Use of RASP inhibitor in preparation of drug for treating or preventing chronic cough, asthma, and alcoholic liver disease

Abstract

Provided is use of a RASP inhibitor in the treatment of chronic cough, asthma, and alcoholic liver disease. The RASP inhibitor comprises a compound represented by formula (II) or a pharmaceutically acceptable salt or crystal form thereof. The RASP inhibitor has good efficacy in animal model tests of chronic cough, asthma, alcoholic liver disease, and other diseases.

Description

Application of RASP inhibitors in the preparation of drugs for treating or preventing chronic cough, asthma and alcoholic liver disease Technical Field

[0001] The present invention relates to the field of pharmaceuticals, and in particular to the use of a RASP inhibitor in the preparation of a medicament for treating or preventing chronic cough, asthma and alcoholic liver disease. Background Art

[0002] Chronic cough is categorized as unexplained chronic cough and refractory chronic cough. Cough is typically the sole or primary symptom, with a duration of >8 weeks and no significant chest abnormalities on chest X-ray (for children, duration >4 weeks). Cough variant asthma, upper airway cough syndrome, eosinophilic bronchitis, allergic cough, and gastroesophageal reflux cough account for 70% to 95% of the causes of chronic cough. A key pathophysiological characteristic of chronic cough is cough hypersensitivity, which is associated with dysregulation of channels and receptors in peripheral and central nervous systems. Various stimuli in peripheral nerves activate cough receptors, transmitting signals along nerve fibers. Inflammatory mediators can sensitize cough receptors, increasing their excitability, thereby lowering the cough threshold and increasing peripheral impulse input to the cough center, inducing cough.

[0003] The existing treatments for chronic cough include drug therapy and non-drug therapy. Drug therapy includes central antitussive drugs, peripheral antitussive drugs and traditional Chinese medicine. Non-drug treatments include speech pathology therapy and cough suppressant physiotherapy, which have shown certain effects in improving patients' cough-related quality of life, reducing cough sensitivity and cough frequency. Targeting different targets in the cough reflex process, some receptor antagonists have shown certain potential in clinical studies for the treatment of chronic cough. Potential regulatory targets of peripheral nerves include: P2X3 receptors and transient receptor potential (TRP) channels; potential regulatory targets of central nerves include: voltage-gated sodium channels (Nav), neurokinin-1 receptor (NK-1R), α7 acetylcholine receptor (α7AchR) and γ-aminobutyric acid receptor (GABAR), etc.

[0004] Asthma, commonly known as asthma, is a chronic, heterogeneous airway disease with a high incidence worldwide. Its clinical manifestations primarily include recurrent episodes of wheezing, shortness of breath, chest tightness, or cough. Airway pathology manifests as airway hyperresponsiveness, variable airflow limitation, and airway remodeling. The pathogenesis of asthma is complex, driven by both genetic and environmental factors and involving multiple systems, including the nervous system, endocrine system, and immune system. Asthma is broadly categorized into allergic and non-allergic asthma. Allergic asthma is a subtype of allergen-induced asthma characterized by eosinophilic airway inflammation, bronchial hyperresponsiveness, and elevated immunoglobulin E (IgE) levels. Allergic asthma is characterized by the synthesis of specific immunoglobulin antibodies following initial exposure to the allergen. Subsequent exposure to the allergen leads to crosslinking of high-affinity IgA receptors on tissue mast cells, resulting in degranulation of mast cells and basophils, leading to bronchoconstriction, and subsequent eosinophil recruitment and inflammatory responses. Asthma episodes are often accompanied by significant behavioral changes, such as nasal scratching and coughing and wheezing. Existing therapeutic drugs include maintenance anti-inflammatory drugs, including inhaled corticosteroids (ICS), systemic hormones, leukotriene modifiers, long-acting inhale bete2-agonist (LABA), sustained-release theophylline, supelalast tolyl, sodium cromoglycate, etc. In addition, there are drugs that can quickly relieve bronchospasm and thus relieve asthma symptoms, including fast-acting inhaled and short-acting oral beta2 receptor agonists, inhaled anticholinergics, short-acting theophylline and systemic hormones, etc. There are additional therapeutic drugs for severe asthma: mainly biological targeted drugs, such as anti-IgE monoclonal antibodies, anti-IL-5 monoclonal antibodies, anti-IL-5 receptor monoclonal antibodies and anti-IL-4 receptor monoclonal antibodies, etc., and others include macrolide drugs, etc. However, inhaled and oral steroids have numerous adverse reactions and are not suitable for long-term use. Leukotriene modifiers can also cause psychiatric symptoms. Theophylline-based medications have significant individual variability and are only used as supplemental maintenance therapy. Other medications also have limitations in efficacy and safety. Therefore, there is a significant unmet clinical need for asthma treatments.

[0005] Alcoholic liver disease (ALD) is a chronic liver disease caused by long-term, heavy drinking. It typically presents initially with hepatocyte fatty degeneration, which can progress to alcoholic hepatitis and further to alcoholic liver fibrosis and cirrhosis. The liver is one of the most complex organs in the human body, performing functions such as detoxification, digestion, and regulating glucose and lipid levels. Under normal circumstances, liver cells possess a robust capacity for repair and regeneration. However, chronic alcohol abuse can lead to persistent damage to liver cells, reducing their regenerative capacity and potentially causing severe liver impairment, ultimately leading to ALD. Alcoholic hepatitis is a severe inflammatory response syndrome associated with alcohol-related liver disease. It often occurs in patients with persistent, heavy drinking. Its main clinical features are nausea, vomiting, jaundice, and hepatomegaly and tenderness, which can be complicated by liver failure and upper gastrointestinal bleeding. Severe alcohol abuse can induce hepatocellular necrosis and even lead to liver failure. The pathogenesis of AHD is complex, involving both direct and indirect effects of alcohol and its metabolites on the liver. Oxidative stress is a typical mechanism of AHD. Oxidative stress can trigger numerous cascading injury responses and synergize with other factors to accelerate liver damage. Furthermore, alcohol-induced Kupffer cell activation, leading to the massive release of inflammatory factors and inhibition of autophagy, is another key factor in the development of alcoholic fatty liver disease. Currently, there is no clear, targeted treatment for alcoholic fatty liver disease and alcoholic hepatitis; alcohol abstinence, psychological counseling, and infection prevention remain the cornerstones of treatment. When patients have a good appetite and normal serum creatinine levels, corticosteroids remain the mainstay of treatment, while early liver transplantation is the only option for patients unresponsive to steroids. While there is currently no cure for alcoholic liver fibrosis and alcoholic cirrhosis, controlling and reversing the progression of alcoholic fatty liver disease and alcoholic hepatitis can delay the development of severe alcoholic fibrosis and cirrhosis. Therefore, the search for safe and effective antioxidant and anti-inflammatory substances is of great clinical significance in addressing the development of alcoholic liver disease.

[0006] RASP is a pro-cytokine mediator of inflammation. It enhances cytokine release and activates inflammasomes by binding to thiol and amine residues on proteins. It can also bind to the class A scavenger receptor (SR-A1 / CD204) to exert a pro-inflammatory effect. The compound of the present invention, as a new RASP inhibitor, can complex with aldehydes in the body, thereby reducing aldehyde toxicity and inflammation. It has good water solubility, good permeability, and excellent pharmacokinetic properties. Efficacy studies of the compound in citric acid-induced chronic cough in guinea pigs, in a Gao-binge diet-induced alcoholic hepatitis model in mice, and in an OVA-induced asthma model in mice have demonstrated that RASP inhibitors are effective in treating chronic cough, asthma, and alcoholic liver disease.

[0007] WO2020125659A1 discloses a compound for retinal diseases, which provides an aldehyde binder. WO2022063325 discloses a crystalline form of a pyridinephenyl compound and a preparation method thereof, as well as the use of the crystalline form in the preparation of treatments for related diseases.

[0008] Currently, there is no application of RASP inhibitors or pharmaceutically acceptable salts or crystal forms thereof for the preparation of drugs for treating diseases such as chronic cough, asthma or alcoholic liver disease. Summary of the Invention

[0009] The present invention provides a use of a RASP inhibitor in the preparation of a medicament for treating chronic cough, asthma, alcoholic liver disease, and the like, wherein the RASP inhibitor comprises a compound represented by the following formula (II) or a pharmaceutically acceptable salt or crystalline form thereof:

[0010] in,

[0011] is selected from single bonds and double bonds;

[0012] T1, T2, T3 and T4 are each independently selected from N, C or CR1;

[0013] T5 is selected from C, CR5 or C=O;

[0014] T6 is selected from C, CR6 or N;

[0015] T7 is selected from N or CR7;

[0016] When T5 is selected from C=O and T6 is selected from N, is selected from single bonds;

[0017] L is selected from a single bond, -O-, -S-, -NR2- or -(CR3R4) n -;

[0018] R1 is selected from H, F, Cl, Br, I, OH or NH2;

[0019] R2 is selected from H, or optionally 1, 2 or 3 R a Substituted C 1-3 alkyl;

[0020] R3 or R4 are independently selected from H, F, Cl, Br, I, OH, NH2, CN, or optionally 1, 2 or 3 R b Substituted C 1-3 alkyl;

[0021] R 5、 R6 or R7 are independently selected from H, F, Cl, Br or I;

[0022] n is selected from 1, 2 or 3;

[0023] R a or R b Each is independently selected from H, F, Cl, Br, I, OH, NH2, CN or CH3.

[0024] In the application of the present invention, as one embodiment, in the compound of formula (II), R2 is selected from H, CH3 or CH2CH3, and the CH3 or CH2CH3 is optionally replaced by 1, 2 or 3 R a Substitution, other variables are as defined in the present invention.

[0025] In the application of the present invention, as one embodiment, in the compound of formula (II), R2 is selected from H, CH3 or CH2CH3, and other variables are as defined in the present invention.

[0026] In the application of the present invention, as one embodiment, R3 or R4 in the compound of formula (II) are independently selected from H, F, Cl, Br, I, OH, NH2, CN, CH3 or CH2CH3, and the CH3 or CH2CH3 is optionally replaced by 1, 2 or 3 R b Substitution, other variables are as defined in the present invention.

[0027] In the application of the present invention, as one embodiment, R3 or R4 in the compound of formula (II) is independently selected from H, F, Cl, Br, I, OH, NH2, CN, CH3 or CH2CH3, and other variables are as defined in the present invention.

[0028] In the application of the present invention, as one of the embodiments, L in the compound of formula (II) is selected from a single bond, -O-, -S-, -NH-, -(CH2)2- or -CH2-, and other variables are as defined in the present invention.

[0029] Some other solutions of the present invention are obtained by arbitrarily combining the above variables.

[0030] In the application of the present invention, as one of the embodiments, the alcoholic liver disease includes alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis or cirrhosis.

[0031] In the application of the present invention, as one of the embodiments, the application is the use of the compound of formula (II) or its pharmaceutically acceptable salt or crystal form in the preparation of a preparation for treating alcoholic fatty liver.

[0032] In the application of the present invention, as one of the embodiments, the application is the use of the compound of formula (II) or its pharmaceutically acceptable salt or crystal form in the preparation of a drug for treating alcoholic hepatitis.

[0033] In the application of the present invention, as one of the embodiments, the application is the use of the compound of formula (II) or its pharmaceutically acceptable salt or crystal form in the preparation of a preparation for treating alcoholic liver fibrosis.

[0034] In the application of the present invention, as one of the embodiments, the application is the use of the compound of formula (II) or its pharmaceutically acceptable salt or crystal form in the preparation of a preparation for treating liver cirrhosis.

[0035] In the application of the present invention, as one of the embodiments, the compound or its pharmaceutically acceptable salt or crystal form is selected from

[0036] in,

[0037] T3 and T4 are each independently selected from N or CR1;

[0038] R1 and L are as defined herein.

[0039] In the application of the present invention, as one of the embodiments, the compound or its pharmaceutically acceptable salt or crystal form is selected from

[0040] in,

[0041] R1 and L are as defined herein.

[0042] The present invention also provides a compound represented by the following formula or a pharmaceutically acceptable salt or crystal form thereof, which is selected from:

[0043] In the application of the present invention, as one of the embodiments, there is also provided a compound of formula (III) or a pharmaceutically acceptable salt or crystal form thereof,

[0044] In some embodiments of the present invention, in the above-mentioned application, the RASP inhibitor-related drug is a drug used for chronic cough, asthma and alcoholic liver disease.

[0045] Definition and Description

[0046] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered as undefined or unclear in the absence of a specific definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient. The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment without excessive toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0047] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, prepared by reacting the compounds of the present invention with relatively nontoxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, bisulfate, hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; and salts of amino acids (such as arginine) and organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and can be converted into either base or acid addition salts.

[0048] Pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture of the two.

[0049] In addition to the form of salts, the compounds provided by the present invention also exist in prodrug form. The prodrugs of the compounds described herein easily undergo chemical changes under physiological conditions to be converted into the compounds of the present invention. In addition, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an in vivo environment.

[0050] Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrates. In general, the solvated forms are equivalent to the unsolvated forms and are encompassed within the scope of the present invention.

[0051] The terms "optional" or "optionally" mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0052] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may be substituted or unsubstituted, and unless otherwise specified, the type and number of substituents can be any on the basis of chemical achievable.

[0053] When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may be optionally substituted with up to two Rs, with each occurrence of R being an independent choice. Furthermore, combinations of substituents and / or their variants are permissible only if such combinations result in stable compounds.

[0054] When the number of a linking group is 0, such as -(CRR)0-, it means that the linking group is a single bond.

[0055] When one of the variables is selected from a single bond, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a single bond, it means that the structure is actually AZ.

[0056] When a substituent is vacant, it means that the substituent does not exist. For example, when X in AX is vacant, it means that the structure is actually A. When the listed substituents do not specify which atom it is connected to the substituted group through, the substituent can be bonded through any atom of the substituent. For example, a pyridyl substituent can be connected to the substituted group through any carbon atom on the pyridine ring. When the listed linking group does not specify its connection direction, its connection direction is arbitrary. For example, The connecting group L is -MW-, in which case -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form You can also connect ring A and ring B in the opposite direction of reading from left to right to form Combinations of linkers, substituents, and / or variations thereof are permissible only if such combinations result in stable compounds.

[0057] Unless otherwise specified, the term “C 1-6 "Alkyl" is used to represent a straight or branched chain saturated hydrocarbon group consisting of 1 to 6 carbon atoms. 1-6 Alkyl groups include C 1-5 、C 1-4 、C 1-3 、C1-2 、C 2-6 、C 2-4 , C6 and C5 alkyl, etc.; which can be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). 1-6 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl, and the like.

[0058] Unless otherwise specified, the term “C 1-3 "Alkyl" is used to represent a straight or branched chain saturated hydrocarbon group consisting of 1 to 3 carbon atoms. 1-3 Alkyl groups include C 1-2 and C 2-3 Alkyl, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). 1-3 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

[0059] Unless otherwise specified, the term “C 1-3 "Alkoxy" refers to those alkyl groups containing 1 to 3 carbon atoms which are attached to the rest of the molecule via an oxygen atom. The C 1-3 Alkoxy groups include C 1-2 、C 2-3 , C3 and C2 alkoxy, etc. 1-3 Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.

[0060] Unless otherwise specified, C n-n+m or C n -C n+m Any specific case including n to n+m carbons, such as C 1-12 Including C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 、C 11 , and C 12 , also includes any range from n to n+m, such as C 1-12 Including C 1-3 、C 1-6 、C 1-9 、C 3-6 、C 3-9 、C 3-12 、C 6-9 、C 6-12 , and C 9-12Similarly, n-membered to n+m-membered means that the number of atoms in the ring is n to n+m, for example, a 3-12-membered ring includes a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-membered ring, a 9-membered ring, a 10-membered ring, an 11-membered ring, and a 12-membered ring, and also includes any range from n to n+m, for example, a 3-12-membered ring includes a 3-6-membered ring, a 3-9-membered ring, a 5-6-membered ring, a 5-7-membered ring, a 6-7-membered ring, a 6-8-membered ring, and a 6-10-membered ring, etc.

[0061] The term "leaving group" refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (e.g., an affine substitution reaction). For example, representative leaving groups include trifluoromethanesulfonate; chlorine, bromine, iodine; sulfonate groups such as methanesulfonate, toluenesulfonate, p-bromobenzenesulfonate, p-toluenesulfonate, etc.; acyloxy groups such as acetoxy and trifluoroacetoxy, etc.

[0062] The term "protecting group" includes, but is not limited to, an "amino protecting group," a "hydroxy protecting group," or a "thiol protecting group." The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the amino nitrogen position. Representative amino protecting groups include, but are not limited to, formyl; acyl, such as alkanoyl (e.g., acetyl, trichloroacetyl, or trifluoroacetyl); alkoxycarbonyl, such as tert-butyloxycarbonyl (Boc); arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-bis-(4'-methoxyphenyl)methyl; silyl, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), and the like. The term "hydroxy protecting group" refers to a protecting group suitable for preventing side reactions at the hydroxyl group. Representative hydroxy protecting groups include, but are not limited to, alkyl groups such as methyl, ethyl and tert-butyl; acyl groups such as alkanoyl (e.g., acetyl); arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), and the like.

[0063] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0064] The solvent used in the present invention is commercially available. The present invention uses the following abbreviations: aq represents water; HATU represents O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; EDC represents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; m-CPBA represents 3-chloroperoxybenzoic acid; eq represents equivalent; CDI represents carbonyldiimidazole; DCM represents dichloromethane; PE represents PE; DIAD represents diisopropyl azodicarboxylate; DMF represents N,N-dimethylformamide; DMSO represents dimethyl sulfoxide; EtOAc represents ethyl acetate; EtOH represents ethanol; MeOH represents methanol; CBz represents benzyloxycarbonyl, which is an amine protecting group; BOC represents tert-butyloxycarbonyl, which is an amine protecting group; HOAc represents acetic acid; NaCNBH3 represents sodium cyanoborohydride; rt represents room temperature; O / N represents overnight; THF represents tetrahydrofuran; Boc2O represents di- tert-Butyl dicarbonate; TFA stands for trifluoroacetic acid; DIPEA stands for diisopropylethylamine; SOCl2 stands for thionyl chloride; CS2 stands for carbon disulfide; TsOH stands for p-toluenesulfonic acid; NFSI stands for N-fluoro-N-(phenylsulfonyl)benzenesulfonamide; NCS stands for N-chlorosuccinimide; n-Bu4NF stands for tetrabutylammonium fluoride; iPrOH stands for 2-propanol; mp stands for melting point; LDA stands for lithium diisopropylamide; LiHMD S represents lithium hexamethyldisilazide; Xantphos represents 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene; LiAlH4 represents lithium aluminum tetrahydride; Pd(dba)2 represents tris(dibenzylideneacetone)dipalladium; mCPBA represents meta-chloroperbenzoic acid; pd(dppf)Cl2 represents [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium; and DBU represents 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0065] The compounds were named according to conventional nomenclature in the art or using ChemDraw software, and commercially available compounds were named according to the supplier's catalog name.

[0066] The compound of the present invention has excellent aldehyde complexing ability, good water solubility, good permeability, and excellent pharmacokinetic properties; through efficacy tests on the compound in citric acid-induced chronic cough in guinea pigs, efficacy tests in Gao-binge diet-induced alcoholic hepatitis models in mice, and efficacy tests on OVA-induced asthma models in mice, it is shown that the compound is effective in treating chronic cough, asthma, and alcoholic liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1: In vitro aldehyde capture ability test results;

[0068] Figure 2 Coughing rate in each group on day 8 ***p<0.001vs Model;

[0069] Figure 3. Number of coughs in guinea pigs on day 12. * p<0.05vs Model, ** p<0.01vs Model, *** p<0.001vs Model;

[0070] Figure 4: Coughing number of guinea pigs on day 15. * p<0.05vs Model, ** p<0.01vs Model, *** p<0.001vs Model;

[0071] Figure 5. Mouse righting reflex. (A) Latency of righting reflex loss. (B) Recovery period of righting reflex loss. ** p<0.01vs Model, *** p<0.001vs Model;

[0072] Figure 6 Mouse liver index. *** p<0.001vs Control;

[0073] Figure 7. Serum alanine aminotransferase (ALT) levels in mice. * p<0.05vs Model, ## p<0.01vs Control;

[0074] Figure 8 Pathological changes of mouse liver tissue. (H&E staining, Oil Red O staining, ×200);

[0075] Figure 9: Number of times mice scratched their noses on day 28;

[0076] Figure 10: Number of coughs and wheezes in mice on day 28. * p<0.05vs Model, *** p<0.001vs Model;

[0077] Figure 11: The incubation period of cough and asthma in mice on day 28. *** p<0.001vs Model;

[0078] Figure 12 Behavioral scores of mice panting on day 28, ** p<0.01vs Model, *** p<0.001vs Model;

[0079] Figure 13 Total cell counts in mouse BALF on day 28, * p<0.05vs Model, **p < 0.01 vs Model;

[0080] Figure 14 Eosinophil counts in mouse BALF on day 28, * p<0.05vs Model, ** p<0.01vs Model, *** p<0.001vs Model;

[0081] Figure 15: The number of basophils in BALF of mice on day 28. ** p<0.01vs Model. DETAILED DESCRIPTION

[0082] The present invention is described in detail below by way of examples, but is not intended to limit the present invention in any way. While the present invention has been described in detail herein, and specific embodiments thereof have been disclosed, it will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0083] Example 1: Synthesis of Compound 1

[0084] Synthesis route of compound 1:

[0085] Step 1: Synthesis of compound 1-2

[0086] Compound 1-1 (1.5 g, 6.12 mmol, 1 eq), hexamethyltin (1.60 g, 4.90 mmol, 1.02 mL, 0.8 eq), and ditri-tert-butylphosphine palladium (938.39 mg, 1.84 mmol, 0.3 eq) were dissolved in toluene (20 mL) and stirred at 80°C for 14 hours. TLC (dichloromethane:methanol = 10:1) confirmed the reaction was complete, and the sample was directly concentrated and purified using a flash silica gel column (mobile phase: 0-10% dichloromethane / methanol). Compound 1-2 was obtained. [M+1] + =330.9

[0087] Step 2: Synthesis of compound 1

[0088] Substrate 1-2 (200 mg, 605 μmol, 1 eq) was dissolved in tetrahydrofuran (20 mL). Methylmagnesium bromide (3 M, 4.04 mL, 20 eq) was slowly added dropwise at 0°C. The reaction was stirred at 0°C for 2 hours. Water (20 mL) was added to quench the reaction, followed by extraction with ethyl acetate (50 mL x 3). The organic phase was concentrated. The mixture was dissolved in dimethylformamide (DMF) and purified by HPLC (neutral). Compound 1 was obtained.

[0089] 1 H NMR(400MHz, DMSO-d6)δ7.80(d,J=8.3Hz,2H),7.00(d,J=8.3Hz,2H),5.56(s,4H),5.47(s,2H),1.56(s,12H); LCMS:[M+H] + =302.9

[0090] Example 2: Synthesis of Compound 2

[0091] Synthesis route of compound 2:

[0092] Step 1: Synthesis of compound 2-3

[0093] Substrate 2-1 (702.07 mg, 2.55 mmol, 1.5 eq) and 2-2 (310 mg, 1.70 mmol, 1 eq) were dissolved in acetonitrile (20 mL) and cesium carbonate (1.11 g, 3.40 mmol, 2 eq) was added. The reaction was stirred at 50°C for 3 hours. After the reaction, the reaction solution was directly filtered and the filtrate was dried to obtain the crude product. The crude product was purified on a flash silica gel column (petroleum ether:ethyl acetate = 100:0 to 60:40) to obtain compound 2-3.

[0094] 1 H NMR (400MHz, CDCl3) δ8.42(d,J=9.03Hz,1H),7.24(d,J=4.02Hz,1H),7.15-7.20(m,1H),7.07-7.13(m,1H),5.82(br s, 2H), 4.39 (dq, J = 4.27, 7.11Hz, 4H), 1.35 (td, J = 7.15, 18.57Hz, 6H).

[0095] Step 2: Synthesis of Compound 2-4

[0096] Compound 2-3 (360 mg, 956.63 μmol, 1 eq) and hydrochloric acid (12 M, 478.32 μL, 6 eq) were dissolved in ethanol (20 mL) and water (5 mL). Reduced iron powder (534.23 mg, 9.57 mmol, 10 eq) was added and stirred at 25°C for 2 hours. After the reaction, the pH of the reaction solution was adjusted to 9 with saturated sodium carbonate solution (30 mL) and extracted with ethyl acetate (30 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and spin-dried to obtain the crude product. The crude product was purified on a flash silica gel column (petroleum ether:ethyl acetate = 100:0 to 40:60) to obtain compound 2-4.

[0097] 1 H NMR (400MHz, CDCl3) δ7.17-7.24(m,2H),7.09-7.16(m,2H),5.64(br s,4H),4.37(q,J=7.03Hz,4H),1.37(t,J=7.03Hz,6H).

[0098] Step 3: Synthesis of compound 2

[0099] Compound 2-4 (120 mg, 346.48 μmol, 1 eq) was dissolved in tetrahydrofuran (10 mL) and cooled to 0°C. A solution of methylmagnesium bromide (3 M, 3.00 mL, 26 eq) in 2-methyltetrahydrofuran was added dropwise at 0°C. The reaction was stirred at 0°C for 30 minutes and then at 25°C for 2 hours. After completion, saturated aqueous ammonium chloride (40 mL) was added to quench the reaction and the mixture was extracted with ethyl acetate (100 mL). The organic phase was dried over anhydrous sodium sulfate and spin-dried to obtain the crude product. The crude product was purified by HPLC (column model: Waters Xbridge 150*25 mm 5 μm; mobile phase: [water (10 mM ammonium bicarbonate solution)-acetonitrile]; B (acetonitrile) %: 0%-50%, 10 min) to obtain compound 2.

[0100] LCMS: [MS+H + ]=318.9.

[0101] 1 H NMR (400MHz, DMSO-d6) δ7.03(d,J=8.28Hz,2H),6.57(d,J=8.53Hz,2H),5.37(s,2H),5.27(s,4H),1.38(s,12H).

[0102] Example 3: Synthesis of Compound 3 (also referred to as the compound of formula (III) in the present invention)

[0103] Synthesis route of compound 3:

[0104] Step 1: Synthesis of compound 3-3

[0105] Compound 3-1 (300 mg, 1.30 mmol, 1 eq), 3-2 (379.73 mg, 1.69 mmol, 1.3 eq), potassium phosphate (551.24 mg, 2.60 mmol, 2 eq), and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride / dichloromethane (530.18 mg, 649.22 μmol, 0.5 eq) were dissolved in dichloroethane (15 mL) and stirred at 80°C for 15 hours. After the reaction, the reaction mixture was filtered and the filtrate was dried to obtain the crude product. The crude product was purified using a flash silica gel column (petroleum ether:ethyl acetate = 100:0 to 50:50) to obtain compound 3-3.

[0106] 1 H NMR (400MHz, CDCl3) δ8.29(s,1H),8.08(s,1H),7.88(s,2H),7.25(s,1H),5.95(br s,2H),4.03(s,3H),3.96(s,3H).

[0107] Step 2: Synthesis of compound 3-4

[0108] 3-3 (250 mg, 754.65 μmol, 1 eq) and hydrochloric acid (12 M, 1.26 mL, 20 eq) were dissolved in methanol (12 mL) and water (3 mL). Reduced iron powder (421.43 mg, 7.55 mmol, 10 eq) was added and stirred at 25°C for 1 day and 15 hours. After the reaction, the pH of the reaction solution was adjusted to 9 with saturated aqueous sodium carbonate (50 mL) and extracted with ethyl acetate (60 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and spin-dried to obtain the crude product. The crude product was purified on a flash silica gel column (petroleum ether:ethyl acetate = 100:0 to 50:50) to obtain compound 3-4.

[0109] 1 H NMR (400MHz, DMSO-d6) δ8.09(d,J=2.01Hz,1H),7.82(d,J=8.28Hz,1H),7.43(d,J =2.01Hz,1H),7.08(d,J=1.51Hz,1H),6.71-6.86(m,5H),3.83(d,J=5.77Hz,6H).

[0110] Step 3: Synthesis of compound 3

[0111] 3-4 (80 mg, 265.52 μmol, 1 eq) was dissolved in tetrahydrofuran (10 mL) and cooled to 0°C. A solution of methylmagnesium bromide (33 M, 2 mL, 22.60 eq) in 2-methyltetrahydrofuran was added dropwise at 0°C. The reaction was stirred at 0°C for 30 minutes and then at 25°C for 15 hours. After completion of the reaction, saturated aqueous ammonium chloride (20 mL) was added to quench the reaction and the mixture was extracted with ethyl acetate (50 mL). The organic phase was dried over anhydrous sodium sulfate and spin-dried to obtain the crude product. The crude product was purified by thin-layer chromatography on silica gel (developing solvent: petroleum ether:ethyl acetate = 2:5) to obtain compound 3.

[0112] LCMS: [MS+H + ]=301.9.

[0113] 1 H NMR (400MHz, CDCl3) δ8.12(d,J=1.76Hz,1H),7.20(d,J=8.03Hz,1H),7.07(d,J=2.01Hz ,1H),6.85(dd,J=2.01,8.03Hz,1H),6.81(d,J=2.01Hz,1H),4.31-5.07(m,4H),3.32(br s,2H),1.71(d,J=5.27Hz,12H).

[0114] Example 4: Synthesis of Compound 4

[0115] Synthesis route of compound 4:

[0116] Step 1: Synthesis of compound 4-2

[0117] Dissolve 3-1 (3 g, 12.98 mmol, 1 eq) in tetrahydrofuran (10 mL). Lower the reaction temperature to -78°C. Slowly add methyllithium (1 M, 64.92 mL, 5 eq) dropwise and stir at -78°C for 1 hour. TLC monitoring (petroleum ether:ethyl acetate = 3:1) reveals the formation of new reaction spots. Quench the reaction by adding water (50 mL) and extract with ethyl acetate (50 mL x 2). The organic phase is spin-dried and purified using a flash silica gel column (petroleum ether / ethyl acetate = 3:1 to 1:1). This yields compound 4-2.

[0118] 1H NMR (400MHz, CDCl3) δ7.93 (d, J = 2.0Hz, 1H), 7.06 (d, J = 2.0Hz, 1H), 4.67 (br s, 2H), 1.64 (s, 6H)

[0119] Step 2: Synthesis of compound 4

[0120] 4-2 (500 mg, 2.16 mmol), hexamethyltin (1.74 mmol, 360 mL), and di-tri-tert-butylphosphine palladium (330 mg, 645.73 μmol) were added to toluene (15 mL). The mixture was stirred at 80°C under nitrogen for 12 hours. Thin-layer chromatography (dichloromethane:methanol = 10:1) revealed the formation of a highly polar new spot. After the reaction was complete, methanol (10 mL) and dichloromethane (100 mL) were added to the reaction mixture. The solid dissolved, filtered, and the filtrate concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain an impure product, which was then separated by preparative HPLC (neutral column: Waters Xbridge 150*25mm 5μm; mobile phase: [water (10mM ammonium bicarbonate)-acetonitrile]; B (acetonitrile) %: 10%-35%, 8.2 min) to afford compound 4.

[0121] LCMS(ESI):[M+H] + =303.

[0122] 1 H NMR (400MHz, DMSO-d6) δ7.91 (d, J = 2.0 Hz, 2H), 7.12 (d, J = 2.0 Hz, 2H), 5.68 (s, 4H), 5.48 (br s, 2H), 1.52 (m, 12H).

[0123] Example 5: Synthesis of Compound 5

[0124] Synthesis route of compound 5:

[0125] Step 1: Synthesis of compound 5-3

[0126] Substrates 5-1 (3 g, 17.74 mmol, 1 eq) and 5-2 (4.03 g, 17.91 mmol, 1.01 eq) were dissolved in acetic acid (50 mL) and stirred at 25°C for 2 hours. After the reaction, the reaction solution was directly dried by rotary evaporation. The crude product was dissolved in ethyl acetate (130 mL) and washed once with saturated aqueous sodium carbonate (100 mL), sodium thiosulfate (100 mL, 1 M), and saturated brine (100 mL). The organic phase was dried over anhydrous sodium sulfate and rotary evaporation to obtain the crude product. The crude product was purified on a flash silica gel column (petroleum ether:ethyl acetate = 100:0 to 98:2) to obtain 5-3.

[0127] 1 H NMR (400MHz, CDCl3) δ7.59-7.67(m,2H),6.22(br s,2H),3.90(s,3H).

[0128] Step 2: Synthesis of compound 5-5

[0129] Compound 5-3 (1 g, 3.39 mmol, 1 eq), 5-4 (860.66 mg, 3.39 mmol, 1 eq), and potassium acetate (332.62 mg, 3.39 mmol, 1 eq) were dissolved in toluene (15 mL). Under nitrogen, the mixture was stirred at 25°C for 10 minutes, followed by the addition of [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) in dichloromethane [Pd(dppf)Cl₂.CH₂Cl₂] (2.77 g, 3.39 mmol, 1 eq). The reaction mixture was heated to 100°C and stirred for 15 hours. After completion, the reaction was quenched with saturated aqueous sodium carbonate (60 mL) and extracted with ethyl acetate (60 mL). The organic phase was washed with saturated brine (60 mL), dried over anhydrous sodium sulfate, and spin-dried to obtain the crude product. The crude product was purified by flash silica gel column (petroleum ether:ethyl acetate=100:0-98.8:1.2) to obtain compound 5-5.

[0130] LCMS: [MS+H + ]=295.9.

[0131] Step 3: Synthesis of Compound 5-6

[0132] Compound 2-1 (1.5 g, 5.45 mmol, 1 eq), 5-5 (2.09 g, 7.09 mmol, 1.3 eq), potassium phosphate (3.47 g, 16.36 mmol, 3 eq), and Pd(dppf)Cl2.CH2Cl2 (1.34 g, 1.64 mmol, 0.3 eq) were dissolved in ethylene glycol dimethyl ether (60 mL) and stirred at 80°C for 15 hours. After the reaction, the reaction solution was directly filtered and the filtrate was dried to obtain the crude product. The crude product was purified using a flash silica gel column (petroleum ether:ethyl acetate = 100:0 to 60:40) to obtain compound 5-6.

[0133] 1 H NMR (400MHz, CDCl3) δ8.46 (d, J = 8.78Hz, 1H), 8.12 (br s,2H),7.88(d,J=8.78Hz,1H),7.81(dd,J=3.01,9.03Hz,1H),7.51(dd,J=3.01, 9.03Hz, 1H), 4.53 (q, J = 7.03Hz, 2H), 3.87-3.94 (m, 3H), 1.44 (t, J = 7.15Hz, 3H).

[0134] Step 4: Synthesis of compounds 5-7

[0135] Compound 5-6 (890 mg, 2.45 mmol, 1 eq) was dissolved in ethyl acetate (100 mL). After nitrogen protection, Pd / C (500 mg, 10% purity) was added. The gas was replaced three times with a hydrogen balloon (15 psi). The mixture was stirred at 25°C for 15 hours and then at 65°C for 15 hours. After completion of the reaction, the reaction solution was filtered through celite. The filtrate was directly dried to obtain compound 5-7.

[0136] 1 H NMR (400MHz, CDCl3) δ8.54(br s,2H),7.60-7.68(m,2H),7.45(dd,J=3.14,9.66Hz,1H),7.16(d,J=8.78Hz,1H),5.84(br s, 2H), 4.42 (q, J = 7.03Hz, 2H), 3.90 (s, 3H), 1.45 (t, J = 7.15Hz, 3H).

[0137] Step 5: Synthesis of Compound 5-8

[0138] Compound 5-7 (500 mg, 1.50 mmol, 1 eq) was dissolved in tetrahydrofuran (50 mL) and cooled to 0°C. A solution of methylmagnesium bromide (3 M, 7.50 mL, 15 eq) in 2-methyltetrahydrofuran was added dropwise at 0°C. The reaction was stirred at 0°C for 1.5 hours. After completion, saturated aqueous ammonium chloride (100 mL) was added to quench the reaction, followed by extraction with ethyl acetate (100 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and spin-dried to obtain the crude product. The crude product was purified by HPLC (column model: Boston Uni C18 40 x 150 mm x 5 μm; mobile phase: [water (10 mM ammonium bicarbonate solution) - acetonitrile]; B (acetonitrile) %: 28%-58%, 10 min) to obtain compound 5-8.

[0139] 1 H NMR (400MHz, CDCl3) δ7.30 (d, J = 8.28Hz, 1H), 6.99-7.10 (m, 2H), 6.89 (dd, J = 2.89, 10.16Hz, 1H), 1.71 (d, J = 1.76Hz, 12H).

[0140] Step 6: Synthesis of compound 5

[0141] Compound 5-8 (100 mg, 313.11 μmol, 1 eq) was dissolved in acetonitrile (5 mL) and then separated and purified by HPLC (column model: Phenomenex Gemini-NX 150*30 mm*5 μm; mobile phase: [water (0.04% ammonia water + 10 mM ammonium bicarbonate solution) - acetonitrile]; B (acetonitrile) %: 39%-49%, 8 min) to obtain compound 5.

[0142] 1 H NMR(400MHz,DMSO-d6)δ7.36(d,J=8.53Hz,1H),7.05-7.13(m,2H),6.89(s,2H),6.84(dd ,J=3.01,10.54Hz,1H),5.72(s,2H),5.55(s,1H),5.47(s,1H),1.53(d,J=4.02Hz,12H).

[0143] Example 6: Synthesis of Compound 6

[0144] Synthesis route of compound 6:

[0145] Step 1: Synthesis of compound 6-2

[0146] Compound 2-1 (10 g, 36.36 mmol, 1 eq) and benzyl alcohol (7.86 g, 72.71 mmol, 7.56 mL, 2 eq) were dissolved in acetonitrile (100 mL). Cesium carbonate (23.69 g, 72.71 mmol, 2 eq) was added at 20°C and stirred at 20°C for 12 hours. After completion of the reaction, the reaction solution was quenched with water (40 mL) and extracted with ethyl acetate (80 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain the crude product. The crude product was purified using a flash silica gel column (SiO2, petroleum ether / ethyl acetate = 5:1 to 3:1) to obtain compound 6-2.

[0147] 1 H NMR (400MHz, CDCl3): δ8.39-8.33(m,1H),7.48-7.38(m,5H),6.99-6.93(m,1H),5.52-5.48(m,2H),4.57-4.48(m,2H),1.48-1.41(m,3H).

[0148] Step 2: Synthesis of compound 6-3

[0149] Compound 6-2 (4.9 g, 16.21 mmol, 1 eq) was dissolved in ethanol (90 mL). After nitrogen protection, palladium on carbon (Pd / C, 0.5 g, 16.21 mmol, 10% purity, 1 eq) was added. The atmosphere was replaced three times with a hydrogen balloon, and the mixture was stirred at 30°C for 12 hours. After the reaction, the reaction mixture was filtered, and the filtrate was dried to obtain a crude product. The crude product was purified using a flash silica gel column (SiO2, dichloromethane:methanol = 10:0 to 10:1) to obtain compound 6-3.

[0150] 1 H NMR (400MHz, CDCl3): δ9.17 (br s, 1H), 7.10 (d, J = 9.8Hz, 1H), 6.73 (d, J = 9.8Hz, 1H), 5.41 (br s, 2H), 4.36 (q, J = 7.0Hz, 2H), 1.38 (t, J = 7.2Hz, 3H).

[0151] Step 3: Synthesis of compound 6-5

[0152] Compound 6-4 (1 g, 5.34 mmol, 1 eq) and 6-3 (974.4 mg, 5.35 mmol, 1 eq) were dissolved in DMSO (20 mL), potassium phosphate (1.70 g, 8.00 mmol, 1.5 eq) was added, and the mixture was stirred at 100°C for 12 hours. After the reaction, the mixture was diluted with ethyl acetate (50 mL) and washed sequentially with water (30 mL x 2) and saturated brine (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and dried to obtain the crude product. The crude product was purified using a flash silica gel column (SiO2, petroleum ether / ethyl acetate = 5:1 to 2:1). Compound 6-5 was obtained.

[0153] 1 H NMR (400MHz, CDCl3) δ7.64 (d, J = 11.8Hz, 1H), 7.21-7.13 (m, 1H), 7.02-6.97 (m, 1 H), 6.56 (d, J = 6.8Hz, 1H), 4.44-4.31 (m, 2H), 3.89 (s, 3H), 1.40 (t, J = 7.2Hz, 3H).

[0154] Step 4: Synthesis of compound 6

[0155] Compound 6-5 (200 mg, 572.55 μmol, 1 eq) was dissolved in tetrahydrofuran (40 mL) and cooled to 0°C. A solution of methylmagnesium bromide (3 M, 5 mL, 26.20 eq) in 2-methyltetrahydrofuran was added dropwise at 0°C. The reaction was stirred at 0°C for 1 hour and then at 15°C for 15 hours. After completion of the reaction, saturated aqueous ammonium chloride (50 mL) was added to quench the reaction and the mixture was extracted with ethyl acetate (60 mL). The organic phase was dried over anhydrous sodium sulfate and spin-dried to obtain the crude product. The crude product was purified by HPLC (column model: Waters Xbridge 150*25 mm 5 μm; mobile phase: [water (10 mM ammonium bicarbonate solution)-acetonitrile]; B (acetonitrile) %: 15%-45%, 10 min) to obtain compound 6.

[0156] LCMS: [MS+H+]=336.0.

[0157] 1H NMR (400MHz, DMSO-d6) δ7.06(d,J=8.28Hz,1H),6.88(d,J=13.05Hz,1H),6.55(d,J=8.28Hz,1H),6.33(d,J=7.53Hz,1H),5.37(s,1H),5.30(br d,J=3.01Hz,4H),5.26(s,1H),1.48(s,6H),1.38(s,6H).

[0158] Example 7: Synthesis of Compound 7

[0159] Synthesis route of compound 7:

[0160] Step 1: Synthesis of compound 7-2

[0161] To a solution of compound 7-1 (2 g, 7.19 mmol, 1 eq) in toluene (30 mL) were added 5-4 (3.65 g, 14.39 mmol, 2 eq), Pd(dppf)Cl2 (526.35 mg, 719.34 μmol, 0.1 eq), and potassium acetate (1.41 g, 14.39 mmol, 2 eq). The mixture was reacted at 110°C for 3 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to obtain crude product 7-2.

[0162] Step 2: Synthesis of compound 7-3

[0163] To a solution of compound 7-2 (1.63 g, 7.07 mmol, 1 eq) in toluene (50 mL) and water (10 mL) were added compound 3-1 (2.3 g, 7.07 mmol, 1 eq), cesium carbonate (4.61 g, 14.14 mmol, 2 eq), and Pd(dppf)Cl2 (517.32 mg, 707.00 μmol, 0.1 eq). The mixture was reacted at 110°C under nitrogen for 6 hours. After completion of the reaction, the reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was separated by silica gel column chromatography (SiO2, petroleum ether / ethyl acetate = 5:1 to 0:1). This afforded compound 7-3.

[0164] LCMS: 350.1[M+1] + .

[0165] Step 3: Synthesis of compound 7-4

[0166] To a solution of compound 7-3 (1.58 g, 4.52 mmol, 1 eq) in methanol (50 mL) and ethyl acetate (50 mL) was added Pd / C (1.7 g, 5% purity). The mixture was reacted under a hydrogen atmosphere at 15 psi and 20°C for 3 hours. After completion of the reaction, the reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure to obtain compound 7-4.

[0167] LCMS: 320[M+1] + .

[0168] 1H NMR(400MHz,DMSO-d6)δppm 7.96(t,J=1.76Hz,1H),7.54(d,J=11.84Hz,1H),7.39(s,1H),6.95(d,J=6.58Hz,1H),6.82(s,2H),6.66(s,2H),3.83(d,J=3.96Hz,6H).

[0169] Step 4: Synthesis of compound 7

[0170] To a solution of compound 7-4 (1.5 g, 4.70 mmol, 1 eq) in tetrahydrofuran (150 mL) at 0°C, methylmagnesium bromide (3 M, 31.32 mL, 20 eq) was added. The mixture was allowed to react at 20°C for 3 hours. After completion of the reaction, the reaction mixture was poured into a saturated ammonium chloride solution and separated by adding 50 mL of ethyl acetate. The aqueous phase was extracted with ethyl acetate (50 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was separated by HPLC (column model: Xtimate C18 150 x 25 mm x 5 μm; mobile phase: [water (10 mM ammonium bicarbonate solution) - acetonitrile]; B (acetonitrile) %: 25%-45%, 10.5 min). Compound 7 was obtained.

[0171] LCMS: 320.0[M+1] + ;

[0172] 1 H NMR(400MHz,DMSO-d6)δppm 7.77(s,1H),7.05(s,1H),6.91(d,J=12.28Hz,1H),6.67(d,J=7.46Hz,1H),5.63(s,2H),5.27-5.48(m,4H),1.51(s,12H)

[0173] Example 8: Synthesis of Compound 8

[0174] Synthesis route of compound 8:

[0175] Step 1: Synthesis of compound 8-1

[0176] Compound 3-4 (1 g, 3.32 mmol, 1 eq) and NCS (509.67 mg, 3.82 mmol, 1.15 eq) were dissolved in glacial acetic acid (60 mL) and stirred at 25°C for 15 hours. After the reaction, the reaction mixture was adjusted to pH 9 with saturated aqueous sodium carbonate (200 mL) and extracted with ethyl acetate (150 mL). The organic phase was washed once with saturated brine (50 mL), dried over anhydrous sodium sulfate, and spin-dried to obtain a crude product. The crude product was purified using a flash silica gel column (petroleum ether:ethyl acetate = 100:0 to 70:30) to obtain compound 8-1.

[0177] 1 H NMR (400MHz, CDCl3) δ8.10(d,J=1.76Hz,1H),7.97(s,1H),7.12(d,J=1.76Hz,1H),6.63(s,1H),5.83(br s,4H),4.01(s,3H),3.91(s,3H).

[0178] Step 2: Synthesis of compound 8

[0179] Compound 8-1 (265 mg, 789.30 μmol, 1 eq) was dissolved in tetrahydrofuran (50 mL) and cooled to 0°C. A solution of methylmagnesium bromide (3 M, 4 mL, 15.20 eq) in 2-methyltetrahydrofuran was added dropwise at 0°C. The reaction was stirred at 0°C for 2 hours and then at 25°C for 15 hours. After completion, saturated aqueous ammonium chloride (50 mL) was added to quench the reaction, followed by extraction with ethyl acetate (60 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and spin-dried to obtain the crude product. The crude product was purified by HPLC (column model: Waters Xbridge 150*25 mm*5 μm; mobile phase: [water (10 mM ammonium bicarbonate solution)-acetonitrile]; B (acetonitrile) %: 25%-48%, 7.8 min) to obtain compound 8.

[0180] LCMS: [MS+H+]=336.0;

[0181] 1 H NMR (400MHz, DMSO-d6) δ7.66(d,J=2.01Hz,1H),7.07(s,1H),6.96(d,J=2.01Hz,1H),6.61(s,1H),5.61(br d,J=17.32Hz,4H),5.47(s,1H),5.34(s,1H),1.51(s,12H).

[0182] Example 9: Synthesis of Compound 9

[0183] Synthesis route of compound 9:

[0184] Step 1: Synthesis of compound 9-2

[0185] Compound 9-1 (24.8 g, 120.37 mmol, 1 eq) and benzylamine (15.48 g, 144.45 mmol, 15.75 mL, 1.2 eq) were dissolved in DMF (200 mL). Triethylamine (36.54 g, 361.12 mmol, 50.26 mL, 3 eq) was added at 20°C. The reaction was stirred at 20°C for 21 hours. Upon completion, water (250 mL) was added to quench the reaction, followed by extraction with ethyl acetate (500 mL x 2). The organic phase was spin-dried and purified using a flash silica gel column (SiO2, petroleum ether / ethyl acetate = 5:1 to 3:1) to obtain compound 9-2.

[0186] LCMS(ESI):[M+H] + :277.1.

[0187] 1 H NMR (400MHz, CDCl3) δ8.71 (s, 1H), 8.55 (br s, 1H), 7.36-7.43 (m, 2H), 7.29-7.35 (m, 3H), 6.56 (s, 1H), 4.44 (d, J = 5.52Hz, 2H), 3.88-3.92 (m, 3H).

[0188] Step 2: Synthesis of compound 9-3

[0189] Dissolve compound 9-2 (5 g, 18.07 mmol, 1 eq) and sodium methoxide (9.76 g, 180.69 mmol, 10 eq) in MeOH (50 mL). Stir and react at 90°C under nitrogen for 15 hours. After completion of the reaction, purify the product using a flash silica gel column (SiO2, petroleum ether / ethyl acetate = 5:1 to 3:1) to obtain compound 9-3.

[0190] LCMS (ESI): [M+H]+: 273.

[0191] Step 3: Synthesis of compound 9-4

[0192] Compound 9-3 (3.26 g, 11.97 mmol, 1 eq), trimethylsilyl chloride (5.20 g, 47.89 mmol, 6.08 mL, 4 eq), and NaI (7.18 g, 47.89 mmol, 4 eq) were dissolved in acetonitrile (50 mL) and stirred at 80°C under nitrogen for 15 hours. After completion of the reaction, the solution was adjusted to pH 7 with sodium bicarbonate. The solution was then quenched with water (20 mL) and extracted twice with dichloromethane (100 mL). The organic phase was combined and dried on a rotary evaporator. The crude product was purified on a flash silica gel column (SiO2, petroleum ether / ethyl acetate = 5 / 1 to 3:1) to yield compound 9-4.

[0193] LCMS(ESI):[M+H] + :259.1

[0194] 1 H NMR (400MHz, CDCl3) δ7.47 (br s, 1H), 6.47-6.58 (m, 5H), 4.65 (s, 1H), 3.55 (br d, J = 5.27Hz, 2H), 3.00-3.08 (m, 3H)

[0195] Step 4: Synthesis of compound 9-6

[0196] Compound 9-4 (2.29 g, 8.87 mmol, 1 eq), compound 9-5 (2.77 g, 10.64 mmol, 1.2 eq), cuprous iodide (337.73 mg, 1.77 mmol, 0.2 eq), potassium carbonate (2.45 g, 17.73 mmol, 2 eq), and (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (252.24 mg, 1.77 mmol, 0.2 eq) were dissolved in toluene (30 mL) and stirred at 110°C under nitrogen for 13 hours. After the reaction, the solution was filtered and dried on a rotary evaporator. Compound 9-6 was obtained by purification on a flash silica gel column (SiO2, petroleum ether / ethyl acetate = 1:1 to 0:1).

[0197] LCMS(ESI):[M+H] + :438.1.

[0198] Step 5: Synthesis of compound 9-7

[0199] Compound 9-6 (1 g, 2.29 mmol, 1 eq) was dissolved in tetrahydrofuran (30 mL). Palladium on carbon (1.5 g, 10% purity) was added to the mixed solution under a nitrogen atmosphere. Stir at 65°C for 12 hours under a hydrogen atmosphere. After the reaction, the reaction mixture was filtered and dried. Purification on a flash silica gel column (SiO2, petroleum ether / ethyl acetate = 1:1 to 0:1) afforded compound 9-7.

[0200] LCMS(ESI):[M+H] + :318.1

[0201] 1H NMR: 1 H NMR (400MHz, DMSO-d6) δ8.12 (s, 1H), 7.76 (d, J = 8.53Hz, 1H), 7.06 (br s,2H),6.84(s,2H),6.77(d,J=1.76Hz,1H),6.51(dd,J=2.01,8.53Hz,1H),5.42(s,1H),3.81(s,3H),3.74(s,3H)

[0202] Step 6: Synthesis of compound 9

[0203] Compound 9-7 (200 mg, 630.33 μmol, 1 eq) was dissolved in tetrahydrofuran (20 mL). Under a nitrogen atmosphere, the reaction solution was cooled to -78°C in a dry ice-ethanol bath and methyllithium (1.6 M, 5.91 mL, 15 eq) was slowly added dropwise. After the addition, the reaction solution was slowly warmed to 0°C and stirred for 30 minutes. After the reaction was completed, the reaction solution was quenched with water (10 mL) at 0°C, diluted with water (5 mL), and extracted with THF (40 mL). The organic phase was combined and dried over anhydrous sodium sulfate, filtered, and dried to obtain compound 9.

[0204] LCMS(ESI):[M+H] + :318.

[0205] 1 H NMR (400MHz, DMSO-d6) δ6.20(d,J=8.28Hz,1H),6.14(s,1H),5.69(d,J=2.26Hz,1H),5.53(dd,J=2.13,8 .16Hz,1H),5.45(s,2H),4.73(s,2H),4.57(s,1H),4.53(s,1H),4.41(s,1H),0.67(s,6H),0.60(s,6H).

[0206] Experimental Example 1: In vitro aldehyde capture ability experiment

[0207] 1. Experimental Purpose and Process

[0208] Purpose: Dry eye is caused by inflammation within the eye, accompanied by the production of reactive aldehydes. If these aldehydes are not promptly eliminated, they accelerate the symptoms of inflammation and worsen the condition. This study simulated an environment with high levels of reactive aldehydes in the body and selected relatively optimal compounds based on their complexing ability with aldehydes in the body (as shown in Table 2).

[0209] Procedure: Dissolve sulfobutyl-B-cyclodextrin (310 mg) in phosphate buffer (1.25 ml) to prepare a solution.

[0210] At room temperature, nonanal (5.0 mg, 32 μmol, 1.0 eq) and triolein (300 mg) were added to the reaction flask. After adding the above-prepared solution, linoleic acid (300 mg) was added, and finally a solution of the compound of the present invention (32 μmol, 1.0 eq) in dimethyl sulfoxide (0.15 ml) was added. The reaction solution was reacted at 20-23°C.

[0211] After stirring and reacting for 10 minutes, 100 minutes, 200 minutes, and 300 minutes, the solution was allowed to stand for 2 minutes to separate the layers and then sampled for high performance liquid chromatography (HPLC) detection.

[0212] Sampling method: Use a pipette to sample 25 μl of the upper emulsion layer and 50 μl of the lower aqueous phase, and dilute with 1 ml of methanol.

[0213] 2. Experimental Results

[0214] Nonanal has weak UV absorption at a wavelength of 254nm, which has little overall effect on the content of the complex product. Therefore, the percentage content of the complex at 254nm was compared by HPLC to observe the ability of the compound's aldehyde to capture the complexed aldehyde. See Figure 1 and Table 1:

[0215] Table 1 Statistics of compound aldehyde capture ability test results

[0216] HPLC analysis method: XBRIGE 2.5μm, 3.0*100mm 5-95CD_XBEH_12min_0.8.1cm or XBRIGE 2.5μm, 3.0*100mm 5-80CD_XBEH_12min_0.8.1cm

[0217] Specific conditions: XBRIGE 2.5μm, 3.0*100mm 5-80CD_XBEH_12min_0.8.1cm

[0218] XBRIGE 2.5μm,3.0*100mm 5-95CD_XBEH_12min_0.8.lcm

[0219] The specific HPLC data of the percentage content of the complex product of the compound of the present invention is as follows Table 2:

[0220] Table 2 Specific HPLC data of the percentage content of the complex products of the compounds

[0221] Conclusion: The experiment shows that compound 3 of the present invention has very significant ability and speed of complexing aldehydes.

[0222] Experimental Example 2: In vitro evaluation

[0223] Objective: To study the inhibitory effect of compounds on human liver microsomal cytochrome P450

[0224] Experimental procedure: Compound 3 was used to study the inhibitory activity of human liver microsomal cytochrome P450 (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Mixed human liver microsomes were used as the CYP450 enzyme source. Different concentrations of compound 3 (10, 5, 1.5, 0.5, 0.15, 0.05, and 0.015 mM) were incubated with probe substrates of five CYP enzymes (two substrates for CYP3A4) and a cofactor (NADPH). The IC values ​​of the compound for each CYP enzyme were determined. 50 The test results are as follows:

[0225] Table 3 Test results of the inhibitory effect of the compounds of the present invention on human liver microsomal cytochrome P450 isoenzymes

[0226] Conclusion: The present invention has high safety, has no inhibitory effect on major CYP isoenzymes, and has a low possibility of drug-drug interaction.

[0227] Experimental Example 3: Pharmacokinetic Evaluation of Compounds

[0228] 1. Five-day toxicity study of test compound 3 in SD rats

[0229] Experimental materials: SD rats (male, 200-300 g, 7-9 weeks old, Shanghai Lingchang)

[0230] Experimental operation:

[0231] The pharmacokinetic characteristics of the compounds were tested in rodents following intravenous administration. Compound 3 was prepared as a clear solution and administered to SD rats as a single intravenous injection at 20 mg / kg for five consecutive days. The intravenous injection vehicle was a 10% aqueous hydroxypropyl β-cyclodextrin solution. Whole blood samples were collected at 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after dosing on the first and fifth days of administration. The supernatant was separated by centrifugation at 3000 r / min for 10 minutes to obtain plasma samples. 15 μL of plasma sample was mixed with 300 μL of acetonitrile solution containing an internal standard to precipitate protein. The supernatant was centrifuged and 2 μL of the supernatant was injected. Plasma concentrations were quantified by LC-MS / MS analysis, and pharmacokinetic parameters such as clearance, half-life, and area under the concentration-time curve were calculated.

[0232] Experimental results:

[0233] Table 4 Pharmacokinetic test results

[0234] Conclusion: There is no risk of drug accumulation when compound 3 of the present invention is administered by injection for 5 consecutive days.

[0235] 2. Pharmacokinetic Study of Test Compound 3 in Beagle Dogs

[0236] Experimental materials: Beagle dogs (male, 8-11 kg, 6 months or older, Mas)

[0237] Experimental operation:

[0238] To test the pharmacokinetic profile of the compound following intravenous administration in rodents, a clear solution of compound 3 was administered intravenously as a single 1 mg / kg injection to beagle dogs. The intravenous injection vehicle was 10% aqueous hydroxypropyl β-cyclodextrin. Whole blood was collected at 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours after incubation. The blood was centrifuged at 3000 r / min for 10 minutes. The supernatant was separated to obtain plasma samples. 20 μL of plasma sample was mixed with 400 μL of acetonitrile solution containing an internal standard to precipitate protein. After centrifugation, 2 μL of the supernatant was injected. Plasma concentrations were quantified by LC-MS / MS analysis, and pharmacokinetic parameters such as clearance, half-life, and area under the concentration-time curve (AUC) were calculated.

[0239] Experimental results:

[0240] Table 5 Pharmacokinetic test results

[0241] Conclusion: Compound 3 of the present invention has high clearance rate, moderate half-life and good pharmacokinetic characteristics.

[0242] 3. Pharmacokinetic Study of Test Compounds in Cynomolgus Monkeys

[0243] Experimental materials: Cynomolgus macaques (male, 2.5-4 kg, ≥2 years old, Jingang, Hainan)

[0244] Experimental operation:

[0245] To characterize the pharmacokinetic profile of the compound following intravenous administration in monkeys, a single 1 mg / kg intravenous injection of the candidate compound was administered to cynomolgus monkeys as a clear solution. The intravenous injection vehicle was 10% hydroxypropyl β-cyclodextrin in water. Whole blood was collected at 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-inoculation. The supernatant was separated and plasma samples were obtained by centrifugation at 3000 r / min for 10 minutes. 20 μL of plasma sample was mixed with 400 μL of acetonitrile solution containing an internal standard to precipitate protein. 2 μL of the supernatant was then centrifuged and injected. LC-MS / MS analysis was used to quantify plasma concentrations, and pharmacokinetic parameters such as clearance, half-life, and area under the concentration-time curve (AUC) were calculated.

[0246] Experimental results:

[0247] Table 6 Pharmacokinetic test results

[0248] Conclusion: Compound 3 of the present invention has high clearance rate, moderate half-life and good pharmacokinetic characteristics.

[0249] Experimental Example 4: Study on the efficacy of the compound in the citric acid-induced chronic cough model in guinea pigs

[0250] In this study, a chronic cough guinea pig model was established by repeated inhalation of aerosol citric acid, and the pharmacodynamic effect of the compound of formula (III) (also referred to as compound 3 in the present invention) in the citric acid-induced chronic cough model in guinea pigs was evaluated.

[0251] Experimental process:

[0252] 1) Modeling and Grouping: A total of 38 male guinea pigs were used in this study. Modeling was induced with 0.4 M citric acid.

[0253] 2) Administration of Test Substances: All subjects were administered orally. The normal control group (G1) and the model group (G2) were administered 0.9% saline. Group 3 (G3) was administered benproperine at a dose of 10 mg / kg by intraperitoneal injection once daily; Group 4 (G4) was administered a suspension of the Form B of the compound of Formula (III) at a dose of 25 mg / kg by gavage twice daily; and Group 5 (G5) was administered a suspension of the Form B of the compound of Formula (III) at a dose of 50 mg / kg by gavage twice daily. Dosing began 8 days after citric acid induction. The dosing cycle was 7 days.

[0254] 3) Detection index: number of guinea pig coughs in 5 minutes.

[0255] Table 7 Modeling, grouping and drug administration

[0256] 4) Index detection: On the 8th, 12th and 15th days, the guinea pigs were induced to cough for 3 minutes using 0.4M citric acid, and the number of coughs over 5 minutes (including the 3 minutes of cough induction and the 2 minutes after cough induction) was recorded.

[0257] 5) Statistical Analysis: Data are presented as mean ± standard error. Statistical significance was analyzed using t-test, one-way ANOVA, and post-hoc Dunnett's test. When the number of participants within a group was small or did not conform to a normal distribution, the Mann-Whitney nonparametric test was used. P < 0.05 was considered significant.

[0258] Experimental results:

[0259] 1. Detection of Coughing Number of Guinea Pigs on Day 8

[0260] On the 8th day after citric acid modeling, the number of coughs of the animals was recorded. According to the number of coughs of the animals (Figure 2; Table 8), the animals in group 2 of the model group (G2) were randomly divided into four groups (groups 2 to 5; G2-G5), and drug administration began on the 9th day.

[0261] Table 8 Statistics of guinea pig coughing on day 8

[0262] Note: Mean ± standard error; *** p<0.001 vs G2; One-way ANOVA

[0263] 2. Coughing test of guinea pigs on the 12th day

[0264] On the 12th day of the study, the number of coughs in the model group guinea pigs increased significantly compared with the control group ( *** p<0.001; Figure 3; Table 9). Compared with the model group G2, the number of coughs in the guinea pigs in the positive drug group was significantly reduced ( ** p<0.01; Figure 3; Table 9). Compared with the model group 2 (G2), the test compound of formula (III) at a dose of 25 mg / kg, administered orally twice a day, significantly reduced the number of coughs in guinea pigs ( * p<0.01; Figure 3; Table 9). When the compound of formula (III) was administered orally at a dose of 50 mg / kg twice daily, the number of coughs in guinea pigs did not change significantly (Figure 3; Table 9).

[0265] Table 9 Statistics of the number of guinea pig coughs on day 12

[0266] Note: Mean ± standard error; * p<0.05vs G2;** p<0.01vs G2; *** p<0.001 vs G2; One-way ANOVA

[0267] 3. End point of the experiment: Detection of the number of guinea pig coughs

[0268] At the end of the study, the number of coughs in the model group guinea pigs increased significantly compared with the control group ( *** p<0.001; Figure 4; Table 10). Compared with the model group G2, the number of coughs in the positive drug group was significantly reduced ( *** p<0.001; Figure 4; Table 10). Compared with the model group 2 (G2), the test compound of formula (III) at a dose of 25 mg / kg, administered orally twice a day, significantly reduced the number of coughs in guinea pigs ( *** p<0.001; Figure 4; Table 10). When the compound of formula (III) was administered orally at a dose of 50 mg / kg twice a day, the number of coughs in guinea pigs was significantly reduced ( ** p < 0.01; Figure 4; Table 10).

[0269] Table 10 Statistics of guinea pig coughing on day 15

[0270] Note: mean ± standard error; * p<0.05vs G2; ** p<0.01vs G2; *** p<0.001 vs G2; One-way ANOVA

[0271] Experimental conclusion:

[0272] Compared with the normal control group, the number of coughs in the guinea pigs in the chronic cough model group increased significantly, indicating that the model was successfully established.

[0273] Compared with the model group, the number of coughs was significantly reduced on the 12th and 15th days of the study when benproperine was administered orally at a dose of 10 mg / kg once a day, indicating that the positive drug benproperine can significantly suppress coughs.

[0274] Compared with the model group, the compound of formula (III) at a dose of 25 mg / kg, administered orally twice a day, significantly reduced the number of coughs in the model animals on day 12 and at the end of the study.

[0275] Compared with the model group, the compound of formula (III) at a dose of 50 mg / kg, administered orally twice daily, showed a trend of decreasing coughing in the model animals on day 12 after modeling (day 4 of administration), but there was no statistical difference. It significantly reduced the coughing in the model animals at the end of the study.

[0276] In summary, the compound of formula (III) demonstrated significant efficacy in treating coughing behavior in a guinea pig model of chronic cough. It significantly reduced the number of coughs in the model animals, and 25 mpk of the compound of formula (III) exhibited significant cough suppression effects as early as four days after treatment. This suggests that the compound of formula (III) can reduce the number of coughs in cough model mice and has the potential to treat chronic cough.

[0277] Experimental Example 5: Study on the efficacy of compounds in a mouse alcoholic hepatitis model

[0278] In this study, the Gao-binge model of mice was established to evaluate the pharmacological effects of the compounds in alcoholic hepatitis.

[0279] Experimental process:

[0280] 1) Modeling and Grouping: A total of 59 female C57BL / 6 mice were randomly divided into groups. The modeling method used was an alcoholic hepatitis model induced by a short-term alcohol liquid diet (Gao-binge model).

[0281] 2) Administration of Test Articles: All test articles were administered orally. The control group (Control group) and the model group (Model group) were gavaged with 0.9% saline twice daily. The drug-treated group (Compound of Formula (III) at 45 / 90 / 180 mg / kg) was gavaged with a suspension of Form B of the compound of Formula (III) at doses of 45 mg / kg, 90 mg / kg, and 180 mg / kg, respectively, twice daily. The modeling and drug-treatment cycle was 21 days, and samples were collected at the experimental endpoint on the 22nd day.

[0282] Table 11 Modeling, grouping and drug administration

[0283] 3) Detection indicators: latency period of righting reflex disappearance, recovery period of righting reflex disappearance

[0284] 4) Detection indicators: Liver index

[0285] 5) Detection indicators: serum alanine aminotransferase (ALT)

[0286] 6) Detection indicators: serum total cholesterol (TC), triglyceride (TG)

[0287] 7) Detection indicators: H&E staining of liver tissue

[0288] 8) Detection index: Oil red O staining of liver tissue

[0289] 9) Statistical Analysis: Data are presented as mean ± SEM. Statistical significance was analyzed using t-test, one-way ANOVA, and post-hoc Dunnett's test. When the number of participants within a group was small or did not conform to a normal distribution, the Mann-Whitney nonparametric test was used. P < 0.05 was considered significant.

[0290] Experimental results:

[0291] 1. Latency period of righting reflex disappearance and recovery period of righting reflex disappearance

[0292] On the 22nd day, when the experiment reached its end point, the mice were gavaged with 20 μL / g body weight of alcohol and then tested for righting reflex. The control group mice were gavaged with 9 g / kg of maltodextrin, and the model group and the drug-treated group were gavaged with 5 g / kg of 31.5% alcohol. The corresponding indicators of righting reflex were recorded and blood and liver were taken. The experimental results showed that compared with the model group (Model group), when the dose of the drug-treated group was 45 mg / kg [45 mg / kg of the compound of formula (III)], there was no significant difference in the latency of righting reflex disappearance and the recovery period of righting reflex disappearance in mice (Figure 5A, B; Table 12); when the dose of the drug-treated group was 90 mg / kg [90 mg / kg group of the compound of formula (III)], there was no significant difference in the latency of righting reflex disappearance in mice (Figure 5A; Table 12), and the recovery period of righting reflex disappearance was significantly reduced ( ** p<0.01; Figure 5B; Table 12); When the dose of the drug group was 180 mg / kg [the compound of formula (III) 180 mg / kg group], the latency of the mice to righting reflex was significantly increased ( *** p<0.001; Figure 5A; Table 12), and the recovery period of the righting reflex loss was significantly reduced ( *** p<0.001; Figure 5B, Table 12).

[0293] Table 12 Statistics of righting reflex in mice

[0294] Note: mean ± standard error; ** p<0.01vs Model; *** p<0.001 vs Model; One-way ANOVA

[0295] 2. Liver index

[0296] On the 22nd day, when the experiment reached the end point, the body weight and liver weight of the mice were recorded. The experimental results showed that compared with the normal control group (Control group), the liver index of the mice in the model group (Model group) and the drug-treated group [45 / 90 / 180 mg / kg of the compound of formula (III)] administered with the compound of formula (III) at doses of 45 mg / kg, 90 mg / kg, and 180 mg / kg were significantly increased ( ### p<0.001; Figure 6; Table 13).

[0297] Table 13 Statistics of mouse liver index

[0298] Note: mean ± standard error; ### p<0.001 vs Control; One-way ANOVA

[0299] 3. Serum alanine aminotransferase (ALT)

[0300] The experimental results showed that compared with the control group, the serum ALT level of mice in the model group was significantly increased ( ## p<0.01; Figure 7; Table 14); Compared with the model group (Model group), the serum ALT level of mice in the 45 mg / kg group (45 mg / kg group of the compound of formula (III)) was significantly reduced ( * p<0.05; Figure 7; Table 14); The serum ALT level of mice in the 90 mg / kg group [90 mg / kg group of compound (III)] was significantly decreased ( * There was no significant difference in the serum ALT levels of mice in the 180 mg / kg dose group [compound (III) 180 mg / kg group] ( Figure 7 ; Table 14 ).

[0301] Table 14 Statistics of ALT levels in mouse serum

[0302] Note: mean ± standard error; * p<0.05vs Model; ## p<0.001 vs Control; One-way ANOVA

[0303] 4. Liver Histopathological Analysis (H&E Staining, Oil Red O Staining)

[0304] At the end of the experiment on day 22, liver tissues were obtained for fixation, embedding, sectioning, staining, and then histopathological analysis.

[0305] The results of H&E staining showed that compared with the control group (Control), the hepatic cords in the liver tissue of the model group (Model) and the three-dose administration groups [45 / 90 / 180 mg / kg of Formula (III) compound] were irregularly arranged, and there were a large number of lipid droplet vacuoles and macrovesicular fatty degeneration; compared with the model group (Model), the number of fat vacuoles in the liver of mice in the three-dose administration groups [45 / 90 / 180 mg / kg of Formula (III) compound] was significantly reduced, among which the degree of vacuolar fatty lesions in the liver of mice in the administration group with a dose of 180 mg / kg [180 mg / kg of Formula (III) compound] was greatly improved (Figure 8).

[0306] The results of Oil Red O staining showed that compared with the control group (Control), the Oil Red O-stained areas in the livers of the mice in the model group (Model) and the three-dose administration groups [45 / 90 / 180 mg / kg of Formula (III) compound] were significantly increased, and obvious lipid accumulation and lipid droplet accumulation were observed; compared with the model group (Model), the number of lipid droplets in the livers of the mice in the three-dose administration groups [45 / 90 / 180 mg / kg of Formula (III) compound] was significantly reduced, among which the lipid droplets in the livers of the mice in the administration group with a dose of 180 mg / kg [180 mg / kg of Formula (III) compound] were small and the degree of lipid droplet accumulation was greatly improved (Figure 8).

[0307] Experimental conclusion:

[0308] An alcoholic hepatitis model (Gao-binge model) was induced by short-term alcohol liquid feed. The experimental results showed that compared with the normal control group, the liver index of the model group and the three dose groups was significantly increased. The results showed that feeding with alcohol liquid feed and a single gavage of alcohol (5g / kg) caused liver enlargement and lesions.

[0309] Compared with the model group, the latency of loss of righting reflex in mice treated with 90 mg / kg of the compound (Formula (III)) showed an upward trend, but no significant difference, and the recovery period of loss of righting reflex was significantly reduced. The latency of loss of righting reflex in mice treated with 180 mg / kg of the compound (Formula (III)) was significantly increased, and the recovery period of loss of righting reflex was significantly reduced. This suggests that the compound (III) can delay the onset of intoxication in mice and shorten the recovery time of the righting reflex in mice, demonstrating its effectiveness in preventing and treating alcohol intoxication.

[0310] Compared with the normal control group, serum ALT levels in the model group and the three dose-administered groups showed an upward trend, indicating that alcoholic hepatitis caused varying degrees of liver damage in mice. Compared with the model group, serum ALT levels in mice treated with 45 mg / kg (45 mg / kg of Formula (III) compound) and 90 mg / kg (90 mg / kg of Formula (III) compound) were significantly reduced. Serum ALT levels in mice treated with 180 mg / kg (180 mg / kg of Formula (III) compound) showed a downward trend, but no significant differences were observed. This suggests that Formula (III) compound can reduce serum ALT levels in mice and, to a certain extent, alleviate alcohol-induced liver damage in mice.

[0311] Pathological H&E staining and Oil Red O staining results showed that compared with the liver tissue of normal control mice, the liver tissue of the model group and the three dose-treated groups had irregular arrangement of hepatic cords and the presence of a large number of lipid droplet vacuoles or lipid droplet and macrovesicular steatosis, indicating that feeding with alcohol liquid feed and a single oral administration of alcohol (5g / kg) caused liver damage, steatosis, and varying degrees of lipid accumulation in mice. After treatment with three different doses of the compound of formula (III), the number of fat vacuoles in the mouse liver decreased and the degree of lipid droplet accumulation decreased, indicating that the compound of formula (III) has an ameliorative effect on alcohol-induced liver steatosis in mice.

[0312] In summary, the compound of formula (III) demonstrated significant efficacy in the alcoholic hepatitis model. It significantly shortened the righting reflex recovery time in mice, reduced serum ALT levels in mice, alleviated alcohol-induced liver damage in mice, and improved liver lipid accumulation in mice, indicating that the compound of formula (III) has a protective effect against alcoholic hepatitis.

[0313] Experimental Example 6: Study on the efficacy of the compound of formula (III) on OVA-induced asthma model in mice

[0314] Purpose of the experiment:

[0315] In this study, an asthma model was established in BALB / c mice by inducing ovalbumin (OVA), and the pharmacodynamic effect of the compound of formula (III) was evaluated in the OVA-induced asthma model in mice.

[0316] Experimental process:

[0317] 1) Modeling and Grouping: A total of 72 female BALB / c mice were used in this study. Modeling procedures: The sensitization phase involved intraperitoneal injection of 0.2 mL of OVA (20 μg of OVA + 1 mg of Al(OH)3) on days 1, 7, and 14. The challenge phase began on day 21 with nebulized inhalation of a 5% OVA solution (30 mL for 30 minutes) for 7 days.

[0318] 2) Administration of the test substance: All administration methods were oral. The normal control group (G1) and the model group (G2) were administered with 0.9% normal saline. Group 3 (G3) was administered with dexamethasone sodium phosphate at a dose of 3 mg / kg, intraperitoneally, once a day; Group 4 (G4) was administered with a suspension of the B form of the compound of formula (III) at a dose of 45 mg / kg, by gavage, twice a day; Group 5 (G5) was administered with a suspension of the B form of the compound of formula (III) at a dose of 90 mg / kg, by gavage, twice a day; Group 6 (G6) was administered with a suspension of the B form of the compound of formula (III) at a dose of 180 mg / kg, by gavage, twice a day. The administration cycle was 28 days.

[0319] Table 15 Modeling, grouping and drug administration

[0320] 3) Detection indicators: behavioral testing and BALF bronchoalveolar lavage fluid cell analysis.

[0321] 4) Indicator testing: Behavioral assessment of cough and wheeze indicators includes cough and wheeze latency and frequency; morphological scoring includes nasal scratching frequency and wheezing severity. BALF bronchoalveolar lavage fluid cellular analysis includes analysis of total cells, eosinophils, and basophils.

[0322] After the aerosol challenge phase, mice were placed in an acrylic box with the sides covered with black stickers to create a black background for easy observation of their behavior. Videos were recorded to assess behavioral indicators, including the number of nose scratches and coughs within 10 minutes, the cough-wheeze latency (the time it took for the mouse to first cough or wheeze), and the severity of the wheeze. The scoring criteria are shown in Table 16.

[0323] Table 16 Scoring criteria

[0324] After behavioral testing, mice were anesthetized and sacrificed, secured to an operating table, and the neck hair was disinfected with 75% alcohol. Surgical scissors were used to trim the neck fur, the chest cavity was opened, and hemostats were used to fully expose the lungs. The trachea was bluntly dissected and tied with silk suture to prevent the lavage fluid from leaking out of the mouth and nose. Using a 1mL sterile syringe, 0.8mL of normal saline was drawn. The needle was inserted between two cartilaginous rings in the trachea and the alveoli were lavaged 3-5 times to obtain BALF. A 1.5mL EP tube (rinsed with 200μL of 2% glacial acetic acid solution and then discarded) was filled with BALF and gently shaken. The glacial acetic acid was used to lyse the red blood cells. The BALF was centrifuged at 1500 rpm for 10 minutes at 4°C. The supernatant was discarded and the cells were resuspended in phosphate-buffered saline.

[0325] After resuspending the cells, blow the cells evenly and use a hemocytometer to count the number of eosinophils and basophils. The cell counting formula is: total cells = n / 4×10 4 × dilution factor cells / L (n = total number of cells in 4 large squares). Take the remaining mixed cells to make a cell smear and perform Swiss-Giemsa staining as follows:

[0326] (1) Take a smear and let it dry naturally;

[0327] (2) Add 2-3 drops of Swiss-Giemsa complex stain to cover the entire specimen smear and stain for 1-2 minutes;

[0328] (3) Add an equal amount of 0.01 M phosphate buffer solution (pH 6.4-6.8) dropwise, shake gently, mix thoroughly with Swiss-Giemsa staining solution, and stain for 3-5 minutes;

[0329] (4) Wash with water, dry, and examine under a microscope.

[0330] 5) Statistical Analysis: Data are presented as mean ± standard error. One-way ANOVA was used for significance analysis. When the number of groups was small or did not conform to a normal distribution, the Mann-Whitney nonparametric test was used. P < 0.05 was considered significant.

[0331] Experimental results:

[0332] 1. Behavioral investigation of OVA-induced asthmatic mice

[0333] As shown in Figure 9, the number of nose scratching in the model group mice increased but not significantly compared to the control group (Figure 9; Table 17). Compared to the model group, the number of nose scratching in mice in all groups after drug administration showed an improvement trend (Figure 9; Table 17).

[0334] Table 17 Statistics of the number of times mice scratched their noses on day 28

[0335] Note: Mean ± standard error; One-way ANOVA

[0336] Figure 10 shows the number of coughs and asthma in mice on day 28. Compared with the control group, the number of coughs and asthma in the model group was significantly increased ( *** p<0.001; Figure 10; Table 18). Compared with the model group, the number of cough and wheezing in the positive drug group was significantly reduced ( *** p<0.001; Figure 10; Table 18). Compared with the model group, the test compound of formula (III) at a dose of 45 mg / kg and 90 mg / kg was administered orally twice a day, and the number of coughs and asthma in mice was significantly reduced (* p<0.05; Figure 10; Table 18). When the compound of formula (III) was administered orally at a dose of 180 mg / kg twice a day, the number of coughs and wheezing in mice was significantly reduced ( *** p<0.001; Figure 10; Table 18).

[0337] Table 18 Statistics of the number of coughs and asthma in mice on day 28

[0338] Note: mean ± standard error; * p<0.05vs Model; *** p<0.001 vs Model; One-way ANOVA

[0339] Figure 11 shows the incubation period of cough and asthma in mice on day 28. Compared with the control group, the incubation period of cough and asthma in mice in the model group was significantly reduced ( *** p<0.001; Figure 11; Table 19). Compared with the model group, the latency period of cough and asthma in the positive drug group was significantly increased ( *** p<0.001; Figure 11; Table 19). Compared with the model group, the test compound of formula (III) at a dose of 45 mg / kg and 90 mg / kg, administered orally twice a day, showed an increasing trend in the incubation period of cough and asthma in mice (p>0.05, Figure 11; Table 19). The test compound of formula (III) at a dose of 180 mg / kg, administered orally twice a day, showed a significant increasing trend in the incubation period of cough and asthma in mice ( *** p<0.001; Figure 11; Table 19).

[0340] Table 19 Statistics of the incubation period of cough and asthma in mice on day 28

[0341] Note: mean ± standard error; *** p<0.001 vs Model; One-way ANOVA

[0342] Figure 12 shows the behavioral score of asthma severity. The behavioral score was performed by observing the mice's breathing and scratching of their noses. Compared with the control group, the asthma severity of the mice in the model group was significantly increased ( *** p<0.001; Figure 12; Table 20). Compared with the model group, the asthma severity of mice in the positive drug group was significantly reduced ( *** p<0.001; Figure 12; Table 20). Compared with the model group, the test compound of formula (III) at a dose of 45 mg / kg, administered orally twice a day, did not significantly change the asthma severity of the mice (Figure 12; Table 20). The test compound of formula (III) at a dose of 90 mg / kg, administered orally twice a day, significantly reduced the asthma severity of the mice ( **p<0.01; Figure 12; Table 20). When the compound of formula (III) was administered orally at a dose of 180 mg / kg twice a day, the asthma severity of mice was significantly reduced ( *** p<0.001; Figure 12; Table 20).

[0343] Table 20 Statistics of behavioral scores of mice panting degree on day 28

[0344] Note: mean ± standard error; ** p < 0.01 vs Model; *** p<0.001 vs Model; One-way ANOVA

[0345] 2. BALF Cell Analysis of OVA-Induced Asthma Mice

[0346] Figure 13 shows the total cell count in BALF. Compared with the control group, the total cell count in BALF of the model group mice increased significantly ( ** p<0.01; Figure 13; Table 21). Compared with the model group, the total number of cells in the BALF of mice in the positive drug group was significantly reduced ( * p<0.05; Figure 13; Table 21). Compared with the model group, the total cell count in the BALF of mice was reduced in a dose-dependent manner when the compound of formula (III) was administered orally at a dose of 45 mg / kg, 90 mg / kg, and 180 mg / kg twice a day (Figure 13; Table 21). Among them, the total cell count in the BALF of mice was significantly reduced when the compound of formula (III) was administered orally at a dose of 180 mg / kg twice a day ( * p<0.05; Figure 13; Table 21).

[0347] Table 21 Statistics of total cell counts in mouse BALF on day 28

[0348] Figure 14 shows the number of eosinophils in BALF. Compared with the control group, the number of eosinophils in BALF of mice in the model group increased significantly ( *** p<0.001; Figure 14; Table 22). Compared with the model group, the number of eosinophils in the BALF of mice in the positive drug group was significantly reduced ( * p<0.05; Figure 14; Table 22). Compared with the model group, the compound of formula (III) was administered orally at a dose of 180 mg / kg twice a day, and the number of eosinophils in the BALF of mice was significantly reduced ( ** p<0.01; Figure 14; Table 22).

[0349] Table 22 Statistics of eosinophil counts in mouse BALF on day 28

[0350] Figure 15 shows the number of basophils in BALF. Compared with the control group, the number of basophils in BALF of mice in the model group increased significantly ( ** p<0.01; Figure 15; Table 23). Compared with the model group, the number of basophils in the BALF of mice in the positive drug group showed a trend of decrease (p>0.05, Figure 15; Table 23). The test compound of formula (III) at a dose of 180 mg / kg, administered orally twice daily, showed a greater reduction in the number of basophils in the BALF of mice (p>0.05, Figure 15; Table 23).

[0351] Table 23 Statistics of basophil counts in mouse BALF on day 28

[0352] Test conclusion:

[0353] Ovalbumin (OVA) was used to induce BALB / c mice to establish an asthma model. The experimental results showed that compared with the blank group, the number of coughs in the model group mice was significantly increased, the latency period of cough and asthma was significantly prolonged, the behavioral score of asthma severity was significantly increased, and the number of total cells, eosinophils, and basophils in BALF were significantly increased, indicating that the asthma model was successfully induced.

[0354] Compared with the model group, the number of coughs and wheezes in the mice in the positive drug group was significantly reduced. When the compound of formula (III) was administered orally at doses of 45 mg / kg, 90 mg / kg and 180 mg / kg twice a day, the number of coughs and wheezes in the mice was significantly reduced.

[0355] Compared with the model group, the latency of cough and asthma in mice in the positive drug group was significantly increased. When the compound of formula (III) was administered orally at doses of 45 mg / kg and 90 mg / kg twice daily, the latency of cough and asthma in mice showed an increasing trend. When the compound of formula (III) was administered orally at a dose of 180 mg / kg twice daily, the latency of cough and asthma in mice was significantly increased.

[0356] Compared with the model group, the asthma severity score of the mice in the positive drug group was significantly reduced. When the compound of formula (III) was administered orally at a dose of 90 mg / kg and 180 mg / kg twice a day, the asthma severity score of the mice was significantly reduced.

[0357] Compared with the model group, the total cell number in the BALF of mice in the positive drug group was significantly reduced. When the test compound of formula (III) was administered orally at doses of 45 mg / kg, 90 mg / kg and 180 mg / kg twice a day, the total cell number in the BALF of mice decreased in a dose-dependent manner.

[0358] Compared with the model group, the number of eosinophils in the BALF of mice in the positive drug group was significantly reduced. When the compound of formula (III) was administered orally at a dose of 180 mg / kg twice a day, the number of eosinophils in the BALF of mice was significantly reduced.

[0359] Compared with the model group, the number of basophils in the BALF of mice in the positive drug group showed a decreasing trend. When the compound of formula (III) was administered orally at a dose of 180 mg / kg twice a day, the number of basophils in the BALF of mice decreased more.

[0360] In summary, the compound of formula (III) demonstrated excellent efficacy in the OVA-induced asthma model in mice: significantly reducing the number of coughs, cough and wheeze latency, and cough and wheeze scores; and significantly reducing the number of total cells, eosinophils, and basophils in the BALF of asthmatic mice. These experimental results suggest that the compound of formula (III) can improve symptoms in asthma models and has potential for treating asthma.

Claims

1. Use of a RASP inhibitor in the preparation of a medicament for treating chronic cough, asthma and alcoholic liver disease, wherein the RASP inhibitor comprises a compound represented by formula (II) or a pharmaceutically acceptable salt or crystal form thereof, Wherein, selected from a single bond or a double bond; T1, T2, T3 and T4 are each independently selected from N, C or CR1; T5 is selected from C, CR5 or C═O; T6 is selected from C, CR6 or N; T7 is selected from N or CR7; When T5 is selected from C═O and T6 is selected from N, selected from a single bond; L is selected from a single bond, -O-, -S-, -NR2- or -(CR3R4)- n -; each R1 is independently selected from H, F, Cl, Br, I, OH or NH2; R2 is selected from H, or C alkyl optionally substituted by 1, 2 or 3 Rs a alkyl 1-3 optionally substituted by 1, 2 or 3 Rs R3 or R4 are each independently selected from H, F, Cl, Br, I, OH, NH2, CN, or C b alkyl optionally substituted with 1, 2 or 3 R 1-3 groups; R 5、 R6 or R7 is independently selected from H, F, Cl, Br or I; n is selected from 1, 2 or 3; R a or R b are each independently selected from H, F, Cl, Br, I, OH, NH2, CN or CH3.

2. The application according to claim 1, wherein the alcoholic liver disease includes alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis or liver cirrhosis.

3. The application according to claim 2, wherein The application is the application of the compound of formula (II) or a pharmaceutically acceptable salt or crystal form thereof in the preparation of a medicament for treating alcoholic fatty liver.

4. The application according to claim 2, characterized in that, The application is the application of the compound of formula (II) or a pharmaceutically acceptable salt or crystal form thereof in the preparation of a medicament for treating alcoholic hepatitis.

5. The application according to claim 2, wherein The application is the application of the compound of formula (II) or a pharmaceutically acceptable salt or crystal form thereof in the preparation of a medicament for treating alcoholic liver fibrosis.

6. The application according to claim 2, characterized in that, The application is the application of the compound of formula (II) or a pharmaceutically acceptable salt or crystal form thereof in the preparation of a medicament for treating liver cirrhosis.

7. The application according to claim 1, characterized in that In the compound of formula (II), R2 is selected from H, CH3 or CH2CH3, and the CH3 or CH2CH3 is optionally substituted by 1, 2 or 3 R a substituents.

8. The application according to claim 7, characterized in that, In the compound of formula (II), R2 is selected from H, CH3 and CH2CH3.

9. The application according to any one of claims 1, 7, and 8, characterized in that In the compound of formula (II), R3 and R4 are each independently selected from H, F, Cl, Br, I, OH, NH2, CN, CH3 or CH2CH3, and the CH3 or CH2CH3 is optionally substituted with 1, 2 or 3 R b substituents.

10. The application according to claim 9, wherein In the compound of formula (II), R3 and R4 are each independently selected from H, F, Cl, Br, I, OH, NH2, CN, CH3 or CH2CH3.

11. The application according to claim 10, wherein In the compound of formula (II), L is selected from a single bond, -O-, -S-, -NH-, -(CH2)2- or -CH2-.

12. The application according to any one of claims 1, 7 to 11, characterized in that, The compound of formula (II) or a pharmaceutically acceptable salt or crystal form thereof is selected from Wherein, T3 and T4 are each independently selected from N or CR1; R1 and L are as defined in any one of claims 1, 7 to 11.

13. The application according to claim 12, wherein The compound of formula (II) or a pharmaceutically acceptable salt or crystal form thereof is selected from Wherein, R1 and L are as defined in claim 12.

14. The application according to claim 1, wherein, The compound of formula (II) or a pharmaceutically acceptable salt or crystal form thereof is selected from:

15. The application according to claim 14, characterized in that The following compound of formula (III) or a pharmaceutically acceptable salt or crystal form thereof:

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