Pyridazinone compounds that modulate muteins for treatment of respiratory diseases

Abstract

The present disclosure relates to pyridazinone compounds of formula (I): # imgabs0 # for the treatment of respiratory diseases caused by misfolded or misshaped proteins, such as chronic obstructive pulmonary disease (COPD), cystic fibrosis, cancer, long QT syndrome or dravet syndrome

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 400,857, filed on August 25, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] FIELD OF THE DISCLOSURE

[0004] The present application relates to a pyridazinone compound of formula (I), which is used for treating respiratory diseases associated with misfolded or misshaped proteins.

[0005] BACKGROUND OF THE DISCLOSURE

[0006] Cystic fibrosis (CF) is the most common fatal genetic disease in Canadians and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR / ABCC7) gene. This gene encodes the CFTR protein (a plasma membrane channel) that is responsible for the flow of chloride ions across the apical membrane of epithelial cells in hollow organs such as the lungs, pancreas and digestive tract, as well as the skin (Gadsby, et al. (2006) The ABC protein turned chloride channel whose failure causes cystic fibrosis, Nature, 440: 477-483). This maintains the ion balance required for the thin layer of mucus in these organs. Normally, after the CFTR protein is synthesized in the endoplasmic reticulum, it is folded into the correct structural conformation and then escorted to the Golgi apparatus for the addition of complex glycosylation and transported to the plasma membrane.

[0007] CFTR is a member of the ATP-binding cassette (ABC) superfamily of membrane proteins that utilize ATP hydrolysis to carry out biological processes, most commonly active transport of substances across membranes. CFTR is unique in that it is the only member of this family that is an ion channel; however, its structure is similar to other ABC proteins, consisting of two transmembrane domains (MSDs), each connected to a nucleotide binding domain (NBD). The domain unique to CFTR is the regulatory (R) domain, which is structurally disordered and, when phosphorylated, participates in channel gating. CFTR is also regulated by interdomain interactions and the binding and hydrolysis of the ATP binding site located at the NBD1:NBD2 interface. Specifically, ATP-driven dimerization of NBD1 and NBD2 opens the ion channel, while ATP hydrolysis dissociates the dimers and closes the channel (Serohijos, AWR, et al. (2007)).

[0008] The most common mutation present in 70% of alleles is ΔF508 (missing (Δ) the amino acid phenylalanine (Phe, F) at position 508 in 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 at the interface of NBD1 and MSD2 and is believed to mediate interactions between domains to achieve correct folding and channel function. Specifically, the aromatic side chain of F508 is believed to form an aromatic cluster with residues from intracellular loop 4 (ICL4) of MSD2 and other residues from NBD1, playing an indispensable role in the stability of the tertiary structure of CFTR (Serohijos et al., supra.). The absence of this key residue prevents the correct folding of CFTR and forces it to remain in the endoplasmic reticulum, where it is targeted for degradation. In addition, even if the mutant is rescued back to the cell surface, its function is reduced. Loss of F508 may alter the domain:domain interface in the protein, preventing proper channel gating. Overall, the mutation disrupts extracellular ion balance, reduces surface hydration, and leads to thick mucus in many vital organs, such as the lungs and pancreas, which are susceptible to bacterial infections. Many other CFTR mutations are known, such as gating mutations, such as the G551D mutation.

[0009] Small molecules can act as correctors (promoting the onward transport of mutant CFTR proteins to the cell surface) or as potentiators (increasing the channel activity of mutant CFTR proteins that have already reached the plasma membrane).

[0010] Chronic obstructive pulmonary disease (COPD) is a syndrome characterized by airway inflammation induced by environmental toxins, primarily tobacco smoke and household and industrial air pollution. The manifestations of COPD are very heterogeneous and are believed to be a combination of multiple diseases, including chronic bronchitis and emphysema, both of which result in reduced expiratory airflow and are associated with bronchial obstruction. Environmental pollutants cause loss of CFTR channel function on the luminal surface of the airway epithelium, and this loss is thought to lead to mucus obstruction.

[0011] SUMMARY OF THE DISCLOSURE

[0012] The present application relates to pyridazinone compounds of formula (I), and in one aspect of the present disclosure, can be used to treat 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 the defects in activity and folding of mutant proteins associated with respiratory diseases.

[0013] In one embodiment, the compound of formula (I) has the structure

[0014]

[0015] in

[0016] R 1 and R 2 independently or simultaneously H or (C 1 -C 6 )-alkyl;

[0017] R 3 and R 4 independently or simultaneously H or (C 1 -C 6 )-alkyl, or

[0018] R 3 and R 4 Together with the nitrogen atoms to which they are attached, they form (C 5 -C 6 )-heteroaryl or (C 4 -C 6 )-heterocycloalkyl, each of which is optionally substituted by halogen, OH, (C 1 -C 6 )-alkyl or halo-(C 1 -C 6 )-alkyl substituted;

[0019] Ring B is (C 6 -C 10 )-aryl or (C 5 -C 10 )-heteroaryl, each of which is optionally substituted by halogen, OH, (C 1 -C 6 )-alkyl, halo-(C 1 -C 6 )-alkyl, (C 3 -C 6 )-cycloalkyl or halo-(C 3 -C 6 )-One or more substitutions in cycloalkyl;

[0020] W is (C 1 -C 6 )-alkyl or -(C 0 -C 6 )-alkylene-(C 6 -C 10 )-aryl, each of which is optionally substituted by halogen, OH, CN, (C 1 -C 6 )-alkyl, halo-(C 1-C 6 )-alkyl, (C 1 -C 6 )-alkoxy, (C 3 -C 6 )-cycloalkyl or halo-(C 3 -C 6 )-cycloalkyl substituted;

[0021] or any pharmaceutically acceptable salt, stereoisomer or solvate thereof.

[0022] The present disclosure also encompasses 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.

[0023] The present disclosure also encompasses the use of a therapeutically effective amount of a compound of formula (I) as a synergist, for example in the treatment of COPD.

[0024] The present disclosure also includes methods of treating a patient with cystic fibrosis comprising administering a therapeutically effective amount of a compound of formula (I).

[0025] The present disclosure also includes methods of treating a COPD patient comprising administering a therapeutically effective amount of a compound of formula (I).

[0026] In another embodiment, a compound of formula (I) is co-administered with a corrector compound, wherein the corrector compound primarily targets the cellular processing error and transports the protein to the cell surface, while the potentiator compound (formula (I)) helps to restore the function of the protein, for example, by restoring the cAMP-dependent chloride channel activity of the misfolded protein (e.g., CFTR) on the cell surface.

[0027] Through the following detailed description, other features and advantages of the present application will become apparent. However, it should be understood that the detailed description and specific examples, although indicating the preferred embodiments of the present application, are only given as illustrations, because according to the detailed description, various changes and modifications within the spirit and scope of the present application are obvious to those skilled in the art.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In the paragraphs provided below, the present disclosure is described in an exemplary manner in conjunction with the accompanying drawings. The figures provided herein are provided to better understand the example embodiments and to more clearly show how to implement various embodiments. These figures are not intended to limit the present disclosure.

[0030] Figure 1 Microscopic images (A) and (B) of microsphere tracking are shown, and (C) is a graph showing improved microsphere motility after exposure to a compound of the present disclosure.

[0031] Figure 2 The fluorescence-based membrane potential assay was shown to be - The dose response of VX-770 and SK-POT (compounds of the present disclosure) in enhancing the cyclic AMP (forskolin-activated) chloride channel activity of F508del CFTR was measured in FIG.

[0032] Figure 3 (i) shows cell-attached CFTR recordings. a-representative traces recorded in 150 NM DG-Cl base solution, b-supplemented with 1 mM forskolin, c-supplemented with 10 nM SK-POT analog, and d-supplemented with 10 mM inhibitor 172 (I172); the 0 current line is indicated by a gray dashed line; the total number of CFTR channels in the cell-attached patch for the representative traces shown was estimated to be 8. V m = -80 mV; e - open probability for each drug normalized to open probability in the presence of 1 μM forskolin (see Methods section), (ii) Bar graphs show channel open probability normalized to total channel number in the presence of forskolin, forskolin plus SK-POT, or after addition of CFTR Inh-172 at the above concentrations. Error bars are standard deviations; n = 5 for each drug.

[0033] Figure 4 (i) Representative Ussing chamber study plots showing enhancement of SK-POT (1 μM) following forskolin activation (100 nM) in primary bronchial epithelial cultures. (ii). Bar graphs showing forskolin-dependent changes in CFTR-mediated short circuiting following addition of compound 11 or VX-770 (n=3 donors, n=2-3 technical replicates per condition).

[0034] Figure 5Shown are upper panels: Representative traces of forskolin-stimulated (addition indicated by downward black arrows) CFTR-dependent FLiPR signal changes measured in confluent Calu-3 cell cultures pre-exposed to cigarette smoke (CSE) and acutely treated with forskolin (+DMSO (VEH), filled circles). Addition of CFTR inhibitors is indicated by grey arrows. After superposition, we show the effect of acute co-application of VX-770 (open squares) or SK-POT (open triangles) with forskolin. The bar graph (lower panel) shows the inhibitory effect of cigarette smoke extracts (-CSE vs. +CSE) on CFTR channel activity measured in Calu-3 monolayers (3 biological replicates and 3-4 technical replicates) using the FLiPR assay. The bar graph also shows the relative effect of DMSO (VEH), VX-770 (770, 1 μM) or SK-POT (1 μM) "p" values ​​(determined using one-way ANOVA, Sidak's multiple comparison test). In the presence of cigarette smoke extract, only SK-POT rescued CFTR channel function.

[0035] Figure 6 (i-iii) show the trajectory of green microspheres over a 5-second period, captured at 10 frames per second under 20X objective magnification. Cells were chronically pretreated with 2% cigarette smoke extract plus DMSO (VEH), VX-770, or SK-POT for 24 hours, and then acutely treated with 10 μM forskolin (FSK) for 20 minutes at room temperature before recording. ii. Trajectory of moving green microspheres under the same conditions as i., except that 1 μM ivacaftor (VX-770) was co-applied during the chronic treatment. The color of the dots within the trajectory represents the point within the 5-second period, purple (cold color) represents the beginning, and red / warm color represents the end of the period. iv. Summary of the average microsphere velocity of n=4 donors, which compares the velocity without CSE and the velocity with CSE (with or without chronic co-application of VEH, VX-770 (iv) or SK-POT (v)). The dots represent the average microsphere velocity of each video, and n=4-6 videos were obtained for each donor and condition. The horizontal line represents the mean of all videos and the error bars represent the standard deviation. Statistical significance was assessed by one-way ANOVA using Sidak's multiple comparison test.

[0036] Figure 7 Shown are (a) representative Ussing chamber studies of SK-POT (1 μM) enhancement following forskolin activation (100 nM) in primary bronchial epithelial cultures prepared from adult ferret trachea. (a) Bar graphs show forskolin-dependent changes in CFTR-mediated short circuiting following addition of SK-9919 or VX-770 (n=3 donors, n=2-3 technical replicates per condition). Student's "t" test was performed.

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] (I) Definition

[0039] As used herein, the term “(C 1- C n )-alkyl" refers to a straight and / or branched saturated alkyl group containing from 1 to "n" carbon atoms, and includes (depending on the identity of n) methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, 2,2-dimethylbutyl, n-pentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, n-hexyl, and the like, wherein the variable n is an integer representing the maximum number of carbon atoms in the alkyl group.

[0040] As used herein, the term “(C 1- C n ")-alkoxy" refers to a straight and / or branched saturated alkoxy group containing from 1 to "n" carbon atoms, and includes (depending on the identity of n) methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy, and the like, wherein the variable n is an integer representing the maximum number of carbon atoms in the alkyl group.

[0041] As used herein, the term “(C 3 -C 6 ")-cycloalkyl" refers to a monocyclic saturated or partially unsaturated carbocyclic group containing 3 to 6 carbon atoms and includes (depending on the identity of m) cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, and the like.

[0042] The term "heterocycloalkyl" as used herein refers to a monocyclic saturated or partially unsaturated group containing, for example, 5 to 6 ring atoms and including one, two, three or four hetero groups independently selected from N, NH, N(C 1-6 alkyl), O and S, pyrrolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl and tetrahydrofuranyl.

[0043] The term "aryl" as used herein refers to 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.

[0044] The term "heteroaryl" as used herein refers to a monocyclic or bicyclic ring system containing one or two aromatic rings and 5 to 10 atoms, and includes one, two, three or four hetero groups independently selected from N, NH, N(C 1-6 alkyl), O and S, and includes thienyl, thiazolyl, furanyl, pyrrolyl, pyridyl, indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuranyl, benzothiophenyl and the like.

[0045] The term "halo" as used herein refers to halogen and includes chlorine, fluorine, bromine, iodine and the like.

[0046] The term "halogenated" as used herein refers to, for example, a halo-(C 1 -C 6 )-alkyl means that at least one (including all) hydrogen atoms on the referenced group are replaced by halogen atoms.

[0047] The term "stereoisomer" as used herein refers to an isomer having the same constitution as the corresponding stereoisomer but having an arrangement of atoms in space that is different from the corresponding stereoisomer. 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 mixtures thereof in any ratio (e.g., racemic mixtures, non-racemic mixtures).

[0048] The term "solvate" as used herein refers to a pharmaceutically acceptable solvate form of a particular compound of formula (I), for example, formed by the physical association of the compound with one or more solvent molecules, which retains the biological effectiveness of the compound. Examples of solvates include, but are not limited to, combinations of the compounds of the invention with water, 1-propanol, 2-propanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid or ethanolamine. When the solvent is water, the form is referred to as a "hydrate".

[0049] As used herein, the term "therapeutically effective amount" refers to a dose that produces a selected effect. For example, an effective amount of a compound of formula (I) is an amount sufficient to allow the compound to act as a potentiator and, for example, alleviate or ameliorate cystic fibrosis or COPD or a symptom thereof.

[0050] The term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of the compounds of formula (I) and are not biologically or otherwise undesirable. In many cases, the disclosed compounds are capable of forming acid or base salts due to the presence of acidic or basic moieties. The preparation of salts with suitable acids or bases is known in the art.

[0051] (II) Compounds of formula (I)

[0052] The present disclosure relates to pyridazinone compounds of formula (I), and in one aspect of the present disclosure, can be used to treat diseases associated with misfolded or misshaped proteins. In one embodiment, the compound of formula (I) has the following structure

[0053]

[0054] in

[0055] R1 and R 2 independently or simultaneously H or (C 1 -C 6 )-alkyl;

[0056] R 3 and R 4 independently or simultaneously H or (C 1 -C 6 )-alkyl, or

[0057] R 3 and R 4 Together with the nitrogen atoms to which they are attached, they form (C 5 -C 6 )-heteroaryl or (C 4 -C 6 )-heterocycloalkyl, each of which is optionally substituted by halogen, OH, (C 1 -C 6 )-alkyl or halo-(C 1 -C 6 )-alkyl substituted;

[0058] Ring B is (C 6 -C 10 )-aryl or (C 5 -C 10 )-heteroaryl, each of which is optionally substituted by halogen, OH, (C 1 -C 6 )-alkyl, halo-(C 1 -C 6 )-alkyl, (C 3 -C 6 )-cycloalkyl or halo-(C 3 -C 6 )-One or more substitutions in cycloalkyl;

[0059] W is (C 1 -C 6 )-alkyl or -(C 0 -C 6 )-alkylene-(C 6 -C 10 )-aryl, each of which is optionally substituted by halogen, OH, CN, (C 1 -C 6 )-alkyl, halo-(C 1 -C 6 )-alkyl, (C 1 -C 6 )-alkoxy, (C 3 -C 6 )-cycloalkyl or halo-(C 3 -C6 )-One or more substitutions in cycloalkyl;

[0060] or any pharmaceutically acceptable salt, stereoisomer or solvate thereof.

[0061] In one embodiment, R 1 and R 2 independently or simultaneously H or (C 1 -C 3 In another embodiment, R 1 and R 2 H or CH independently or simultaneously 3 In another embodiment, R 1 and R 2 It's H.

[0062] In another embodiment, R 3 and R4 are independently or simultaneously H or (C 1 -C 3 )-alkyl, or R 3 and R 4 Together with the nitrogen atoms to which they are attached, they form (C 5 )-heteroaryl or (C 4 -C 6 )-heterocycloalkyl, each of which is optionally substituted by halogen, OH, (C 1 -C 3 )-alkyl or halo-(C 1 -C 3 )-alkyl substitution.

[0063] In another embodiment, R 3 and R 4 H or CH independently or simultaneously 3 In another embodiment, R 3 and R 4 Yes CH 3 .

[0064] In another embodiment, R 3 and R 4 Together with the nitrogen atom to which they are attached, they form an optionally substituted piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl or pyrazolyl group.

[0065] In another embodiment, Ring B is (C 6 )-aryl or (C 5 -C 6 )-heteroaryl, each of which is optionally substituted by halogen, OH, (C 1 -C 6 )-alkyl, halo-(C 1-C 6 )-alkyl, (C 3 -C 6 )-cycloalkyl or halo-(C 3 -C 6 In another embodiment, ring B is optionally substituted with halogen, (C 1 -C 6 )-alkyl or halo-(C 1 -C 6 )-alkyl. In another embodiment, ring B is phenyl or pyridyl optionally substituted by one or more of fluoro or trifluoromethyl. In a further embodiment, ring B has the following structure:

[0066]

[0067] In another embodiment, W is (C 1 -C 4 )-alkyl or -(C 0 -C 4 )-alkylene-(C 6 -C 10 )-aryl, each of which is optionally substituted by halogen, OH, CN, (C 1 -C 4 )-alkyl, halo-(C 1 -C 4 )-alkyl, (C 1 -C 4 )-alkoxy, (C 3 -C 6 )-cycloalkyl or halo-(C 3 -C 6 In another embodiment, W is (C 1 -C 4 )-alkyl or -(C 0 -C 1 )-alkylene-phenyl, each of which is optionally substituted by halogen, OH, CN, (C 1 -C 4 )-alkyl, halo-(C 1 -C 4 )-alkyl or (C 1 -C 4 In another embodiment, W has the following structure

[0068]

[0069] In one embodiment of the present disclosure, the compound of formula (I) is

[0070]

[0071]

[0072]

[0073]

[0074] In one embodiment, the compound is

[0075]

[0076] The present disclosure also includes pharmaceutical compositions comprising a compound of formula (I) as defined above (compound of the present disclosure), or a pharmaceutically acceptable salt, solvate and prodrug thereof, and a pharmaceutically acceptable carrier or diluent. These compounds are suitably formulated into a pharmaceutical composition, which is administered to a subject, preferably a human, in a biocompatible form suitable for in vivo administration.

[0077] Compositions containing the disclosed compounds can be prepared by known methods for preparing pharmaceutically acceptable compositions that can be applied to subjects, so that an effective amount of the active substance is mixed with a pharmaceutically acceptable vehicle. For example, suitable vehicles are described in Remington's Pharmaceutical Compendium (2003, 20th edition) and the United States Pharmacopeia: National Formulary (USP 24NF19), published in 1999. On this basis, the composition includes, but is not limited to, a solution of a substance combined with one or more pharmaceutically acceptable carriers or diluents, and is contained in a buffer solution having an appropriate pH and isotonic with physiological fluids.

[0078] The compounds of the present disclosure can be used pharmaceutically in free base form, salt form, solvate form and hydrate form. 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, used as a source of the free base form, even if the particular salt itself is only required as an intermediate, for example, when the formation of the salt is 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.

[0079] In one embodiment, the compounds of formula (I) of the present disclosure are formulated into pharmaceutical compositions in a manner familiar to any person 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.

[0080] Suitable carrier materials are not only inorganic carrier materials but also organic carrier materials. Suitable carrier materials for topical preparations are glycerides, semisynthetic and synthetic glycerides, hydrogenated oils, liquid waxes, liquid paraffins, liquid fatty alcohols, sterols, polyethylene glycols and cellulose derivatives.

[0081] Usual stabilizers, preservatives, wetting and emulsifiers, consistency improving agents, salts for varying the osmotic pressure, buffer substances, solubilizers, colorants and antioxidants are considered as pharmaceutical adjuvants.

[0082] In some embodiments, the disclosure includes compositions comprising a compound of formula (I) as a potentiator and another active agent as a corrector for treating a disease associated with misfolded and / or misshaped proteins. In one embodiment, the corrector compound is VRT-534 or VX-809.

[0083] In another embodiment, when the compound of formula (I) is co-administered with a corrector, the active agents (the compound of formula (I) and the corrector) may be administered simultaneously or sequentially.

[0084] 3. Medical treatment methods

[0085] The present disclosure includes medical treatment methods, which include administering formula (I) compounds to mammals. In some embodiments, the present disclosure includes methods for treating respiratory diseases caused by misfolded and / or misshaped proteins. In one embodiment, the present disclosure includes methods for regulating mutant proteins defective in activity and / or folding related to respiratory diseases, which include administering formula (I) compounds. In one embodiment, respiratory diseases are cystic fibrosis or chronic obstructive pulmonary disease (COPD). In another embodiment, the present disclosure includes methods for treating other diseases (e.g., long QT syndrome or Dravet syndrome) associated with misfolded or misshaped proteins.

[0086] In one embodiment, the disclosure includes a method for treating a patient with a respiratory disease caused by misfolded or misshaped proteins, comprising administering a therapeutically effective amount of a compound of formula (I). In one embodiment, the 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, cystic fibrosis is the result of a ΔF508 mutation in the CFTR protein.

[0087] In one embodiment, the disclosure includes a method for treating a patient with respiratory disease caused by misfolded or misshaped proteins, comprising administering a therapeutically effective amount of a compound of formula (I). In one embodiment, the 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 CF, in which CFTR protein function is reduced due to cigarette smoke or other environmental toxins.

[0088] In another embodiment of the present disclosure, a method for treating a disease caused by misfolded or misshaped proteins is also included, comprising administering a therapeutically effective amount of a compound of formula (I) to a subject (e.g., a human being). In one embodiment, the present disclosure includes a method for regulating a mutant protein defective in activity and / or folding associated with respiratory diseases, comprising administering a compound of formula (I). In another embodiment of the present disclosure, a method for treating long QT syndrome is also included, comprising administering a therapeutically effective amount of a compound of formula (I) to a subject (e.g., a human being). In another embodiment of the present disclosure, a method for treating Dravet syndrome (epilepsy) is also included, comprising administering a therapeutically effective amount of a compound of formula (I) to a subject (e.g., a human being). In another embodiment of the present disclosure, a method for treating cancer associated with misfolded or misshaped proteins (e.g., p53 proteins) is also included, comprising administering a therapeutically effective amount of a compound of formula (I) to a subject (e.g., a human being).

[0089] In other embodiments, the disclosure also includes the use of compounds of formula (I) for treating respiratory diseases caused by misfolded or misshaped proteins. In one embodiment, the respiratory disease is cystic fibrosis. In one embodiment, the disease is COPD.

[0090] The dosage of the compound of formula (I) varies within a wide range, depending on the disease to be controlled, the age and personal condition of the patient and the mode of administration, and should of course be adapted to the individual needs of each particular case. For adult patients, a daily dosage of about 1 mg to about 1,000 mg, particularly about 1 mg to about 100 mg, may be considered. Depending on the dosage, the daily dosage may be conveniently administered in several dosage units.

[0091] Although the present disclosure has been described in conjunction with its specific embodiments, it is apparent that many substitutions, modifications and variations will be apparent to those skilled in the art. Therefore, it is intended to cover all such substitutions, modifications and variations that fall within the spirit and broad scope of the appended claims. In addition, the citation or mentioning of any reference in this application should not be construed as an admission that such reference may serve as prior art to the present disclosure. Example

[0092] The implementation of the present disclosure is illustrated by the following representative examples. It is obvious to those skilled in the art that many details of the examples can be changed while still being able to implement the disclosure described herein.

[0093] Materials and methods

[0094] Cell culture: HEK293 cells stably expressing human ΔF508 CFTR were cultured in DMEM / F12 medium containing 10% fetal bovine serum (FBS) and 600 μg / mL geneticin. For functional CFTR assays, frozen cells were quickly thawed in a 37°C water bath, diluted with warm DMEM / F12 medium containing 10% FBS, centrifuged at 300×g for 5 min, and resuspended in the same medium at a density of 300,000 cells / mL. The cystic fibrosis bronchial derived cell line (“CFBE”) supplemented with 4.7Kb ΔF508 CFTR cDNA was obtained from Dr. Dieter Gruenert with permission from UCSF. These cells were cultured in MEM containing Earle's salts (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 300 μg / mL hygromycin (Millipore). CFBE cells were grown on flasks coated with a fibronectin solution containing LHC basal medium (Invitrogen), 0.001% bovine serum albumin (Sigma), 1% v / v Vitrogen 100 (BD Bioscience), and 10 μg / mL human fibronectin (Life Technologies). Unless specified for testing, all cells were maintained at 37°C and 5% CO. 2 Down growth.

[0095] HEK293ΔF508 CFTR cell potentiator assay: Cells were plated at a density of 15,000 cells per well in 384-well black poly-D-lysine-coated plates (Greiner) and placed at 37°C, 5% CO 2 The cell plate was then placed in an incubator at 30°C and 5% CO 2 Incubator for 20 to 24 hours to allow temperature to rescue delF508-CFTR expression. Before the assay, the cell culture medium was removed and 20 μL / well assay buffer containing blue membrane potential dye (10 ml diluted dye per 200 ml assay buffer, Molecular Devices) was added. The assay buffer was a modified Tyrode buffer containing 140 mM sodium gluconate, 0.5 mM potassium gluconate, 2 mM calcium gluconate, 2 mM magnesium gluconate, 10 mM HEPES, 12 mM NaHCO3 , pH 7.4.

[0096] The cells were then incubated with the buffer at 30°C and 5% CO 2 Incubate incubator for 45min to 60min. Then measure DelF508 CFTR activity on FLIPR fluorescence microplate reader (Molecular Devices). Add assay buffer containing 90nM genistein (Sigma) and compound, 90nM genistein and DMSO (1.6%, negative control) or 90nM genistein plus 30μmol forskolin (positive control, Sigma) to trigger activity with 10μL / well. Use the following filter settings to measure fluorescence changes, excitation wavelength: 510nm-545nm, emission wavelength: 565nm-625nm.

[0097] HEK293ΔF508 CFTR Cell Corrector Assay: Cells were plated at a density of 15,000 cells per well in 384-well black poly-D-lysine coated plates (Greiner) containing 0.5 μL DMSO-diluted compound, DMSO alone (negative control), or DMSO mixed with 1 μmol VX-661 (positive control, Selleckchem) per well and placed at 37°C, 5% CO 2 Incubate the cells in an incubator for 18-24 hours. Before the assay, remove the cell culture medium and add 20 μL / well of modified Tyrode assay buffer containing a blue membrane potential dye. Incubate the cells with this buffer at 37°C, 5% CO 2 Incubate in an incubator at 40 °C for 45 to 60 min. DelF508-CFTR activity is then measured on the FLIPR. Activity is triggered by adding 10 μL / well of assay buffer containing 30 μmol genistein and 30 μmol forskolin. Changes in fluorescence are measured as described above.

[0098] CFBE potentiator assay: CFBE cells were dissociated for 10 min at 37°C using Hank's balanced salt solution containing 0.6 mM EDTA and 10% of 0.25% trypsin / EDTA solution (both from Life Technologies). An equal amount of CFBE cell growth medium (see cell culture above) was then added to the flask and the cells were centrifuged at 500 x g for 10 min. The cells were resuspended in cell culture medium at a density of 150,000 cells / mL and plated in 384-well black poly-D-lysine coated plates (7,500 cells / well). The cells were then incubated at 37°C, 5% CO 2Grow for 6-8 days to allow differentiation. Change the cell culture medium every 2-3 days. One day before each experiment, remove the culture medium, add fresh culture medium, and place the cells at 30°C, 5% CO 2 The cells were incubated for 18-24 hours to allow temperature to rescue delF508 CFTR. The enhancement activity of HEK cells was measured as described above, except that the cells were stained for a strict 60 min, the compounds (or negative control DMSO) were mixed with assay buffer containing 3 μmol forskolin, and the positive control wells had assay buffer containing 3 μmol forskolin and 9 μmol VX-770 (Selleckchem). The results are shown in Table 1.

[0099] CFBE calibrator assay: CFBE cells were dissociated and grown in 384-well plates for 6-8 days as described above. One day before each experiment, cell growth medium was removed, 50 μL of medium containing compound, 50 μL of 0.1% DMSO (negative control) or 50 μL of 1 μmol VX-809 (positive control, Selleckchem) was added, and the cells were placed at 30°C, 5% CO 2 The cells were then stained for 18-24 hrs to allow temperature rescue of delF508 CFTR. Correction activity was measured as described above for HEK cells, except that cells were stained for a stringent 60 min and activity was triggered by the addition of 10 μL / well of assay buffer containing 3 μmol forskolin and 9 μmol VX-770.

[0100] Data Analysis: CFTR activity was defined as the maximum fluorescence signal after addition minus baseline fluorescence. All data were normalized to the positive and negative controls for each plate and expressed as a percentage of response. Concentration response curves were analyzed by fitting the data to a 4-parameter logistic equation in Microcal's Origin or IDBS' Activity Base software.

[0101] Example 1 - Synthesis of Intermediate A

[0102]

[0103] Synthesis of 5,6-dichloropyridazin-3(2H)-one (2):

[0104] 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 temperature (130°C). After stirring for 3 h, the reaction mixture was cooled to RT and poured into ice-cold water (500 mL). The precipitated solid was filtered, washed with ice-cold water (50 mL x 2), and dried under vacuum to give an off-white solid 2 (10.3 g, 38%).1 HNMR (400MHz, CDCl 3 ): δ11.19(bs,1H),7.16(s,1H); LCMS(ESI): m / z 162.9[MH + ]; 95.3%; RT = 1.92 min (XBrigde C18 column, 5 mM ammonium bicarbonate in water and MeCN).

[0105] Synthesis of 6-chloro-5-(piperidin-1-yl)pyridazin-3(2H)-one (3):

[0106] At RT, under an inert atmosphere, the 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). 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 RT 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) and washed with anhydrous Na 2 SO 4 Dry and concentrate under reduced pressure. The crude product was triturated with pentane (100 mL) to give 3 (25 g, 77%) as a brown solid. 1 HNMR (400 MHz, DMSO-d 6 ):δ12.68(brs,1H;D 2 O exchangeable), 6.13 (s, 1H), 2.93-3.11 (m, 4H), 1.44-1.75 (m, 6H); LCMS (ESI): m / z 214.1 [M+H + ]; 98.3%; RT = 1.81 min (AcquityBEH C18 column, 0.1% formic acid / MeCN containing 0.1% formic acid.

[0107] Synthesis of 6-chloro-4-nitro-5-(piperidin-1-yl)pyridazin-3(2H)-one (A):

[0108] At 0 °C, 3 (3.0 g, 14.04 mmol) was added to CH 2 Cl 2 Concentrated H (50 mL) was added to the stirred solution. 2 SO 4 (18 mL), then added fuming nitric acid (5 mL), and stirred for 1 h. The reaction was monitored by TLC. The reaction mixture was diluted with ice-cold water (200 mL) and washed with CH 2 Cl2 (2X 100mL) extraction. The combined organic layers were washed with anhydrous Na 2 SO 4 The mixture was dried and concentrated under reduced pressure. Note: The reaction was repeated in four batches and the resulting crude products were combined and purified by silica gel column chromatography using 20% ​​EtOAc petroleum ether to give a yellow solid A (4.6 g, 25%). 1 HNMR (400MHz, CDCl 3 ): δ10.74(br s,1H),3.26-3.21(m,4H),1.80-1.65(m,6H); LCMS(ESI):m / z 257.1[M+H + ]; 93.0%; RT = 2.98 min (Acquity BEH C18 column, 0.05% formic acid / MeCN containing 0.05% formic acid.

[0109] Example 2-Synthesis of Intermediate B

[0110]

[0111] Synthesis of 1-bromo-3-(methoxymethoxy)benzene (5):

[0112] To a stirred solution of 3-bromophenol (4) (20.0 g, 115.6 mmol) in DMF (300 mL) at 0°C under an inert atmosphere was added NaH (5.1 g, 127.5 mmol; 60% mineral oil) followed by MOM chloride (9.66 mL, 127.18 mmol). 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 mixture was precipitated with anhydrous Na 2 SO 4 The combined organic layers were dried and concentrated under reduced pressure to give 5 (25.0 g) as a brown gum, which was used in the next reaction without purification. 1 HNMR (400MHz, CDCl 3 ): 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).

[0113] Synthesis of (3-(methoxymethoxy)phenyl)boronic acid (B):

[0114] To a stirred solution of 5 (25.0 g, 115.17 mmol) in THF (250 mL) was added n-butyl lithium (50.8 mL; 126.7 mmol; 2.5 M hexane solution) at -78 °C and stirred at the same temperature for 1 h. Triisopropyl borate (30 mL, 138.21 mmol) was added dropwise and kept at room temperature for 4 h. After the reaction was completed (monitored by TLC), the reaction mixture was quenched with 1N HCl aqueous solution (40 mL) and extracted with EtOAc (150 mL). The organic layer was washed with anhydrous Na 2 SO 4 Dry and concentrate 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 (400 MHz, DMSO-d 6 ): 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).

[0115] Example 3 - Synthesis of Intermediate C

[0116]

[0117] Synthesis of 5-bromo-2-tert-butylphenol (6):

[0118] To a stirred solution of 3-bromophenol (4) (20 g, 115.6 mmol) in tBuCl (200 mL) was added AlCl 3 (30.83 g, 231.2 mmol) and kept 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 (2X 200 mL). The combined organic layers were washed with anhydrous Na 2 SO 4 Dried 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. 1 HNMR (400MHz, CDCl 3 ): 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 / z 226.8[MH +]; 70.6%; RT = 3.57 min (Acquity BEHC18 column, 0.05% formic acid / MeCN containing 0.05% formic acid).

[0119] Synthesis of 4-bromo-1-(tert-butyl)-2-methoxybenzene (7):

[0120] 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) at 0°C under an inert atmosphere. Methyl iodide (2.72 mL, 43.67 mmol) was added 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 washed with anhydrous Na 2 SO 4 Drying and concentration under reduced pressure gave brown liquid 7 (4.5 g 85%). 1 HNMR (400MHz, CDCl 3 ): 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 / z 222.1[M + ]; 80.9%; RT=8.85min (ZB-5MS column, 100℃ / 1min, 20*C / min / 310*C / 5.5min.

[0121] Synthesis of 4-(tert-butyl)-3-methoxyphenylboronic acid (C):

[0122] To a stirred solution of 7 (4.5 g, 18.5 mmol) in THF (90 mL) was added n-butyl lithium (12.7 mL, 20.45 mmol; 1.6 M hexane solution) at -78 °C and stirred at the same temperature for 1 h. Triisopropyl borate (4.19 g, 22.3 mmol) was added dropwise at -78 °C. The reaction was stirred at room temperature for 16 h. After the reaction was completed (monitored by TLC), the reaction was quenched with 5N HCl aqueous solution (150 mL) and stirred for 1 h. The reaction solution was extracted with EtOAc (2×150 mL). The combined organic layers were washed with anhydrous Na 2 SO 4 Dry and concentrate under reduced pressure.The crude product was purified by silica gel column chromatography using 25% EtOAc in hexanes as 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 / z 207.1[MH + ]; 88.9%; RT = 2.37 min (Acquity BEH C18 column, 0.1% formic acid / MeCN containing 0.1% formic acid).

[0123] Example 4-Synthesis of 4-amino-2-(4-(tert-butyl)-3-hydroxyphenyl)-6-(4-fluorophenyl)-5-(piperidin-1-yl)pyridazine-3(2H)-one (11):

[0124]

[0125] Synthesis of 2-(4-(tert-butyl)-3-methoxyphenyl)-6-chloro-4-nitro-5-(piperidin-1-yl)pyridazin-3(2H)-one (8):

[0126] At room temperature, CH 2 Cl 2 C (1.6 g, 7.69 mmol), Cu(OAc) and 2 (1.4 g, 7.70 mmol), triethylamine (782 mg, 7.74 mmol) and pyridine (613 mg, 7.75 mmol) were added and stirred for 2 h. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with 2N HCl aqueous solution (100 mL) and washed with CH 2 Cl 2 The combined organic layers were washed with water (2X 200 mL) and washed with anhydrous Na 2 SO 4 Dried and concentrated under reduced pressure.The crude product was purified by silica gel column chromatography using 10% EtOAc in hexanes as eluent to give 8 (600 mg, 36%) as a yellow solid. 1 HNMR (400MHz, CDCl 3 ): 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 / z 421.2[M+H +]; 96.4%; RT = 3.19 min (Acquity BEH C18 column, 0.05% formic acid / MeCN containing 0.05% formic acid).

[0127] Synthesis of 4-amino-2-(4-(tert-butyl)-3-methoxyphenyl)-6-chloro-5-(piperidin-1-yl)pyridazin-3(2H)-one (9):

[0128] To a stirred solution of 8 (600 mg, 1.42 mmol) in ethanol (18 mL) were added Fe powder (239.3 mg, 4.28 mmol), NH 4 Cl (457.7 mg, 8.57 mmol) and H 2 O (6 mL). 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 small piece of celite. The filtrate was concentrated in vacuo and diluted with EtOAc (200 mL). The resulting solution was washed with water (300 mL) and washed with anhydrous Na 2 SO 4 Dried and concentrated under reduced pressure.The crude product was purified by silica gel column chromatography using 10% EtOAc in hexanes to give 9 (310 mg, 55%) as a brown solid. 1 HNMR (400MHz, CDCl 3 ): 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 / z 391.0[M+H + ]; 96.7%; RT = 4.16 min (Acquity BEH C18 column, 0.05% formic acid / MeCN containing 0.05% formic acid).

[0129] Synthesis of 4-amino-2-(4-(tert-butyl)-3-methoxyphenyl)-6-(4-fluorophenyl)-5-(piperidin-1-yl)pyridazin-3(2H)-one (10):

[0130] The reaction tube was charged with 9 (300 mg, 0.76 mmol), 1,4-dioxane (15 mL), Na 2 CO 3 (163 mg, 1.53 mmol), 4-fluorophenylboronic acid (215 mg, 1.53 mmol), and purged with argon for 15 min. Pd(PPh 3 ) 4(88.8 mg, 0.076 mmol) and H 2 O (9 mL) and cap the tube. The reaction was heated to 110 ° C and stirred for 7 h. After the reaction was completed (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 washed with anhydrous Na 2 SO 4 Dried and concentrated under reduced pressure.The crude product was purified by silica gel column chromatography using 20% ​​EtOAc in hexanes to give 10 as a brown solid (200 mg, 58%). 1 HNMR (400MHz, CDCl 3 ): 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 / z 451.0[M+H + ]; 84.1%; RT = 4.13 min (Acquity BEH C18 column, 0.05% formic acid / MeCN containing 0.05% formic acid).

[0131] Synthesis of 4-amino-2-(4-(tert-butyl)-3-hydroxyphenyl)-6-(4-fluorophenyl)-5-(piperidin-1-yl)pyridazin-3(2H)-one (11):

[0132] At 0 °C under inert atmosphere, 10 (200 mg, 0.44 mmol) of CH 2 Cl 2 Add BBr (20 mL) dropwise to the stirred solution 3 (0.23 mL, 2.22 mmol). The reaction was placed at room temperature and stirred for 4 h. After the reaction was complete (monitored by TLC), saturated NaHCO 3 solution (10 mL; pH up to 8) to basify the reaction mixture and add CH 2 Cl 2 (2X 50mL) extraction. The combined organic layers were washed with anhydrous Na 2 SO 4 The crude product was purified by preparative HPLC (Kromasil C 18 column; 5 mM ammonium bicarbonate in MeCN) to give 10 (71 mg, 36%) as a pale yellow solid. 1 HNMR (400 MHz, DMSO-d 6 ):δ9.57(s,1H;D 2O exchangeable), 7.58-7.44 (m, 2H), 7.25 (t, J = 8.9 Hz, 2H), 7.18 (d, J = 8.3 Hz, 1H), 7.02 (d, J = 2.2 Hz, 1H), 6.96 (dd, J = 2.2, 8.3 Hz, 1H), 5.81 (s, 2H; D 2 O exchangeable),2.73–2.68(m,4H),1.48-1.28(m,15H); LCMS(ESI):m / z 437.2[M+H + ]; 98.9%; RT = 2.94min (Acquity BEH C18 column, 0.05% formic acid / MeCN containing 0.05% formic acid); FT-IR (KBr): 3477 (OH), 3369 (1 o NH 2 ),1604(C=O)cm -1 .CFBE potentiator assay, potentiator (pot.) pEC 50 =8.0.

[0133] Example 5-Synthesis of 4-amino-2-(3-hydroxyphenyl)-5-(piperidin-1-yl)-6-(o-tolyl)pyridazine-3(2H)-one (15):

[0134]

[0135] Synthesis of 6-chloro-2-(3-methoxyphenyl)-4-nitro-5-(piperidin-1-yl)pyridazin-3(2H)-one (12):

[0136] At room temperature, A (800 mg, 3.09 mmol) and 3-methoxyphenylboronic acid (700 mg, 4.64 mmol) were added to CH 2 Cl 2 (20 mL) was added to the stirred solution in sequence: Cu(OAc) 2 (840 mg, 4.64 mmol), triethylamine (0.86 mL, 6.18 mmol), pyridine (0.5 mL, 6.18 mmol) and Molecular sieves (150 mg) were added and stirred for 16 h. After completion of the reaction (monitored by TLC), the reaction mixture was filtered through a celite pad and washed with CH 2 Cl 2 The filtrate was washed with water (50 mL) and then with CH 2 Cl 2 The aqueous layer was extracted with (2×50 mL). The combined organic layers were washed with anhydrous Na 2 SO 4Dried and concentrated under reduced pressure.The crude product was purified by silica gel column chromatography using 25% EtOAc in petroleum ether as eluent to afford 11 (600 mg, 53%) as a yellow solid. 1 HNMR (400MHz, CDCl 3 ): δ7.36(t,J=8.3Hz,1H),7.19-7.13(m,2H),6.93-6.89(m,1H),3.81(s,3H),3.24-3.21(m,4H),1.78-1.68(m,6H); LCMS(ESI):m / z 365.1[M+H + ]; 95.1%; RT = 8.39 min (XSelect CSH C18 column, 5 mM ammonium acetate aqueous solution containing MeCN).

[0137] Synthesis of 4-amino-6-chloro-2-(3-methoxyphenyl)-5-(piperidin-1-yl)pyridazin-3(2H)-one (13):

[0138] To a stirred solution of 12 (650 mg, 1.78 mmol) in ethanol (13 mL) were added Fe powder (300 mg, 5.34 mmol), NH 4 Cl (570 mg, 10.68 mmol) and H 2 O (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 small piece of celite. The filtrate was concentrated under reduced pressure. The resulting residue was diluted with water (20 mL) and washed with CH 2 Cl 2 (2×25 mL) extraction. The combined organic layers were washed with anhydrous Na 2 SO 4 Dried and concentrated under reduced pressure.The crude product was purified by silica gel column chromatography using 20% ​​EtOAc in petroleum ether to give 13 (280 mg, 47%) as a brown solid. 1 HNMR (400MHz, CDCl 3 ): 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 / z 335.1[M+H + ]; 92.8%; RT = 3.67 min (Acquity BEH C18 column, 0.05% formic acid / MeCN containing 0.05% formic acid).

[0139] Synthesis of 4-amino-2-(3-methoxyphenyl)-5-(piperidin-1-yl)-6-(o-tolyl)pyridazin-3(2H)-one (14):

[0140] The reaction tube was charged with 13 (400 mg, 1.19 mmol), o-tolylboronic acid (490 mg, 3.58 mmol), Na 2 CO 3 (380 mg, 3.58 mm), 1,4-dioxane (8 mL), H 2 O (0.8 mL) and degassed by purging with argon for 10 min. Pd(PPh 3 ) 4 (83 mg, 0.071 mmol) and purged with argon again for 15 min. The reaction tube was capped and stirred at 110 ° C for 16 h. 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 washed with anhydrous Na 2 SO 4 Dried and concentrated under reduced pressure.The crude product was purified by silica gel column chromatography using 30% EtOAc in petroleum ether to give brown semisolid 14 (280 mg, 65% pure), which was used directly in the next reaction without further purification. 1 HNMR (400MHz, CDCl 3 ): 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 / z 391.2[M+H + ]; 65.6%; RT = 2.79 min (Acquity BEH C18 column, 0.05% formic acid / MeCN containing 0.05% formic acid).

[0141] Synthesis of 4-amino-2-(3-hydroxyphenyl)-5-(piperidin-1-yl)-6-(o-tolyl)pyridazin-3(2H)-one (15):

[0142] At 0 °C under inert atmosphere, 14 (280 mg) was added to CH 2 Cl 2 Add BBr (10 mL) dropwise to the stirred solution 3(3.5 mL, 3.58 mmol; 1M CH 2 Cl 2 The reaction mixture was diluted with water (5 mL) and saturated with NaHCO 3 The solution (10 mL; pH up to 8) was basified with CH 2 Cl 2 (2X 15mL) extraction. The combined organic layers were washed with anhydrous Na 2 SO 4 The crude product was purified by preparative HPLC (Xbridge C18 column; 10 mM ammonium bicarbonate in MeCN) to give 15 (98 mg, 22% from 12) as a white solid. 1 HNMR (400 MHz, DMSO-d 6 ):δ9.62(br s,1H;D 2 O 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, 8.2 Hz, 1H), 5.78 (br s, 2H; D 2 O exchangeable),2.63–2.58(m,4H),2.21(s,3H),1.40-1.18(m,6H); LCMS(ESI):m / z 377.1[M+H + ]; 99.1%; RT = 2.50min (Acquity BEH C18 column, 0.05% formic acid / MeCN containing 0.05% formic acid); FT-IR (KBr): 3483 (OH), 3368 (1 o NH), 1594(C=O)cm -1 .CFBE potentiator assay, potentiator (pot.) pEC 50 =7.8.

[0143] Example 6-Synthesis of 4-amino-5-(dimethylamino)-2-(3-hydroxyphenyl)-6-phenylpyridazine-3(2H)-one (21):

[0144]

[0145] Synthesis of 6-chloro-5-(dimethylamino)pyridazin-3(2H)-one (16):

[0146] At RT, under an inert atmosphere, 2 (5.0 g, 30.5 mmol), dimethylamine (30.4 mL, 2M THF solution, 61.0 mmol), diisopropylethylamine (26.5 mL, 152.5 mmol) and ethanol (60 mL) were charged into the reaction tube. The reaction tube was capped and stirred at 110 ° C for 16 h. After the starting material was completely consumed (monitored by TLC), the reaction mixture was cooled to RT and concentrated under reduced pressure. The resulting residue was diluted with water (200 mL) and filtered. The solid was washed with ether (50 mL) to give an off-white solid 16 (3.2 g, 60%). 1 HNMR (400 MHz, DMSO-d 6 ): 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 containing 0.05% formic acid).

[0147] Synthesis of 6-chloro-5-(dimethylamino)-4-nitropyridazin-3(2H)-one (17):

[0148] At 0 °C, 16 (1.5 g, 8.64 mmol) was added to CH 2 Cl 2 Concentrated H (25 mL) was added to the stirred solution. 2 SO 4 (9 mL), then fuming nitric acid (2.4 mL) was added dropwise 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 washed with CH 2 Cl 2 The combined organic layers were washed with water (2X 50 mL), anhydrous Na 2 SO 4 Drying and concentration under reduced pressure gave 17 as a yellow solid (460 mg 24%). 1 HNMR (400MHz, CDCl 3 ): δ10.80(br s,1H),3.04(s,6H); LCMS(ESI):m / z 218.9[M+H + ]; 86.2%; RT = 2.31 min (Acquity BEH C18 column, 0.05% formic acid / MeCN containing 0.05% formic acid).

[0149] Synthesis of 6-chloro-5-(dimethylamino)-2-(3-(methoxymethoxy)phenyl)-4-nitropyridazin-3(2H)-one (18):

[0150] At room temperature, 17 (2.0 g, 9.15 mmol) was added to CH 2 Cl 2 To the stirred solution (40 mL) were added (3-(methoxymethoxy)phenyl)boronic acid (3.33 g, 18.3 mmol), Cu(OAc) 2 (3.33g, 18.3mmol), triethylamine (2.6mL, 18.3mmol), pyridine (1.5mL, 1.83mmol), and stirred for 16h. After the reaction was completed (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 (100mL) and extracted with EtOAc (50mL). The EtOAc layer was washed with anhydrous Na 2 SO 4 The obtained crude product was purified by silica gel column chromatography using a 10%-15% EtOAc gradient in petroleum ether to afford 18 (800 mg, 25%) as a yellow solid. 1 HNMR (400 MHz, DMSO-d 6 ): 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 containing 0.05% formic acid).

[0151] Synthesis of 4-amino-6-chloro-5-(dimethylamino)-2-(3-(methoxymethoxy)phenyl)pyridazin-3(2H)-one (19):

[0152] To a stirred solution of 18 (800 mg, 2.25 mmol) in ethanol (16 mL) were added Fe powder (380 mg, 6.75 mmol), NH 4 Cl (720 mg, 13.5 mmol) and H 2 O (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 small piece of celite. The filtrate was concentrated under reduced pressure. The resulting residue was diluted with water (250 mL) and extracted with EtOAc (2×30 mL). The combined organic layers were washed with anhydrous Na 2 SO 4 Drying and concentration under reduced pressure gave 19 as a brown solid (400 mg, 54%). 1 HNMR (400 MHz, DMSO-d 6): 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 / z 325.1[M+H + ]; 93.6%; RT = 2.31 min (Acquity BEH C18 column, 0.05% formic acid / MeCN containing 0.05% formic acid).

[0153] Synthesis of 4-amino-5-(dimethylamino)-2-(3-(methoxymethoxy)phenyl)-6-nitropyridazin-3(2H)-one (20):

[0154] The reaction tube was charged with 19 (400 mg, 1.23 mmol), 1,4-dioxane (8 mL), Na 2 CO 3 (260 mg, 2.46 mmol), phenylboronic acid (300 mg, 2.46 mol), Pd(PPh 3 ) 4 (142 mg, 0.123 mmol) and H 2 O (4mL) and purged with argon for 10min. The tube was capped and stirred at 110°C for 16h. After the reaction was completed (monitored by TLC), the reaction mixture was cooled to room temperature and filtered through a celite pad. The filtrate was concentrated under reduced pressure. The resulting residue was diluted with water (50mL) and extracted with EtOAc (2×20mL). The combined organic layers were washed with anhydrous Na 2 SO 4 The crude product was purified by silica gel column chromatography using 50% EtOAc in petroleum ether to afford 300 mg of 20 (purity about 45%; brown solid) as a mixture with triphenylphosphineoxide (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 containing 0.05% formic acid).

[0155] Synthesis of 4-amino-5-(dimethylamino)-2-(3-hydroxyphenyl)-6-phenylpyridazin-3(2H)-one (21):

[0156] To a stirred solution of 20 (300 mg, purity about 45%) in methanol (3 mL) was added a 3M HCl solution in methanol (3 mL) at 0°C under an inert atmosphere. The reaction was stirred at room temperature for 2 h. After the reaction was completed (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 washed with anhydrous Na 2 SO 4 The crude product was purified by preparative HPLC (Kinetex phenylhexyl C18 column; 5 mM ammonium bicarbonate in MeCN) to give 21 as a brown solid (85 mg, 11% from 5 steps). 1 HNMR (400 MHz, DMSO-d 6 ):δ9.61(br s,1H,D 2 Oexchangeable),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;D 2 O exchangeable),2.46(s,6H); LCMS(ESI):m / z323.1[M+H + ]; 99.8%; RT = 2.05 min (Acquity BEH C188 column, 0.05% formic acid / MeCN containing 0.05% formic acid). CFBE potentiator assay, potentiator (pot.) pEC 50 =7.7.

[0157] Compound 11 - In vitro model of chronic obstructive pulmonary disease

[0158]

[0159] CFTR chloride channel is located on the luminal membrane of the airway surface epithelium, and is used to enhance the fluidity of airway surface liquid, thereby preventing mucus obstruction. Tobacco smoke reduces the functional expression of epithelial surface CFTR, aggravates mucus accumulation 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 causes CFTR deficiency, also referred to as "acquired cystic fibrosis", and small molecules that enhance the activity of remaining CFTR function will reverse the initial stage of COPD pathogenesis. Ivacaftor is an approved CFTR potentiator that has shown effects in reversing mucus accumulation in certain tissue culture models, and a newer CFTR potentiator called icenticaftor has shown early promise in restoring lung function in clinical trials in patients with 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).

[0160] Compound 11 was tested in an in vitro COPD model. In this model, primary differentiated human bronchial epithelial cultures were exposed to cigarette smoke extract (4, 5). Exposure to cigarette smoke extract (CSE) resulted in mucus accumulation and slowed mucociliary motility ( Figure 1 (A) and (B)). We found that when compound 11 was added to differentiated airway epithelial cultures, it prevented mucus accumulation induced by cigarette smoke extract ( Figure 1 (C)). Changes in mucociliary motility were determined as described in our previous publication (7) by tracking the velocity of fluorescent microspheres added to the surface of airway cultures. The relative rescue efficacy of compound 11 was superior to that of a competitor, an FDA-approved CFTR potentiator marketed as ivacaftor (VX-770).

[0161] Figure 1The compound is shown to enhance mucociliary motility on the surface of primary bronchial cultures previously exposed to cigarette smoke extract. The images on the left ((A) and (B)) show the negative effect of cigarette smoke extract (CSE) on fluorescent microsphere tracking, reflecting the development of viscous mucus on the surface fluid on primary bronchial cultures. In the right panel (C), it shows the quantification of microsphere velocity on the surface of primary bronchial airway cultures under control conditions (cultures from 4 donors with vehicle only, DMSO, open circles). Ivacaftor (VX-770, circles) or compound 11 (circles) were added at 24 hours of vehicle incubation. Under control conditions, neither VX-770 nor compound 11 had any effect on microsphere velocity. Treatment with CSE (2% / volume, solid black symbols) resulted in a significant decrease in microsphere velocity in cultures from 5 donors. The microsphere velocity on bronchial cultures exposed to CSE treated with compound 11 potentiator (solid symbols) was rescued to control values. Each symbol represents the average microsphere velocity from 5 videos taken of a randomly selected area on each airway culture.

[0162] A novel potentiator of Wt-CFTR channel activity.

[0163] Materials and methods

[0164] Cell-attached patch clamp studies of the enhancement of CFTR channels by novel potentiator compounds.

[0165] In patch clamp studies of single-channel activity, HEK-293 cells stably expressing wild-type human CFTR were used. Cells were generously donated by D.Rotin (SickKids Hospital, Toronto). CFTR Cl was recorded in cell-attached patches using an Axopatch 200A patch clamp amplifier and pCLAMP software (both from Molecular Devices, Sunnyvale, CA). - Channel. Pipette and reservoir (extracellular) solution contained 140 mM N-methyl-d-glucamine, 140 mM aspartic acid, 5 mM CaCl 2 、2mmMgSO 4 and 10 mM TES, with Tris ([Cl - ]: 10 mM and adjust the pH to 7.3. Keep the tank at room temperature.

[0166] To activate Wt-CFTR channels, 10 μM forskolin was added to the tank. CFTR-mediated channel opening was inhibited by applying CFTRInh-172 to the tank. CFTR Cl was enhanced by adding SK-POT (Compound 11) (10 μM) to the tank. -channels. To determine the number of channels, we used the maximum number of simultaneous channel openings observed during the experiment. Single-channel recordings were filtered and data were digitized as described previously. To measure single-channel current amplitudes, a Gaussian distribution was fit to the current amplitude histogram. To measure P o , lists of open and close times were created using the half-amplitude crossing criterion for event detection, and dwell time histograms were constructed and fitted. For illustration purposes, single-channel recordings were filtered at 500 Hz.

[0167] Calu-3 CSE Experiment

[0168] 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. The cells were then subjected to chronic treatment with toxins and potentiators. Toxin treatment consisted of cigarette smoke extract (CSE) prepared in 100% DMSO (University of Alabama at Birmingham) dissolved in Calu-3 culture medium at a concentration of 2% (v / v), with an equivalent concentration of DMSO alone used as a toxin control. Long-term drug treatment was co-applied with toxins, and drugs included VX-770 or compound 11 (both prepared in 100% DMSO) dissolved in Calu-3 culture medium at a final concentration of 1 μM. VX-770 (Vertex) and compound 11 solutions were prepared from 1 mM stock solutions. The control for drug treatment was an equal volume of vehicle DMSO.Two toxin treatments and three drug treatments were combined for a total of 6 chronic treatments of 4 wells per condition, for a total of 24 wells that received chronic treatments.

[0169] After long-term treatment application, cells were cultured for an additional 24 hours and then incubated with FLiPR assay buffer. FLiPR assay buffer consists of blue FLiPR dye (Molecular Devices) dissolved in chloride-free buffer (150 mM NMDG, 150 mM gluconolactone, 3 mM potassium gluconate, 300 mOsm, and pH 7.38) at a concentration of 0.5 mg / mL. The buffer was incubated with cells at 37 degrees and 5% CO. 2Incubate for 35 minutes at 4 °C. CFTR function was assessed using the SpectraMax i3x multi-function microplate reader by measuring changes in fluorescence activity after acute CFTR channel activation by adding the cAMP agonist forskolin dissolved in a chloride-free buffer at a final concentration of 1 μM. Fluorescence readings (excitation 530 nm; emission 560 nm) were read for each well within 5 minutes (baseline) or 10 minutes (activation) at 30 second intervals. CFTR activity was then terminated by adding CFTR-inhibitor 172 dissolved in a chloride-free buffer at a concentration of 10 μM to further verify the specificity of the response to CFTR activity. Fluorescence changes were recorded every 30 seconds for 10 minutes. In the analysis, all fluorescence values ​​for each well were normalized to the final reading before the addition of forskolin and expressed as a percentage of that reading.

[0170] Microsphere tracer test of mucociliary motility

[0171] Mature bronchial epithelial cultures seeded on 24-well transwell inserts from nonsmoker donors of varying ages were received from the University of Iowa and cultured at the air-liquid interface (ALI) using UltraG medium. A total of 6 inserts per donor were cultured, and all inserts from a single donor were selected to receive long-term drug and toxin treatments. Prior to long-term treatment, cultures were treated with mucomist to reduce the volume and viscosity of the mucus layer prior to imaging. Cultures were first washed twice with 200 μL HBSS (Wisent) applied top to the transwell, each at 37 degrees and 5% CO. 2 The cells were then incubated with 10 μM N-acetylcysteine ​​(source?) dissolved in HBSS (Wisent) for 30 minutes, followed by a final HBSS wash as described above. The toxin treatment consisted of cigarette smoke extract (CSE) dissolved in UltraG medium at a concentration of 2% (v / v) prepared in 100% DMSO (University of Alabama at Birmingham) and DMSO alone at an equivalent concentration as a control. Long-term drug treatments were applied together with toxin treatments, and the drugs included VX-770 (Vertex) or SK-POT, aka compound 11 (both prepared in 100% DMSO) dissolved in UltraG medium at a final concentration of 1 μm. There were a total of 6 long-term treatments, and each of the 6 inserts of each donor received a different long-term treatment. At 37°C and 5% CO2, bronchial epithelial cells were incubated with the long-term treatments applied on the top for 24 hours. After 24 hours, green fluorescent microspheres (brand?) dissolved in HBSS at a concentration of 0.05% (v / v) were applied apically; the cultures were incubated with microspheres for 45 minutes. Toxin and potentiator treatments were introduced into the microsphere solution.

[0172] The inserts were then imaged by epifluorescence microscopy (Zeiss or Olympus) at room temperature. A basic replicate of the assay consisted of a 5 second video captured in the green channel and 40-60 frames per second; the Z position was adjusted before each video to ensure capture of the microspheres in the airway surface liquid layer just above the cilia. Five videos were captured per insert / condition, representing the center and 4 corners of the insert. Five videos were captured per insert / condition, 2 sets: one at baseline and one 20 minutes after CFTR stimulation with forskolin at a concentration of 10 μM. After the addition of forskolin, the inserts were incubated at room temperature.

[0173] Videos were captured using Volocity software and exported as one TIF file per frame. Frames were then recompiled into complete videos using Arivis4D software. Microspheres were highlighted using an algorithm previously described in Wu et al. 2017 by adjusting two parameters (fluorescence intensity and diameter). Parameters were adjusted until the software identified only single microspheres like this one; success in identifying single microspheres was manually verified at the beginning, middle, and end of each video. After highlighting the microspheres, the algorithm generated a displacement value for each microsphere, as well as a frame value for the algorithm tracking the microsphere. Microspheres that were tracked for less than 5 frames were excluded from the analysis. The velocity (in μm / s) was then calculated (for each microsphere analyzed) by dividing the resulting velocity by the number of frames tracked and then multiplying by the number of frames per second. The velocity values ​​were compiled and the average velocity was calculated for each video.

[0174] Calu-3 Western Blot

[0175] Calu3 was lysed in radioimmunoprecipitation (RIPA) buffer (50 mM Tris–HCl, 1 mM EDTA, 150 mM NaCl, pH 7.4) with 0.1% SDS, 0.1% Triton X-100, and a 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 a single well by scraping with a micropipette tip and then centrifuged at 4 degrees Celsius and 15,000 rpm for 10 minutes. The lysate was then analyzed by SDS-PAGE (Invitrogen) using 6% Tris-glycine gel; transferred to a nitrocellulose membrane (Bio-Rad) at 100 mV for 1 hour. After blocking with 5% (w / v) skim milk dissolved in PBS-Tween, CFTR was probed overnight at 4 degrees Celsius with the primary antibody CFTR-NBD2-specific mouse mAb 596 dissolved in blocking buffer at a dilution of 1:2000. The loading control calnexin was probed with rat anti-calnexin dissolved in blocking buffer at a dilution of 1:10,000. Horseradish peroxidase (HRP)-labeled anti-mouse and anti-rat antibodies, both diluted 1:5,000 in blocking buffer, were used as secondary antibodies and incubated at room temperature for 1 hour. The blots were developed using ECL reagent (Bio-Rad) and imaged using Li-Cor Odyssey Fc (Li-Cor Biosciences) with an exposure of 2 minutes. Relative CFTR levels were quantified using ImageStudioLite (LI-COR Biosciences).

[0176] Results and discussion

[0177] Figure 2 The dose response of SK-POT (Compound 11) activity relative to VX-770, a highly potentiator compound used to treat cystic fibrosis, is shown. In these studies, F508del-CFTR was endogenously expressed in CFBE41o- cells, and its transport defect was corrected by incubation at low temperature (27 degrees Celsius). The fold increase in cyclic AMP-dependent F508del-CFTR-mediated chloride channel activity induced by potentiator treatment was determined by using a previously described fluorescence-based membrane potential difference assay. Figure 1 As shown, compound 11 was found to have a potency of 5 nM relative to 32 nM as determined for VX-770.

[0178] The activity of this new class of small molecules in enhancing Wt-CFTR was analyzed in detail in patch clamp studies in HEK-293 cells stably expressing the gene. Figure 3In (i), tracks obtained in cell attachment mode are shown, where cells were sequentially exposed to forskolin, then a structural analog of compound 11, followed by the CFTR channel inhibitor CFTRInh172 (10 μM). While only one activated channel was evident in the presence of forskolin, a total of eight channels were activated in the presence of the potentiator compounds. The number of activated channels was significantly reduced upon addition of CFTRinh-172. Figure 3 The bar graph in (ii) shows CFTR channel activity normalized to the total number of channels (n=8). As expected for a CFTR potentiator, the channel open probability increased significantly after the addition of the novel compound.

[0179] SK-POT (Compound 11) was then tested for its ability to enhance Wt-CFTR-mediated transepithelial chloride conduction in primary human bronchial epithelial cultures. Figure 4 As shown, in Ussing chamber studies, SK-POT (1 μM) enhanced forskolin-activated, Wt-CFTR-mediated short-circuit currents in primary bronchial epithelial cultures. Representative traces are shown on the left, and the scatter plots on the right show that these results are reproducible and statistically significant. Interestingly, the enhancement induced by 1 μM SK-POT is similar to that achieved by 10 μM ivacaftor (VX-770).

[0180] We then determined whether SK-POT compounds could ameliorate the adverse effects of cigarette smoke (CSE) on Wt-CFTR channel function. The cell line Calu-3 has been widely used for studies of Wt-CFTR regulation, as this airway epithelial cell line expresses the channel endogenously following its differentiation. CFTR channel activity was measured using FLIPR (a fluorescence-based microplate reader assay). First, we recapitulated previously published studies on the deleterious effects of cigarette smoke extracts on CFTR channel activity ( Figure 5 i, upper and lower panels). In paired studies (3 biological replicates, 3-4 technical replicates), VX-770 and SK-POT (both at 1 μM) were compared for their ability to enhance CFTR activity after CSE exposure. Figure 5 As shown in the bar graph, only (SK-POT) treatment resulted in a significant increase in forskolin-activated CFTR channel activity.

[0181] Mucus accumulation, a major defect in COPD, has been simulated in vitro as reduced motility of fluorescent nanoparticles seeded in partially mucus-containing fluid on well-differentiated tracheal airway 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).

[0182] Fluorescent nanoparticles were seeded onto the airway surface and the movement of the microspheres was tracked. Figure 6 The upper panels (a.i and ii) show that, as expected, microsphere displacement was reduced after exposure to CSE. We then evaluated the effect of pretreatment with the potentiators VX-770 (1 μM) or SK-POT (1 μM) on CSE-altered mucociliary motility (Fig. a.iii-v) and Figure 6 Microsphere velocity was assessed in (b) following pretreatment with specific potentiators. In 5 biological replicates and 4-5 technical replicates (or videos), SK-POT pretreatment produced a significant increase in microsphere velocity in CSE-treated primary bronchial cultures. Interestingly, in these studies, no significant increase in forskolin-induced responses was observed in CSE-treated cultures following addition of VX-770.

[0183] discuss

[0184] The current study demonstrates that potentiators of CFTR channel activity have the potential to ameliorate cigarette smoke extract (CSE)-induced mucus accumulation on human bronchial epithelial cells. Furthermore, the results suggest that potentiators that promote better CFTR channel activity in CSE-exposed epithelial cells, such as the potentiators described herein, are also more effective in preventing CSE-associated mucus accumulation. In conclusion, these studies support future in vivo evaluation of SK-POT as a therapeutic intervention for COPD-associated bronchitis.

[0185] The study found that acute treatment with 1 micromolar SK-POT compounds was superior to VX-770 in enhancing CFTR channel activity in epithelial cells exposed to cigarette smoke (EC90s were similar for both). The superior effect of SK-POT compounds may reflect a different molecular mechanism of action relative to VX-770. In the current study, although we did observe a significant decrease in CFTR channel function in Calu-3 cells after CSE pretreatment, no significant decrease in the steady-state abundance of mature C-bands was observed.

[0186] The positive effect of SK-POT compounds in enhancing CFTR channel activity in epithelial cells exposed to CSE translated into efficacy in rescuing impaired mucociliary motility in primary bronchial cultures exposed to CSE ( Figure 6 This observation is consistent with the proposal that increasing CFTR function has the potential to improve mucus aggregation and obstruction in COPD. Interestingly, there was considerable variability in microsphere velocity among donor-specific cultures ( Figure 6 ). This variability may reflect donor-specific differences in the transport properties of primary bronchial cultures as well as some heterogeneity in the relative proportion of ciliated cells in each culture.

[0187] In summary, these data support that CFTR channel activity will prevent mucus aggregation and mucus accumulation in tissue culture models of COPD. Although the efficacy of SK-POT in preventing mucus accumulation on the surface of human bronchial epithelial cultures varied among donor-specific cultures, the mean response was comparable to that of ivacaftor.

[0188] Figure 7 The SK-POT compound was shown to be effective in enhancing CFTR channel function in ferret bronchial tissue as well—the animal model of choice for preclinical studies of interventions for airway diseases (N. Kaza, VY Lin et al. Eur Respir J. 2022 Jul 13; 60(1): 2101581).

[0189] Table 1: pEC values ​​of compounds of the present disclosure 50 value

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

Claims

1. A compound of formula (I) in R 1 and R 2 independently or simultaneously H or (C 1 -C 6 )-alkyl; R 3 and R 4 independently or simultaneously H or (C 1 -C 6 )-alkyl, or R 3 and R 4 Together with the nitrogen atoms to which they are attached, they form (C 5 -C 6 )-heteroaryl or (C 4 -C 6 )-heterocycloalkyl, each of which is optionally substituted by halogen, OH, (C 1 -C 6 )-alkyl or halo-(C 1 -C 6 )-alkyl substituted; Ring B is (C 6 -C 10 )-aryl or (C 5 -C 10 )-heteroaryl, each of which is optionally substituted by halogen, OH, (C 1 -C 6 )-alkyl, halo-(C 1 -C 6 )-alkyl, (C 3 -C 6 )-cycloalkyl or halo-(C 3 -C 6 )-One or more substitutions in cycloalkyl; W is (C 1 -C 6 )-alkyl or -(C 0 -C 6 )-alkylene-(C 6 -C 10 )-aryl, each of which is optionally substituted by halogen, OH, CN, (C 1 -C 6 )-alkyl, halo-(C 1 -C 6 )-alkyl, (C 1 -C 6 )-alkoxy, (C 3 -C 6 )-cycloalkyl or halo-(C 3 -C 6 )-One or more substitutions in cycloalkyl; or any pharmaceutically acceptable salt, stereoisomer or solvate thereof.

2. The compound of formula (I) according to claim 1, wherein R 1 and R 2 independently or simultaneously H or (C 1 -C 3 )-alkyl.

3. The compound of formula (I) according to claim 1 or 2, wherein R 1 and R 2 It's H.

4. A compound of formula (I) according to any one of claims 1 to 3, wherein R 3 and R4 are independently or simultaneously H or (C 1 -C 3 )-alkyl, or R 3 and R 4 Together with the nitrogen atoms to which they are attached, they form (C 5 )-heteroaryl or (C 4 -C 6 )-heterocycloalkyl, each of which is optionally substituted by halogen, OH, (C 1 -C 3 )-alkyl or halo-(C 1 -C 3 )-alkyl substitution.

5. The compound of formula (I) according to claim 4, wherein R 3 and R 4 independently or simultaneously H or CH 3 .

6. The compound of formula (I) according to claim 4, wherein R 3 and R 4 Together with the nitrogen atom to which they are attached, they form an optionally substituted piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl or pyrazolyl.

7. A compound of formula (I) according to any one of claims 1 to 6, wherein ring B is (C 6 )-aryl or (C 5 -C 6 )-heteroaryl, each of which is optionally substituted by halogen, OH, (C 1 -C 6 )-alkyl, halo-(C 1 -C 6 )-alkyl, (C 3 -C 6 )-cycloalkyl or halo-(C 3 -C 6 )-cycloalkyl.

8. The compound of formula (I) according to claim 7, wherein ring B is optionally substituted with halogen, (C 1 -C 6 )-alkyl or halo-(C 1 -C 6 )-alkyl, one or more phenyl or pyridyl substituted.

9. The compound of formula (I) according to claim 8, wherein ring B is phenyl or pyridyl optionally substituted with one or more of fluoro or trifluoromethyl.

10. The compound of formula (I) according to claim 9, wherein ring B has the following structure:

11. A compound of formula (I) according to any one of claims 1 to 9, wherein W is (C 1 -C 4 )-alkyl or -(C 0 -C 4 )-alkylene-(C 6 -C 10 )-aryl, each of which is optionally substituted by halogen, OH, CN, (C 1 -C 4 )-alkyl, halo-(C 1 -C 4 )-alkyl, (C 1 -C 4 )-alkoxy, (C 3 -C 6 )-cycloalkyl or halo-(C 3 -C 6 )-cycloalkyl substitution.

12. The compound of formula (I) according to claim 10, wherein W is (C 1 -C 4 )-alkyl or -(C 0 -C 1 )-alkylene-phenyl, each of which is optionally substituted by halogen, OH, CN, (C 1 -C 4 )-alkyl, halo-(C 1 -C 4 )-alkyl or (C 1 -C 4 )-alkoxy with one or more substitutions.

13. The compound of formula (I) according to claim 11, wherein W has the following structure:

14. A compound of formula (I) according to any one of claims 1 to 12, wherein the compound of formula (I) is 15. The compound of formula (I) according to claim 14, wherein the compound is 16. The compound of formula (I) according to claim 15, wherein the compound is 17. A method for treating a disease associated with misfolded proteins or misshaped proteins, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) according to any one of claims 1 to 16.

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

19. The method of claim 18, wherein the disease is cystic fibrosis or COPD.

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

21. Use of a therapeutically effective amount of a compound of formula (I) according to any one of claims 1 to 16 for treating diseases associated with misfolded proteins or misshaped proteins.

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

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