Triazolopyridine Polymorph A

Abstract

The present invention relates to Form A polymorph of triazolopyridine compounds, pharmaceutical compositions thereof, and methods of use.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Application No. 17 / 838,838, filed Jun. 13, 2022. The entire teachings of the above application are incorporated herein by reference.

Background Art

[0002] Background Mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene result in the absence or dysfunction of a protein that controls ion transport across the apical cell membrane at the surface of certain epithelia. CFTR functions primarily as a chloride channel but has many other roles such as inhibition of sodium transport through epithelial sodium channels, outwardly rectifying chloride channels, ATP channels, control of intracellular vesicular trafficking, and inhibition of endogenous calcium-activated chloride channels. CFTR is also a bicarbonate channel that has several conformations that enhance bicarbonate conductance. Defects in bicarbonate secretion result in poor solubility and aggregation of luminal mucins in several organs with pancreas, sinuses, vas deferens, and other tissues that depend on CFTR-mediated bicarbonate secretion. Reduction of CFTR function such as bicarbonate conductance results in recurrent acute pancreatitis (RAP) or chronic pancreatitis (CP) by one or more mechanisms (LaRusch et al PLoS Genetics. 2014;10(7):e1004376). In the lung, CFTR dysfunction results in depletion of airway surface liquid (ASL) and thick viscous mucus that adheres to the airway surface. The result is a decrease in mucociliary clearance (MCC) and a deficit in host defense. The dehydrated thick secretions result in a worsened inflammatory response that leads to bronchial infections by a limited spectrum of characteristic bacteria, mainly Staphylococcus aureus and Pseudomonas aeruginosa, as well as the development of bronchiectasis and progressive obstructive airway disease. Lung dysfunction is the cause of most CF-related deaths. (Cohen-Cymberknoh, M et al., Am. J. Respir. Crit. Care Med. 1463-1471, 2011).

[0003] The prognosis for the treatment of cystic fibrosis (CF) and other CFTR-mediated diseases has improved over the past 40 years. This has been achieved by improving pancreatic enzyme replacement, replacing drugs designed to treat loss of exocrine pancreatic function and lung infections, reducing inflammation, and enhancing mucociliary clearance. Currently, the therapeutic challenges are to correct the biochemical defect in CF and to identify effective treatments for chronic respiratory infections. (Frerichs C. et al., Expert Opin Pharmacother. 10(7), 1191-202, 2009). SUMMARY OF THE INVENTION

[0004] Summary The present invention relates to novel solid forms or polymorphs of a compound

Chemical formula

[0005] In one aspect, the present application provides a crystalline form of a compound, such as Form A, characterized by X-ray powder diffraction (XRPD) comprising one or more peaks at about 18.5, 19.5, 21.5, 23 and 24° θ using CuKα radiation. Preferably, Form A exhibits a differential scanning calorimetry (DSC) thermogram comprising an endothermic peak starting at about 203°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Brief Summary of the Drawings

Figure 1

[0007] Detailed Description of the Invention The present invention has the structure: [Chem.] relates to a stable solid form or polymorph of a compound having

[0008] The compounds of the present invention can be prepared by the method described in U.S. Patent 10,472,357 (Cole et al., issued November 12, 2019).

[0009] The compounds of the present invention are useful as modulators of CFTR and treat CFTR-mediated diseases or disorders, such as cystic fibrosis, constipation, distal intestinal obstruction syndrome, asthma, sinusitis, sinus obstruction, acute pancreatitis, recurrent acute pancreatitis, chronic pancreatitis, pancreatic exocrine insufficiency, gastrointestinal disorders, liver disorders, bile duct disorders, gallbladder disorders, infertility, duodenal inflammation, peptic ulcer disease, hereditary emphysema, hereditary hemochromatosis, coagulation-fibrinolysis deficiency, such as protein C deficiency, hereditary angioedema type 1, lipid processing deficiency, such as familial hypercholesterolemia, type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage disease, such as I-cell disease (I-cell disease) / pseudo-Hurler, mucopolysaccharidosis, Sandhoff / Tay-Sachs, Crigler-Najjar type II, multiple endocrine neoplasia / hyperinsulinemia, true diabetes, Laron dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, glycanosis CDG type 1, hereditary emphysema, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency, diabetes insipidus (DI), neurophyseal DI, renal DI, Charcot-Marie-Tooth syndrome, Pelizaeus-Merzbacher disease, neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy (plasy), Pick's disease, some polyglutamine neurological disorders, such as Huntington's, spinocerebellar ataxia type I, spinal bulbar muscular atrophy, dentatorubal pallidoluysian and myotonic dystrophy, and spongiform encephalopathy, such as hereditary Creutzfeldt-Jakob disease (due to prion protein processing deficiency), Fabry disease and Schröder-Shincker syndrome, etc. are useful for the treatment of diseases, disorders or conditions.

[0010] The compounds of the present invention can be administered in combination with antibiotics, anti-inflammatory drugs, bronchodilators, mucus-thinning drugs, proton pump inhibitors, histamine-2 receptor antagonists, and pancreatic enzyme replacement therapies. In particular, antibiotics for the treatment of bacterial mucoid Pseudomonas can be used in combination with the compounds of the present invention. Inhaled antibiotics such as tobramycin, colistin, and aztreonam can be used in combination with the treatment using the compounds of the present invention. Anti-inflammatory drugs can also be used in combination with the compounds of the present invention for treating CFTR-related diseases. Bronchodilators can be used in combination with the compounds of the present invention for treating CFTR-related diseases (CFTR-RD).

[0011] In one aspect, the present invention relates to a combination therapy comprising a compound of the present invention and other pharmaceutical agents useful for the treatment of CF and CFTR-RD. In a preferred aspect, the aminoglycoside gentamicin can be used. In a preferred aspect, a CFTR potentiator, enhancer or corrector can be used in combination with the compound of the present invention.

[0012] In one aspect, the present invention relates to a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable carrier. The composition can comprise a compound of the present invention, and optionally a pharmaceutically acceptable carrier, adjuvant or vehicle. In certain aspects, these compositions optionally further comprise one or more additional therapeutic agents useful for the treatment of CFTR-related diseases or disorders.

[0013] Pharmaceutical composition The pharmaceutical composition of the present invention comprises a therapeutically effective amount of a compound of the present invention formulated together with one or more pharmaceutically acceptable carriers or excipients.

[0014] As used herein, the term "pharmaceutically acceptable carrier or excipient" means any kind of non-toxic and inert solid, semi-solid, gel or liquid filler, diluent, encapsulating material or formulation aid. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; cyclodextrins such as alpha-(α), beta-(β) and gamma-(γ) cyclodextrins; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and coloring agents, release agents, coating agents, sweetening agents, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition at the discretion of the formulator.

[0015] The pharmaceutical compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. In a preferred embodiment, the administration is parenteral administration by injection.

[0016] The pharmaceutical compositions of the present invention can include any conventional non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle. In some cases, the pH of the formulation can be adjusted using a pharmaceutically acceptable acid, base or buffer to enhance the stability of the formulated compound or its delivery form. The term parenteral, as used herein, includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0017] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage forms may include inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, EtOAc, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, tuber oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof. In addition to the inert diluent, the oral composition may also include adjuvants such as wetting agents, emulsifiers and suspending agents, sweetening agents, flavoring and perfuming agents.

[0018] Injectable formulations, such as sterile injectable aqueous or oily suspensions, can be formulated according to known art using suitable dispersing or wetting agents and suspending agents. Sterile injectable formulations can also be sterile injectable suspensions or emulsions, such as solutions in INTRALIPID®, LIPOSYN® or OMEGAVEN®, or in a sterile non-parenterally acceptable diluent or solvent, such as a solution in 1,3 - butanediol. INTRALIPID® is an intravenous lipid emulsion containing 10 - 30% soybean oil, 1 - 10% egg yolk phospholipid, 1 - 10% glycerin and water. LIPOSYN® is also an intravenous lipid emulsion containing 2 - 15% safflower oil, 2 - 15% soybean oil, 0.5 - 5% egg phospholipid, 1 - 10% glycerin and water. Omegaven® is an injectable emulsion containing about 5 - 25% fish oil, 0.5 - 10% egg phospholipid, 1 - 10% glycerin and water. In particular, acceptable vehicles and solvents that can be used are water, Ringer's solution, USP and isotonic sodium chloride solution. Also, a sterile fixed oil is commonly used as a solvent or suspending medium. For this purpose, any low - irritation fixed oil such as synthetic mono - or diglycerides can be used. Also, fatty acids such as oleic acid are used in the preparation of injectable substances.

[0019] Injectable formulations can be sterilized, for example, by filtration through a bacteria - retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0020] Compositions for rectal or vaginal administration can be preferably prepared by mixing a compound of the present invention with a suitable non - irritating excipient or carrier, such as cocoa butter, polyethylene glycol, in the form of a suppository or a suppository wax that is solid at room temperature but liquid at body temperature and thus melts in the rectal or vaginal cavity to release the active compound.

[0021] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or; a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption promoters such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) adsorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also include buffering agents.

[0022] Similar types of solid compositions may also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol.

[0023] Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may optionally contain opacifying agents and may also be compositions that release only or preferentially the active ingredient(s) in a optionally delayed manner in a specific part of the intestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0024] Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservative or buffer, if required. Ophthalmic preparations, ear drops, ophthalmic ointments, powders and solutions are also contemplated as being within the scope of the present invention.

[0025] Ointments, pastes, creams and gels may contain, in addition to the active compounds of the present invention, excipients such as animal and vegetable lipids, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide or mixtures thereof.

[0026] Powders and sprays may contain, in addition to the compounds of the present invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder or mixtures of these substances. Sprays may further contain conventional high-pressure gases such as chlorofluorohydrocarbons.

[0027] Transdermal patches have the further advantage of providing controlled delivery of the compound to the body. Such dosage forms may be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers may also be used to increase the flow rate of the compound through the skin. The rate may be controlled either by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0028] For pulmonary delivery, the therapeutic compositions of the present invention are formulated in solid or liquid particulate form and administered to a patient by direct administration, such as inhalation into the respiratory system. Solid or liquid particulate forms of the active compounds prepared for practicing the present invention include particles of respirable size: i.e., particles of a size small enough to pass through the mouth and larynx upon inhalation and reach the alveoli of the bronchi and lungs. Delivery of aerosolized therapeutic agents is known in the art (see, e.g., U.S. Patent No. 5,767,068 to VanDevanter et al., U.S. Patent No. 5,508,269 to Smith et al., and WO 98 / 43650 by Montgomery).

[0029] The compositions described herein may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unit doses for the subject to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or for multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose. The amount of the active compound in the unit dosage form will vary, for example, depending on the host to be treated and the particular mode of administration. In one aspect, the unit dosage form may have one of the compounds of the present invention in an amount of about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 750 mg, 800 mg, 900 mg, 1000 mg or 1,250 mg as the active ingredient.

[0030] In some aspects, the compounds of the present invention may be administered at a dosage of at least about 10 mg / day to at least about 1500 mg / day. In some aspects, the compounds of the present invention are administered at a dosage of at least about 300 mg (e.g., at least about 450 mg, at least about 500 mg, at least about 750 mg, at least about 1,000 mg, at least about 1250 mg or at least about 1500 mg).

[0031] Dose adjustment can be made for patients with mild, moderate or severe liver impairment (Child-Pugh class A). Further, dose adjustment can be made for patients taking one or more cytochrome P450 inhibitors and inducers, particularly CYP3A4, CYP2D6, CYP2C9, CYP2C19 and CYP2B6 inhibitors and inducers. Dose adjustment can also be made for patients with impaired cytochrome P450 function such as poor, intermediate, extensive and ultra-rapid metabolizers.

[0032] Definitions Definitions of various terms used to describe the present invention are listed below. These definitions apply to those terms as they are used throughout this specification and the claims, unless in a specific instance it is otherwise limited, either individually or as part of a larger group.

[0033] As used herein, the word "about" is intended to refer to a peak that is within + / - 0.5 of the provided value with respect to XRPD peaks, particularly when adjusted so as not to conflict with the XPRD pattern shown in the figures.

[0034] As used herein, the term "effective amount" of a compound of interest refers to the amount of the compound of interest that, when delivered as part of a desired dosing regimen, results in the management of a disease or disorder relative to clinically acceptable standards for the desired method of treatment.

[0035] "Treatment" or "treating" refers to an approach for obtaining a beneficial or desired clinical outcome in a patient. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, the following: reduction of symptoms, diminishment of the degree of a disease, stabilization of a disease state (i.e., does not worsen), prevention of the spread (i.e., metastasis) of a disease, prevention of the onset or recurrence of a disease, delay or slowing of disease progression, improvement of a disease state, and one or more of remission (partial or total).

[0036] List of abbreviations: All temperatures are in degrees Celsius. CF - Cystic Fibrosis CFTR - Cystic Fibrosis Transmembrane Conductance Regulator CFTR - RD Cystic Fibrosis Transmembrane Conductance Regulator - Related Disorders DIPEA - N,N - Diisopropylethylamine DMF - Dimethylformamide DMSO - Dimethyl Sulfoxide ENaC - Epithelial Sodium Channel Et2O - Diethyl Ether Et3N - Triethylamine EtOAc - Ethyl Acetate h - hour H2O - Water HATU - (1 - [Bis(dimethylamino)methylene] - 1H - 1,2,3 - triazolo[4,5 - b]pyridinium 3 - oxide hexafluorophosphate) HBS - Hepes Buffered Saline HCl - Hydrochloric Acid HOAc - Acetic Acid HPLC - High Performance Liquid Chromatography hr - hour HTS - High - Throughput Screen MDC - Methylene Dichloride Na2SO4 - Sodium Sulfate NaH - Sodium Hydride NaHCO3 - Sodium Bicarbonate NAUC - Area Under the Curve NH4Cl - Ammonium Chloride NMR - Nuclear Magnetic Resonance PBS - Phosphate Buffered Saline POCl3 - Phosphorus Oxychloride rt - Room Temperature TEA - Triethylamine TFA - Trifluoroacetic Acid Tetrakis - (Triphenylphosphine)palladium(0) THF - Tetrahydrofuran YFP - Yellow Fluorescent Protein

Example

[0037] Example

Chem.

[0038]

Chem.

[0039]

Chem.

[0040]

Chemical formula

[0041]

Chemical formula

[0042] [Chemical Structure] 2-Chloro-6-(phenylsulfonyl)-[1,2,4]triazolo[1,5-a]pyridine: To a stirred solution of 6-(phenylsulfonyl)-[1,2,4]triazolo[1,5-a]pyridin-2-amine (52.0 g, 189.4 mmol) in conc HCl (625 mL) was added copper(II) chloride dihydrate (8.39 g, 49.24 mmol) at 25 °C. The reaction mixture was cooled to 0 - 5 °C and sodium nitrite (15.68 g, 227.0 mmol) in water (293 mL) was added dropwise at 0 - 5 °C over 30 min, and the reaction was stirred at 25 °C for 16 h. The reaction mass was diluted with water (3000 mL) and stirred for 1 h. The solid was filtered and dried under vacuum to give the crude product, which was purified by column chromatography (2% methanol in MDC) to give 2-chloro-6-(phenylsulfonyl)-[1,2,4]triazolo[1,5-a]pyridine (44.0 g). 1H NMR: (400 MHz, DMSO) δ: 7.638 - 7.686 (m, 2H), 7.726 - 7.769 (m, 1H), 7.967 - 8.006 (m, 1H), 8.092 - 8.125 (m, 3H), 9.741 (s, 1H).

[0043] [Chemical Structure] (N-(4-Bromobenzyl)-6-(phenylsulfonyl)-[1,2,4]triazolo[1,5-a]pyridin-2-amine: A stirred solution of 2-chloro-6-(phenylsulfonyl)-[1,2,4]triazolo[1,5-a]pyridine (40.0 g, 136.1 mmol) in (4-bromophenyl)methanamine (40 g, 214.9 mmol) was heated at 140 °C for 16 h. The reaction mixture was cooled, diluted with methanol, and stirred for 1 h. The solid was filtered off and dried under vacuum to give (N-(4-bromobenzyl)-6-(phenylsulfonyl)-[1,2,4]triazolo[1,5-a]pyridin-2-amine (38.0 g, 444.9 [M+1]). 1H NMR: (400 MHz, DMSO) δ: 4.437 - 4.453 (d, J = 6.4, 2H), 7.286 - 7.307 (d, J = 8.4, 2H), 7.482 - 7.520 (m, 3H), 7.611 - 7.649 (t, 2H), 7.688 - 7.738 (q, 2H), 7.783 - 7.811 (d, J = 9.2, 1H), 8.042 - 8.063 (d, J = 8.4, 2H), 9.287 - 9.290 (d, J = 1.2, 1H).

[0044] [Chemical formula] 6-(Phenylsulfonyl)-N-(4-(pyridin-2-yl)phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-amine: A solution of 6-(phenylsulfonyl)-[1,2,4]triazolo[1,5-a]pyridin-2-amine (300 mg, 1.09 mmol), 2-(4-bromophenyl)pyridine (304 mg, 1.31 mmol), davephos (68 mg, 0.17 mmol) and Cs2CO3 (708 mg, 2.18 mmol) in dry 1,4-dioxane (15 mL) was prepared. The reaction was degassed under nitrogen and vacuum for 10 minutes. Pd(OAc)2 (39 mg, 0.17 mmol) was added and then the reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled, diluted with water (50 mL) and extracted with EtOAc (50 mL x 2). The organic layer was washed with brine (30 mL), dried over anhydrous sodium sulfate and evaporated to dryness to give the crude product, which was purified by flash chromatography (0 - 5% MDC in methanol) to give pure 6-(phenylsulfonyl)-N-(4-(pyridin-2-yl)phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-amine (10 mg, 428.09 [M+H]). 1 1H NMR: (400 MHz, DMSO) (3716) δ: 7.27 - 7.30 (m, 1H), 7.66 - 7.68 (m, 2H), 7.71 - 7.75 (m, 3H), 7.81 - 7.86 (m, 2H), 7.91 - 7.95 (m, 2H), 8.06 - 8.12 (m, 4H), 8.62 - 8.63 (d, 1H), 9.545 - 9.548 (d, J = 1.2 Hz, 1H), 10.197 (s, 1H).

[0045] An alternative protocol is as follows:

Chemical Structure

[0046] Product 6-(Phenylthiol)-[1,2,4]triazolo[1,5-a]pyridin-2-amine (150 g, 619 mmol) was added to a round-bottom flask equipped with a mechanical stirrer, a cooler, and a thermocouple. After adding acetic acid (10 volumes) and heating, NaBO3 (285 g, 1.9 mol) was added and stirred for 2 h. The reaction was cooled to 35 °C and quenched with 15 volumes of 10% aqueous Na2S2O3. The reaction mixture was filtered through a PPFC and a Buchner funnel and dried in an oven to a constant mass (153 g, 91% crude yield).

[0047] 6-(Phenylsulfonyl)-[1,2,4]triazolo[1,5-a]pyridin-2-amine (153 g) was added to a round-bottom flask equipped with an overhead stirrer, a cooler, N2, and a temperature probe, and then 4-(pyridin-2-yl)benzaldehyde was added. The mixture was diluted with 10 volumes of THF, and then 2,2,2-trifluoroacetic acid (4 equivalents) was added. Sodium triacetoxyborohydride was added (1.5 equivalents), and the mixture was stirred overnight at room temperature. At completion, the reaction was quenched with water (12 volumes), and the pH was adjusted to 6 using 40% NaOH. The precipitate was filtered off, collected, washed twice with a THF / water mixture, and then dried overnight in a vacuum oven to give 216 g (88%) of the desired material.

[0048] Form A Form A was examined for solubility by slurrying at room temperature in various organic solvents and solvent mixtures and examined by XPRD. Form A was identified, for example, in acetone, ethanol, ethyl acetate, methanol, water, dichloromethane, and tetrahydrofuran. The saturated solvent-mediated polymorph screen experiment consisted of dissolving the compound in or near the selected organic solvent to achieve supersaturation and allowing the precipitation of the solid upon returning to ambient temperature. The solid was first examined by PLM, and in the case of birefringence, subsequently examined by FTIR and XRPD for polymorph determination. Birefringent anisotropic needles were formed. The figure provides an overlay of several polymorphs that were recovered. The bottom line represents the starting material.

[0049] Assay for Detecting and Measuring the Effect of Compounds on the dF508-CFTR Channel CFRT-YFP High-Throughput Assay: Collector Assay: The following protocol is designed to selectively screen small molecule compounds for other protein-modifying gene variants of F508del and wild-type CFTR collector activity in the HTS YFP flux assay. In this protocol, cells are incubated with test compounds for 24 hours, washed with PBS, stimulated with forskolin and standard potentiators, and read on a 384-well HTS plate reader, such as the Hamamatsu FDDD-6000.

[0050] YFP fluorescence intensity is acquired rapidly before and after injecting iodide buffer into the assay cells. Iodide enters the cells through active CFTR channels in the cell membrane and quenches YFP fluorescence. The rate of fluorescence quenching is related proportionally to the total CFTR activity in the cell membrane. The dF508-CFTR collector accelerates YFP quenching by increasing the number of CFTR molecules in the test cell membrane.

[0051] This method was first developed for a benchtop plate reader (Galietta, 2001) and adapted to the HTS format (Sui, 2010).

[0052] Fisher rat thyroid (FRT) cells stably expressing both human ΔF508-CFTR and halide-sensitive yellow fluorescent protein (YFP-H148Q / I152L 25, 22) (Galietta, 2001) were cultured on plastic surfaces in Coon's modified Ham's F12 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin. G418 (0.75 - 1.0 mg / mL) and zeocin (3.2 μg / mL) were used for the selection of FRT cells expressing ΔF508-CFTR and YFP. For primary screening, FRT cells were plated at a cell density of 20,000 - 40,000 cells per well in 384-well, black-wall, clear-bottom microtiter plates (Costar; Corning Inc.). Test compounds were applied to the cells at varying concentrations in the range of 2 nM - 40 nM in either a 2-fold or 3-fold dilution series. The cells were incubated for 24 - 26 h at 37 °C with 5% CO2 in a cell culture incubator. The assay plates were washed with DPBS medium (Thermo, cat#SH30028.02) to remove unbound cells and compounds. Stimulation medium (25 μL) containing 20 μM forskolin and 30 μM P3 [6-(ethyl-phenyl-sulfonyl)-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid 2-methoxy-benzylamide] in Hams F-12 coon's modified medium was added to the plate wells and incubated at room temperature for 60 - 120 min. Then 25 μL of HEPES-PBS-I buffer (10 mM HEPES, 1 mM MgCl2, 3 mM KCl, 1 mM CaCl2, 150 mM NaI) was added and the fluorescence quenching curve (excitation 500 nm / emission 540 nm; exposure 136 ms) was immediately recorded with an FDSS-6000 plate reader (Hamamatsu). The quenching rate was derived from the least-squares fitting of the data (Sui, 2010).

[0053] Enhancement factor assay: The following protocol is designed to selectively screen small molecule compounds for F508del, other protein-modifying gene variants, and wild-type CFTR potentiator activity in the HTS YFP flux assay. In this protocol, cells are incubated at low temperature for uniformly accelerated dF508-CFTR expression in the cell membrane and then incubated at 27 °C for 24 hours, washed with PBS, stimulated with forskolin, and read on a 384-well HTS plate reader, such as the Hamamatsu FDDD-6000.

[0054] YFP fluorescence intensity is acquired rapidly before and after injection of iodide buffer into the assay cells. Iodide enters the cells through the active CFTR channels in the cell membrane and quenches the YFP fluorescence. The rate of fluorescence quenching is proportionally related to the total CFTR activity in the cell membrane. dF508del-CFTR potentiators accelerate YFP quenching by increasing CFTR activity in the test cell membrane.

[0055] This method was first developed for a benchtop plate reader (Galietta, 2001) and adapted to the HTS format (Sui, 2010).

[0056] Fisher rat thyroid (FRT) cells (Galietta, 2001) that stably express both human ΔF508-CFTR and halide-sensitive yellow fluorescent protein (YFP-H148Q / I152L 25, 22) were cultured on plastic surfaces in Coon's modified Ham's F12 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin. For the selection of FRT cells expressing ΔF508-CFTR and YFP, G418 (0.75 - 1.0 mg / mL) and zeocin (3.2 μg / mL) were used. For the primary screening, FRT cells were plated at a cell density of 20,000 - 40,000 cells per well in 384-well black-walled clear-bottom microtiter plates (Costar; Corning Inc.). The cells were incubated at 37 °C for 24 - 26 h in a cell culture incubator with 5% CO2. The assay plates were washed with DPBS medium (Thermo, cat#SH30028.02) to remove unbound cells. Test compounds were applied to the cells at varying concentrations in the range of 2 nM - 40 nM in either a 2-fold or 3-fold dilution series in DPBS and stimulated with 20 μM forskolin (final concentration) in Hams F-12 coon's modified medium. The plates were incubated at room temperature for 60 - 120 min. Then, 25 μL of HEPES-PBS-I buffer (10 mM HEPES, 1 mM MgCl2, 3 mM KCl, 1 mM CaCl2, 150 mM NaI) was added, and the fluorescence quenching curve (excitation 500 nm / emission 540 nm; exposure 136 ms) was immediately recorded with an FDSS-6000 plate reader (Hamamatsu). The quenching rate was derived from the least-squares fitting of the data (Sui, 2010).

[0057] Reference: Galietta, L. V., Jayaraman, S., and Verkman, A. S. Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists. Am. J. Physiol. Cell Physiol. 281(5), C1734-42, 2001. Sui J., Cotard S., Andersen J., Zhu P., Staunton J., Lee M., Lin S. (2010) Optimization of a Yellow fluorescent protein-based iodide influx high-throughput screening assay for cystic fibrosis transmembrane conductance regulator (CFTR) modulators. Assay Drug Dev. Technol. 2010 Dec;8(6): 656-68.

[0058] Cell culture: Primary CF airway epithelial cells were obtained from the UNC Cystic Fibrosis Tissue Procurement and Cell Culture Core. Cells were grown at 37 °C in a Heracell 150i incubator using growth medium (BEGM, Fischer). Cells were then transferred to differentiation medium (ALI, UNC) on coated Costar Snapwells for at least 4 weeks. Two days before the Ussing assay, the mucosa on the apical surface of the cells was aspirated after at least 30 (30) minutes of incubation with 200 μL of differentiation medium. One day before the Ussing assay, test compounds were added to the basolateral surface of the cells at various test concentrations dissolved in DMSO. Correctors at the same concentration were added to 3 or 4 wells to yield a protocol with n = 3 or n = 4.

[0059] Ussing assay: The Ussing chamber and associated voltage clamp were obtained from Physiologic Instruments (San Diego, CA). The Ussing assay was performed at 37 °C. HEPES-buffered saline (HB-PS) was used in the apical and basolateral chambers, and glucose was added to the basolateral solution. Prior to application of the voltage clamp, the epithelium was equilibrated in the chamber for 15 minutes while stabilizing and adjusting the bath temperature and transepithelial voltage.

[0060] The compounds were added in the following order: [Table 1]

[0061] The short-circuit current and resistance (typically > 300 Ω-cm2) from each chamber were recorded every 10 seconds and stored on a PC using Acquire and Analyze (Physiologic Instruments).

[0062] Analysis: The efficacy of the test compounds was compared using the mean forskolin response and CFTR-172 response of the test compounds divided by the mean forskolin response and CFTR-172 response induced by the positive control. The standardized scores were tabulated for all compounds and concentrations. The compounds showed an Ec50 of 1 - 10 μM.

[0063] The compounds and processes of the present invention are intended as illustrative only and are better understood in connection with the following examples which are not intended to limit the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art, and such changes and modifications, including those related to the chemical structure, substituents, derivatives, formulations and / or methods of the present invention, may be made without departing from the spirit of the invention and the scope of the appended claims.

[0064] The patents and scientific literature referred to in this specification establish knowledge available to those of ordinary skill in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated herein by reference. All other published references, documents, transcripts, and scientific literature cited herein are incorporated herein by reference.

Claims

Claim 1 A compound, characterized by having one or more X-ray powder diffraction peaks at about 18.5, 19.5, 21.5, 23 and / or 24° θ using CuKα radiation: 【Chemical Formula 1】 Polymorph Form A. Claim 2 The Form A polymorph according to claim 1, characterized by having two or more X-ray powder diffraction peaks at about 18.5, 19.5, 21.5, 23 and / or 24° θ using CuKα radiation. Claim 3 The Form A polymorph according to claim 1, characterized by having three or more X-ray powder diffraction peaks at about 18.5, 19.5, 21.5, 23 and / or 24° θ using CuKα radiation. Claim 4 The Form A polymorph according to claim 1, characterized by having four or more X-ray powder diffraction peaks at about 18.5, 19.5, 21.5, 23 and / or 24° θ using CuKα radiation. Claim 5 The Form A polymorph according to claim 1, characterized by having an X-ray powder diffraction peak at about 18.5, 19.5, 21.5, 23 and 24° θ using CuKα radiation. Claim 6 The Form A polymorph according to claim 1, showing a differential scanning calorimetry (DSC) thermogram including an endothermic peak starting at about 203°C. Claim 7 A pharmaceutical composition comprising the compound according to any of the preceding claims and one or more of a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable vehicle or a pharmaceutically acceptable excipient. Claim 8 A method for treating a disease or disorder mediated by cystic fibrosis transmembrane conductance regulator (CFTR), comprising the step of administering a therapeutically effective amount of the compound according to any of claims 1 to 6 or the composition according to claim 7 to a patient in need of treatment for a disease or disorder mediated by cystic fibrosis transmembrane conductance regulator (CFTR). Claim 9 The method according to claim 8, wherein the disease or disorder is selected from cystic fibrosis, hereditary emphysema, distal intestinal obstruction disorder, asthma, sinusitis, sinus obstruction, acute pancreatitis, recurrent acute pancreatitis, chronic pancreatitis, pancreatic exocrine insufficiency, gastrointestinal disorders, liver disorders, bile duct disorders, gallbladder disorders, infertility, duodenal inflammation, peptic ulcer disease, hereditary hemochromatosis, constipation, coagulation-fibrinolysis deficiency, type 1 hereditary angioedema, lipid processing deficiency, such as familial hypercholesterolemia, type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage disease, such as I-cell disease / pseudo-Hurler, mucopolysaccharidosis, Sandhoff / Tay-Sachs, Crigler-Najjar type II, multiple endocrine neoplasia / hypoinsulinemia, true diabetes, Laron dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, glycanosis CDG type 1, hereditary emphysema, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency, diabetes insipidus (DI), neurophyseal DI, nephrogenic DI, Charcot-Marie-Tooth syndrome, Pelizaeus-Merzbacher disease, neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, Pick's disease, some polyglutamine neurological disorders, spongiform and myotonic dystrophy.

10. A method for treating cystic fibrosis or a symptom thereof, comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 6 to a subject in need of treatment for cystic fibrosis or a symptom thereof.

11. A method for treating pancreatitis or a symptom thereof, comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 6 to a subject in need of treatment for pancreatitis or a symptom thereof.

12. A method for treating a disease, dysfunction or symptom thereof of the liver, bile duct or gallbladder, comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 6 to a subject in need of treatment for a disease, dysfunction or symptom thereof of the liver, bile duct or gallbladder.

13. A method for treating a disease, dysfunction or symptom thereof of the intestine, comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 6 to a subject in need of treatment for a disease, dysfunction or symptom thereof of the intestine.

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