Pyridazinone compounds that modulate mutant proteins for treating respiratory diseases

Abstract

The present disclosure relates to pyridazinone compounds of formula (I) for the treatment of respiratory diseases that arise as a result of misfolded or misshaped proteins, such as chronic obstructive pulmonary disease (COPD), cystic fibrosis, cancer, long QT syndrome or Dravet syndrome. [Formula 1] TIFF2025529902000034.tif62159

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 400,857, filed August 25, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] The present application relates to pyridazinone compounds of formula (I) for the treatment of respiratory diseases associated with misfolded or misshaped proteins. [Background technology]

[0003] Cystic fibrosis (CF), the most common fatal genetic disease in Canadians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR / ABCC7) gene. This gene encodes the CFTR protein, a plasma membrane channel responsible for chloride ion flow across the apical membranes of epithelial cells in hollow organs such as the lungs, pancreas, and gastrointestinal tract, as well as in skin (Gadsby, et al. (2006) The ABC protein turned chloride channel whose failure causes cystic fibrosis, Nature, 440: 477-483). This maintains the ionic balance necessary for the thin layer of mucus in these organs. Normally, the CFTR protein is synthesized in the endoplasmic reticulum, folded into a precise structural conformation, and then transported to the Golgi apparatus for complex glycosylation before reaching the plasma membrane.

[0004] CFTR is a member of the ATP-binding cassette (ABC) superfamily of membrane proteins that utilizes ATP hydrolysis to carry out biological processes, most commonly to actively transport substrates across membranes. CFTR is unique in that it is the only member of the family that is an ion channel; however, its structure is similar to other ABC proteins, consisting of two transmembrane domains (MSDs), each of which binds to a nucleotide-binding domain (NBD). A unique domain in CFTR is the structurally disordered regulatory (R) domain, which, when phosphorylated, is involved in channel gating. CFTR is also regulated by interdomain interactions as well as ATP binding and hydrolysis at the ATP-binding site located at the NBD1:NBD2 interface. Specifically, ATP-driven dimerization of NBD1 and NBD2 allows the ion channel to open, whereas ATP hydrolysis dissociates the dimers and closes the gate (Serohijos, AWR, et al. (2007)).

[0005] The most common mutation, present in 70% of alleles, is ΔF508, a deletion (Δ) of the amino acid phenylalanine (Phe, F) at position 508 within the CFTR protein (Welsh, MJ, et al. (1993), Dysfunction of CFTR bearing the delta F508 mutation. J. Cell Sci. Suppl., 17: 235-239). F508 is located in NBD1 at its interface with MSD2 and is thought to mediate interdomain interactions for proper folding and channel function. Specifically, the aromatic side chain of F508 is thought to form an aromatic cluster with residues from intracellular loop 4 (ICL4) of MSD2 and other residues from NBD1, which plays an essential role in the stability of the tertiary structure of CFTR (Serohijos et al., supra). Deletion of this essential residue prevents proper folding of CFTR and forces its retention in the endoplasmic reticulum, where it becomes a target for degradation. Furthermore, this mutant has reduced function even when rescued to the cell surface. The loss of F508 likely alters the domain:domain interface of the protein, preventing proper channel gating. Overall, this mutation disrupts the extracellular ionic balance, reducing surface hydration and resulting in thick, sticky mucus in many vital organs, such as the lungs and pancreas, which predisposes to bacterial infections. Many other CFTR mutations, such as the G551D mutation, are known to be gating mutations.

[0006] Small molecules can act as collectors that promote the forward transport of mutant CFTR protein to the cell surface or as enhancers that increase the channel activity of mutant CFTR protein that has reached the plasma membrane.

[0007] Chronic obstructive pulmonary disease (COPD) is a disease syndrome characterized by airway inflammation induced by environmental toxins, primarily cigarette smoke and indoor and industrial air pollution. The symptoms of COPD are highly variable and are thought to be a combination of disorders including chronic bronchitis and emphysema, both of which cause reduced airflow during expiration and are associated with obstructed bronchi. Environmental pollutants are thought to cause a loss of CFTR channel function at the luminal surface of airway epithelia, leading to mucus obstruction. Summary of the Invention

[0008] The present application is directed to pyridazinone compounds of formula (I), which in one aspect of the present disclosure are useful for treating respiratory diseases associated with misfolded or misshaped proteins, such as cystic fibrosis. In one embodiment, the disease is COPD. In one embodiment, the compounds of formula (I) modulate mutant proteins with defective activity and folding associated with respiratory diseases.

[0009] In one embodiment, the compound of formula (I) has the structure:

[0010] [ka]

[0011] (In the formula, R1 and R2 are independently or simultaneously H or (C1-C6)-alkyl; R3 and R4 are independently or simultaneously H or (C1-C6)-alkyl; or R3 and R4 together with the nitrogen atom to which they are attached form a (C5-C6)-heteroaryl or a (C4-C6)-heterocycloalkyl, each optionally substituted with halo, OH, (C1-C6)-alkyl, or halogenated (C1-C6)-alkyl; Each ring B is optionally substituted with one or more of halo, OH, (C1-C6)-alkyl, halogenated (C1-C6)-alkyl, (C3-C6)-cycloalkyl, or halogenated (C3-C6)-cycloalkyl (C6-C 10 )-aryl or (C5-C 10 )-heteroaryl; W is (C1-C6)-alkyl or -(C0-C6)-alkylene-(C6-C), each optionally substituted with halo, OH, CN, (C1-C6)-alkyl, halogenated (C1-C6)-alkyl, (C1-C6)-alkoxy, (C3-C6)-cycloalkyl, or halogenated (C3-C6)-cycloalkyl. 10 )-aryl) or any pharmaceutically acceptable salt, stereoisomer or solvate thereof It has.

[0012] The present disclosure also includes the use of a therapeutically effective amount of a compound of formula (I) as a potentiating agent, for example, in the treatment of cystic fibrosis.

[0013] The present disclosure also includes the use of a therapeutically effective amount of a compound of formula (I) as a potentiator, for example, in the treatment of COPD.

[0014] The present disclosure also includes a method for treating a patient with cystic fibrosis, comprising administering a therapeutically effective amount of a compound of formula (I).

[0015] The present disclosure also includes a method for treating a patient with COPD comprising administering a therapeutically effective amount of a compound of formula (I).

[0016] In another embodiment, a compound of formula (I) is co-administered with a collector compound, where the collector compound primarily targets cellular processing errors and trafficking of proteins to the cell surface, while the potentiator compound (of formula (I)) helps restore protein function, for example, by restoring cAMP-dependent chloride channel activity for misfolded proteins (such as CFTR) at the cell surface.

[0017] Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments herein, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present application will become apparent to those skilled in the art from this detailed description.

[0018] The present disclosure is described in the paragraphs given below by way of example with reference to the accompanying drawings. The drawings provided herein are provided for a better understanding of the exemplary embodiments and to more clearly show how various embodiments may be practiced. The drawings are not intended to limit the present disclosure. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows (A) and (B) micrographs of bead tracking and (C) a graph showing improved bead motility upon exposure to compounds of the present disclosure. [Figure 2] FIG. 2 shows the dose response for VX-770 and SK-POT (a compound of the disclosure) in potentiating cyclic AMP (forskolin-activated) chloride channel activity by F508del-CFTR as measured in CFBE41o- using a fluorescence-based membrane potential assay. [Figure 3]Figure 3(i) shows cell-attached CFTR recordings. Representative traces are shown for recordings made with a 150 NMDG-Cl base solution, b with 1 mM forskolin added, c with 10 nM SK-POT analog added, and d with 10 mM inhibitor 172 (I172); the zero current line is represented by a gray dashed line. The total number of CFTR channels in the cell-attached patch used for the representative traces shown was estimated to be 8. Vm = -80 mV; e with the open probability for each drug normalized to the open probability in the presence of 1 μM forskolin (see Methods). (ii) Bar graphs show the channel open probability normalized to the total number of channels in the presence of forskolin, forskolin + SK-POT, or after the addition of CFTR Inh-172 at the concentrations mentioned above. Error bars represent standard deviation; n = 5 for each drug. [Figure 4] Figure 4(i) shows a graph of a representative Ussing chamber assay of SK-POT (1 µM) potentiation after forskolin activation (100 nM) in primary bronchial epithelial cell cultures. (ii) The bar graph shows forskolin-dependent changes in CFTR-mediated shunting upon addition of compound 11 or VX-770 (donors n = 3, technical replicates n = 2–3 for each condition). [Figure 5]Figure 5, upper panel: Representative traces showing changes in forskolin-stimulated (addition indicated as downward-pointing black arrows) CFTR-dependent FLiPR signal measured in confluent Calu-3 cell cultures pre-exposed to cigarette smoke (CSE) and also briefly treated with forskolin (+DMSO (VEH), filled circles). Addition of CFTR inhibitors is indicated by gray arrows. Overlaid, the effects of short-term co-application of VX-770 (open squares) or SK-POT (open triangles) with forskolin are shown. Bar graphs (bottom) show the inhibitory effects of cigarette smoke extract (-CSE vs. +CSE) on CFTR channel activity, measured using the FLiPR assay in Calu-3 monolayers (three biological replicates and three to four technical replicates). The bar graph also shows the relative effects of DMSO (VEH), VX-770 (770, 1 μM), or SK-POT (1 μM), with "p" values ​​determined using One-way Anova and Sidak's multiple comparison test. Only SK-POT rescued CFTR channel function in the presence of cigarette smoke extract. [Figure 6]Figure 6 (i-iii) shows traces of motile green microspheres over a 5-second period captured at 10 frames per second using 20x objective magnification. Cells were pretreated for 24 hours with 2% cigarette smoke extract + DMSO (VEH), VX-770, or SK-POT, followed by a short-term treatment with 10 μM forskolin (FSK) for 20 minutes at room temperature before recording. ii. Traces of motile green microspheres under the same conditions as in i, except that 1 μM ivacaftor (VX-770) was co-applied during the long-term treatment. The color of the dots within the traces represents points along the 5-second period, with purple (cool) representing the beginning of the period and red / warm representing the end of the period. iv. Results of the average bead velocity across n=4 donors are tabulated, comparing the velocity without CSE to the velocity with CSE (with or without long-term co-application of VEH, VX-770 (iv), or SK-POT (v)). Points represent the mean bead velocity for individual videos, n = 4–6 videos obtained per donor and condition. Horizontal lines represent the mean across all videos, and error bars represent standard deviations. Statistical significance was assessed by One-Way Anova with Sidak's multiple comparison test. [Figure 7] Figure 7 shows (a) a representative Ussing chamber assay of SK-POT (1 µM) potentiation after forskolin activation (100 nM) in primary bronchial epithelial cell cultures prepared from the trachea of ​​adult ferrets. (b) Bar graphs show forskolin-dependent changes in CFTR-mediated shunting upon addition of SK-9919 or VX-770 (donors n = 3, technical replicates n = 2–3 for each condition). Student's t-test was performed. DETAILED DESCRIPTION OF THE INVENTION

[0020] (I) Definition As used herein, the term "(C1-C n")-alkyl" means a straight- and / or branched-chain saturated alkyl radical containing from 1 to "n" carbon atoms, including (depending on the magnitude of n) methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, 2,2-dimethylbutyl, n-pentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, n-hexyl, etc., where the variable n is an integer representing the maximum number of carbon atoms in the alkyl radical.

[0021] As used herein, the term "(C1-C n ")-alkoxy" means a straight and / or branched chain saturated alkoxy radical containing from 1 to "n" carbon atoms, including (depending on the value of n) methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, s-butoxy, isobutoxy, t-butoxy, pentoxy, hexoxy, etc., where the variable n is an integer representing the maximum number of carbon atoms in the alkyl radical.

[0022] The term "(C3-C6)-cycloalkyl" as used herein means a monocyclic saturated or partially unsaturated carbocyclic group containing 3 to 6 carbon atoms, including (depending on the degree of m) cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, etc.

[0023] As used herein, the term "heterocycloalkyl" refers to a monocyclic saturated or partially unsaturated group containing, for example, 5 to 6 ring atoms, including N, NH, N(C 1-6 alkyl), O and S, pyrrolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

[0024] The term "aryl," as used herein, means a monocyclic or bicyclic aromatic ring system containing at least one aromatic ring and 6 to 10 carbon atoms, and includes phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, indanyl, indenyl, and the like.

[0025] The term "heteroaryl" as used herein means a monocyclic or bicyclic ring system containing one or two aromatic rings and 5 to 10 atoms, including N, NH, N(C 1-6 alkyl), O, and S, and includes 1, 2, 3, or 4 heteromoieties independently selected from thienyl, thiazolyl, furyl, pyrrolyl, pyridinyl, indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl, and the like.

[0026] The term "halo" as used herein means halogen and includes chloro, fluoro, bromo, iodo and the like.

[0027] For example, the term "halogenated" as used herein with respect to a halogenated (C1-C6)-alkyl means that at least one (including all) of the hydrogens on the group in question has been replaced with a halogen atom.

[0028] As used herein, the term "stereoisomer" refers to an isomer that retains the same constitution as a corresponding stereoisomer but differs from the corresponding stereoisomer in the arrangement of its atoms in space. For example, a stereoisomer may be an enantiomer, a diastereomer, and / or a cis-trans (E / Z) isomer. It should be understood that the compound of formula (I) may include a single enantiomer, a single diastereomer, and a mixture thereof in any ratio (e.g., a racemic mixture, a non-racemic mixture).

[0029] As used herein, the term "solvate" refers to a pharmaceutically acceptable solvate form of a particular compound of formula (I) that retains the biological effects of such a compound, for example, resulting from the physical combination of the compound with one or more solvent molecules. Examples of solvates include, but are not limited to, compounds of the present invention combined with water, 1-propanol, 2-propanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine. When the solvent is water, this form is known as a "hydrate."

[0030] As used herein, the term "therapeutically effective amount" refers to a dosage that produces a selected effect. For example, an effective amount of a compound of formula (I) is an amount that is sufficient for the compound to act as a potentiator, for example, to alleviate or improve cystic fibrosis or COPD, or symptoms thereof.

[0031] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the compound of formula (I) and is not biologically or otherwise undesirable.In many cases, the compounds of the present disclosure can form acid or base salts due to the presence of acidic or basic moieties.The preparation of salts and suitable acids or bases is known in the art.

[0032] (II) Compound of formula (I) The present disclosure relates to pyridazinone compounds of formula (I), which in one aspect of the disclosure are useful for treating diseases associated with misfolded or misshaped proteins. In one embodiment, the compounds of formula (I) have the following structure:

[0033] [ka]

[0034] (In the formula, R1 and R2 are independently or simultaneously H or (C1-C6)-alkyl; R3 and R4 are independently or simultaneously H or (C1-C6)-alkyl; or R3 and R4 together with the nitrogen atom to which they are attached form a (C5-C6)-heteroaryl or a (C4-C6)-heterocycloalkyl, each optionally substituted with halo, OH, (C1-C6)-alkyl, or halogenated (C1-C6)-alkyl; Each ring B is optionally substituted with one or more of halo, OH, (C1-C6)-alkyl, halogenated (C1-C6)-alkyl, (C3-C6)-cycloalkyl, or halogenated (C3-C6)-cycloalkyl (C6-C 10 )-aryl or (C5-C 10 )-heteroaryl; W is (C1-C6)-alkyl or -(C0-C6)-alkylene-(C6-C6), each optionally substituted with one or more of halo, OH, CN, (C1-C6)-alkyl, halogenated (C1-C6)-alkyl, (C1-C6)-alkoxy, (C3-C6)-cycloalkyl, or halogenated (C3-C6)-cycloalkyl. 10 )-aryl) or any pharmaceutically acceptable salt, stereoisomer or solvate thereof It has.

[0035] In one embodiment, R1 and R2 are independently or simultaneously H or (C1-C3)-alkyl. In another embodiment, R1 and R2 are independently or simultaneously H or CH3. In another embodiment, R1 and R2 are H.

[0036] In another embodiment, R3 and R4 are independently or simultaneously H or (C1-C3)-alkyl, or R3 and R4 together with the nitrogen atom to which they are attached form a (C5)-heteroaryl or (C4-C6)-heterocycloalkyl, each optionally substituted with halo, OH, (C1-C3)-alkyl, or halogenated (C1-C3)-alkyl.

[0037] In another embodiment, R3 and R4 are independently or simultaneously H or CH3. In another embodiment, R3 and R4 are CH3.

[0038] In another embodiment, R3 and R4, taken together with the nitrogen atom to which they are attached, form an optionally substituted piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl, or pyrazolyl.

[0039] In another embodiment, ring B is (C6)-aryl or (C5-C6)-heteroaryl, each optionally substituted with one or more of halo, OH, (C1-C6)-alkyl, halogenated (C1-C6)-alkyl, (C3-C6)-cycloalkyl, or halogenated (C3-C6)-cycloalkyl. In another embodiment, ring B is phenyl or pyridinyl optionally substituted with one or more of halo, (C1-C6)-alkyl, or halogenated (C1-C6)-alkyl. In another embodiment, ring B is phenyl or pyridinyl optionally substituted with one or more of fluoro or trifluoro-methyl. In a further embodiment, ring B has the following structure:

[0040] [ka]

[0041] It has.

[0042] In another embodiment, W is (C1-C4)-alkyl or -(C0-C4)-alkylene-(C6-C), each optionally substituted with halo, OH, CN, (C1-C4)-alkyl, halogenated (C1-C4)-alkyl, (C1-C4)-alkoxy, (C3-C6)-cycloalkyl, or halogenated (C3-C6)-cycloalkyl. 10 )-aryl. In another embodiment, W is (C1-C4)-alkyl or -(C0-C1)-alkylene-phenyl, each optionally substituted with one or more of halo, OH, CN, (C1-C4)-alkyl, halogenated (C1-C4)-alkyl, or (C1-C4)-alkoxy. In another embodiment, W is a group having the following structure:

[0043] [ka]

[0044] It has.

[0045] In one embodiment of the present disclosure, the compound of formula (I) is

[0046] [ka] TIFF2025529902000007.tif187159TIFF2025529902000008.tif235159TIFF2025529902000009.tif178159

[0047] is.

[0048] In one embodiment, the compound is

[0049] [ka]

[0050] is.

[0051] The present disclosure also includes pharmaceutical compositions comprising a compound of formula (I) as defined above (the compound of the present disclosure), or a pharmaceutically acceptable salt, solvate, or prodrug thereof, and a pharmaceutically acceptable carrier or diluent. The compound is suitably formulated into a pharmaceutical composition for administration to a subject, preferably a human, in a biologically compatible form suitable for in vivo administration.

[0052] Compositions containing the compounds of the present disclosure can be prepared by known methods for preparing pharmaceutically acceptable compositions that can be administered to a subject such that an effective amount of the active substance is mixed with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (2003-20th edition) and the United States Pharmacopeia: National Formulary, 1999 (USP24 NF19). Based on this, compositions include, but are not limited to, solutions of substances associated with one or more pharmaceutically acceptable vehicles or diluents, and are contained in buffers that are isotonic with physiological fluids at an appropriate pH.

[0053] The compounds of the present disclosure can be used pharmacy in the form of free base, salt, solvate or hydrate.All forms are within the scope of the present disclosure.Acid addition salts and base addition salts can be formed with the compounds of the present disclosure for use as the source of free base form, even if a particular salt itself is only desired as an intermediate product, for example, when salts are formed only for the purpose of purification and identification.Therefore, all salts that can be formed with the compounds of the present disclosure are within the scope of the present disclosure.

[0054] In one embodiment, the compounds of formula (I) of the present disclosure are formulated into pharmaceutical compositions in a manner familiar to those skilled in the art by combining the compounds of formula (I) with suitable non-toxic, inert, therapeutically compatible solid, liquid or aerosol carrier materials and, if necessary, conventional pharmaceutical adjuvants.

[0055] Suitable carrier materials include not only inorganic carrier materials but also organic carrier materials. Suitable carrier materials for topical preparations are glycerides, semi-synthetic and synthetic glycerides, hydrogenated oils, liquid waxes, liquid paraffins, liquid fatty alcohols, sterols, polyethylene glycols and cellulose derivatives.

[0056] Conventional stabilizers, preservatives, wetting and emulsifying agents, viscosity-improving agents, salts for varying the osmotic pressure, buffer substances, solubilizers, coloring agents and antioxidants come into consideration as pharmaceutical adjuvants.

[0057] In some embodiments, the present disclosure includes compositions comprising a compound of Formula (I) as a potentiator and another active agent that is a corrector for the treatment of diseases associated with misfolded and / or misshaped proteins. In one embodiment, the corrector compound is VRT-534 or VX-809.

[0058] In another embodiment, when the compound of formula (I) is co-administered with a collector, the active agents (compound of formula (I) and collector) may be administered simultaneously or sequentially.

[0059] (III) Medical treatment methods The present disclosure includes a method of medical treatment comprising administering a compound of Formula (I) to a mammal. In some embodiments, the present disclosure includes a method for treating a respiratory disease resulting from misfolded and / or misshaped proteins. In one embodiment, the present disclosure includes a method for modulating a mutant protein having defects in activity and / or folding associated with a respiratory disease, comprising administering a compound of Formula (I). In one embodiment, the respiratory disease is cystic fibrosis or chronic obstructive pulmonary disease (COPD). In another embodiment, the present disclosure includes a method for treating other diseases associated with misfolded or misshaped proteins, such as long QT syndrome or Dravet syndrome.

[0060] In one embodiment, the present disclosure includes a method for treating a patient with a respiratory disease resulting from misfolded or misshaped proteins, comprising administering a therapeutically effective amount of a compound of Formula (I). In one embodiment, the present disclosure includes a method for treating a patient with cystic fibrosis, comprising administering a therapeutically effective amount of a compound of Formula (I). In another embodiment, the cystic fibrosis is the result of the ΔF508 mutation in the CFTR protein.

[0061] In one embodiment, the present disclosure includes a method for treating a patient with a respiratory disease resulting from misfolded or misshaped proteins, comprising administering a therapeutically effective amount of a compound of Formula (I). In one embodiment, the present disclosure includes a method for treating a patient with COPD, comprising administering a therapeutically effective amount of a compound of Formula (I). In another embodiment, COPD is the result of acquired CFTR, in which CFTR protein function is impaired by tobacco smoke or other environmental toxins.

[0062] Another embodiment of the present disclosure includes a method for treating a disease resulting from a misfolded or misshaped protein, comprising administering a therapeutically effective amount of a compound of Formula (I) to a subject, such as a human. In one embodiment, the present disclosure includes a method for modulating a mutant protein having defects in activity and / or folding associated with a respiratory disease, comprising administering a compound of Formula (I). Another embodiment of the present disclosure includes a method for treating long QT syndrome, comprising administering a therapeutically effective amount of a compound of Formula (I) to a subject, such as a human. Another embodiment of the present disclosure includes a method for treating Dravet syndrome (epilepsy), comprising administering a therapeutically effective amount of a compound of Formula (I) to a subject, such as a human. Another embodiment of the present disclosure includes a method for treating a cancer associated with a misfolded or misshaped protein, such as P53, comprising administering a therapeutically effective amount of a compound of Formula (I) to a subject, such as a human.

[0063] In other embodiments, the present disclosure also encompasses the use of a compound of formula (I) for the treatment of respiratory diseases resulting from misfolded or misshaped proteins. In one embodiment, the respiratory disease is cystic fibrosis. In one embodiment, the disease is COPD.

[0064] The dosage of the compound of formula (I) varies within a wide range depending on the disease to be controlled, the age and individual condition of the patient, and the method of administration, and will, of course, be adapted to the individual requirements of each particular case. For adult patients, a daily dose of about 1 mg to about 1000 mg, particularly about 1 mg to about 100 mg, is considered. Depending on the dosage, it is convenient to administer the daily dose in several dosage units.

[0065] While the present disclosure has been described in conjunction with specific embodiments thereof, it is apparent that numerous alternatives, modifications, and variations will become apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims. Furthermore, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present disclosure.

[0066] Example The operation of the present disclosure is illustrated by the following representative examples. As will be apparent to those skilled in the art, many of the details of the examples can be varied while practicing the disclosure described herein.

[0067] Materials and Methods Cell Culture: HEK293 cells stably expressing human deltaF508CFTR were cultured in DMEM / F12 medium containing 10% fetal bovine serum (FBS) and 600 micrograms / mL of geneticin. For functional CFTR assays, cryopreserved cells were rapidly thawed in a 37°C water bath, diluted in warmed DMEM / F12 medium containing 10% FBS, spun down at 300 x g for 5 minutes, and resuspended in the same medium at a density of 300,000 cells / mL. A cystic fibrosis bronchial cell line complemented with the 4.7 kb deltaF508CFTR cDNA ("CFBE") was obtained from Dr. Dieter Gruenert under license from UCSF. These cells were cultured in Earle's salt-containing MEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 300 micrograms / mL of hygromycin (Millipore). CFBE cells were grown in flasks coated with LHC basal medium (Invitrogen), 0.001% bovine serum albumin (Sigma), 1% v / v Vitrogen 100 (BD Biosciences), and a fibronectin solution containing 10 micrograms / mL human fibronectin (Life Technologies). All cells were grown at 37°C and 5% CO unless otherwise specified for the assay.

[0068] HEK293 deltaF508CFTR cell enhancer assay: Cells were seeded into 384-well black poly-D-lysine-coated plates (Greiner) at a density of 15,000 cells per well and placed in a 37°C, 5% CO2 incubator for 2–3 hours. The cell plates were then placed in a 30°C, 5% CO2 incubator for 20–24 hours to allow temperature rescue of delF508-CFTR expression. Prior to the assay, the cell medium was removed and 20 microliters per well of blue membrane potential dye-containing assay buffer (10 ml pre-diluted dye per 200 ml of assay buffer, Molecular Devices) was added. The assay buffer was modified Tyrode's buffer, pH 7.4, containing 140 mM sodium gluconate, 0.5 mM potassium gluconate, 2 mM calcium gluconate, 2 mM magnesium gluconate, 10 mM HEPES, and 12 mM NaHCO3. Cells were then incubated with this buffer for 45–60 minutes in a 30°C, 5% CO2 incubator. delF508-CFTR activity was then measured using a FLIPR fluorescence plate reader (Molecular Devices). Activity was induced by the addition of 10 microliters / well of assay buffer containing 90 nM genistein (Sigma) and compound, 90 nM genistein and DMSO (1.6%, negative control), or 90 nM genistein plus 30 micromolar forskolin (positive control, Sigma). Changes in fluorescence were measured using the following filter settings: excitation wavelength: 510–545 nm, emission wavelength: 565–625 nm.

[0069] HEK293 deltaF508CFTR cell collector assay: Cells were seeded at a density of 15,000 cells per well into 384-well black poly-D-lysine-coated plates (Greiner) containing 0.5 microliters per well of compound diluted in DMSO, DMSO alone (negative control), or a mixture with 1 micromolar VX-661 (positive control, Selleckchem) and placed in a 37°C, 5% CO2 incubator for 18–24 hours. Prior to the assay, the cell culture medium was removed and 20 microliters / well of modified Tyrode's assay buffer containing blue membrane potential dye was added. Cells were incubated with this buffer for 45–60 minutes in a 37°C, 5% CO2 incubator. delF508-CFTR activity was then measured using a FLIPR. Activity was induced by the addition of 10 microliters / well of assay buffer containing 30 micromolar genistein and 30 micromolar forskolin. Changes in fluorescence were measured as described above.

[0070] CFBE Enhancer Assay: CFBE cells were dissociated at 37°C for 10 minutes using Hank's Balanced Salt Solution containing 0.6 mM EDTA and 10% 0.25% trypsin / EDTA (all from Life Technologies). An equal volume of CFBE cell growth medium (see Cell Culture above) was then added to the flask, and the cells were spun at 500 x g for 10 minutes. The cells were resuspended in cell culture medium at a density of 150,000 cells / mL and seeded into 384-well black poly-D-lysine-coated plates (7,500 cells / well). Cells were then grown and differentiated at 37°C and 5% CO2 for a total of 6–8 days. Cell culture medium was changed every 2–3 days. The day before each experiment, the medium was removed, fresh medium was added, and the cells were incubated at 30°C and 5% CO2 for 18–24 hours to allow temperature rescue of delF508 CFTR. Cells were stained for exactly 60 minutes, and potentiation activity was measured as described above for HEK cells, except that compounds (or DMSO for the negative control) were mixed with assay buffer containing 3 micromolar forskolin, and positive control wells contained assay buffer containing 3 micromolar forskolin and 9 micromolar VX-770 (Selleckchem). The results are shown in Table 1.

[0071] CFBE collector assay: CFBE cells were dissociated and grown in 384-well plates for 6–8 days as described above. The day before each experiment, cell growth medium was removed and 50 microliters of medium containing compound, 0.1% DMSO (negative control), or 1 micromolar VX-809 (positive control, Selleckchem) was added. Cells were incubated at 30°C and 5% CO2 for 18–24 hours to allow temperature rescue of delF508 CFTR. Cells were stained for exactly 60 minutes, and correction activity was measured as described above for HEK cells, except that activity was induced by the addition of 10 microliters / well of assay buffer containing 3 micromolar forskolin and 9 micromolar VX-770.

[0072] Data analysis: CFTR activity was defined as the maximum fluorescence signal after addition minus baseline fluorescence. All data were normalized to positive and negative controls on each plate and expressed as a percentage response. Concentration-response curves were analyzed by fitting data to a four-parameter logistic equation with Microcal's Origin or IDBS's Activity Base software.

[0073] Example 1 – Synthesis of Intermediate A

[0074] [ka]

[0075] Synthesis of 5,6-dichloropyridazin-3(2H)-one (2): A stirred solution of 3,4,6-trichloropyridazine (1) (30.0 g, 163.4 mmol) in glacial acetic acid (120 mL) was heated to reflux (130 °C). After stirring for 3 h, the reaction mixture was cooled to room temperature and poured into ice-cold water (500 mL). The precipitated solid was filtered, washed with ice-cold water (50 mL × 2), and dried under vacuum to give 2 (10.3 g, 38%) as an off-white solid. 1 HNMR(400MHz,CDCl3):δ11.19(bs,1H),7.16(s,1H);LCMS(ESI):m / z162.9[MH + ]; 95.3%; RT=1.92 min (XBrigde C18 column, 5 mM ammonium bicarbonate in water with MeCN).

[0076] Synthesis of 6-chloro-5-(piperidin-1-yl)pyridazin-3(2H)-one (3): Under an inert atmosphere, a reaction tube was charged with 2 (25.0 g, 151.6 mmol), piperidine (26.9 mL, 272.0 mmol), diisopropylethylamine (132.5 mL, 757.6 mmol), and ethanol (125 mL) at room temperature. The reaction tube was capped and stirred at 150 °C for 12 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting residue was diluted with water (250 mL) and extracted with EtOAc (2 × 250 mL). The combined organic layers were washed with 10% aqueous HCl (50 mL), dried over anhydrous NaSO, and concentrated under reduced pressure. The crude product was triturated with pentane (100 mL) to give 3 (25 g, 77%) as a brown solid. 1 HNMR(400MHz,DMSO-d6):δ12.68(brs,1H;D2O exchangeable),6.13(s,1H),2.93-3.11(m,4H),1.44-1.75(m,6H);LCMS(ESI):m / z214.1[M+H + ]; 98.3%; RT=1.81 min (Acquity BEH C18 column, 0.1% formic acid / MeCN with 0.1% formic acid).

[0077] Synthesis of 6-chloro-4-nitro-5-(piperidin-1-yl)pyridazin-3(2H)-one (A): To a stirred solution of 3 (3.0 g, 14.04 mmol) in CHCl (50 mL) was added concentrated HSO (18 mL) followed by fuming nitric acid (5 mL) at 0 °C and stirred for 1 h. The reaction was monitored by TLC. The reaction mixture was diluted with ice-cold water (200 mL) and extracted with CHCl (2 × 100 mL). The combined organic layers were dried over anhydrous NaSO and concentrated under reduced pressure. Note: The reaction was repeated in four batches in parallel, and the resulting crude products were combined and purified by silica gel column chromatography using 20% ​​EtOAc in petroleum ether to give A (4.6 g, 25%) as a yellow solid. 1 HNMR(400MHz,CDCl3):δ10.74(br s,1H),3.26-3.21(m,4H),1.80-1.65(m,6H);LCMS(ESI):m / z257.1[MH +]; 93.0%; RT=2.98 min (Acquity BEH C18 column, 0.05% formic acid / MeCN with 0.05% formic acid).

[0078] Example 2 – Synthesis of Intermediate B

[0079] [ka]

[0080] Synthesis of 1-bromo-3-(methoxymethoxy)benzene (5): To a stirred solution of 3-bromophenol (4) (20.0 g, 115.6 mmol) in DMF (300 mL) was added NaH (5.1 g, 127.5 mmol; 60% in mineral oil) followed by MOM-chloride (9.66 mL, 127.18 mmol) under an inert atmosphere at 0 °C. The reaction was stirred at room temperature for 4 h. The reaction was monitored by TLC. The reaction mixture was diluted with ice-cold water (250 mL) and extracted with EtOAc (150 mL). The combined organic layers were dried over anhydrous NaSO and concentrated under reduced pressure to give 5 (25.0 g) as a brown gum, which was used directly in the next reaction without purification. 1 HNMR (400MHz, CDCl3): δ7.24-7.20 (m, 1H), 7.16-7.11 (m, 2H), 6.99-6.94 (m, 1H), 5.16 (s, 2H), 3.47 (s, 3H).

[0081] Synthesis of (3-(methoxymethoxy)phenyl)boronic acid (B): To a stirred solution of 5 (25.0 g, 115.17 mmol) in THF (250 mL) was added n-butyllithium (50.8 mL; 126.7 mmol; 2.5 M solution in hexane) dropwise at −78 °C and stirred at the same temperature for 1 h. To this was added triisopropyl borate (30 mL, 138.21 mmol) dropwise at −78 °C and maintained at room temperature for 4 h. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with 1 N aqueous HCl (40 mL) and extracted with EtOAc (150 mL). The organic layer was dried over anhydrous NaSO and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using 50% EtOAc in petroleum ether to give B (11.6 g, 55%) as a brown solid. 1 HNMR (400MHz, DMSO-d6): δ8.05 (br s, 2H), 7.45-7.38 (m, 2H), 7.26 (t, J = 7.8Hz, 1H), 7.08-7.01 (m, 1H), 5.18 (s, 2H), 3.37 (s, 3H).

[0082] Example 3 – Synthesis of Intermediate C

[0083] [ka]

[0084] Synthesis of 5-bromo-2-(tert-butyl)phenol (6): To a stirred solution of 3-bromophenol (4) (20 g, 115.6 mmol) in tBuCl (200 mL) was added AlCl (30.83 g, 231.2 mmol) and maintained at room temperature for 4 days. The reaction was monitored by TLC. The reaction mixture was diluted with ice-cold water (200 mL) and extracted with EtOAc (2 × 200 mL). The combined organic layers were dried over anhydrous NaSO and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using 1–2% EtOAc in petroleum ether to give 6 (8.0 g, 30%) as a colorless liquid. 1HNMR(400MHz,CDCl3):δ7.11(d,J=8.3Hz,1H),7.06-6.96(m,1H),6.83(d,J=2.0Hz,1H),4.81(s,1H),1.38(s,9H);LCMS(ESI):m / z226.8[MH + ]; 70.6%; RT=3.57 min (Acquity BEH C18 column, 0.05% formic acid / MeCN with 0.05% formic acid).

[0085] Synthesis of 4-bromo-1-(tert-butyl)-2-methoxybenzene (7): To a stirred solution of NaH (1.75 g, 43.75 mmol; 60% in mineral oil) in THF (40 mL) was added a solution of 6 (5.0 g, 21.82 mmol) in THF (35 mL) under an inert atmosphere at 0 °C. To this was added methyl iodide (2.72 mL, 43.67 mmol) dropwise at 0 °C and stirred at room temperature for 6 h. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with ice-cold water (100 mL) and extracted with EtOAc (2 × 100 mL). The combined organic layers were dried over anhydrous NaSO and concentrated under reduced pressure to give 7 (4.5 g, 85%) as a brown liquid. 1 HNMR(400MHz,CDCl3):δ7.12(d,J=8.3Hz,1H),7.03-6.99(m,1H),6.97(d,J=2.0Hz,1H),3.83(s,3H),1.34(s,9H);GCMS(ESI):m / z222.1[M + ]; 80.9%; RT = 8.85 min (ZB-5MS column, 100 °C / 1 min, 20 °C / min / 310 °C / 5.5 min).

[0086] Synthesis of 4-tert-butyl-3-methoxyphenylboronic acid (C): To a stirred solution of 7 (4.5 g, 18.5 mmol) in THF (90 mL) was added n-butyllithium (12.7 mL, 20.45 mmol; 1.6 M solution in hexane) dropwise at −78° C. and stirred at the same temperature for 1 h. To this was added triisopropyl borate (4.19 g, 22.3 mmol) dropwise at −78° C. The reaction was stirred at room temperature for 16 h. After completion of the reaction (monitored by TLC), the reaction was quenched with 5 N aqueous HCl (150 mL) and stirred for 1 h. The reaction solution was extracted with EtOAc (2 × 150 mL). The combined organic layers were dried over anhydrous NaSO and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using 25% EtOAc in hexane as the eluent to give C (1.8 g, 46%) as a brown solid. 1 HNMR(400MHz,CDCl3):δ7.77(d,J=7.6Hz,1H),7.70(s,1H),7.44(d,J=7.6Hz,1H),3.97(s,3H),1.43(s,9H);LCMS(ESI):m / z207.1[MH + ]; 88.9%; RT=2.37 min (Acquity BEH C18 column, 0.1% formic acid / MeCN with 0.1% formic acid).

[0087] Example 4 - Synthesis of 4-amino-2-(4-(tert-butyl)-3-hydroxyphenyl)-6-(4-fluorophenyl)-5-(piperidin-1-yl)pyridazin-3(2H)-one (11)

[0088] [ka]

[0089] Synthesis of 2-(4-(tert-butyl)-3-methoxyphenyl)-6-chloro-4-nitro-5-(piperidin-1-yl)pyridazin-3(2H)-one (8): To a stirred solution of A (1.0 g, 3.87 mmol) in CHCl (30 mL) was added C (1.6 g, 7.69 mmol), Cu(OAc) (1.4 g, 7.70 mmol), triethylamine (782 mg, 7.74 mmol), and pyridine (613 mg, 7.75 mmol) sequentially at room temperature and stirred for 2 h. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with 2 N aqueous HCl (100 mL) and extracted with CHCl (2 × 150 mL). The combined organic layers were washed with water (2 × 200 mL), dried over anhydrous NaSO, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using 10% EtOAc in hexane as the eluent to give 8 (600 mg, 36%) as a yellow solid. 1 HNMR(400MHz,CDCl3):δ7.33(d,J=8.3Hz,1H),7.10(d,J=2.0Hz,1H),7.09-7.04(m,1H),3 .85(s,3H),3.32-3.17(m,4H),1.81-1.66(m,6H),1.37(s,9H);LCMS(ESI):m / z421.2[M+H + ]; 96.4%; RT=3.19 min (Acquity BEH C18 column, 0.05% formic acid / MeCN with 0.05% formic acid).

[0090] Synthesis of 4-amino-2-(4-(tert-butyl)-3-methoxyphenyl)-6-chloro-5-(piperidin-1-yl)pyridazin-3(2H)-one (9): To a stirred solution of 8 (600 mg, 1.42 mmol) in ethanol (18 mL) was added Fe powder (239.3 mg, 4.28 mmol), NH4Cl (457.7 mg, 8.57 mmol), and HO (6 mL) at room temperature. The reaction was heated to 80 °C and stirred at the same temperature for 4 h. The reaction mixture was cooled to room temperature and filtered through a short pad of Celite. The filtrate was concentrated in vacuo and diluted with EtOAc (200 mL). The resulting solution was washed with water (300 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using 10% EtOAc in hexane to give 9 (310 mg, 55%) as a brown solid. 1 HNMR(400MHz, CDCl3): δ7.33(d,J=8.3Hz,1H),7.13-7.04(m,2H),5.42(br s,2H),3.85(s,3H),3.31-3.27(m,2H),2.91-2.96(m,2H),1.77-1.73(m,6H),1.37(s,9H);LCMS(ESI):m / z391.0[M+H + ]; 96.7%; RT=4.16 min (Acquity BEH C18 column, 0.05% formic acid / MeCN with 0.05% formic acid).

[0091] Synthesis of 4-amino-2-(4-(tert-butyl)-3-methoxyphenyl)-6-(4-fluorophenyl)-5-(piperidin-1-yl)pyridazin-3(2H)-one (10): A reaction tube was charged with 9 (300 mg, 0.76 mmol), 1,4-dioxane (15 mL), Na2CO3 (163 mg, 1.53 mmol), and 4-fluorophenylboronic acid (215 mg, 1.53 mmol) and purged with argon for 15 minutes. To this, Pd(PPh3)4 (88.8 mg, 0.076 mmol) and HO (9 mL) were added, and the tube was capped. The reaction was heated to 110 °C and stirred for 7 hours. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to room temperature, diluted with water (100 mL), and extracted with EtOAc (2 × 100 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using 20% ​​EtOAc in hexane to give 10 (200 mg, 58%) as a brown solid. 1 HNMR (400MHz, CDCl3): δ7.43(dd,J=5.4,8.6Hz,2H),7.31(d,J=8.3Hz,1H),7.16-7.05(m,4H),5.06(br s,2H),3.83(s,3H),2.71-2.68(m,4H),1.56-1.52(m,6H),1.36(s,9H);LCMS(ESI):m / z451.0[M+H + ]; 84.1%; RT=4.13 min (Acquity BEH C18 column, 0.05% formic acid / MeCN with 0.05% formic acid).

[0092] Synthesis of 4-amino-2-(4-(tert-butyl)-3-hydroxyphenyl)-6-(4-fluorophenyl)-5-(piperidin-1-yl)pyridazin-3(2H)-one (11): To a stirred solution of 10 (200 mg, 0.44 mmol) in CHCl (20 mL) was added BBr (0.23 mL, 2.22 mmol) dropwise under an inert atmosphere at 0 °C. The reaction was stirred at room temperature for 4 h. After completion of the reaction (monitored by TLC), the reaction mixture was basified with saturated NaHCO solution (10 mL; to pH ∼8) and extracted with CHCl (2 × 50 mL). The combined organic layers were dried over anhydrous NaSO and concentrated under reduced pressure. The crude product was purified by preparative HPLC (Kromasil C18 column; 5 mM ammonium bicarbonate solution in MeCN) to give 10 (71 mg, 36%) as a pale yellow solid. 1 HNMR(400MHz,DMSO-d6):δ9.57(s,1H;D2O exchangeable),7.58-7.44(m,2H),7.25(t,J=8.9Hz,2H),7.18(d,J=8.3Hz,1H),7.02(d,J=2.2Hz,1H),6.96(dd,J=2.2,8.3Hz,1H),5.81(s,2H;D2O exchangeable),2.73-2.68(m,4H),1.48-1.28(m,15H);LCMS(ESI):m / z437.2[M+H + ]; 98.9%; RT = 2.94 min (Acquity BEH C18 column, 0.05% formic acid / MeCN with 0.05% formic acid); FT-IR (KBr): 3477 (OH), 3369 (1 o NH2), 1604(C=O)cm -1 CFBE enhancer assay pot.pEC 50 =8.0.

[0093] Example 5 - Synthesis of 4-amino-2-(3-hydroxyphenyl)-5-(piperidin-1-yl)-6-(o-tolyl)pyridazin-3(2H)-one (15)

[0094] [ka]

[0095] Synthesis of 6-chloro-2-(3-methoxyphenyl)-4-nitro-5-(piperidin-1-yl)pyridazin-3(2H)-one (12): To a stirred solution of A (800 mg, 3.09 mmol) and 3-methoxyphenylboronic acid (700 mg, 4.64 mmol) in CHCl (20 mL) was added Cu(OAc) (840 mg, 4.64 mmol), triethylamine (0.86 mL, 6.18 mmol), pyridine (0.5 mL, 6.18 mmol), and 4A molecular sieves (150 mg) sequentially at room temperature and stirred for 16 h. After completion of the reaction (monitored by TLC), the reaction mixture was filtered through a pad of Celite and washed with CHCl (10 mL). The filtrate was washed with water (50 mL), and the aqueous layer was extracted with CHCl (2 × 50 mL). The combined organic layers were dried over anhydrous NaSO and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using 25% EtOAc in petroleum ether as the eluent to give 11 (600 mg, 53%) as a yellow solid. 1 HNMR(400MHz, CDCl3): δ7.36(t,J=8.3Hz,1H),7.19-7.13(m,2H),6.93-6.89(m,1 H),3.81(s,3H),3.24-3.21(m,4H),1.78-1.68(m,6H);LCMS(ESI):m / z365.1[M+H + ]; 95.1%; RT=8.39 min (XSelect CSH C18 column, 5 mM ammonium acetate in water with MeCN).

[0096] Synthesis of 4-amino-6-chloro-2-(3-methoxyphenyl)-5-(piperidin-1-yl)pyridazin-3(2H)-one (13): To a stirred solution of 12 (650 mg, 1.78 mmol) in ethanol (13 mL) at room temperature was added Fe powder (300 mg, 5.34 mmol), NH4Cl (570 mg, 10.68 mmol), and HO (6.5 mL). The reaction was heated to 80 °C and stirred for 3 h. The reaction mixture was cooled to room temperature and filtered through a short pad of Celite. The filtrate was concentrated under reduced pressure. The resulting residue was diluted with water (20 mL) and extracted with CHCl2 (2 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using 20% ​​EtOAc in petroleum ether to give 12 (280 mg, 47%) as a brown solid. 1 HNMR(400MHz,CDCl3):δ7.41-7.29(m,1H),7.18(d,J=8.1Hz,1H),7.14(d,J=2.0Hz,1H),6.92(dd,J=2.0,8.1Hz,1H),5.43(br s,2H),3.83(s,3H),3.29-3.26(m,2H),2.92-2.86(m,2H),1.76-1.70(m,6H);LCMS(ESI):m / z335.1[M+H + ]; 92.8%; RT=3.67 min (Acquity BEH C18 column, 0.05% formic acid / MeCN with 0.05% formic acid).

[0097] Synthesis of 4-amino-2-(3-methoxyphenyl)-5-(piperidin-1-yl)-6-(o-tolyl)pyridazin-3(2H)-one (14): A reaction tube was charged with 13 (400 mg, 1.19 mmol), o-tolylboronic acid (490 mg, 3.58 mmol), Na2CO3 (380 mg, 3.58 mmol), 1,4-dioxane (8 mL), and HO (0.8 mL) and degassed by purging with argon for 10 minutes. To this was added Pd(PPh3)4 (83 mg, 0.071 mmol), and the mixture was again purged with argon for 15 minutes. The reaction tube was capped and stirred at 110 °C for 16 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was diluted with water (25 mL) and extracted with EtOAc (2 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using 30% EtOAc in petroleum ether to give 14 (280 mg; 65% purity) as a brown semi-solid, which was used directly in the next reaction without further purification. 1 HNMR (400MHz, CDCl3): δ7.49-7.40(m,1H),7.37-7.29(m,3H),7.20-7.16(m,3H),6.88(dd,J=1.8,8.3Hz,1H),5.08(br s,2H),3.81(s,3H),2.30(s,3H),2.62-2.59(m,4H),1.51-1.43(m,4H),1.39-1.35(m,2H);LCMS(ESI):m / z391.2[M+H + ]; 65.6%; RT=2.79 min (Acquity BEH C18 column, 0.05% formic acid / MeCN with 0.05% formic acid).

[0098] Synthesis of 4-amino-2-(3-hydroxyphenyl)-5-(piperidin-1-yl)-6-(o-tolyl)pyridazin-3(2H)-one (15): To a stirred solution of 14 (280 mg) in CHCl (10 mL) under an inert atmosphere at 0 °C, BBr (3.5 mL, 3.58 mmol; 1 M solution in CHCl) was added dropwise. The reaction was stirred at room temperature for 3 h. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with water (5 mL), basified with saturated NaHCO solution (10 mL; to pH ∼8), and extracted with CHCl (2 × 15 mL). The combined organic layers were dried over anhydrous NaSO and concentrated under reduced pressure. The crude product was purified by preparative HPLC (XBridge C18 column; 10 mM aqueous ammonium bicarbonate in MeCN) to give 15 (98 mg, 22% from 12) as a white solid. 1 HNMR(400MHz,DMSO-d6):δ9.62(br s,1H;D2O exchangeable),7.37-7.26(m,2H),7.26-7.19(m,3H),7.04-6.93(m,2H),6.74(ddd,J=1.0,2.4,8.2Hz,1H),5.78(br s,2H;D2O exchangeable),2.63-2.58(m,4H),2.21(s,3H),1.40-1.18(m,6H);LCMS(ESI):m / z377.1[M+H + ]; 99.1%; RT = 2.50 min (Acquity BEH C18 column, 0.05% formic acid / MeCN with 0.05% formic acid); FT-IR (KBr): 3483 (OH), 3368 (1 o NH), 1594(C=O)cm -1 CFBE enhancer assay pot.pEC 50 =7.8.

[0099] Example 6 - Synthesis of 4-amino-5-(dimethylamino)-2-(3-hydroxyphenyl)-6-phenylpyridazin-3(2H)-one (21):

[0100] [ka]

[0101] Synthesis of 6-chloro-5-(dimethylamino)pyridazin-3(2H)-one (16): A reaction tube was charged with 2 (5.0 g, 30.5 mmol), dimethylamine (30.4 mL, 2 M solution in THF, 61.0 mmol), diisopropylethylamine (26.5 mL, 152.5 mmol), and ethanol (60 mL) under an inert atmosphere at room temperature. The reaction tube was capped and stirred at 110 °C for 16 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting residue was diluted with water (200 mL) and filtered. The solid was washed with diethyl ether (50 mL) to give 16 (3.2 g, 60%) as an off-white solid. 1 HNMR(400MHz,DMSO-d6):δ12.59(br s,1H),6.01(s,1H),2.85(s,6H);LCMS(ESI):m / z173.8[M+H + ]; 99.8%; RT=2.04 min (Acquity BEH C18 column, 0.05% formic acid / MeCN with 0.05% formic acid).

[0102] Synthesis of 6-chloro-5-(dimethylamino)-4-nitropyridazin-3(2H)-one (17): To a stirred solution of 16 (1.5 g, 8.64 mmol) in CHCl (25 mL) was added concentrated HSO (9 mL) dropwise at 0 °C, followed by fuming nitric acid (2.4 mL) and stirred at the same temperature for 1 h. The reaction was monitored by TLC. The reaction mixture was diluted with ice-cold water (100 mL) and extracted with CHCl (2 × 40 mL). The combined organic layers were washed with water (2 × 50 mL), dried over anhydrous NaSO, and concentrated under reduced pressure to give 17 (460 mg, 24%) as a yellow solid. 1 HNMR(400MHz,CDCl3):δ10.80(br s,1H),3.04(s,6H);LCMS(ESI):m / z218.9[M+H + ]; 86.2%; RT=2.31 min (Acquity BEH C18 column, 0.05% formic acid / MeCN with 0.05% formic acid).

[0103] Synthesis of 6-chloro-5-(dimethylamino)-2-(3-(methoxymethoxy)phenyl)-4-nitropyridazin-3(2H)-one (18): To a stirred solution of 17 (2.0 g, 9.15 mmol) in CHCl (40 mL) was added (3-(methoxymethoxy)phenyl)boronic acid (3.33 g, 18.3 mmol), Cu(OAc) (3.33 g, 18.3 mmol), triethylamine (2.6 mL, 18.3 mmol), and pyridine (1.5 mL, 18.3 mmol) sequentially at room temperature and stirred for 16 h. After completion of the reaction (monitored by TLC), the reaction mixture was filtered through a Celite pad, and the filtrate was concentrated under reduced pressure. The residue was diluted with water (100 mL) and extracted with EtOAc (50 mL). The EtOAc layer was dried over anhydrous NaSO and concentrated under reduced pressure. The crude product obtained was purified by silica gel column chromatography using a gradient of 10–15% EtOAc in petroleum ether to give 18 (800 mg, 25%) as a yellow solid. 1 HNMR(400MHz,DMSO-d6):δ7.43(t,J=8.1Hz,1H),7.23-7.09(m,3H),5.22(s,2H),3.39(s,3H),2.99(s,6H);LCMS(ESI):m / z355.15[M+H + ]; 84.9%; RT=2.33 min (Acquity BEH C18 column, 0.05% formic acid / MeCN with 0.05% formic acid).

[0104] Synthesis of 4-amino-6-chloro-5-(dimethylamino)-2-(3-(methoxymethoxy)phenyl)pyridazin-3(2H)-one (19): To a stirred solution of 18 (800 mg, 2.25 mmol) in ethanol (16 mL) at room temperature was added Fe powder (380 mg, 6.75 mmol), NH4Cl (720 mg, 13.5 mmol), and HO (8 mL). The reaction was heated to 80 °C and stirred for 4 h. The reaction mixture was cooled to room temperature and filtered through a short pad of Celite. The filtrate was concentrated under reduced pressure. The resulting residue was diluted with water (50 mL) and extracted with EtOAc (2 × 30 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure to give 19 (400 mg, 54%) as a brown solid. 1 HNMR(400MHz,DMSO-d6):δ7.35-7.49(m,1H),7.13-7.23(m,2H),7.03-7.12(m,1H),6.63(br s,2H),5.22(s,2H),3.39(s,3H),2.73(s,6H);LCMS(ESI):m / z325.1[M+H + ]; 93.6%; RT=2.31 min (Acquity BEH C18 column, 0.05% formic acid / MeCN with 0.05% formic acid).

[0105] Synthesis of 4-amino-5-(dimethylamino)-2-(3-(methoxymethoxy)phenyl)-6-phenylpyridazin-3(2H)-one (20): A reaction tube was charged with 19 (400 mg, 1.23 mmol), 1,4-dioxane (8 mL), Na2CO3 (260 mg, 2.46 mmol), benzeneboronic acid (300 mg, 2.46 mmol), Pd(PPh3)4 (142 mg, 0.123 mmol), and HO (4 mL) and purged with argon for 10 minutes. The tube was capped and stirred at 110 °C for 16 hours. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to room temperature and filtered through a pad of Celite. The filtrate was concentrated under reduced pressure. The resulting residue was diluted with water (50 mL) and extracted with EtOAc (2 × 20 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using 50% EtOAc in petroleum ether to give 300 mg of 20 (purity ca. 45%; brown solid) as a mixture with triphenylphosphine oxide (35%), which was used directly in the next step without further purification. LCMS (ESI): m / z 367.1 [M+H + ]; 45.4%; RT=3.07 min (Acquity BEH C18 column, 0.05% formic acid / MeCN with 0.05% formic acid).

[0106] Synthesis of 4-amino-5-(dimethylamino)-2-(3-hydroxyphenyl)-6-phenylpyridazin-3(2H)-one (21): To a stirred solution of 20 (300 mg; approximately 45% purity) in methanol (3 mL) was added dropwise a 3 M HCl solution in methanol (3 mL) at 0 °C under an inert atmosphere. The reaction was stirred at room temperature for 2 h. After completion of the reaction (monitored by TLC), the methanol was concentrated. The resulting residue was diluted with water (20 mL) and extracted with EtOAc (2 × 10 mL). The combined organic layers were dried over anhydrous NaSO and concentrated under reduced pressure. The crude product was purified by preparative HPLC (Kinetex phenylhexyl C18 column; 5 mM aqueous ammonium bicarbonate in MeCN) to give 21 (85 mg; 5 to 11% over two steps) as a brown solid. 1HNMR(400MHz,DMSO-d6):δ9.61(br s,1H, D2O exchangeable),7.53-7.38(m,5H),7.29-7.17(m,1H),7.07-6.96(m,2H),6.76(ddd,J=8.1,2.4,1.0Hz,1H),5.98(br s,2H;D2O exchangeable),2.46(s,6H);LCMS(ESI):m / z323.1[M+H + ]; 99.8%; RT = 2.05 min (Acquity BEH C18 column, 0.05% formic acid / MeCN with 0.05% formic acid). CFBE Enhancer Assay pot.pEC 50 =7.7.

[0107] Compound 11 – In vitro model of chronic obstructive pulmonary disease

[0108] [ka]

[0109] The CFTR chloride channel is located in the luminal membrane of airway surface epithelium and increases airway surface liquid fluidity, thereby helping to prevent mucus obstruction. Cigarette smoke reduces functional CFTR expression at the epithelial surface, exacerbating mucus congestion and obstruction (A. Rab, SM Rowe, SV Raju, Z. Bebok, S. Matalon, and JF Collawn. Am J Physiol Lung Cell Mol Physiol 2013 Vol. 305 Issue 8 Pages L530-41). These findings support the hypothesis that smoke exposure induces CFTR deficiency, also known as "acquired cystic fibrosis," and that small molecules that enhance the activity of residual CFTR function may reverse early stages of COPD pathogenesis. Ivacaftor, an approved CFTR potentiator, has been shown to reverse mucus congestion in certain tissue culture models, and a novel CFTR potentiator called isenticaftor has shown promise in restoring lung function in clinical trials in people with early COPD (SM Rowe, I. Jones, MT Dransfield, N. Haque, S. Gleason, KA Hayes, et al. Int J Chron Obstruct Pulmon Dis 2020 Vol. 15 Pages 2399-2409).

[0110] Compound 11 was tested in an in vitro model of COPD. In this model, primary differentiated human bronchial epithelial cell cultures were exposed to cigarette smoke extract (4, 5). Exposure to cigarette smoke extract (CSE) resulted in mucus congestion and slowed mucociliary motility (Figure 1(A) and (B)). Compound 11, when added to differentiated airway epithelial cell cultures, prevented the cigarette smoke extract-induced mucus congestion (Figure 1(C)). Changes in mucociliary motility were measured as previously described (7), i.e., by tracking the velocity of fluorescent beads added to the surface of airway cell cultures. The relative rescue effect of compound 11 was superior to that of a competing drug, ivacaftor (VX-770), an FDA-approved CFTR potentiator.

[0111] Figure 1 shows that this compound enhances mucociliary motility on the surface of primary bronchial cell cultures previously exposed to cigarette smoke extract. The photographs on the left ((A) and (B)) show the adverse effect of cigarette smoke extract (CSE) on fluorescent bead tracking, which reflects the development of sticky mucus in the liquid on the surface of primary bronchial cell cultures. The graph on the right (C) shows quantification of bead velocity on the surface of primary bronchial airway cell cultures under control conditions (cell cultures from four donors treated with vehicle-only DMSO, open circles). Ivacaftor (VX-770 circles) or compound 11 (circles) were added simultaneously with vehicle for 24 hours. Neither VX-770 nor compound 11 had any effect on bead velocity under control conditions. Treatment with CSE (2% by volume, filled black symbols) caused a significant decrease in bead velocity in cell cultures from five donors. Bead velocity in bronchial cell cultures exposed to CSE and treated with compound 11 potentiator (filled symbols) was rescued to control values. Each symbol represents the average bead velocity across five videos taken in randomly selected areas of each airway cell culture.

[0112] A novel enhancer of Wt-CFTR channel activity. Materials and Methods Cell-attached patch clamp studies of CFTR channel potentiation by novel potentiator compounds. HEK-293 cells stably expressing wild-type human CFTR were used for patch clamp studies of single channel activity. Cells were kindly provided by D. Rotin (Children's Hospital, Toronto). CFTR Cl channels were measured in cell-attached membrane patches using an Axopatch 200A patch clamp amplifier and pCLAMP software (both from Molecular Devices, Sunnyvale, CA). - The pipette and bath (extracellular) solutions contained 140 mM N-methyl-d-glucamine, 140 mM aspartate, 5 mM CaCl, 2 mM MgSO, and 10 mM TES, and were buffered with Tris([Cl]). - ]:10 mM) to a pH of 7.3. The bath was maintained at room temperature.

[0113] To activate the Wt-CFTR channel, 10 μM forskolin was added to the bath. CFTR-mediated channel opening was inhibited by applying CFTRInh-172 to the bath. - Channels were potentiated by adding SK-POT (compound 11) (10 μM) to the bath. To determine the number of channels, the maximum number of simultaneous channel openings observed during the experimental procedure was used. Single-channel recordings were filtered and digitized as described above. To measure single-channel current amplitudes, Gaussian distributions were fitted to current amplitude histograms. P o To measure this, we generated lists of opening and closing times using a single-amplitude crossing criterion for event detection, and constructed and fitted dwell time histograms. For illustration, single-channel recordings were filtered at 500 Hz.

[0114] Calu-3 CSE experiment Calu-3 cells were cultured in EMEM (Wisent) supplemented with 20% (v / v) FBS and 1x penicillin / streptomycin (Wisent). Calu-3 cells were seeded at a density of 10,000 cells / well in clear-bottom, black-walled 96-well plates (Costar, Corning) and cultured for 2 days after confluence. Cells then underwent long-term treatment with toxins and enhancers. Toxin treatment consisted of either cigarette smoke extract (CSE) (University of Alabama, Birmingham) prepared in 100% DMSO dissolved in Calu-3 medium at a concentration of 2% (v / v), or DMSO alone at the same concentration, which served as a toxin control. Long-term drug treatment was combined with toxins containing either VX-770 or Compound 11 (both prepared in 100% DMSO) dissolved in Calu-3 medium at a final concentration of 1 μM. Solutions of VX-770 (Vertex) and Compound 11 were prepared from 1 mM stock solutions. Controls for drug treatments were an equal volume of the vehicle, DMSO. Two toxin and three drug treatments were combined, with a total of six treatments applied chronically to four wells per condition, for a total of 24 wells receiving chronic treatment.

[0115] Cells were cultured for an additional 24 hours after application of the long-term treatment, after which they were incubated with FLiPR assay buffer. The FLiPR assay buffer consisted of blue FLiPR dye (Molecular Devices) dissolved at a concentration of 0.5 mg / mL in chloride-free buffer (150 mM NMDG, 150 mM gluconolactone, 3 mM potassium gluconate, pH 7.38 at 300 mOsm). The buffer was incubated with cells for 35 minutes at 37°C and 5% CO2. CFTR function was assessed using a SpectraMax i3x multimode plate reader by measuring changes in fluorescence activity after short-term CFTR channel activation by the addition of the cAMP agonist forskolin, dissolved in chloride-free buffer to a final concentration of 1 μM. Fluorescence readings (excitation 530 nm; emission 560 nm) were taken for each well at 30-second intervals for 5 minutes (baseline) or 10 minutes (activation). To further verify the specificity of the response to CFTR activity, CFTR activity was subsequently terminated by the addition of CFTR inhibitor 172 dissolved in chloride-free buffer to a concentration of 10 μM. Fluorescence changes were recorded at 30-second intervals for an additional 10 minutes. For analysis, all fluorescence values ​​were normalized to the final measurement per well before addition of forskolin and expressed as a percentage of this measurement.

[0116] Bead tracking assay of mucociliary motility Mature bronchial epithelial cell cultures from non-smoking donors of various ages seeded in 24-well transwell inserts were received from the University of Iowa and cultured at the air-liquid interface (ALI) using UltraG medium. A total of six inserts were cultured per donor, and all inserts from one donor were selected for long-term drug and toxin treatment. Prior to long-term treatment, the cell cultures were subjected to Mucomyst to reduce the volume and viscosity of the mucus layer before imaging. First, the cell cultures were washed twice with 200 μL of HBSS (Wisent) applied to the tip of the transwell, with each wash performed for 10 min at 37°C and 5% CO2. The cells were then incubated with 10 μM N-acetyl-cysteine ​​(source?) dissolved in HBSS (Wisent) for 30 min, followed by a final HBSS wash as described above. Toxin treatment consisted of either cigarette smoke extract (CSE) (University of Alabama at Birmingham) prepared in 100% DMSO and dissolved in UltraG medium at a concentration of 2% (v / v), or DMSO alone at the same concentration, which served as a control. Long-term drug treatments were combined with toxin treatments containing either VX-770 (Vertex) or SK-POT, also known as compound 11 (both prepared in 100% DMSO) dissolved in UltraG medium at a final concentration of 1 μM. Six long-term treatments were performed, with six inserts per donor receiving a different long-term treatment. Bronchial epithelium was incubated with the apically applied long-term treatment for 24 hours at 37°C and 5% CO2. After 24 hours, green fluorescent microspheres (brand?) dissolved in HBSS at a concentration of 0.05% (v / v) were applied apically, and cell cultures were incubated with the microspheres for 45 minutes. Toxin and enhancer treatments were performed in the microsphere solution.

[0117] The inserts were then imaged at room temperature using an epifluorescence microscope (Zeiss or Olympus). A basic repeat of the assay consisted of 5-second videos taken at 40–60 frames per second in the green channel. The Z-position was adjusted before each video to ensure capture of beads in the liquid layer on the airway surface just above the cilia. Five videos representing the center and four corners of the insert were taken per insert / condition. Two sets of five videos were taken per insert / condition: one set at baseline and one set 20 minutes after CFTR stimulation with 10 μM forskolin. The inserts were incubated at room temperature after forskolin addition.

[0118] Videos were captured using Volocity software and exported as a single .TIF file per frame. The frames were then recompiled into a complete video using Arivis 4D software. Using an algorithm previously described by Wu et al. (2017), microspheres were highlighted by adjusting two parameters: fluorescence intensity and diameter. The parameters were adjusted until the software identified only unique beads. Therefore, successful identification of unique beads was manually checked at the beginning, middle, and end of each video. After highlighting the beads, the algorithm generated a displacement value for each bead along with a value for the number of frames the algorithm tracked the bead. Beads tracked for fewer than five frames were excluded from analysis. The generated velocity was then divided by the number of frames tracked and then multiplied by the frames per second to calculate the velocity (μm / s) (per bead analyzed). The velocity values ​​were then tallied, and an average velocity was calculated for each video.

[0119] Calu-3 Western blot Calu3 was lysed in radioimmunoprecipitation (RIPA) buffer (50 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, pH 7.4) supplemented with 0.1% SDS, 0.1% Triton X-100, and protease inhibitor cocktail (Roche) (1x). Lysis was performed on ice for 5 minutes using 40 μL of RIPA buffer as described. Lysed cells were collected from individual wells by scraping with a micropipette tip followed by centrifugation at 15,000 rpm for 10 minutes at 4°C. Lysates were then analyzed by SDS-PAGE using 6% Tris-glycine gels (Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad) at 100 mV for 1 hour. After blocking with 5% (w / v) nonfat milk in PBS-Tween, CFTR was probed overnight at 4°C with the primary antibody CFTR-NBD2-specific mouse mAb 596 in blocking buffer at a dilution of 1:2000. Calnexin, a loading control, was probed with rat anti-calnexin in blocking buffer at a dilution of 1:10,000. Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rat antibodies, both diluted 1:5000 in blocking buffer, were used as secondary antibodies and incubated for 1 hour at room temperature. Blots were developed using ECL reagent (Bio-Rad) and imaged with a 2-minute exposure using Li-Cor Odyssey Fc (LI-COR Biosciences). Relative CFTR abundance was quantified using ImageStudioLite (LI-COR Biosciences).

[0120] Results and Discussion Figure 2 shows the dose response of SK-POT (compound 11) activity compared to VX-770, a highly effective potentiator compound used in the treatment of cystic fibrosis. In these studies, F508del-CFTR inhibited CFBE41o -It was endogenously expressed in cells, and its trafficking defect was corrected by incubation at low temperature (27°C). The fold increase in cyclic AMP-dependent F508del-CFTR-mediated chloride channel activity induced by potentiator treatment was determined using a fluorescence-based membrane potential assay as described previously. As shown in Figure 1, the potency of compound 11 was found to be 5 nM, compared with 32 nM determined with VX-770.

[0121] Detailed analysis of the activity of this novel group of small molecules in potentiating wild-type CFTR was performed using patch-clamp experiments in HEK-293 cells stably expressing this gene. Figure 3(i) shows traces obtained in cell-attached mode, in which cells were sequentially exposed to forskolin, then a structural analog of compound 11, and then the CFTR channel inhibitor CFTRInh172 (10 μM). While only one active channel was evident in the presence of forskolin, a total of eight channels were active in the presence of the potentiator compounds. After the addition of CFTRInh-172, the number of active channels was significantly reduced. The bar graph in Figure 3(ii) shows CFTR channel activity normalized to the total number of channels (n = 8). As expected for CFTR potentiators, the addition of the novel compounds clearly increased the channel open probability.

[0122] We then tested the effect of SK-POT (compound 11) in enhancing Wt-CFTR-mediated transepithelial chloride conductance in primary human bronchial epithelial cell cultures. As shown in Figure 4, in an Ussing chamber assay, SK-POT (1 μM) enhanced forskolin-activated short-circuit currents mediated by Wt-CFTR in primary bronchial epithelial cell cultures. Representative traces are shown in the left panel, and the scatter plots on the right demonstrate that these results are reproducible and statistically significant. Interestingly, the enhancement induced by 1 μM SK-POT was similar to that achieved by 10 μM ivacaftor (VX-77).

[0123] We then determined whether the SK-POT compound could ameliorate the negative effects of cigarette smoke (CSE) on Wt-CFTR channel function. The Calu-3 cell line has been widely used in studies on the regulation of Wt-CFTR because this airway epithelial cell line endogenously expresses this channel upon differentiation. CFTR channel activity was measured using FLIPR (a fluorescence-based plate reader assay). First, we reproduced the previously published adverse effects of cigarette smoke extract on CFTR channel activity (Figure 5i, upper and lower panels). In a paired study (three biological replicates and three to four technical replicates), we compared the effects of VX-770 and SK-POT, both of which enhance CFTR activity after CSE exposure at 1 μM. As shown in the bar graph in Figure 5, only SK-POT treatment resulted in a significant increase in forskolin-activated CFTR channel activity.

[0124] Mucus stasis, the primary defect in COPD, has been modeled in vitro as reduced motility of fluorescent nanoparticles seeded in the upper mucus-containing fluid of fully differentiated tracheal airway cell cultures (YS Wu, J. Jiang, S. Ahmadi, A. Lew, O. Laselva, S. Xia, et al. Mol Pharmacol 2019 Vol. 96 Issue 4 Pages 515-525).

[0125] Fluorescent nanoparticles were seeded onto the airway surface, and bead movement was tracked. Bead trajectories are shown in the images in Figure 6. The top images (a, i, and ii) show that bead displacement decreases after exposure to CSE, as expected. We then evaluated the effect of pretreatment with the potentiators VX-770 (1 μM) or SK-POT (1 μM) on CSE-induced alterations in mucociliary motility (panels a.iii-v). Figure 6(b) assessed bead velocity in response to pretreatment with specific potentiators. In five biological replicates and four to five technical replicates (or videos), pretreatment with SK-POT resulted in a significant increase in bead velocity in CSE-treated primary bronchial cell cultures. Interestingly, in these studies, no significant increase in forskolin-induced responses in CSE-treated cell cultures was observed after VX-770 addition.

[0126] Consideration The current study demonstrates that potentiators of CFTR channel activity can ameliorate mucus congestion induced by cigarette smoke extract (CSE) in human bronchial epithelial cells. Furthermore, the findings suggest that potentiators such as those described herein that promote superior CFTR channel activity in CSE-exposed epithelial cells may also be more effective in preventing CSE-associated mucus congestion. Collectively, these studies support further in vivo evaluation of SK-POT as a therapeutic intervention for COPD-associated bronchitis.

[0127] We found that short-term treatment with SK-POT compounds was superior to 1 micromolar VX-770 in enhancing CFTR channel activity in epithelial cells exposed to cigarette smoke (both with approximate EC90 values). The superior efficacy of SK-POT compounds may reflect a different molecular mechanism of action than VX-770. In this study, we observed a significant decrease in CFTR channel function in Calu-3 cells after CSE pretreatment, but no significant decrease in the steady-state abundance of mature band C.

[0128] The positive effect of the SK-POT compound in enhancing CFTR channel activity in epithelial cells exposed to CSE translated into efficacy in rescuing defective mucociliary motility in primary bronchial cell cultures exposed to CSE (Figure 6). This observation is consistent with the proposal that enhancing CFTR function may improve mucus aggregation and obstruction in COPD. Interestingly, there was considerable variability in bead velocity among donor-specific cell cultures (Figure 6). This variability may reflect donor-specific differences in primary bronchial cell culture transport properties and certain heterogeneity within each cell culture regarding the proportion of ciliated cells.

[0129] In summary, these data support that CFTR channel activity prevents mucus aggregation and mucus stasis in tissue culture models of COPD. The effectiveness of SK-POT in preventing mucus stasis on the surface of human bronchial epithelial cell cultures varied depending on the donor-specific cell culture, but the average response was substantial compared to ivacaftor.

[0130] Figure 7 shows that the SK-POT compound is also effective in enhancing CFTR channel function in bronchial tissue of ferrets, a preferred animal model for preclinical testing of interventions targeting airway diseases (N. Kaza, VY Lin et al. Eur Respir J . 2022 Jul 13;60(1):2101581).

[0131] [Table 1] TIFF2025529902000019.tif223159TIFF2025529902000020.tif234159TIFF2025529902000021.tif234159 TIFF2025529902000022.tif234159TIFF2025529902000023.tif241159TIFF2025529902000024.tif223159

Claims

1. Equation (I): 【Chemistry 1】 A compound of or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein in the formula, R １ and R ２ H or (C) independently or simultaneously １ ~C ６ ) - Alkyl; R ３ and R ４ are, independently or simultaneously, H or (C １ ~C ６ )-alkyl, or R ３ and R ４ combine together with the nitrogen atom to which they are attached to form (C5-C6)-heteroaryl or (C4-C6)-heterocycloalkyl, and the (C5-C6)-heteroaryl or (C4-C6)-heterocycloalkyl may each be substituted with halo, OH, (C １ ~C ６ )-alkyl, or halogenated (C １ ~C ６ )-alkyl; Ring B is (C6-C10)-aryl or (C5-C10)-heteroaryl, and the (C6-C10)-aryl or (C5-C10)-heteroaryl is respectively a halo, OH, (C １ ~C ６ )-alkyl, halogenated (C １ ~C ６ )-alkyl, (C ３ ~C ６ )-cycloalkyl, or halogenated (C ３ ~C ６ ) - May be substituted with one or more cycloalkyl groups; W is (C1-C6)-alkyl or -(C0-C6)-alkylene-(C6-C10)-aryl, and the (C1-C6)-alkyl or -(C0-C6)-alkylene-(C6-C10)-aryl are respectively halo, OH, CN, and (C １ ~C ６ )-alkyl, halogenated (C １ ~C ６ )-alkyl, (C １ ~C ６ )-alkoxy, (C ３ ~C ６ )-cycloalkyl, or halogenated (C ３ ~C ６ ) - May be substituted with one or more cycloalkyl groups; A compound or its pharmaceutically acceptable salt, stereoisomer, or solvate.

2. R １ and R ２ However, independently or simultaneously, H or (C １ ~C ３ A compound of formula (I) according to claim 1, which is alkyl, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof.

3. R １ and R ２ A compound of formula (I) according to claim 1, wherein H is present, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof.

4. R ３ and R ４ However, independently or simultaneously, H or (C １ ~C ３ ) - Alkyl or R ３ and R ４ These combine, along with the nitrogen atoms to which they are bonded, to form (C5)-heteroaryl or (C4-C6)-heterocycloalkyl groups, and each of these (C5)-heteroaryl or (C4-C6)-heterocycloalkyl groups is a halo, OH, (C １ ~C ３ )-alkyl, or halogenated (C １ ~C ３ A compound of formula (I) according to claim 1, which may be substituted with an alkyl group, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof.

5. R ３ and R ４ However, independently or simultaneously, H or CH ３ The compound of formula (I) according to claim 4, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof.

6. R ３ and R ４ A compound of formula (I) according to claim 4 or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein the compounds combine to form, together with the nitrogen atoms to which they are bonded, a substituted piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl, or pyrazolyl.

7. Ring B is (C6)-aryl or (C5-C6)-heteroaryl, and the (C6)-aryl or (C5-C6)-heteroaryl is, respectively, a halo, an OH group, and (C １ ~C ６ )-alkyl, halogenated (C １ ~C ６ )-alkyl, (C ３ ~C ６ )-cycloalkyl, or halogenated (C ３ ~C ６ A compound of formula (I) according to claim 1, which may be substituted with one or more cycloalkyl groups, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof.

8. Ring B is, Haro, (C １ ~C ６ )-alkyl, or halogenated (C １ ~C ６ A compound of formula (I) according to claim 7, which is phenyl or pyridinyl, which may be substituted with one or more alkyl groups, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof.

9. A compound of formula (I) according to claim 8, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein ring B is a phenyl or pyridinyl, which may be substituted with one or more of fluoro or trifluoromethyl groups.

10. Ring B has the following structure: 【Chemistry 2】 A compound of formula (I) according to claim 9, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, having the above.

11. W is (C1-C4)-alkyl or -(C0-C4)-alkylene-(C6-C10)-aryl, and the (C1-C4)-alkyl or -(C0-C4)-alkylene-(C6-C10)-aryl are respectively halo, OH, CN, (C １ ~C ４ )-alkyl, halogenated (C １ ~C ４ )-alkyl, (C １ ~C ４ )-alkoxy, (C ３ ~C ６ )-cycloalkyl, or halogenated (C ３ ~C ６ A compound of formula (I) according to claim 1, which may be substituted with a cycloalkyl group, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof.

12. W is (C1-C4)-alkyl or -(C0-C1)-alkylene-phenyl, and the (C1-C4)-alkyl or -(C0-C1)-alkylene-phenyl is, respectively, halo, OH, CN, (C １ ~C ４ )-alkyl, halogenated (C １ ~C ４ )-alkyl, or (C １ ~C ４ A compound of formula (I) according to claim 10, which may be substituted with one or more of the alkoxys, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof.

13. W has the following structure 【Transformation 3】 A compound of formula (I) according to claim 11, having the above, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof.

14. The compound of formula (I) is 【Chemistry 4】 【change】 【change】 【change】 The compound of formula (I) described in claim 1 or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof.

15. The compound is 【Transformation 5】 The compound of formula (I) according to claim 14, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof.

16. The compound is 【Transformation 6】 The compound of formula (I) according to claim 15, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof.

17. A composition for use in the treatment of diseases associated with misfolded or misshaped proteins, comprising a compound of formula (I) according to any one of claims 1 to 16 or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof.

18. The composition according to claim 17, wherein the disease is cystic fibrosis, cancer, long QT syndrome, chronic obstructive pulmonary disease, or Dravet syndrome (epilepsy).

19. The composition according to claim 18, wherein the disease is cystic fibrosis or COPD.

20. The composition according to claim 19, wherein the cystic fibrosis is the result of a ΔF508 mutation in the CFTR protein.

21. Use of a compound of formula (I) according to any one of claims 1 to 16 or a pharmaceutically acceptable salt, stereoisomer or solvate thereof in the manufacture of a pharmaceutical for the treatment of diseases associated with misfolded or misshaped proteins.

22. A pharmaceutical composition comprising a compound of formula (I) according to any one of claims 1 to 16 or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, and a pharmaceutically acceptable excipient.

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