile solution of 3-(benzyloxy)propan-2-ol was used in the next procedure (cyclization) without further manipulation of 156806.doc -48-201204353. -Aminos(7-benzyloxy·14-dimethylethyl)·6-fluoropterin

Diacetonitrile di-palladium (Oi equivalent) and CuI (〇i equivalent) were charged into the reactor, followed by suspension of the above (and) amine group 2_(4_(benzyloxy)_3,3-dimethyl A solution of butyl hydrazide (1 eq) in acetonitrile (9.5 vol. in total) in the solution of keto-1-ynyl-1-yl)-5-fluorophenyl)amino)-3-(phenoxy)propan-2-ol (1 eq.) . The mixture was purged with nitrogen for 1 hour and then heated to 80 °C. The progress of the reaction was monitored by HPLC and the reaction was usually completed in 1 to 3 hours. The mixture was passed through Shixia, and the filter cake was washed with acetonitrile. The solvent was changed to ethyl acetate (75 volumes). The ethyl acetate solution was washed successively with a NHrNt^Cl aqueous solution (2 x 2.5 vol) and 10% brine (2.5 vol). The ethyl acetate solution was then stirred with tannin (丨.8 equivalent) and Si-TMT (0.1 equivalent) for 6 hours. After filtration, the resulting solution was concentrated. The residual oil was dissolved in DCM / heptane (4 vol) and then purified by column chromatography. The oil thus obtained was then crystallized from 25% EtOAc / heptane (4 vol). Crystallization is usually obtained (i〇-l-(5-amino-2-(1-(benzyloxy)-2-methylpropan-2-yl)-6-fluoro-1H-inden-1-yl) -3-(benzyloxy)propan-2-ol, yield 27% - 38%. 4 NMR (400 MHz, DMSO) δ 7.38-7.34 (m, 4 Η), 7.32-7.23 (m, 6 Η), 7.21 (d, 1 Η, J=12.8 156B06.doc -49- 201204353

Hz), 6.77 (d, 1H, J=9.0 Hz), 6.06 (s, 1 H), 5.13 (d, 1H, J=A.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, 7=9.8, 4.4 Hz), 3.43 (dd, 1H, *7=9.8, 5_7 Hz), 1.40 〇, 6 H). Synthesis of iV-benzyl glycolate-5-amino-2-(2-stupylmethoxy-14-didecylethyl)-6-fluoroanthr

2. (MeCN) 2PdCl2 MeCN, 80 °C 3. Filtration of acetic acid I bar (33 g, 0.04 equivalent), dppb (94 g, 0.06 equivalent) and potassium carbonate (1.5 kg, 3.0 equivalent) into the reactor . The free-alkaliated oil, phenylhydrinyl glycolate 4-ammonium-2-bromo-5-fluoroaniline (1.5 kg, 1.0 eq.) was dissolved in acetonitrile (8.2 L' 4.1 vol) and then added to the reactor . The mixture was purged with nitrogen for 1 hour for use in NLT. A solution of 4-stupylmethoxy-3,3-dimethyl-butan-1-yne (70%, 1.1 kg, 1.05 equivalent) in acetonitrile was added to the mixture' then the mixture was purged with nitrogen for 1 hour. In NLT. The mixture was heated to 80 ° C' and then stirred overnight. The lpc was carried out by Hplc and the reaction was completed after 16 hours. The mixture was cooled to ambient temperature and then filtered through a pad of celite (228 g). The reaction benefit and Shixiazao soil were washed with acetonitrile (2Χ2 [, 2 body 156806.doc -50- 201204353 product). The combined phases were concentrated using a 22 L rotary evaporator until 8 l of solvent was collected and the crude product was taken in 7L (3.5 vol.) acetonitrile. Diacetonitrile dipalladium (144 g, 0.15 equivalent) was charged to the reactor. The crude solution was transferred back to the reactor and the rotary evaporator ball was washed with acetonitrile (4 L, 2 vol). The combined solution was purged with nitrogen for 1 hour for use in NLT. The reaction mixture was heated to 80 ° C for 16 hours for use in NLT. Process control by HpLC showed complete depletion of the starting material. The reaction mixture was filtered through celite (300 g). The reactor and filter cake were washed with acetonitrile (3 Torr, 15 vol). The combined filtrate was concentrated to an oil by rotary evaporation. This oil was dissolved in ethyl acetate (8.8 L, 4.4 vol). The solution was washed successively with 20% ammonium hydride (5 L, 2.5 vol) and 5% brine (5 L·, 2-4 vol). Silicone (3.5 kg '1.8 equivalents) was added to the organic phase and stirred overnight. A Deloxan® II metal scavenger (358 g) and a heptane (17.6 L) were added and the resulting mixture was mixed for 3 hours for NLT. The mixture was filtered through a sintered glass funnel. The filter cake was washed with heptane (25 L) containing 30% ethyl acetate. Concentration of the combined filtrate under reduced pressure' to give a benzyl glycolate as a brown paste. _5_Amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindole哚 (14 kg). Synthesis of Compound 1 Synthesis of Benzyl Protected Compound 1

156806.doc •51· 201204353

this. Cooling (i?)-l-(5-Amino-2-(1-(benzyloxy)_2-methylpropan-2-yl)_6_fluoro-1H-indenyl)_3_(benzyloxy) A solution of propan-2-ol (1 equivalent) and triethylamine (3 equivalents) in DCM (4 vol). Add acid chloride toluene solution (1 volume) while maintaining the material temperature below 10 &lt; t. The progress of the I reaction is monitored by HpLC and the reaction is usually completed in a few minutes. Warm up to Μ. After the reaction, the reaction mixture was washed with 5% NaHC.sub.3 (3.5 vol.), 1 M Na?H (35 vol.) and 1 M HCl (5 vol). The solvent was changed to methanol (2 volumes) and the obtained (i?)-N-(l-(3-(benzophenoxy)_2-hydroxypropyl)_2_(丨_(benzyloxy)_2_methylpropyl -2-yl)-6-fluoro-li/-吲哚_5_yl)fluorobenzo[£/][13]dioxocyclopenten-5-yl)cyclopropanecarbamide in decyl alcohol The solution was used in the next step (hydrogenolysis) without further manipulation. Synthetic compound 1

Will be 5 /. Ji/charcoal (about 5 〇% humidity, 〇丨 〇丨 equivalent) filled into a suitable hydrogenation vessel 156806.doc -52- 201204353

in. Carefully added (Λ)_Ν_(1_(3_(benzyloxy) 2 hydroxypropyl)-2-(1_(phenoxy)_2-methylpropan-2-yl)-6-fluoro obtained above a solution of 1Η-吲哚_5_yl)-1_(2,2-difluorobenzopyrene]anthracene, 3]dioxycyclopenten-5-yl)cyclopropanecarnitine in methanol (2 vol) Then, a 3 M HCl solution in methanol was added. The vessel was purged with nitrogen and hydrogen successively. The mixture was vigorously disrupted until the reaction was completed as determined by HPLC analysis. The typical reaction time is 3_5 hours. The reaction mixture was filtered through celite and the filter cake was washed with methanol (2 vol). The agent was changed to isopropanol (3 volumes). The crude νχ·661 was crystallized from 75% ΙΡΑ-heptane (4 volumes, ie 1 volume of heptane added to 3 volumes of ruthenium) and the resulting crystals were added at 50% ΙΡΑ-heptane (ie 2 vol. Matured in the mixture). A typical yield of compound 4 in a two-step deuteration/hydrogenolysis procedure is from 68% to 84°/. Within the scope. Compound 4 can be recrystallized from ιρΑ_heptane according to the same procedure as just described. Compound 1 can also be prepared by one of several synthetic routes disclosed in U.S. Patent Application Serial No. US2009013 1492, which is incorporated herein by reference. Table 10 below presents the analytical data for Compound 1. Table 10. !H NMR (400.0 MHz, CD3CN) d 7.69 (d, J=7.7 Hz, 1H), 7.44 (d, J=1.6 Hz, 1H), 7.39 (dd, J=1.7, 8.3 Hz, 1H) , 7.31 (s, 1H), 7.27 (d, J=8.3 Hz, 1H), 7.20 (d, J=12.0 Hz, 1H), 1 521.5 1.69 6.34 (s, 1H), 4.32 (d, J=6.8 Hz , 2H), 4.15-4.09 (m, 1H), 3.89 (dd, J=6.0, 11.5 Hz, 1H), 3.63-3.52 (m, 3H), 3.42 (d5 J=4.6 Hz, 1H), 3.21 (dd , J=6.2, 7.2 Hz, 1H), 3.04 (t, J=5.8 Hz, 1H), 1.59 (dd, J=3.8, 6.8 Hz, 2H), 1.44 (s, 3H), 1.33 (s, 3H) And 1.18 (dd, J = 3.7, 6.8 Hz, 2H) ppm. 156806.doc -53- 201204353 Preparation of Compound 1 Formulation Preparation 40 g of Formulation Containing 50 mg of Compound 1 Preparation of 40 g of aqueous formulation having the following composition and amount: Component (g) Compound 1 0.05 PEG 400 19.95 Vitamin E TPGS 4.48 Water 15.52 A 22.4% by weight Vitamin E TPGS solution was prepared separately by adding Vitamin E TPGS (4.48 g) to a clear glass bottle equipped with a magnetic stir bar. Water (15.52 mL) was added and the bottle was sealed and agitated at a rate such that the vortex depth was about 2/3 of the liquid depth. The mixture of vitamin E TPGS and water was stirred at room temperature until all of the vitamin E TPGS was dissolved and no visible solid particles were present. Depending on the size of the vitamin E TPGS solid particles, this step can be carried out for more than 4 hours. The solution can be stirred overnight in a closed container. To accelerate the dissolution of the vitamin E TPGS, the dissolution can be carried out in a water bath at 30 ° C to 35 ° C. The stirred solution was stopped and filtered using a disposable filter unit. 19.95 g of PEG 400 was weighed into another bottle and the bottle was placed in a water bath and warmed at 40 ° C ± 5 ° C. Increase the agitation rate until a turbulent vortex forms in the center of the liquid. 50 mg of Compound 1 was weighed, the bottle was sealed and the formulation was stirred at 40 °C ± 5 °C until all Compound 1 was completely dissolved (about 1 hour). The formulation was removed from the water bath and stirred at room temperature for at least 1 hour to reach equilibrium. Add the first bottle of Vitamin E TPGS solution and mix the formulation by hand until a homogeneous solution is formed. 156806.doc -54- 201204353 Preparation of 40 g of Formulation Containing 100 mg of Compound 1 Preparation of 40 g of aqueous formulation having the following composition and amount: Component (g) Compound 1 0.10 PEG 400 19.90 Vitamin ETPGS 4.48 Water 15.52 A 22.4% by weight vitamin E TPGS solution was prepared.

19.90 g of PEG 400 was weighed into another bottle and the bottle was placed in a water bath and warmed at 40 °C ± 5 °C. Increase the agitation rate until a vortex is formed in the center of the liquid. 100 mg of Compound 1 was weighed, the bottle was sealed and the formulation was stirred at 40 °C ± 5 °C until all Compound 1 was completely dissolved (about 1 hour). The formulation was removed from the water bath and stirred at room temperature for at least 1 hour to reach equilibrium. Add the first bottle of vitamin ETPGS solution and mix the formulation by hand until a homogeneous solution is formed. Preparation of a 40 g formulation containing 200 mg of Compound 1

A 40 g aqueous formulation having the following composition and amount was prepared: Component Amount (g) Compound 1 0.20 PEG 400 19.80 Vitamin ETPGS 4.48 Water 15.52 A 22.4 wt% vitamin E TPGS solution was prepared as described above. 19.80 g of PEG 400 was weighed into another bottle and the bottle was placed in a water bath and warmed at 40 ° C ± 5 ° C. Increase the agitation rate until the center of the liquid forms a vortex 156806.doc •55- 201204353 vortex. 200 mg of Compound 1 was weighed, the bottle was sealed and the formulation was stirred at 40 ° C ± 5 ° C until all Compound 1 was completely dissolved (about 1 hour). The formulation was removed from the water bath and stirred at room temperature for at least 1 hour to reach equilibrium. Add the first bottle of Vitamin E TPGS solution and mix the formulation by hand until a homogeneous solution is formed. A 40 g formulation containing 300 mg of Compound 1 was prepared to prepare a 40 g aqueous formulation having the following composition and amount: Component (g) Compound 1 0.30 PEG 400 19.70 Vitamin E TPGS 4.48 Water 15.52 Preparation of 22.4% by weight of vitamins as described above E TPGS solution. 19.70 g of PEG 400 was weighed into another bottle and the bottle was placed in a water bath and warmed at 40 °C ± 5 °C. Increase the agitation rate until a vortex is formed in the center of the liquid. 300 mg of Compound 1 was weighed into the bottle, the bottle was sealed and the formulation was stirred at 40 ° C ± 5 ° C until all Compound 1 was completely dissolved (about 1 hour). The formulation was removed from the water bath and stirred at room temperature for at least 1 hour to reach equilibrium. Add the first bottle of Vitamin E TPGS solution and mix the formulation by hand until a homogeneous solution is formed. Characterization of AF508-CFTR calibration properties for detection and measurement of compounds. Membrane potential optics for the determination of AF508-CFTR modulating properties of compounds 156806.doc •56· 201204353 Optical membrane potential assay using pressure sensitive features described by Gonzalez and Tsien FRET sensor (see Gonzalez, JE and RY Tsien (1995) "Voltage sensing by fluorescence resonance energy transfer in single cells" Biophys J 69(4): 1272-80, and Gonzalez, JE and RY Tsien (1997) "Improved Indicators of cell membrane potential that use fluorescence resonance energy transfer" Chem Biol 4(4): 269-77) and instruments for measuring fluorescence changes (such as voltage/ion probe readers (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) ° These pressure-sensitive assays are based on membranes Fluorescence resonance energy transfer (FRET) change between the soluble pressure sensitive dye DiSBAC2 (3) and the fluorescent phospholipid CC2-DMPE attached to the outer layer of the plasma membrane and acting as a FRET donor. The change in membrane potential (Vm) causes the negatively charged DiSBAC2 (3) to redistribute across the plasma membrane and the energy transmitted from the CC2-DMPE changes accordingly. Monitoring of Fluorescence Emissions Using VIPRTM II' VIPRTM II is an integrated liquid handler and fluorescence detector designed to perform cell-based screening in 96-well or 384-well microtiter plates. 1. Identification of Calibration Compounds To identify small molecules that correct for transfer defects associated with AF508-CFTR, a single addition HTS assay format was developed. The cells were incubated for 16 hours at 37 ° C in serum-free medium in the presence or absence (negative control) of the test compound. As a positive control, cells coated in 384-well plates were incubated at 27 ° C for 16 hours with "temperature-corrected" AF508-CFTR. The cells were then washed 3 times with Krebslin 156806.doc • 57-201204353 Krebs Ringers solution and loaded with pressure sensitive dye. To activate ΔΡ508-ΟΡΤΙΙ, 10 μΜ of forskolin and CFTR potentiator genistein (20 μΜ) and a Cl-free medium were added to each well. The addition of Cl-free medium promoted the outflow of Cl_ in response to activation of AFSOS-CFTR and optically monitored the resulting membrane depolarization using a FRET based voltage sensor dye. 2. Identification of synergist compounds To identify synergists of ΔΡ508-€ΡΤίΙ, develop a dual-addition HTS assay format. During the first addition, cry-free medium with or without test compound was added to each well. After 22 seconds, a second Cl-free medium containing 2-10 μM forskolin was added to activate AF508-CFTR. The extracellular Cr concentration was 28 mM after two additions, which promoted Cl_ efflux in response to ΔΡ508-ΟΡΤΙΙ activation and optically monitored membrane depolarization using a FRET-based voltage sensor dye. 3. Dissolved in solution Electrolyte #1: (mM) NaCl 160, KC1 4.5, CaCl2 2, MgCl2 1, HEPES 10, pH 7.4 (using NaOH). Chlorine-free electrolyte: replace the chloride salt in electrolyte #1 with gluconate. CC2-DMPE: Prepared as a 10 mM stock solution in DMSO and stored at -20 °C.

DiSBAC2 (3): Prepared as a 10 mM stock solution in DMSO and stored at -20 °C. 4. Cell culture NIH3T3 mouse fibroblasts stably expressing AF508-CFTR were used for optical measurement of membrane potential. The cells were maintained in a 1 75 cm2 flask at 37 ° C, 5% 156806.doc -58 - 201204353 C02 and 90% humidity supplemented with 2 mM branic acid, 10% fetal bovine serum, 1 X NEAA, β-ΜΕ , 1 X penicillin/streptomycin and 25 mM HEPES in Dulbecco's modified Eagle's medium. For all optical assays, cells were seeded at 30,000 per well in Matrigel-coated 384-well plates and incubated for 2 hours at 37 °C, followed by 24 hours at 27 °C for potentiator assays. For calibration assays, cells are cultured for 16-24 hours at 27 ° C or 37 ° C in the presence and absence of compounds. Electrophysiological assay for the characterization of AFS08-CFTR regulatory properties of compounds 1. Ussing Chamber Assay Eus perfusion chamber experiments were performed on polarized epithelial cells expressing AF508-CFTR to further characterize the optical assay Identification of the AF508-CFTR modulator. The FRTAF5G8-cftr epithelial cells grown on a Costar Snap well cell culture insert were mounted in a Uss perfusion chamber (Physiologic Instruments, Inc., San Diego, CA) using a voltage clamp system (Department of Bioengineering, University of Iowa). , IA and Physiologic Instruments, Inc., San Diego, CA) continuously shorted a single layer. The transepithelial resistance was measured by applying a 2 mV pulse. Under these conditions, the FRT epithelium shows a resistance of 4 Κ Ω/cm 2 or more than 4 Κ Ω/cm 2 . The solution was maintained at 27 ° C and bubbled with air. Use a cell-free insert to correct the electrode offset potential and fluid resistance. Under these conditions, the current reflects the flow of Cl_ through the ΔF508-CFTR represented in the apical membrane. Use MP100A-CE interface and AcqKnowledge software (v3.2.6; BIOPAC Systems, Santa 156806.doc •59- 201204353

Barbara, CA) obtained Isc in digital form. 2. Identification of Calibration Compounds A typical protocol utilizes a Cl_ concentration gradient from the basal membrane to the apical membrane. To establish this gradient, a normal Ringer's solution was used on the base side membrane, while the top NaCl was replaced with an equivalent of sodium molybdate (titrated to pH 7.4 with NaOH) to obtain a large Cl_ concentration gradient across the epithelium. All experiments were performed in a complete single layer. To fully activate ΔF508-CFTR, apply foscomin (10 μΜ) and PDE inhibitor ΙΒΜΧ (100 μΜ) followed by the CFTR synergist genistein (50 μM). As observed in other cell types, incubation of FRT cells stably expressing ΔF5 08-CFTR at low temperatures increased the functional density of CFTR in the oral membrane. To determine the activity of the calibrated compound, cells were incubated with 10 μΜ of the test compound for 24 hours at 37 ° C, followed by washing 3 times, and then recorded. The cAMP and genistein-mediated Isc in the compound-treated cells were corrected relative to the 27 ° C and 37 ° C controls and expressed as percent activity. Pre-incubation of cells with the calibration compound significantly increased cAMP and genistein-mediated Isc compared to the 37 °C control. 3. Identify synergist compounds

A typical approach utilizes a Cl_ concentration gradient from the basal membrane to the apical membrane. To establish this gradient, normal Ringer's solution was used on the basal side membrane and permeabilized with nystatin (360 pg/ml), while the top NaCl was treated with sodium oleate. The NaOH was titrated to pH 7.4) to achieve a large Cl_ concentration gradient across the epithelium. All experiments were carried out 30 minutes after the serotonin was permeabilized. Forskolin (10 μΜ) and all test compounds were added to both sides of the cell culture insert. Comparative presumptive AF508-CFTR 156806.doc -60- 201204353 The synergistic effect of the synergist with the known synergist genistein. 4. Solution side solution (mM): NaCl (135), CaCl2 (1.2), MgCl2 (1.2), K2HP〇4 (2.4), KHP〇4 (〇.6), N-2-hydroxyethyl Piperazine-indole-2-ethanesulfonic acid (HEPES) (10) and dextrose (10). The solution was titrated with NaOH to pH 7.4. Top solution (mM): same as the base side solution] where NaCl was replaced with sodium gluconate (135). 5. Fine Cannon Culture Fischer rat epithelial (FRT) cells (FRTAF5Q84ftr) expressing AF508-CFTR were used in the Ussing perfusion chamber experiment of the putative ΔΡ508-CFTR regulator identified by our optical assay. Cells were cultured on Costar Snapwell cell culture inserts and supplemented with 5% fetal bovine serum, 100 U/ml penicillin and 100 pg/ml streptomycin in Coon's modified Ham's F-12 medium (Coon's modified Ham's F-12 Medium) Incubate for 5 days at 37 ° C and 5% C02. The cells were incubated at 27 °C for 16-48 hours to correct for AF508-CFTR prior to characterizing the potentiator activity of the compounds. To determine the activity of the calibration compound, cells were incubated for 24 hours at 27 ° C or 37 ° C in the presence and absence of compound. 6. Whole-cell recording The macroscopic AF508-CFTR current (ΙΔΡ508) in NIH3T3 cells calibrated for temperature and test compound calibration was monitored using a perforated patch whole-cell recording. Briefly, using Axopatch 200B Patch Clamp Amplifier (Axon Instruments Inc., Foster City, CA) at room temperature 156806.doc -61- 201204353

The voltage sweet record of IaF5Q8. All records were obtained at a 10 kHz sampling frequency and low pass filtered at 1 kHz. When filled with an intracellular solution, the resistance of the drip tube is 5-6 ΜΩ. Under these recording conditions, the calculated reversal potential (EC1) of Cl_ at room temperature was -28 mV. All recorded tight resistance &gt; 20 GQ and series resistance &lt; 15 Μ Ω. Pulse generation, data acquisition and analysis were performed using a PC equipped with a Digidata 1320 A/D interface and Clampex 8 (Axon Instruments Inc.). The tank contained less than 250 μL of saline and was continuously infused at a rate of 2 ml/min using a gravity-driven perfusion system. 7. Identification of the calibration compound To determine the activity of the calibration compound to increase the density of the functional ΔΡ 508-ΟΡΤΙΙ in the plasma membrane, the current density after the compound treatment was corrected for 24 hours using the above-described perforated membrane recording technique. To fully activate AFSOS-CFTR, 10 μΜ forskolin and 20 μΜ genistein were added to the cells. Under the conditions recorded by us, the current density after incubation at 27 ° C for 24 hours was higher than that observed after incubation at 37 ° C for 24 hours. These results are consistent with the effect of known low temperature incubation on the density of AF508-CFTR in the plasma membrane. To determine the effect of the corrected compound on CFTR current density, cells were incubated with 10 μΜ of the test compound for 24 hours at 37 ° C and compared to 27 ° C and 37 ° C for comparison of current density (% active). Prior to recording, cells were washed 3 times with extracellular recording medium to remove any residual test compound. Pre-incubation with 10 μM of the calibration compound significantly increased cAMP and genistein-dependent currents compared to the 37 °C control. 8. Identification of potentiator compounds also using perforated patch recording techniques to study the ability of AF508-CFTR potentiators to increase 156806.doc -62- 201204353 Stable performance of AF508-CFTR in NIH3T3 cells with macroscopic ΔΡ508-€ Cl current (Iaf508) . Synergists identified by optical assays caused a dose-dependent increase in IaF508, with similar potency and efficacy observed in optical assays. In all of the test cells, the reversal potential before and during the application of the synergist was about -30 mV, which is the EC1 calculated value (-28 mV). 9. Solution intracellular solution (mM): aspartic acid planer (90), CsCl (50), MgCl2 (1), HEPES (IO) and 240 pg/ml amphotericin-B (amphotericin-B) ( Adjust to pH 7.35 with CsOH. Extracellular solution (mM): thiol-D-reducing glucosamine (NMDG)-Cl (150), MgCl2(2), CaCl2(2), HEPES(10) (adjusted to pH 7.35 with HC1) ° 10. Cell cultures NIH3T3 mouse fibroblasts stably expressing AF5 08-CFTR were used for whole cell recording. The cells were maintained in a 175 cm2 culture flask supplemented with 2 mM glutamic acid, 10% fetal bovine serum, 1 X NEAA, β-ΜΕ, 1 X penicillin/streptomycin at 37 ° C, 5% CO 2 and 90% humidity. And 25 mM HEPES in Dubeck modified Eagle's medium. For whole cell recording, 2,500-5,000 cells were seeded on poly-L-lysine coated glass coverslips and incubated at 27 °C for 24-48 hours, followed by testing for potentiator activity; Incubation at 37 ° C in the presence or absence of a calibration compound for measuring the activity of the calibrator. 11. Single-channel recording The inner membrane valgus slice was used to observe the single-channel activity of the temperature-corrected ΔF508-CFTR and the activity of the synergist chelating agent in NIH3T3 cells 156806.doc -63- 201204353. Briefly, single channel active voltage clamp recordings were performed at room temperature using an Axopatch 200B patch clamp amplifier (ΑΧσϊ1 Instruments Inc.). All records were taken at a 10 kHz sampling frequency and low pass filtered at 400 Hz. The membrane drop tube was made by Corning Kovar Sealing #7052 (World Precision Instruments, Inc., Sarasota, FL). When filled with an extracellular solution, the electrical resistance was 5-8 ΜΩ. AF508-CFTR was activated after sectioning by the addition of 1 mM Mg-ATP and 75 nM cAMP-dependent protein kinase catalytic subunit (PKA; Promega Corp. Madison, WI). After the channel activity is stabilized, the membrane is infused using a gravity driven microperfusion system. The influent is placed adjacent to the membrane so that complete solution exchange takes place within 1-2 seconds. To maintain AF508-CFTR activity during rapid perfusion, a non-specific phosphatase inhibitor F' (10 mM NaF) was added to the cell solution. Under these recording conditions, channel activity remained constant throughout the membrane recording period (up to 60 minutes). The current generated by the positive charge (the anion moves in the opposite direction) moving from the intracellular solution to the extracellular solution is shown as a positive current. The drop tube potential (Vp) was maintained at 80 mV. Channel activity was analyzed from a membrane containing S2 active channels. The maximum number of simultaneous openings determines the number of active channels during the course of the experiment. To measure the single-channel current amplitude 'offline' at 120 Hz, filter the 120-second record from ΔΡ5〇8-CFTR activity, then use it to construct a full-point amplitude histogram and use Bio-Patch to analyze the software (Bio-Logic Comp France) Fitting these histograms with a multi-Gaussian function. The total macro current and the open probability (P.) were measured from the channel activity of 120 seconds. Use the Bi〇_patch software or by the 156806.doc •64· 201204353 system? . =1/丨(&gt;〇determination p., where ι = average current, i = single channel current amplitude, and N = number of active channels in the membrane. 12. Solution extracellular solution (mM): NMDG (150) Aspartic acid (150), CaCl2 - (5), MgCl2 (2) and HEPES (10) (adjusted to pH 7.35 with Tris base). 'Intracellular solution (mM): NMDG-C1 (150), MgCl2 (2), EGTA (5), TES (10) and Tris test (14) (adjusted to pH 7.35 with HC1) 13. Cell culture 〇 stable expression of AF508-CFTR NIH3T3 mouse fibroblasts for resection of the membrane The pliers were recorded. The cells were maintained in a 175 cm2 flask at 37 ° C, 5% CO 2 and 90% humidity supplemented with 2 mM branic acid, 10% fetal bovine serum, 1 X NEAA, β-ΜΕ, 1 X penicillin. /Streptomycin and 25 mM HEPES in Dubeck modified Eagle's medium. For single channel recording, 2,500-5,000 cells were seeded onto poly-L-lysine coated glass coverslips and before use Incubate for 24-48 hours at 27 ° C. q The activity of Compound 1 (i.e., IC50) was measured using the procedure described above and is shown in Table 11. Table 11. rt -, νε 1 +++ +++ . 156806.doc -65-

Claims (1)

201204353 VII. Patent application scope: 1_ A formulation containing (i?)_l-(2,2-difluorobenzo[d][i,3]dioxol-5-yl)-N -(l-(2,3-dihydroxypropyl)·6-fluoro-2-(1-hydroxy-2-indolyl-2-yl)-1Η-π-β-to-5-yl)cyclopropane Aromatic amine (Compound 1), water and • Polyethylene glycol (PEG). • 2. As requested! A formulation wherein the polyethylene glycol is selected from the group consisting of PEG 3(R), PEG 400, PEG 500, PEG 600, PEG 700, PEG 800, PEG 900 or PEG 1000. Π ^ 3_ The formulation of claim 1, wherein the polyethylene glycol is PEG 400. 4. The formulation of claim 1, wherein the concentration of the compound 1 is from about 5% by weight to about 3% by weight. 5. The formulation of claim 1, wherein the concentration of the compound 1 is from about 5% by weight to about 2% by weight. 6. The formulation of claim 1, wherein the concentration of the compound 1 is about 12% by weight, 0.25% by weight, or 5% by weight. 7. The formulation of claim 1, wherein the concentration of the polyethylene glycol is from about 4% by weight to about 60% by weight. 8. The formulation of claim 1 wherein the concentration of the polyethylene glycol is from about 45 weight percent to about 55 weight percent. 9. The formulation of claim 1, wherein the concentration of the polyethylene glycol is about 5% by weight. 10. The formulation of claim 1, wherein the concentration of the compound 1 is from about 5% by weight to about 3% by weight: and the concentration of the polyethylene glycol is from about 40% by weight to about 60% by weight. 156806.doc 201204353 11. The formulation of claim 1 wherein the concentration of the compound 1 is from about 1 weight to about 2 weight and the concentration of the polyethylene glycol is about 45 weight. Up to about 5% by weight. 12. The formulation of claim 1 wherein the concentration of the compound 1 is about 〇 η wt%, 0.25 wt. /. Or 0.50% by weight; and the concentration of the polyethylene glycol is about 50% by weight. 13. The formulation of claim 1 wherein the concentration of the compound 1 is about ο 重量 %, 0.25 wt% or 0.50 wt%; and the polyethylene glycol is about 5 〇 wt % 2 PEG 400. 14. The formulation of claim 1 further comprising a surfactant. 15. The formulation of claim 14, wherein the surfactant is an anionic, cationic or nonionic surfactant. 16. The formulation of claim 14 wherein the surfactant is a nonionic surfactant selected from the group consisting of vitamin E d-α-tocopheryl PEG 1000 succinate (vitamin E TPGS), Polysorbate 2〇, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, calcined poly(ethylene oxide), poloxamine (p〇i〇xainine) ), hospital based polyglucose, octyl glucomannan, mercapto maltoside, fatty alcohol, cetyl alcohol, oleic acid, cocoamine MEA, cocoamine DEA, polyethylene glycol stearate 15 (Solutol HS 15) and cocoamine TEA. The formulation of claim 14, wherein the surfactant is vitamin E TPGS ° 1 8. The formulation of claim 14, wherein the concentration of the surfactant is from about 5% by weight to about 15% by weight. 156806.doc 201204353 19. The formulation of claim 14 wherein the concentration of the surfactant is from about 1% by weight to about 12% by weight. 20. The formulation of claim 14, wherein the concentration of the surfactant is about 重量 wt%. 21. The formulation of claim 14, wherein the surfactant is about 5% by weight of vitamin E TPGS. 22. The formulation of claim 14 wherein the concentration of the compound 1 is from about 5% by weight to about 3 parts by weight. The concentration of the polyethylene glycol is from about 4% by weight to about 60% by weight and the concentration of the surfactant is from about 5% by weight to about 15% by weight. 23. The formulation of claim 22, wherein the compound! The concentration is from about 5% by weight to about 3 parts by weight. The polydiamide is from about 40% by weight to about 6% by weight of 2 PEG 400, and the surfactant is from about 5% by weight to about 15% by weight of Vitamin E TPGS. 24_ The formulation of claim 1 wherein the formulation is from about 3 〇g to 5 〇g 〇 25. The formulation of claim 1 wherein the formulation is from about 35 g to 45 g 26. A formulation of 1 wherein the formulation is about 4 g. 27. The formulation of claim 14 wherein the formulation comprises the following: component content (g) compound 1 0.05 PEG 400 19.95 vitamin E TPGS 4.48 water 15.52 28. The formulation of claim 14 wherein the formulation comprises The following: 156806.doc 201204353 Component content (g) Compound 1 0.10 PEG 400 19.90 Vitamin ETPGS 4.48 Water 15.52 29. The formulation of claim 14, wherein the formulation comprises the following: Component content (g) Compound 1 0.20 PEG 400 19.80 Vitamin ETPGS 4.48 Water 15.52 3 0. The formulation of claim 14, wherein the formulation comprises the following: Component content (g) Compound 1 0.30 PEG 400 19.70 Vitamin ETPGS 4.48 Water 15.52 3 1. As requested in claim 14 a formulation wherein the formulation comprises the following: Component content (% by weight) Compound 1 0.10-0.80 PEG 400 48-51 Vitamin ETPGS 10-12 Water 37-40 3 2. Formulation of claim 14, wherein the formulation The inclusions include the following: Component content (% by weight) Compound 1 0.20-0.60 156806.doc 201204353 PEG 400 49-50 Vitamin ETPGS 10-12 Water 37-40 33. As requested in item 14 Wherein the formulation comprises the following: Component content (% by weight) Compound 1 0.25 PEG 400 49.75 Vitamin ETPGS 11.20 Water 38.80 ^ 34. The formulation of claim 14, wherein the formulation comprises the following: Component content (% by weight) Compound 1 0.50 PEG 400 49.50 Vitamin ETPGS 11.20 Water 38.80 3 5. The formulation of claim 1 further comprising a taste masking agent. 3. The formulation of claim 1, which further comprises other therapeutic agents. 37. The formulation of claim 36, wherein the additional therapeutic agent is selected from the group consisting of a mucolytic agent, a bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR modulator other than Compound 1, or a nutrient. 3. The use of a formulation according to claim 1 for the manufacture of a medicament for the treatment of a CFTR-mediated disease selected from the group consisting of cystic fibrosis, COPD, emphysema, dry eye or bone Looseness. 39. The use of claim 38, wherein the CFTR mediated disease is cystic fibrosis. 156806.doc 201204353 40. The use of claim 38, wherein the medicament comprises an additional therapeutic agent. 41. The use of claim 40 wherein the other therapeutic agent is selected from the group consisting of a mucolytic agent, a bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR modulator other than Compound 1, or a nutrient. 42. A medical package or kit that contains as requested! &amp; Item 1 formulation and its instruction manual. 156806.doc 201204353 IV. Designated representative map: (1) The representative representative of the case is: (none) (2) The symbolic symbol of the representative figure is simple: 5. If there is a chemical formula in this case, please reveal the best indication of the characteristics of the invention. Chemical formula: Compound 1 156806.doc -2-