A benzopentazocine compound, a preparation method therefor and medical use thereof

Abstract

Provided are a benzopentazocine compound, a preparation method therefor, and medical uses. Specifically provided are a benzopentazocine compound as shown in formula I, or a pharmaceutically acceptable salt, isomer, isotopically labeled compound, prodrug, polymorph, or solvate thereof, a preparation method therefor, and an application thereof in preventing and / or treating a disease related to abnormal HIF-2 signaling pathways.

Description

[0001] This application is based on and claims priority to CN application No. 202211040448.7, filed on August 29, 2022, the disclosure of which is incorporated herein in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of medicinal chemistry, in particular to a benzopentazocane compound, a preparation method and medical use thereof. BACKGROUND

[0003] Kidney is an important organ of human body, in addition to having the basic function of urinary system, it can also secrete erythropoietin (EPO) to promote erythropoiesis, and is the main place of EPO production. The incidence of chronic kidney disease (CKD) is more than 10% in the world, which has become a global public health problem. Renal anemia is one of the main complications of CKD, which is mainly caused by insufficient EPO production after kidney damage. At present, the main treatment for renal anemia is to inject recombinant human EPO (rhEPO) and its related products, i.e. erythropoiesis-stimulating agents (ESAs). ESAs can increase the hemoglobin level of patients with renal anemia, reduce the demand for blood transfusion of patients, and greatly improve the quality of life of patients. However, clinical studies have shown that injection of rhEPO in patients with CKD can lead to a higher risk of cardiovascular disease and mortality, and the price of rhEPO is relatively high, so its application has limitations. Therefore, it is of great significance to find oral, easily absorbed, low-cost and low-side-effect ESAs.

[0004] Another strategy for treating renal anemia is to inhibit the ubiquitination pathway of hypoxia-inducible factor (HIF) 2a by small molecule inhibitors of prolyl hydroxylase domain proteins (PHDs) to promote the expression of EPO. Studies have found that the activity of HIF in CKD patients may be inhibited by factors such as oxidative stress and uremia, thereby leading to renal anemia. Clinical data show that PHD inhibitors such as Roxadustat, Vadadustat and Daprodustat can effectively increase the level of hemoglobin in the blood of patients. At present, Roxadustat has been approved as a class I new drug for listing. Since PHD inhibitors are mostly structural analogues of 2-oxoglutarate (2-OG), they may affect the activity of other enzymes in the body that depend on 2-OG, so the clinical pharmacological mechanism of such compounds needs further study.

[0005] The reported HIF-2a agonist (Wu DL et al. Nat. Chem. Biol. 2019, 15, 367) can directly act on the HIF-2a target, activate the HIF-2 pathway, and positively regulate the protein expression level of the downstream gene EPO. However, it is known that the agonist can only activate the HIF-2 pathway at a relatively high compound concentration. Therefore, it is of great clinical value and significance to develop a better HIF-2a agonist with better activity. SUMMARY

[0006] The present application provides a benzopentazole compound as shown in formula I, or a pharmaceutically acceptable salt, isomer, isotopically labeled compound, prodrug, polymorph or solvate thereof and a preparation method thereof. The compound can activate the HIF-2a signal pathway and has a significant regulatory effect on the transcription or expression of the downstream genes (such as EPO, VGEF, Glut1, NDRG1) of HIF-2a.

[0007] Specifically, in the first aspect, the present application provides a benzopentazole compound as shown in formula I, or a pharmaceutically acceptable salt, isomer, isotopically labeled compound, prodrug, polymorph or solvate thereof:

[0008]

[0009] wherein,

[0010] X, Y and Z are each independently selected from C, N, O and S(O) m wherein m = 0, 1 or 2;

[0011] each R1is independently selected from H, D, halo, cyano, C 1-6 alkyl, haloC 1-6 alkyl, C 1-6 alkoxy, haloC 1-6 alkoxy, -NRR', C 3-8 cycloalkyl and 3-8 membered heterocyclyl, each C 1-6 alkyl, haloC 1-6 alkyl, C 1-6 alkoxy, haloC 1-6 alkoxy, -NRR', C 3-8 cycloalkyl and 3-8 membered heterocyclyl, each independently substituted with one or more (e.g., 1, 2, 3, 4, or 5) groups independently selected from oxo (=0), halo, -NHCOR, C 1-6 alkyl, haloC 1-6 alkyl, C 1-6 alkoxy and haloC 1-6 alkoxy;

[0012] each R and R' is independently selected from hydrogen, halo, C 1-6 alkyl, haloC 1-6 alkyl, C 1-6 alkoxy, haloC 1-6 alkoxy, C 3-8 cycloalkyl and 3-8 membered heterocyclyl;

[0013] R2is selected from H, D, halo, cyano, C 1-6 alkyl, haloC 1-6 alkyl, C 1-6 alkoxy and haloC 1-6 alkoxy;

[0014] R3is absent or selected from H, D, C 1-6 alkyl, C 3-6 cycloalkyl, 6-10 membered aryl and 6-10 membered aryl-C 1-4 alkylene;

[0015] n = 1, 2, 3, 4, or 5.

[0016] In some embodiments, X in Formula I is N.

[0017] In some embodiments, X in Formula I is N; R3is selected from H, D, C 1-4 alkyl, C 3-6 cycloalkyl, 6-10 membered aryl and 6-10 membered aryl-C 1-2 alkylene, preferably, R3is selected from H, D, C 1-2 alkyl, C 3-6cycloalkyl, phenyl and benzyl, preferably R3is selected from H, D, methyl, cyclopropyl and phenyl, preferably R3is selected from H, D and cyclopropyl, preferably R3is H or D.

[0018] In some embodiments, -X(R3)- in Formula I is -NH-.

[0019] In some embodiments, each R1is independently selected from halogen (e.g., fluorine, chlorine, bromine), cyano, C 1-6 alkyl, haloC 1-4 alkyl, C 1-4 alkoxy, -NRR' and 3-8 membered heterocyclyl, each of which is independently substituted with 1, 2, or 3 groups independently selected from oxo (=0), halogen, -NHCOR, and C 1-6 alkyl, haloC 1-4 alkyl, C 1-4 alkoxy, -NRR' and 3-8 membered heterocyclyl, each of which is independently substituted with 1, 2, or 3 groups independently selected from oxo (=0), halogen, -NHCOR, and C 1-6 alkyl; R and R' are each independently selected from hydrogen, C 1-4 alkyl and C 3-6 cycloalkyl; n = 1, 2, 3, 4, or 5.

[0020] In some embodiments, each R1is independently selected from fluorine, cyano, C 1-4 alkyl, haloC 1-2 alkyl, C 1-2 alkoxy, -NRR' and 3-6 membered heterocyclyl, each of which is independently substituted with 1, 2, or 3 groups independently selected from oxo (=0), fluorine, -NHCOR, and C 1-4 alkyl, -NRR' and 3-6 membered heterocyclyl, each of which is independently substituted with 1, 2, or 3 groups independently selected from oxo (=0), fluorine, -NHCOR, and C 1-2 alkyl; R and R' are each independently selected from hydrogen, C 1-2 alkyl and C 3-6 cycloalkyl; n = 1, 2, 3, 4, or 5.

[0021] In some embodiments, each R1is independently selected from fluorine, cyano, methyl, isopropyl, difluoromethyl, trifluoromethyl, methoxy, pyrrolidinyl, piperidinyl, (R)-2-methylpyrrolidinyl, (S)-2-methylpyrrolidinyl, (R)-3-fluoropyrrolidinyl, (S)-3-fluoropyrrolidinyl, 3,3-difluoropyrrolidinyl, butyrolactamyl, succinimidy, morpholinyl, 4H-[1,2,4]triazolyl; n = 1 or 2.

[0022] In some embodiments, each R1is independently selected from cyano, trifluoromethyl, methoxy, pyrrolidinyl, piperidinyl, (R)-3-fluoropyrrolidinyl, (S)-3-fluoropyrrolidinyl, and morpholinyl; n = 1 or 2.

[0023] In some embodiments, each R1is independently selected from cyano, trifluoromethyl, and methoxy, n = 1 or 2.

[0024] In some embodiments, R2is selected from H, D, halogen (e.g., fluorine, chlorine, bromine), C 1-4 alkyl, and C 1-4 alkoxy.

[0025] In some embodiments, R2is selected from H, D, fluorine, chlorine, bromine, C 1-2 alkyl, and C 1-2 alkoxy.

[0026] In some embodiments, R2is selected from H, D, fluorine, chlorine, bromine, and methoxy.

[0027] In some embodiments, R2is selected from H, D, fluorine, and bromine.

[0028] In some embodiments, R2is selected from H and bromine.

[0029] In some embodiments, R2is H or D.

[0030] In some embodiments, R2is fluorine.

[0031] In some embodiments, the compound has the structure of Formula II:

[0032]

[0033] wherein each group is as defined in any of the preceding embodiments.

[0034] In some embodiments, X is selected from N, S, and O;

[0035] each R1is independently selected from H, D, halogen, cyano, C 1-6 alkyl, haloC 1-6 alkyl, C 1-6 alkoxy, and haloC 1-6 alkoxy;

[0036] R2is selected from H, D, halogen, cyano, C 1-6 alkyl, haloC 1-6 alkyl, C 1-6 alkoxy, and haloC 1-6 alkoxy;

[0037] R3is selected from H, D, C 1-6 alkyl, C 3-6 cycloalkyl, 6-10 membered aryl, and 6-10 membered aryl-C 1-4 alkylene;

[0038] n = 1, 2, 3, 4, or 5.

[0039] In some embodiments, X is N in the compound of Formula II.

[0040] In some embodiments, each R1is independently selected from halogen (e.g., fluorine, chlorine, bromine), cyano, haloC 1-4 alkyl, and C 1-4 alkoxy; n = 1, 2, 3, 4, or 5.

[0041] In some embodiments, each R1is independently selected from fluorine, cyano, difluoromethyl, trifluoromethyl, and methoxy, n = 1 or 2. 1-2 alkyl, and C 1-2 alkoxy; n = 1, 2, 3, 4, or 5.

[0042] In some embodiments, each R1is independently selected from fluorine, cyano, difluoromethyl, trifluoromethyl, and methoxy, n = 1 or 2.

[0043] In some embodiments, each R1is trifluoromethyl, n is 2.

[0044] In some embodiments, R2is selected from H, D, halogen (e.g., fluorine, chlorine, bromine), C 1-4 alkyl, and C 1-4 alkoxy.

[0045] In some embodiments, R2is selected from H, D, fluorine, chlorine, bromine, C 1-2 alkyl, and C 1-2 alkoxy.

[0046] In some embodiments, R2is selected from H, D, fluorine, chlorine, bromine, and methoxy.

[0047] In some embodiments, R2is H or D.

[0048] In some embodiments, R3is selected from H, D, C 1-4 alkyl, C 3-6 cycloalkyl, 6-10 membered aryl, and 6-10 membered aryl-C 1-2 alkylene.

[0049] In some embodiments, R3is selected from H, D, C 1-2 alkyl, C 3-6 cycloalkyl, phenyl, and benzyl.

[0050] In some embodiments, R3is selected from H, D, methyl, cyclopropyl, and phenyl.

[0051] In some embodiments, R3in the compound of Formula II is H or D.

[0052] In some embodiments, the compound has the structure of Formula III:

[0053]

[0054] each group is as defined in any of the preceding embodiments.

[0055] In some embodiments, R2is halogen, preferably fluorine.

[0056] In some embodiments, each R1is independently selected from halogen, cyano, C 1-6 alkyl, haloC 1-6 alkyl, C 1-6 alkoxy, haloC 1-6 alkyl, -NRR', C 3-8 cycloalkyl, and 3-8 membered heterocyclyl, each of which is independently substituted with one or more (e.g., 1, 2, 3, 4, or 5) groups independently selected from oxo (=0), halogen, -NHCOR, C 1-6 alkyl, haloC 1-6 alkyl, C 1-6 alkoxy, haloC 1-6 alkyl, -NRR', C 3-8 cycloalkyl, and 3-8 membered heterocyclyl, each of which is independently substituted with one or more (e.g., 1, 2, 3, 4, or 5) groups independently selected from oxo (=0), halogen, -NHCOR, C 1-6 alkyl, haloC 1-6 alkyl, C 1-6 alkoxy, and haloC 1-6 alkoxy;

[0057] R and R' are each independently selected from hydrogen, halogen, C 1-6 alkyl, haloC 1-6 alkyl, C 1-6 alkoxy, haloC 1-6 alkyl, C 3-8 cycloalkyl, and 3-8 membered heterocyclyl;

[0058] n = 1, 2, or 3.

[0059] In some embodiments, each R1is independently selected from fluorine, cyano, C 1-4 alkyl, haloC 1-2 alkyl, C 1-2 alkoxy, -NRR', and 3-6 membered heterocyclyl, each of which is independently substituted with 1, 2, or 3 groups independently selected from oxo (=0), fluorine, -NHCOR, and C 1-4 alkyl, -NRR', and 3-6 membered heterocyclyl, each of which is independently substituted with 1, 2, or 3 groups independently selected from oxo (=0), fluorine, -NHCOR, and C 1-2 alkyl, and C 1-2 alkyl, and C3-6 cycloalkyl; n = 1, 2, 3, 4 or 5;

[0060] In some embodiments, each R1is independently selected from the group consisting of fluoro, cyano, methyl, isopropyl, difluoromethyl, trifluoromethyl, methoxy, pyrrolidinyl, piperidinyl, (R)-2-methylpyrrolidinyl, (S)-2-methylpyrrolidinyl, (R)-3-fluoropyrrolidinyl, (S)-3-fluoropyrrolidinyl, 3,3-difluoropyrrolidinyl, butyrolactamyl, succinimidy, morpholinyl, 4H-[1,2,4]triazolyl; n = 1 or 2;

[0061] In some embodiments, each R1is independently selected from the group consisting of trifluoromethyl, methoxy, pyrrolidinyl, piperidinyl, (R)-3-fluoropyrrolidinyl, (S)-3-fluoropyrrolidinyl and morpholinyl; n = 1 or 2.

[0062] In some embodiments, X in Formula III is N; R3is selected from the group consisting of H, D, C 1-4 alkyl, C 3-6 cycloalkyl, 6-10 membered aryl and 6-10 membered aryl-C 1-2 alkylene, preferably R3is selected from the group consisting of H, D, C 1-2 alkyl, C 3-6 cycloalkyl, phenyl and benzyl, preferably R3is selected from the group consisting of H, D, methyl, cyclopropyl and phenyl, preferably R3is selected from the group consisting of H, D and cyclopropyl, preferably R3is H or D.

[0063] In some embodiments, -X(R3)- in Formula III is -NH-.

[0064] In some embodiments, X is selected from the group consisting of N, S and O;

[0065] each R1is independently selected from the group consisting of H, D, halogen, cyano, C 1-6 alkyl, haloC 1-6 alkyl, C 1-6 alkoxy and haloC 1-6 alkoxy;

[0066] R3is fluoro;

[0067] R3is selected from the group consisting of H, D, C 1-6 alkyl, C 3-6 cycloalkyl, 6-10 membered aryl and 6-10 membered aryl-C 1-4 alkylene;

[0068] n = 1, 2, 3, 4 or 5.

[0069] In some embodiments, the compound is selected from the group consisting of:

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078] In a second aspect, the present application provides a method of preparing a benzazepine compound of any one of the preceding, or a pharmaceutically acceptable salt, isomer, isotopically-labeled compound, prodrug, polymorph, or solvate thereof.

[0079] When Y = O, X = N, and Z = C, the method comprises the steps of:

[0080]

[0081] Step 1 : reacting compound 1a with hydroxylamine hydrochloride to obtain compound 1b;

[0082] Step 2: halogenating compound 1b to obtain compound 1c;

[0083] Step 3: reacting compound 1c with compound B to obtain compound 1d;

[0084] Step 4: cyclizing compound 1d to obtain the compound of Formula II;

[0085] wherein, R 1 , R 2 , R 3 and n are as defined in any one of the first aspect of the present application.

[0086] In some embodiments, the present application provides a method of preparing a benzisoxazole compound of Formula I (i.e., X-R 3 NH), or a pharmaceutically acceptable salt, isomer, isotopically-labeled compound, prodrug, polymorph, or solvate thereof, comprising the steps of:

[0087]

[0088] Step 1 : reacting compound 1a with hydroxylamine hydrochloride to obtain compound 1b;

[0089] Step 2: halogenating compound 1b to obtain compound 1c;

[0090] Step 3: reacting compound 1c with compound B to obtain compound 1d;

[0091] Step 4: cyclizing compound 1d to obtain the compound of formula I';

[0092] wherein, R 1 , R 2 and n are as defined in any one of the first aspects of the application.

[0093] In some specific embodiments, step 1 comprises the following operations:

[0094]

[0095] Compound 1a (5 mmol) was dissolved in 17 mL of an ethanol solution, then hydroxylamine hydrochloride aqueous solution (5 mL) was added to the reaction system, and the reaction liquid was reacted at room temperature overnight. After the reaction was completed, part of the solvent was concentrated under reduced pressure, 50 mL of water was added to dilute the reaction liquid, and extraction was performed with ethyl acetate. The organic layer was washed with water in sequence, then washed with saturated NaCl solution, dried with anhydrous sodium sulfate, and concentrated under reduced pressure in a vacuum to obtain a colorless oily liquid.

[0096] In some specific embodiments, step 2 comprises the following operations:

[0097]

[0098] Compound 1b (5 mmol) was dissolved in anhydrous DMF (12 mL), then NCS (10 mmol) was added to the reaction system (added in two portions with an interval of 10 min), and the reaction was performed at room temperature. The reaction was completed after about 0.5 h, then 80 mL of water was added to the reaction liquid to dilute the reaction liquid, and extraction was performed with ethyl acetate. The organic layer was washed with a large amount of water, saturated sodium chloride solution, and dried with anhydrous sodium sulfate, and concentrated under reduced pressure in a vacuum to obtain a white solid.

[0099] In some specific embodiments, step 3 comprises the following operations:

[0100]

[0101] Step 3: Compound lc (4.6 mmol) was dissolved in anhydrous THF (14 mL), then substituted aniline (6.9 mmol) was added, the reaction was refluxed and stirred, after about 48 h, TLC monitoring showed that there was no raw material left, after the reaction was completed, 60 mL of water was added to dilute the reaction solution, and ethyl acetate was used for extraction, the organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then separated by silica gel column chromatography to obtain solid compound Id.

[0102] In some specific embodiments, step 4 comprises the following operations:

[0103]

[0104] Step 4: Compound Id (0.71 mmol) was dissolved in NMP (7 mL), potassium tert-butoxide was added, and stirred at 100°C for about 2.5 h until the reaction was completed. After the reaction was completed, 40 mL of water was added to dilute the reaction solution, and ethyl acetate was used for extraction, the organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then separated by silica gel column chromatography to obtain solid compound, i.e. benzisoxazole compound.

[0105] When R2 = F, Y = SO2, and X = N, the method comprises the following steps:

[0106]

[0107] Step 1: Compound 2a was reacted with sodium nitrate, copper chloride, and sulfur dioxide to obtain compound 2b;

[0108] Step 2: Compound 2b was ammoniated to obtain compound 2c;

[0109] Step 3: Compound 2c was chlorinated to obtain compound 2d;

[0110] Step 4: Compound 2d was reacted with compound B' to obtain the compound of formula III';

[0111] wherein, R 1 , R 3 and n are as defined in any one of the preceding.

[0112] In some specific embodiments, step 1 comprises the following operations:

[0113]

[0114] To a solution of 2-amino-6-fluorobenzoate (1.8 g, 10.6 mmol) in acetic acid (6 mL) and hydrochloric acid (20 mL) was added an aqueous solution of sodium nitrate (1.46 g, 21.2 mmol) in water (5.0 mL) at 00C. The mixture was stirred at 00C for 1 h, then copper dichloride (1.86 g, 13.7 mmol) was added at 00C, followed by stirring with sulfur dioxide in acetic acid (31.8 mL, 63.6 mmol, 2.0 M). The mixture was gradually warmed to 250C and stirred at this temperature for 12 h. The mixture was poured into ice water (500 mL) and extracted twice with ether (250 mL each). The organic layer was concentrated and the residue was purified by silica gel chromatography eluting with ether / petroleum ether (0-10% ether in 20 min) to give 2-(chlorosulfonyl)-6-fluorobenzoate as a yellow solid (1.5 g, 52% yield).

[0115] In some particular embodiments, step 2 comprises the following operations:

[0116]

[0117] A mixture of 2-(chlorosulfonyl)-6-fluorobenzoate (1.5 g, 5.9 mmol), aqueous ammonia (2.07 g, 59 mmol) and tetrahydrofuran (40.0 mL) was heated to 40 °C and stirred at this temperature for 12 h. The mixture was concentrated and filtered after washing with 1 N hydrochloric acid (20 mL) to give 4-fluorobenzo[d]isothiazol-3(2H)-one 1,1-dioxide as a white solid (980 mg, 83% yield).

[0118] In some particular embodiments, step 3 comprises the following operations:

[0119]

[0120] To a solution of 4-fluorobenzo[d]isothiazol-3(2H)-one 1,1-dioxide (980 mg, 4.87 mmol) and N,N-dimethylformamide (73.0 mg, 1.0 mmol) was added dioxane (20.0 mL), followed by dropwise addition of sulfoxyl chloride (869 mg, 7.3 mmol) at 250C. The mixture was heated to 100 °C and stirred at this temperature for 12 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by recrystallization using petroleum ether / dichloromethane (PE / DCM = 5:1, 40 mL) to give 3-chloro-4-fluorobenzo[d]isothiazole 1,1-dioxide as a yellow solid (720 mg, 64% yield).

[0121] In some particular embodiments, step 4 comprises the following operations:

[0122]

[0123] First, 3-chloro-4-fluorobenzo[d]isothiazole 1,1-dioxide (1,5 g, 6,83 mmol) was dissolved in 20 mL of dichloromethane and the solution was cooled to 0 degrees Celsius, then 2-(pyrrolidin-1-yl)aniline (1.11 g, 6.83 mmol) and ethyldiisopropylamine (1.32 g, 10.25 mmol) were slowly added to the above solution and warmed to room temperature, the reaction was stirred at room temperature for 2 hours, liquid quality monitoring raw material was completely converted, 10 mL of distilled water was added to the reaction liquid, after separation, the organic phase was washed with distilled water (5 mL*2), the organic phase was collected and dried with anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product, which was purified by silica gel column chromatography to obtain 4-fluoro-3-((2-(pyrrolidin-1-yl)phenyl)amino)benzo[d]isothiazole 1,1-dioxide (1,32 g, 53% yield).

[0124] In a third aspect, the present application provides a pharmaceutical composition comprising the benzofive-membered nitrogen ring compound or a pharmaceutically acceptable salt, isomer, isotopically labeled compound, prodrug, polymorph or solvate thereof of any one of the first aspect, and optionally a carrier and / or excipient.

[0125] In the pharmaceutical composition, the compound can be a single effective ingredient, or can be combined with other active ingredients to form a combined preparation. The other active ingredients can be other various drugs that can be used for chronic kidney disease, renal anemia, dyslipidemia and hypercholesterolemia. The content of the active ingredients in the composition is usually a safe and effective amount, which should be adjustable for those skilled in the art. In some embodiments, the pharmaceutical composition further comprises a prolyl hydroxylase inhibitor. In some embodiments, the prolyl hydroxylase inhibitor is selected from the group consisting of Roxadustat, Vadadustat, Deferasirox, Enasidenib and Molidustat.

[0126] In a fourth aspect, the present application provides a vaccine adjuvant comprising the benzofive-membered nitrogen ring compound or a pharmaceutically acceptable salt, isomer, isotopically labeled compound, prodrug, polymorph or solvate thereof of any one of the first aspect, or the pharmaceutical composition of any one of the third aspect.

[0127] In a fifth aspect, the present application provides an immunogenic or immunostimulatory composition comprising the vaccine adjuvant of any one of the fourth aspect.

[0128] The compounds of the present application or pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof have significant enhancing activity on the expression of EPO, VGEF, Glut1, NDRG1 and other mRNA and protein downstream of hypoxia-inducible factor HIF-2. Therefore, the present application further provides the use of the compounds in medicine.

[0129] In another aspect, the present application provides the use of the benzofive nitrogen ring compound of any one of the first aspect or a pharmaceutically acceptable salt, isomer, isotopically labeled compound, prodrug, polymorph or solvate thereof, or the pharmaceutical composition of any one of the third aspect in the manufacture of a medicament for use as a hypoxia-inducible factor HIF-2 agonist, an immunomodulator, or for the treatment and / or prevention of:

[0130] (1) a HIF-2 signaling pathway abnormality related disease; preferably, the HIF-2 signaling pathway abnormality related disease is selected from chronic kidney disease, dyslipidemia, high cholesterol, or a disease and / or disorder associated with low EPO or EPO receptor activity, or characterized by EPO deficiency or red blood cell deficiency or defect; preferably, the disease and / or disorder associated with low EPO or EPO receptor activity, or characterized by EPO deficiency or red blood cell deficiency or defect is an ischemic disease, for example, renal anemia, chronic renal anemia, anemia of reduced erythropoiesis, ischemia-induced stroke or myocardial ischemia;

[0131] (2) kidney failure, hypertension, coronary heart disease, or aging.

[0132] In another aspect, the present application provides a method of activating HIF-2 signaling pathway of a cell of a subject, comprising the step of contacting the cell with the benzofive nitrogen ring compound of any one of the first aspect or a pharmaceutically acceptable salt, isomer, isotopically labeled compound, prodrug, polymorph or solvate thereof, or the pharmaceutical composition of any one of the third aspect.

[0133] In another aspect, the present application provides an immunomodulation method, comprising administering to a subject in need thereof an effective amount of the benzofive nitrogen ring compound of any one of the first aspect or a pharmaceutically acceptable salt, isomer, isotopically labeled compound, prodrug, polymorph or solvate thereof, or the pharmaceutical composition of any one of the third aspect.

[0134] In another aspect, the present application provides a method of treating and / or preventing a disease and / or disorder, comprising administering to a subject in need thereof an effective amount of the benzofive nitrogen ring compound of any one of the first aspect or a pharmaceutically acceptable salt, isomer, isotopically labeled compound, prodrug, polymorph or solvate thereof, or the pharmaceutical composition of any one of the third aspect, the disease and / or disorder being as previously described.

[0135] In a first aspect, the present application provides a benzoguaazepine compound of Formula (I), or a pharmaceutically acceptable salt, isomer, isotopically-labeled compound, prodrug, polymorph, or solvate thereof:

[0136] In some embodiments, the compound or a pharmaceutically acceptable salt, isomer, isotopically-labeled compound, prodrug, polymorph, or solvate thereof stimulates (e.g., initiates or enhances) an immune response in a subject.

[0137] In some embodiments, the immune response is a non-specific immune response.

[0138] In some embodiments, the immune response is an antigen-specific immune response.

[0139] In some embodiments, the immune response comprises activation of B cells, activation of T cells, production of antibodies, and / or release of cytokines.

[0140] In another aspect, the present application provides the use of the immunogenic or immunostimulatory composition of any one of the fifth aspect as a vaccine or in the manufacture of a vaccine.

[0141] Definitions of terms

[0142] In the present application, unless otherwise indicated, the scientific and technical terms used herein have the meanings that would be commonly understood by one of ordinary skill in the art. Also, the experimental procedures for cell culture, biochemistry, nucleic acid chemistry, immunology, etc. used herein are conventional in the respective fields unless otherwise indicated. Also, for better understanding of the present application, the definitions and explanations of the relevant terms are provided below.

[0143] As used herein, the term "salt" should be understood to mean any form of active compound used in this invention, wherein the compound may be in ionic form or charged or coupled to a counterion (cation or anion) or in solution. The term "pharmaceutically acceptable salt" generally refers to any salt that is physiologically tolerable when used in a suitable manner for therapeutic purposes (particularly when applied or used in humans and / or mammals). Pharmaceutically acceptable salts of the compounds of this invention include their acid addition salts and base addition salts. Suitable acid addition salts are formed by acids that form pharmaceutically acceptable salts. Suitable base addition salts are formed by bases that form pharmaceutically acceptable salts. Examples include hydrochlorides, trifluoroacetates and other similar salts. For a review of suitable salts, see Stahl and Wermuth's "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of this invention are known to those skilled in the art.

[0144] As used herein, the term "isomer" refers to compounds with the same molecular formula but different structures, including structural isomers, stereoisomers, and electronic tautomers. It should be understood that the scope of this invention covers any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) of the said isomers or mixtures thereof.

[0145] The present invention also includes pharmaceutically acceptable isotopically labeled compounds, identical to the compounds of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the compounds of the present invention include (but are not limited to) isotopes of hydrogen (e.g., deuterium). 2 H), tritium ( 3 H); carbon isotopes (e.g., H); 11 C 13 C and 14 C); isotopes of chlorine (e.g. 36 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); isotopes of phosphorus (e.g., O); phosphorus isotopes ... 32 P); and isotopes of sulfur (e.g., ...35 S).

[0146] The present application further includes within its scope prodrugs of the compounds, which are certain derivatives of the compounds of the application which can have little or no pharmacological activity themselves but, upon administration, are converted by metabolic processes into compounds of the present application that are pharma- cologically active. The term "prodrug" is used in its broadest sense and encompasses those derivatives that are biotransformed into the compounds of the present application in vivo. Typically such prodrugs will be functional derivatives of the compounds that readily undergo interconversion into the desired therapeutically active compound in vivo. Further information on the use of prodrugs can be found in "Pro-drugs as Novel Delivery Systems", Vol. 14 of the A.C.S. Symposium Series; T. Higuchi and V. Stella. Prodrugs of the present application can be prepared by replacing appropriate functionalities present in the compounds of the application with certain moieties known to those skilled in the art as "pro-moieties" (for example as described in "Design of Prodrugs", H. Bundgaard (Elsevier, 1985).

[0147] The present application encompasses all possible crystalline forms or polymorphs of the compounds, which can be a single polymorph or a mixture of more than one polymorph in any ratio.

[0148] As used herein, the term "solvate" generally refers to any form of the active compounds according to the present application in combination with another molecule (typically a polar solvent) by non-covalent bonds, specifically can be hydrates and alcoholates, for example methanolate, including but not limited to, without limitation.

[0149] As used herein, the term "alkyl" generally refers to saturated aliphatic hydrocarbon groups, which can be straight-chained or branched. For example, C 1-6 Alkyl generally refers to alkyl groups comprising 1, 2, 3, 4, 5, or 6 carbon atoms, including C 1-4 Alkyl, C 1-2 Alkyl, C

[0150] As used herein, the term "haloalkyl" generally refers to groups resulting from the substitution of one or more (for example, 1, 2, 3, 4, 5, or 6, etc.) halogens (for example, fluorine, chlorine, bromine, or iodine) on the alkyl groups described previously. For example, haloC 1-6 Alkyl, C 1-4 Alkyl, C 1-2 Alkyl, C 1-6alkyl, difluoroC 1-6 alkyl, trifluoroC 1-6 alkyl, fluoroC 1-4 alkyl, difluoroC 1-4 alkyl, trifluoroC 1-4 alkyl, fluoroC 1-2 alkyl, difluoroC 1-2 alkyl, trifluoroC 1-2 alkyl, etc. Specific examples of haloalkyl groups include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, etc.

[0151] As used herein, the term "alkoxy" refers to a group having the structure alkyl-O-, wherein alkyl is as previously defined. For example, C 1-6 alkoxy, C 1-4 alkoxy, C 1-2 alkoxy, etc. Specific examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, etc.

[0152] As used herein, the term "haloalkoxy" refers generally to a group resulting from the substitution of one or more (e.g., 1, 2, 3, 4, 5, or 6, etc.) halogens (e.g., fluorine, chlorine, bromine, or iodine) on an alkoxy group as previously described. For example, haloC 1-6 alkoxy, haloC 1-4 alkoxy, haloC 1-2 alkoxy, fluoroC 1-6 alkoxy, difluoroC 1-6 alkoxy, trifluoroC 1-6 alkoxy, fluoroC 1-4 alkoxy, difluoroC 1-4 alkoxy, trifluoroC 1-4 alkoxy, fluoroC 1-2 alkoxy, difluoroC 1-2 alkoxy, trifluoroC 1-2 alkoxy, etc.

[0153] As used herein, the term "cycloalkyl" refers to a saturated monocyclic or polycyclic (such as bicyclic) hydrocarbon radical. For example, "C 3-6 cycloalkyl". Specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0154] As used herein, the term "heterocyclyl" refers to a saturated or unsaturated ring structure whose ring atoms are composed of carbon atoms and at least one (e.g., 1, 2, or 3) heteroatom selected from nitrogen, oxygen, and sulfur. The term "3-8 membered heterocyclyl" refers to a heterocyclyl group having 3 to 8 ring atoms, including 3-membered heterocyclyl, 4-membered heterocyclyl, 5-membered heterocyclyl, 6-membered heterocyclyl, 7-membered heterocyclyl, or 8-membered heterocyclyl, including azepinyl, oxepinyl, and the like. Specific examples include, but are not limited to, pyrrolidinyl, piperidinyl, morpholinyl, butyrolactamyl, succinimideyl, 4H-[l,2,4]triazolyl, or l,3-oxazepin-2-one, and the like.

[0155] As used herein, the term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group that has lost one hydrogen atom. For example, "6-10 membered aryl." Specific examples include, but are not limited to, phenyl or naphthyl.

[0156] As used herein, the term "substituted" means that one or more (e.g., one, two, three, or four) hydrogens on the designated atom is / are replaced with a selection from the indicated group, provided that the designated atom's normal valency is not exceeded, and that the substitution results in a stable compound. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds. If a substituent is described as "optionally substituted" then the substituent can be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more of a list of substituents, then one or more hydrogens on the carbon (to the extent of any hydrogen present) can be replaced with an independently selected optional substituent, alone or in combination. If a nitrogen of a substituent is described as being optionally substituted with one or more of a list of substituents, then one or more hydrogens on the nitrogen (to the extent of any hydrogen present) can each be replaced with an independently selected optional substituent. If a substituent is described as "independently selected from" a group of substituents, then each substituent is selected independently of the other(s). Thus, each substituent can be the same or different from another (other) substituent.

[0157] As used herein, the term "one or more" means 1 or more than 1, e.g., 2, 3, 4, 5, or 10, under reasonable conditions.

[0158] As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to a carrier and / or excipient that is compatible, philosophically and / or physiologically, with the subject and the active ingredient, is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusting agents, surfactants, ionic strength enhancers, agents to maintain osmotic pressure, agents to retard absorption, diluents, adjuvants, preservatives, and the like. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents to retard absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thiomersal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. In certain embodiments, the pharmaceutically acceptable carrier or excipient is a sterile isotonic aqueous or nonaqueous solution (e.g., an

[0159] As used herein, the term "vaccine" is a composition that is administered to produce or artificially increase immunity to a particular antigen. As used herein, the terms "immunogenic composition," "immunostimulatory composition" are compositions that are capable of producing an immune response in vivo when administered to an individual. Thus, it is to be understood that the terms "immunogenic composition," "immunostimulatory composition," and "vaccine" are synonymous terms. In some embodiments, the individual is preferably a mammal, more preferably a human, although other mammals are also possible, e.g., the composition can induce immunity in cattle (including cows), sheep, goats, or horses, or in a pet, such as a dog or cat.

[0160] As used herein, the term "vaccine adjuvant," "adjuvant" refers to a substance that is capable of modifying or enhancing an immune response to an antigen. In other words, the immune response to an antigen in the presence of an adjuvant can be higher or different than when the adjuvant is not present (including when the response is modified, e.g., the subset of T cells activated in the presence of an adjuvant is different than the subset activated in the absence of the adjuvant).

[0161] As used herein, the term "prevention" includes inhibition and delay of the onset of a disease, and includes not only prevention before the development of a disease, but also prevention of the recurrence of a disease after treatment.

[0162] As used herein, the term "treatment" means reversing, alleviating, or inhibiting the progress of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

[0163] As used herein, the term "effective amount" means the amount that is sufficient to achieve the desired prophylactic or therapeutic effect, e.g., the amount that achieves a reduction in one or more symptoms associated with the disease being treated.

[0164] Dosage regimens can be adjusted to provide the optimum desired response. For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is to be noted that dosage values can vary with the type and severity of the condition to be alleviated, and can include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

[0165] The amount administered depends on the subject being treated, the severity of the disorder or condition, the rate of the administration, the disposition of the compound and the judgment of the prescribing physician. In general, an effective dose is in the range of from about 0.0001 to about 50 mg per kg body weight per day, e.g. about 0.01 to about 10 mg / kg / day (single or divided doses). For a 70 kg human, this would amount to about 0.007 mg / day to about 3500 mg / day, e.g. about 0.7 mg / day to about 700 mg / day. In some instances, dosage levels less than the lower limit of the aforesaid range can be more than adequate, while in other cases still larger doses can be employed without causing any harmful side effects, provided that they are divided into several small doses for administration throughout the day.

[0166] As used herein, the term "subject" includes a human or non-human animal. Exemplary human subjects include a human subject afflicted with a disease (e.g., a disease described herein) (referred to as a patient) or a normal subject. "Non-human animals" in the present application include all vertebrates, e.g., non-mammals (e.g., birds, amphibians, reptiles), and mammals, e.g., non-human primates, domestic animals, and / or laboratory animals, e.g., sheep, dogs, cats, cows, pigs, etc.

[0167] The compound provided by the present application can be adapted to any form of administration, which can be oral or parenteral administration, for example, can be pulmonary, nasal, rectal and / or intravenous injection, more specifically can be intradermal, subcutaneous, intramuscular, intra-articular, intraperitoneal, pulmonary, oral, sublingual, nasal, transdermal, vaginal, oral or parenteral administration.

[0168] Advantages of the invention

[0169] The benzopentazole compound has better HIF-2 agonistic activity than the control compound, has significant enhancing activity on the expression of EPO, VGEF, Glut1, NDRG1 and other mRNAs and proteins downstream of HIF-2, and has good industrialization prospects. BRIEF DESCRIPTION OF DRAWINGS

[0170] The accompanying drawings, which are included to provide a further understanding of the present application, form a part of the present application and illustrate the illustrative embodiments of the present application and together with the description serve to explain the present application. In the drawings:

[0171] Figure 1 The figure shows the effect of the compound of the present application on the transcription of NDRG1, a gene downstream of HIF-2α in 786-O cell line.

[0172] Figure 2 The figure shows the effect of the compound of the present application on the transcription of VEGFA, a gene downstream of HIF-2α in 786-O cell line.

[0173] Figure 3 The figure shows the effect of the compound of the present application on the expression of EPO, a gene downstream of HIF-2α. DETAILED DESCRIPTION

[0174] The embodiments of the present application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions are not specified in the examples, which are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, which are all conventional products that can be obtained by purchase.

[0175] Example 1 Preparation of N-(3,5-bis(trifluoromethyl)phenyl)benzo[d]isoxazol-3-amine (1)

[0176]

[0177] Synthesis of compound 1b: Compound 1a (621 mg, 5 mmol) was dissolved in 17 mL of ethanol solution, then hydroxylamine hydrochloride (3.48 g, 5 mL) was added to the reaction system. The reaction solution was reacted at room temperature overnight, and after the reaction was completed, the solvent was concentrated under reduced pressure. 50 mL of water was added to dilute the reaction solution and extracted with ethyl acetate. The organic layer was washed with water, then washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure in a vacuum to obtain a colorless oily liquid (700 mg, yield 100%).

[0178]

[0179] Synthesis of compound 1c: Compound 1b (700 mg, 5 mmol) was dissolved in anhydrous DMF (12 mL), then NCS (1.34 g, 10 mmol) was added to the reaction system (added in two portions, with an interval of 10 min), and reacted at room temperature for about 0.5 h. After the reaction was completed, 80 mL of water was added to dilute the reaction solution, and extracted with ethyl acetate. The organic layer was washed with a large amount of water, saturated sodium chloride solution, and dried over anhydrous sodium sulfate, and concentrated under reduced pressure in a vacuum to obtain a white solid (800 mg, yield 92%).

[0180]

[0181] Synthesis of compound 1d: Compound 1c (800 mg, 4.6 mmol) was dissolved in anhydrous THF (14 mL), then 3,5-bistrifluoromethylaniline (1.58 g, 6.9 mmol) was added, and the reaction solution was refluxed and stirred, and after about 48 h, TLC monitoring showed that there was no raw material left. After the reaction was completed, 60 mL of water was added to dilute the reaction solution, and extracted with ethyl acetate, and the organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and then separated by silica gel column chromatography (PE:EA = 50:1-20:1) to obtain solid compound 1d, yield 16%. 1 H-NMR (500 MHz, CDC13) δ (ppm): 7.79-7.61 (bra, 1H), 7.55 (dt, J = 8.0, 4.0 Hz, 1H), 7.53-7.46 (m, 1H), 7.44 (s, 1H), 7.31-7.27 (m, 1H), 7.08-6.94 (m, 3H). 13 C-NMR (125 MHz, CDC13) δ (ppm): 160.73, 158.73, 146.70, 140.52, 132.75, 132.22 x 2, 130.82, 125.02, 122.85 x 2, 119.54, 118.03, 116.36, 116.16.

[0182]

[0183] Synthesis of compound 1 : Compound 1d (260 mg, 0.71 mmol) was dissolved in NMP (7 mL) and potassium tert-butoxide was added. The reaction was stirred at 100 °C for about 2.5 h until it was completed. After the reaction was completed, 40 mL of water was added to the reaction solution to dilute the reaction solution, and extraction was performed with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure in vacuo. After this, column chromatography was performed using silica gel (PE:EA = 50:1) to isolate a white solid compound (272 mg, yield 57%). 1 H-NMR (500 MHz, CDC13) δ (ppm): 8.11 (d, J = 1.5 Hz, 2H), 7.66-7.58 (m, 2H), 7.57-7.51 (m, 2H), 7.35 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 6.88 (s, 1H). 13 C-NMR (125 MHz, CDC13) δ (ppm): 162.21, 154.31, 141.11, 132.63 x 2, 130.80, 124.23, 123,20 x 2, 119.21, 117.42, 115.76, 110.46, 110.44. HRMS (ESI): m / z [M+H] + calcd for C 1s H9F6N2O 347.0619; found 347.0614.

[0184] Example 2 Preparation of N-(3,5-dimethoxyphenyl)benzo[d]isoxazol-3-amine (2)

[0185]

[0186] Synthesis of compound 2d: Compound 1c (865 mg, 5 mmol) was dissolved in anhydrous THF (14 mL), and then 3,5-dimethoxyaniline (1.1 g, 7.5 mmol) was added. The reaction solution was refluxed and stirred for about 48 h until TLC monitoring showed that no starting material remained. After the reaction was completed, 60 mL of water was added to the reaction solution to dilute the reaction solution, and extraction was performed with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. After this, column chromatography was performed using silica gel to isolate a solid compound 2d.

[0187]

[0188] Synthesis of compound 2: Compound 2d (290 mg, 1 mmol) was dissolved in NMP (7 mL) and potassium tert-butoxide was added. The reaction was stirred at 100 °C for about 2.5 h until it was completed. After the reaction was completed, 40 mL of water was added to the reaction solution to dilute the reaction solution, and extraction was performed with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure in vacuo. After this, column chromatography was performed using silica gel (PE:EA = 50:1) to obtain a white solid compound (162 mg, yield 60%). 1 H-NMR (500 MHz, CDC13) δ (ppm): 8.11 (dd, J = 8.0, 2.0 Hz, 1H), 7.66-7.59 (m, 2H), 7.40 (dd, J = 8.0, 2.0 Hz), 7.32 (s, 2H), 6.89 (s, 1H), 6.21 (s, 1H), 3.9 (s, 6H). 13 C-NMR (125 MHz, CDC13) δ (ppm): 164.9, 160.6, 160.3, 147.1, 144.4, 130.6, 123.0, 122.2, 121.9, 109.6, 91.7, 91.3, 90.4, 55.3, 55.8. HRMS (ESI): m / z [M+H] + calcd for C 15 H 15 N2O3 270.1004; found 270.1006.

[0189] Example 3 Preparation of N-(3,5-difluorophenyl)benzo[d]isoxazol-3-amine (3)

[0190]

[0191] Synthesis of compound 3d: Compound 1c (865 mg, 5 mmol) was dissolved in anhydrous THF (14 mL), and then 3,5-difluoroaniline (0.97 g, 7.5 mmol) was added. The reaction solution was refluxed and stirred, and after about 48 h, TLC monitoring showed that no starting material remained. After the reaction was completed, 60 mL of water was added to the reaction solution to dilute the reaction solution, and extraction was performed with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. After this, column chromatography was performed using silica gel to obtain a solid compound 3d at a yield of 24%.

[0192]

[0193] Synthesis of compound 3: Compound 3d (532 mg, 2 mmol) was dissolved in NMP (15 mL) and potassium tert-butoxide was added. The reaction was stirred at 100 °C for about 2.5 h until it was completed. After the reaction was completed, 40 mL of water was added to the reaction solution to dilute the reaction solution, and extraction was performed with ethyl acetate. The organic layer was washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure in vacuo. After this, column chromatography was performed using silica gel to isolate a solid compound (290 mg, yield 60%). 1 H-NMR (500 MHz, CDC13) δ (ppm): 8.32 (dd, J = 8.0, 2.0 Hz), 8.00 (m, 1H), 7.72-7.53 (m, 3H), 6.88 (s, 1H), 6.79 (s, 1H). 13 C-NMR (125 MHz, CDC13) s ) δ (ppm): 165.3, 158.3, 158.1, 147.1, 145.6, 130.6, 123.0, 122.2, 121.9, 109.6, 100.4, 100.4, 94.7. HRMS (ESI): m / z [M+H] + calcd for C 13 H9F2N2O 246.0605; found 246.0609.

[0194] Example 4 Preparation of N-(3-trifluoromethylphenyl)benzo[d]isoxazol-3-amine (4)

[0195]

[0196] Synthesis of compound 4d: Compound 1c (800 mg, 4.6 mmol) was dissolved in anhydrous THF (14 mL), and then 3-trifluoromethylaniline (1.1 g, 6.9 mmol) was added. The reaction solution was refluxed and stirred, and after about 48 h, TLC monitoring revealed that no starting material remained. After the reaction was completed, 60 mL of water was added to the reaction solution to dilute the reaction solution, and extraction was performed with ethyl acetate. The organic layer was washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then column chromatography was performed using silica gel to isolate a solid compound 4d, yield 23%.

[0197]

[0198] Synthesis of compound 4: Compound 4d (298 mg, 1 mmol) was dissolved in NMP (10 mL) and potassium tert-butoxide was added. The reaction was stirred at 100 °C for about 2.5 h until it was completed. After the reaction was completed, 40 mL of water was added to the reaction solution to dilute the reaction solution, and extraction was performed with ethyl acetate. The organic layer was washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure in vacuo. After this, column chromatography was performed using silica gel to isolate a white solid compound (164 mg, yield 59%). 1 H-NMR (500 MHz, CDC13) δ (ppm): 8.11 (dd, J = 8.0, 2.0 Hz), 7.66-7.58 (m, 2H), 7.63-7.56 (m, 2H), 7.23 (m, 1H), 7.13 (m, 1H), 6.86 (s, 1H). 13 C-NMR (125 MHz, CDC13) δ (ppm): 165.0, 147.1, 141.1, 131.8, 130.6, 129.8, 124.1, 123.0, 122.2, 121.9, 121.2, 116.0, 115.3, 108.9. HRMS (ESI): m / z [M+H] + calcd for C 14 H10F3N2O 278.0667; found 278.0663.

[0199] Example 5 Preparation of N-(3-cyanophenyl)benzo[d]isoxazol-3-amine (5)

[0200]

[0201] Synthesis of compound 5d: Compound 1c (0.5 g, 3 mmol) was dissolved in anhydrous THF (20 mL), and then 3-cyanophenylamine (0.53 g, 4.5 mmol) was added. The reaction solution was refluxed and stirred, and after about 48 h, TLC monitoring showed that no starting material remained. After the reaction was completed, 60 mL of water was added to the reaction solution to dilute the reaction solution, and extraction was performed with ethyl acetate. The organic layer was washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then column chromatography was performed using silica gel to isolate a solid compound 5d (153 mg) at a yield of 20%.

[0202]

[0203] Synthesis of compound 5: Compound 5d (153 mg, 0.6 mmol) was dissolved in NMP (5 mL) and potassium tert-butoxide was added. The reaction was stirred at 100 °C for about 2.5 h until it was completed. After the reaction was completed, 40 mL of water was added to the reaction solution to dilute the reaction solution, and extraction was performed with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure in vacuo. After this, column chromatography was performed on silica gel to isolate a white solid compound (80 mg, yield 57%). 1 H-NMR (500 MHz, CDC13) δ (ppm): 8.32 (m, 1H), 7.91-7.71 (m, 2H), 7.52-7.48 (m, 2H), 7.29-7.23 (m, 3H), 6.86 (s, 1H). 13 C-NMR (125 MHz, CDC13) δ (ppm): 164.8, 147.4, 143.3, 130.6, 130.2, 127.8, 123.0, 122.2, 122.1, 121.9, 121.0, 118.4, 113.4, 109.6. HRMS (ESI): m / z [M+H] + calcd for C 14 H 10 N3O 235.0746; found 235.0739.

[0204] Example 6 Preparation of 3-(benzo[d]isoxazol-3-amino)-5-(trifluoromethyl)benzonitrile (6)

[0205]

[0206] Synthesis of compound 6d: Compound 1c (865 mg, 5 mmol) was dissolved in anhydrous THF (20 mL), and then 3-cyano-5-trifluoromethylaniline (1.4 g, 7.5 mmol) was added. The reaction solution was refluxed and stirred, and after about 48 h, TLC monitoring showed that no starting material remained. After the reaction was completed, water was added to the reaction solution to dilute the reaction solution, and extraction was performed with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then column chromatography was performed on silica gel to isolate a solid compound 6d (306 mg) at a yield of 19%.

[0207]

[0208] Synthesis of compound 6: Compound 6d (306 mg, 0.95 mmol) was dissolved in NMP (10 mL) and potassium tert-butoxide was added. The reaction was stirred at 100 °C for about 2.5 h until it was completed. After the reaction was completed, water was added to the reaction solution to dilute the reaction solution, and the reaction solution was extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure in vacuo. The residue was separated by column chromatography on silica gel to obtain compound 6 as a white solid (138 mg, 48% yield). 1 H-NMR (500 MHz, CDC13) δ (ppm): 8.32 (dd, J = 8.0, 2.0, 1H), 7.91-7.83 (m, 2H), 7.64 (dd, J = 8.0, 8.0, 1H), 7.51-7.27 (m, 3H), 6.89 (s, 1H). 13 C-NMR (125 MHz, CDC13) δ (ppm): 164.7, 147.0, 143.5, 132.5, 130.6, 124.6, 123.4, 122.2, 121.9, 120.3, 119.3, 118.6, 113.8, 109.1. HRMS (ESI): m / z [M+H] + calcd for C 15 H9F3N3O 303.0619; found 303.0614.

[0209] Example 7 Preparation of N-(3,5-bis(trifluoromethyl)phenyl)-N-methylbenzo[d]isoxazol-3-amine (7)

[0210]

[0211] Synthesis of compound 7a: 3,5-bistrifluoromethyl aniline (0.65 g, 4 mmol) was dissolved in DMF (20 mL), and potassium carbonate (1.38 g, 10 mmol) and iodomethane (0.25 mL, 4 mmol) were added. After stirring at room temperature for 2 h, it was stirred at 70 °C for 24 h. After confirming completion of the reaction by thin layer chromatography (TLC) (PE:EtOAc = 15:1), it was cooled to room temperature, poured into 80 mL of water, and extracted with 150 mL of DCM three times. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was column chromatographed (PE:EtOAc = 300:1) to obtain the target intermediate 7a (0.29 g) as a yellow oil in a yield of 30%.

[0212]

[0213] Synthesis of compound 7b: Compound 1c (138 mg, 0.8 mmol) was dissolved in dry THF (5 mL) followed by addition of compound 7a (0.29 g, 1.2 mmol) and the reaction was refluxed and stirred, TLC monitored for the absence of starting material after about 48 h. After completion of the reaction, the reaction was diluted with 60 mL of water and extracted with ethyl acetate, the organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure and purified by silica gel column chromatography to obtain compound 7b as a solid in 30% yield.

[0214]

[0215] Synthesis of compound 7: Compound 7b (91 mg, 0.24 mmol) was dissolved in NMP (3 mL) and potassium tert-butoxide was added and stirred at 100 °C for about 2.5 h. After completion of the reaction, the reaction was diluted with 10 mL of water and extracted with ethyl acetate, the organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure and purified by silica gel column chromatography to obtain compound 7 as a white solid (53 mg, 62% yield). 1 H-NMR (500 MHz, CDC13) δ (ppm): 8.11 (m, 1H), 7.66-7.58 (m, 2H), 7.57-7.51 (m, 2H), 7.35 (m, 2H), 3.31 (s, 3H). 13 C-NMR (125 MHz, CDC13) δ (ppm): 164.5, 150.0, 147.3, 132.3, 132.1, 130.6, 124.4, 124.1, 123.0, 122.4, 121.9, 112.8, 112.6, 111.5, 40.9. HRMS (ESI): m / z [M+H] + calcd for C 16 H 11 F6N2O 360.0697; found 360.0699.

[0216] Example 8 Preparation of N-(3,5-bis(trifluoromethyl)phenyl)-N- cyclopropylbenzo[d]isoxazol-3-amine (8)

[0217]

[0218] Synthesis of compound 8b: Compound 8a (200 mg, 1.2 mmol) and 3,5-ditrifluoromethylaniline (229 mg, 1 mmol) were dissolved in methanol (4 mL), and acetic acid (5 mL) was added. The mixture was stirred at 80 °C for 2.5 h, and sodium cyanoborohydride (126 mg, 2 mmol) was added at room temperature. The mixture was stirred at 80 °C for 3 h. After the reaction was completed, the mixture was cooled to room temperature, quenched with saturated sodium bicarbonate solution, and then extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and then subjected to column chromatography (PE:EtOAc = 120:1) to give a yellow oily substance in 55% yield. 1 H NMR (CDCl3, 500MHz) δ (ppm): 7.25 (s, 1H), 7.05 (s, 2H), 2.25 (m, 1H), 0.90-0.80 (m, 2H), 0.69-0.60 (m, 2H).

[0219]

[0220] Synthesis of compound 8c: Compound 1c (76 mg, 0.44 mmol) was dissolved in anhydrous THF (3 mL), followed by the addition of compound 8b (177 mg, 0.66 mmol). The reaction mixture was refluxed and stirred. After approximately 48 h, TLC monitoring showed no starting material remaining. After the reaction was complete, the reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography (PE:EA = 50:1-20:1) to give solid compound 8c in 25% yield.

[0221]

[0222] Synthesis of Compound 8: Compound 8c (44 mg, 0.08 mmol) was dissolved in NMP (1 mL), potassium tert-butoxide was added, and the mixture was stirred at 100 °C for approximately 2.5 h until the reaction was complete. After the reaction was complete, water was added to dilute the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under vacuum, and separated by silica gel column chromatography (PE:EA = 40:1) to give a white solid compound (22 mg, yield 67%). 1 H-NMR (500MHz, CDCl3) δ (ppm): 8.11 (m, 1H), 7.66-7.58 (m, 2H), 7.57-7.51 (m, 2H), 7.35 (m, 2H), 2.25 (m, 1H), 0.90-0.80 (m, 2H), 0.69-0.60 (m, 2H). 13C-NMR (125 MHz, CDC13) δ (ppm): 164.6, 150.0, 147.1, 132.1, 132.1, 130.6, 124.4, 124.4, 123.0, 122.2, 121.9, 112.6, 112.6, 111.5, 109.6, 40.2, 5.5, 5.2. HRMS (ESI): m / z [M+H] Calcd for C + calcd for C 1s H 13 F6N2O 386.0854; found 386.0858.

[0223] Example 9 Preparation of N-(3,5-bis(trifluoromethyl)phenyl)-N- phenylbenzo[d]isoxazol-3-amine (9)

[0224]

[0225] Synthesis of compound 9a: Palladium acetate (11 mg, 0.05 mmol) and DPPE (39 mg, 0.1 mmol) were dissolved in toluene and stirred at 110 °C for ten minutes. A mixture of iodobenzene (204 mg, 1 mmol) and 3,5-bistrifluoromethyl aniline (229 mg, 1 mmol) was dissolved in toluene (5 mL) and added to the mixture. Sodium methoxide (59.4 mg, 1.1 mmol) was finally added and the reaction was allowed to proceed for 24 h. After the reaction was completed, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. Column chromatography (PE:EtOAc = 120:1) gave an oil with a yield of 70%. 1 H NMR (500 MHz, CDC13) δ (ppm): 7.56 (s, 2H), 7.49 (s, 1H), 7.40-7.33 (m, 2H), 7.21-7.01 (m, 3H).

[0226]

[0227] Synthesis of compound 9b: Compound 1c (81 mg, 0.47 mmol) was dissolved in anhydrous THF (2 mL) and compound 9a (0.21 g, 0.7 mmol) was added. The reaction was refluxed and stirred. After about 48 h, TLC monitoring showed that no starting material remained. After the reaction was completed, water was added to the reaction mixture to dilute the reaction mixture and extract with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography on silica gel to obtain solid compound 9b with a yield of 21%.

[0228]

[0229] Synthesis of compound 9: Compound 9b (44 mg, 0.1 mmol) was dissolved in NMP (1 mL) and potassium tert-butoxide was added. The reaction was stirred at 100 °C for about 2.5 h until it was completed. After the reaction was completed, water was added to the reaction solution to dilute the reaction solution, and ethyl acetate was used to extract it. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure in a vacuum, and then separated using a silica gel column to obtain a white solid compound (27 mg, yield 65%). 1 H-NMR (500 MHz, CDC13) δ (ppm): 8.11 (m, 1H), 7.66-7.58 (m, 2H), 7.57-7.51 (m, 2H), 7.40-7.33 (m, 2H), 7.35 (m, 2H), 7.21-7.01 (m, 3H). 13 C-NMR (125 MHz, CDC13) δ (ppm): 164.7, 147.1, 143.2, 139.5, 132.3, 132.2, 130.5, 129.6, 129.8, 129.8, 129.6, 129.6, 129.6, 124.4, 124.4, 123.0, 122.2, 121.9, 121.8, 112.6, 109.6. HRMS (ESI): m / z [M+H] + calcd for C 21 H 13 F6N2O 422.0854; found 422.0849.

[0230] Example 10 Preparation of N-(3,5-bis(trifluoromethyl)phenyl)-5-bromobenzo[d]isoxazol-3-amine (10)

[0231]

[0232] Synthesis of compound 10b: Compound 10a (1 g, 5 mmol) was dissolved in 20 mL of an ethanol solution, and then hydroxylamine hydrochloride aqueous solution (3.48 g, 5 mL) was added to the reaction system. The reaction solution was allowed to react at room temperature overnight, and after the reaction was completed, the solvent was partially concentrated under reduced pressure. Water (50 mL) was added to dilute the reaction solution and ethyl acetate was used to extract it. The organic layer was washed with water, and then with saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure in a vacuum to obtain a colorless oily liquid (1.1 g, yield 100%).

[0233]

[0234] Synthesis of compound 10c: Compound 10b (1.1 g, 5 mmol) was dissolved in dry DMF (12 mL) followed by addition of NCS (1.34 g, 10 mmol) in two portions at an interval of 10 min and the reaction was allowed to proceed at room temperature for about 0.5 h. After completion of the reaction, the reaction mixture was diluted with 80 mL of water and extracted with ethyl acetate. The organic layer was washed with copious amount of water, saturated sodium chloride solution and dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a white solid (1.1 g, yield 90%).

[0235]

[0236] Synthesis of compound 10c: Compound 10b (1.1 g, 5 mmol) was dissolved in dry DMF (12 mL) followed by addition of NCS (1.34 g, 10 mmol) in two portions at an interval of 10 min and the reaction was allowed to proceed at room temperature for about 0.5 h. After completion of the reaction, the reaction mixture was diluted with 80 mL of water and extracted with ethyl acetate. The organic layer was washed with copious amount of water, saturated sodium chloride solution and dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a white solid (1.1 g, yield 90%).

[0237]

[0238] Synthesis of compound 10: Compound 10d (379 mg, 0.85 mmol) was dissolved in NMP (10 mL) followed by addition of potassium tert-butoxide and the reaction was allowed to proceed at 100 °C for about 2.5 h. After completion of the reaction, the reaction mixture was diluted with 40 mL of water and extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure in vacuum. The compound was isolated by column chromatography on silica gel to obtain compound 10 as a white solid (183 mg, yield 54%). 1 H-NMR (500 MHz, CDC13) δ (ppm): 7.57-7.51 (m, 4H), 7.35 (s, 1H), 7.11 (d, J = 8 Hz, 1H), 6.87 (s, 1H). 13 C-NMR (125 MHz, CDC13) δ (ppm): 163.8, 147.2, 140.7, 133.9. 132.2, 132.2, 127.6, 124.4, 124.5, 124.4, 119.3, 119.3, 117.6, 113.1, 111.8. HRMS (ESI): m / z [M+H] + calcd for C 15 H8BrF6N2O 423.9646; found 423.9644.

[0239] Example 11 Preparation of N-(3,5-bis(trifluoromethyl)phenyl)-5-fluorobenzo[d]isoxazol-3- amine (11)

[0240]

[0241] Synthesis of compound 11b: Compound 11a (710 mg, 5 mmol) was dissolved in 20 mL of ethanol solution, then hydroxylamine hydrochloride (3.48 g, 5 mL) was added to the reaction system. The reaction solution was reacted at room temperature overnight, and after the reaction was completed, part of the solvent was concentrated under reduced pressure. 50 mL of water was added to dilute the reaction solution and extracted with ethyl acetate. The organic layer was washed with water in turn, then washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure in vacuum to obtain a colorless oily liquid (780 mg, yield 100%).

[0242]

[0243] Synthesis of compound 11c: Compound 11b (780 mg, 5 mmol) was dissolved in anhydrous DMF (12 mL), then NCS (1.34 g, 10 mmol) was added to the reaction system (added in two times, interval 10 min), and reacted at room temperature, about 0.5 h after the reaction was completed. After the reaction was completed, 80 mL of water was added to the reaction solution to dilute the reaction solution, and extracted with ethyl acetate. The organic layer was washed with a large amount of water, saturated sodium chloride solution, and dried over anhydrous sodium sulfate, and concentrated under reduced pressure in vacuum to obtain a white solid (850 mg, yield 89%).

[0244]

[0245] Synthesis of compound 11d: Compound 11c (850 mg, 4.4 mmol) was dissolved in anhydrous THF (15 mL), then 3,5-bistrifluoromethyl aniline (1.5 g, 6.6 mmol) was added, and the reaction solution was refluxed and stirred, and after about 48 h, TLC monitoring showed that there was no raw material left. After the reaction was completed, 60 mL of water was added to the reaction solution to dilute the reaction solution, and extracted with ethyl acetate, and the organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and then separated by silica gel column chromatography (PE:EA = 50:1-20:1) to obtain solid compound 11d, yield 25%.

[0246]

[0247] Synthesis of compound 11: Compound 11d (422 mg, 1.1 mmol) was dissolved in NMP (15 mL) and potassium tert-butoxide was added. The reaction was stirred at 100 °C for about 2.5 h until it was completed. After the reaction was completed, 50 mL of water was added to dilute the reaction solution, and extraction was performed with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure in vacuum, and then separated by silica gel column chromatography to obtain a white solid (216 mg, yield 54%). 1 H-NMR (500 MHz, CDC13) δ (ppm): 7.75 (m, 1H), 7.57-7.51 (m, 2H), 7.35 (dd, J = 8.0, 8.0 Hz, 1H), 7.20-7.18 (m, 2H), 6.89 (s, 1H). 13 C-NMR (125 MHz, CDC13) δ (ppm): 160.5, 156.6, 147.1, 140.7, 132.1, 132.1, 124.4, 124.4, 123.8, 119.3, 119.3, 117.6, 113.1, 111.3, 111.2. HRMS (ESI): m / z [M+H] + calcd for C 15 H8F7N2O 364.0447; found 364.0451.

[0248] Example 12 Preparation of N-(3,5-bis(trifluoromethyl)phenyl)-5-methoxybenzo[d]isoxazol-3-amine (12)

[0249]

[0250] Synthesis of compound 12b: Compound 12a (770 mg, 5 mmol) was dissolved in 20 mL of an ethanol solution, and then an aqueous solution of hydroxylamine hydrochloride (3.48 g, 5 mL) was added to the reaction system. The reaction solution was reacted at room temperature overnight, and after the reaction was completed, the solvent was partially concentrated under reduced pressure. The reaction solution was diluted with 50 mL of water and extracted with ethyl acetate. The organic layer was washed with water, and then with saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure in vacuum to obtain a colorless oily liquid (845 mg, yield 100%).

[0251]

[0252] Synthesis of compound 12c: Compound 12b (845 mg, 5 mmol) was dissolved in dry DMF (15 mL) followed by the addition of NCS (1.34 g, 10 mmol) in two portions at an interval of 10 min. The reaction was completed in about 0.5 h at room temperature. After completion of the reaction, the reaction mixture was diluted with 80 mL of water and extracted with ethyl acetate. The organic layer was washed with copious amount of water, saturated sodium chloride solution and dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a white solid (1 g, yield 98%).

[0253]

[0254] Synthesis of compound 12d: Compound 12c (1 g, 4.9 mmol) was dissolved in dry THF (15 mL) followed by the addition of 3,5-bistrifluoromethylaniline (1.68 g, 7.35 mmol). The reaction mixture was refluxed and stirred for about 48 h, after which TLC monitoring showed no starting material left. After completion of the reaction, the reaction mixture was diluted with 60 mL of water and extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The compound 12d was isolated as a solid by column chromatography using silica gel (PE:EA = 50:1) in 22% yield.

[0255]

[0256] Synthesis of compound 12: Compound 12d (396 mg, 1.0 mmol) was dissolved in NMP (10 mL) followed by the addition of potassium tert-butoxide. The reaction was completed in about 2.5 h at 100 °C. After completion of the reaction, the reaction mixture was diluted with 40 mL of water and extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The compound 12 was isolated as a white solid by column chromatography using silica gel (PE:EA = 50:1) in 45% yield. 1 H-NMR (500 MHz, CDC13) δ (ppm): 7.82 (dd, J = 8.0, 2.0 Hz, 1H), 7.58-7.53 (m, 2H), 7.36 (s, 1H), 7.00 (dd, J = 8.0, 2.0 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H), 6.86 (s, 1H). 13 C-NMR (125 MHz, CDC13) δ (ppm): 157.2, 156.5, 147.2, 140.7, 132.1, 132.1, 124.4, 124.4, 123.2, 119.3, 119.3, 117.8, 111.3, 110.6, 109.9, 55.8. HRMS (ESI): m / z [M+H] +C 16 H 11 F7N2O2 376.0646; found 376.0648.

[0257] Example 13 Preparation of N-(3,5-bis(trifluoromethyl)phenyl)-6-bromobenzo[d]isoxazol-3- amine (13)

[0258]

[0259] Synthesis of compound 13b: Compound 13a (1 g, 5 mmol) was dissolved in 20 mL of ethanol solution, then hydroxylamine hydrochloride (3.48 g, 5 mL) was added to the reaction system. The reaction solution was reacted at room temperature overnight, and after the reaction was completed, part of the solvent was concentrated under reduced pressure. 50 mL of water was added to dilute the reaction solution and extracted with ethyl acetate. The organic layer was washed with water in turn, then washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure in vacuum to obtain a colorless oily liquid (1.08 g, yield 100%).

[0260]

[0261] Synthesis of compound 13c: Compound 13b (1.08 g, 5 mmol) was dissolved in anhydrous DMF (15 mL), then NCS (1.34 g, 10 mmol) was added to the reaction system (added in two times, interval 10 min), and reacted at room temperature, about 0.5 h after the reaction was completed. After the reaction was completed, 80 mL of water was added to the reaction solution to dilute the reaction solution, and extracted with ethyl acetate. The organic layer was washed with a large amount of water, saturated sodium chloride solution, and dried over anhydrous sodium sulfate, and concentrated under reduced pressure in vacuum to obtain a white solid (1.15 g, yield 92%).

[0262]

[0263] Synthesis of compound 13d: Compound 13c (1.15 g, 4.6 mmol) was dissolved in anhydrous THF (15 mL), then 3,5-bistrifluoromethyl aniline (1.58 g, 6.9 mmol) was added, and the reaction solution was refluxed and stirred, and after about 48 h, TLC monitoring showed that there was no raw material left. After the reaction was completed, 60 mL of water was added to the reaction solution to dilute the reaction solution, and extracted with ethyl acetate, and the organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and then separated by silica gel column chromatography (PE:EA = 50:1-20:1) to obtain solid compound 13d, yield 20%.

[0264]

[0265] Synthesis of compound 13: Compound 13d (408 mg, 0.92 mmol) was dissolved in NMP (7 mL) and potassium tert-butoxide was added. The reaction was stirred at 100 °C for about 2.5 h until it was completed. After the reaction was completed, 40 mL of water was added to dilute the reaction solution, and it was extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure in vacuum. Then, it was separated by silica gel column chromatography (PE:EA = 40:1) to obtain compound 13 as a white solid (222 mg, yield 57%). 1 H-NMR (500 MHz, CDC13) δ (ppm): 7.57-7.51 (m, 2H), 7.35-7.33 (m, 3H), 7.22 (d, J = 8.0 Hz, 1H), 6.85 (s, 1H). 13 C-NMR (125 MHz, CDC13) δ (ppm): 167.1, 147.2, 140.7, 132.1, 132.1, 126.3, 125.2, 124.4, 124.4, 124.1, 121.2, 119.4, 119.3, 113.2, 113.1. HRMS (ESI): m / z [M+H] + calcd for C 15 H8BrF6N2O 423.9646; found 423.9644.

[0266] Example 14 Preparation of N-(3,5-bis(trifluoromethyl)phenyl)-6-fluorobenzo[d]isoxazol-3-amine (14)

[0267]

[0268] Synthesis of compound 14b: Compound 14a (710 mg, 5 mmol) was dissolved in 17 mL of an ethanol solution, and then hydroxylamine hydrochloride aqueous solution (3.48 g, 5 mL) was added to the reaction system. The reaction solution was allowed to react at room temperature overnight, and after the reaction was completed, the solvent was partially concentrated under reduced pressure. The reaction solution was diluted with 50 mL of water and extracted with ethyl acetate. The organic layer was washed with water, and then with saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure in vacuum to obtain a colorless oily liquid (780 mg, yield 100%).

[0269]

[0270] Synthesis of compound 14c: Compound 14b (780 mg, 5 mmol) was dissolved in dry DMF (15 mL) followed by the addition of NCS (1.34 g, 10 mmol) in two portions at 10 min interval. The reaction was stirred at room temperature and was complete in ~0.5 h. After completion of the reaction, the reaction mixture was diluted with 80 mL of water and extracted with ethyl acetate. The organic layer was washed with copious amount of water, saturated sodium chloride solution and dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a white solid (850 mg, 89% yield).

[0271]

[0272] Synthesis of compound 14d: Compound 14c (850 mg, 4.4 mmol) was dissolved in dry THF (14 mL) followed by the addition of 3,5-bistrifluoromethylaniline (1.5 g, 6.6 mmol). The reaction mixture was refluxed and stirred for ~48 h, after which TLC monitoring showed no starting material left. After completion of the reaction, the reaction mixture was diluted with 60 mL of water and extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA = 50:1-20:1) to obtain compound 14d as a solid (26% yield).

[0273]

[0274] Synthesis of compound 14: Compound 14d (439 mg, 1.14 mmol) was dissolved in NMP (10 mL) followed by the addition of potassium tert-butoxide. The reaction mixture was stirred at 100 °C and was complete in ~2.5 h. After completion of the reaction, the reaction mixture was diluted with 40 mL of water and extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA = 50:1) to obtain compound 14 as a white solid (220 mg, 53% yield). 1 H-NMR (500 MHz, CDC13) δ (ppm): 7.57-7.51 (m, 2H), 7.35-7.31 (m, 3H), 7.02 (m, 1H), 6.88 (s, 1H), 6.87 (d, J = 8.0 Hz, 1H). 13 C-NMR (125 MHz, CDC13) δ (ppm): 166.5, 164.2, 147.2, 140.7, 132.1, 132.1, 124.4, 124.4, 123.6, 119.3, 119.3, 117.8, 113.1, 110.0, 96.9. HRMS (ESI): m / z [M+H] + calcd for C15 H s F7N2O 364.0447; found 364.0451.

[0275] Example 15 Preparation of N-(3,5-bis(trifluoromethyl)phenyl)-6-methoxybenzo[d]isoxazol-3-amine (15)

[0276]

[0277] Synthesis of compound 15b: Compound 15a (770 mg, 5 mmol) was dissolved in 17 mL of ethanol solution, then hydroxylamine hydrochloride (3.48 g, 5 mL) was added to the reaction system. The reaction solution was reacted at room temperature overnight, and after the reaction was completed, part of the solvent was concentrated under reduced pressure. 50 mL of water was added to dilute the reaction solution and extracted with ethyl acetate. The organic layer was washed with water, then washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure in vacuum to obtain a colorless oily liquid (845 mg, yield 100%).

[0278]

[0279] Synthesis of compound 15c: Compound 15b (845 mg, 5 mmol) was dissolved in anhydrous DMF (15 mL), then NCS (1.34 g, 10 mmol) was added to the reaction system (added in two times, interval 10 min), and reacted at room temperature, about 0.5 h after the reaction was completed. After the reaction was completed, 80 mL of water was added to dilute the reaction solution, and extracted with ethyl acetate. The organic layer was washed with a large amount of water, saturated sodium chloride solution, and dried over anhydrous sodium sulfate, and concentrated under reduced pressure in vacuum to obtain a white solid (964 mg, yield 95%).

[0280]

[0281] Synthesis of compound 15d: Compound 15c (964 mg, 4.6 mmol) was dissolved in anhydrous THF (15 mL), then 3,5-bistrifluoromethyl aniline (1.58 g, 6.9 mmol) was added, and the reaction solution was refluxed and stirred, and after about 48 h, TLC monitoring showed that there was no raw material left. After the reaction was completed, 60 mL of water was added to dilute the reaction solution, and extracted with ethyl acetate, and the organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and then separated by silica gel column chromatography (PE:EA=50:1-20:1) to obtain solid compound 15d, yield 20%.

[0282]

[0283] Synthesis of compound 15: Compound 15d (364 mg, 0.92 mmol) was dissolved in NMP (10 mL), potassium tert-butoxide was added, and stirred at 100 °C for about 2.5 h until the reaction was completed. After the reaction was completed, 40 mL of water was added to the reaction solution to dilute the reaction solution, and extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure in vacuum, and separated by silica gel column chromatography (PE:EA = 60:1) to obtain a white solid compound (183 mg, yield 53%). 1 H-NMR (500 MHz, CDC13) δ (ppm): 7.57-7.51 (m, 2H), 7.35-7.31 (m, 2H), 6.88 (s, 1H), 6.78-6.74 (m, 2H), 3.85 (s, 3H). 13 C-NMR (125 MHz, CDC13) δ (ppm): 165.9, 164.1, 147.1, 140.7, 132.1, 132.1, 124.4, 124.4, 122.9, 119.3, 119.3, 114.5, 113.1, 110.2, 95.5, 55.8. HRMS (ESI): m / z [M+H] + calcd for C 16 H 11 F7N2O2 376.0646; found 376.0648.

[0284] The synthesis method of Examples 16 to 46 is the same as the general synthesis method of Formula III'.

[0285] Example 16 4-Fluoro-3-((2-(pyrrolidin-1-yl)phenyl)amino)benzo[d]isothiazole-1,1-dioxide

[0286]

[0287] To a solution of 3-chloro-4-fluorobenzo[d]isothiazole 1,1-dioxide (1.5 g, 6.83 mmol, prepared according to the procedure described in the previous example 2d) in 20 mL of dichloromethane was added 2-(pyrrolidin-1-yl)aniline (1.11 g, 6.83 mmol) and ethyldiisopropylamine (1.32 g, 10.25 mmol) at 0 °C and the reaction mixture was allowed to warm to room temperature. The reaction mixture was stirred at room temperature for 2 h. The reaction was monitored by LC-MS. The reaction mixture was diluted with 10 mL of distilled water and the organic layer was separated. The organic layer was washed with distilled water (5 mL x 2). The organic layer was collected and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography on silica gel to obtain 4-fluoro-3-((2-(pyrrolidin-1-yl)phenyl)amino)benzo[d]isothiazole 1,1-dioxide (1.32 g, 53% yield).

[0288] 1 1H NMR (400 MHz, DMSO-d6) δ 10.02 (d, J = 6.8 Hz, 1H), 7.95 (p, J = 4.1 Hz, 2H), 7.84 - 7.72 (m, 2H), 7.24 (t, J = 7.8 Hz, 1H), 7.15 (t, J = 6.3 Hz, 1H), 7.03 (td, J = 7.6, 3.8 Hz, 1H), 3.33 (s, 4H), 1.90 (q, J = 6.4, 4.8 Hz, 4H). LC-MS: m / z [M+H] + 346.6

[0289] Example 17 Synthesis of N-(3,5-bis(trifluoromethyl)phenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine

[0290]

[0291] Using 3-chloro-4-fluorobenzo[d]isothiazole 1,1-dioxide and 3,5-bis(trifluoromethyl)aniline as starting materials, N-(3,5-bis(trifluoromethyl)phenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine was obtained by the synthetic procedure of step 4.

[0292] 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 8.55 (d, J = 1.6 Hz, 2H), 8.07 - 7.95 (m, 3H), 7.87 - 7.76 (m, 1H).

[0293] Example 18 Synthesis of N-(3,5-bis(methyloxy)phenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine

[0294]

[0295] Using 3-chloro-4-fluorobenzo[d]isothiazole 1,1-dioxide and 3,5-dimethoxyaniline as starting materials, the synthesis method of Step 4 was used to obtain N-(3,5-bis(methoxy)phenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine.

[0296] 1H NMR (400 MHz, DMSO-d6) δ 9.71 (s, 1H), 7.96 (q, J = 3.1, 2.3 Hz, 2H), 7.78 (dp, J = 13.4, 4.5, 3.9 Hz, 1H), 7.04 (d, J = 2.2 Hz, 2H), 6.49 - 6.43 (m, 1H), 3.78 (s, 6H).

[0297] Example 19 Synthesis of N-(3-trifluoromethyl-5-methoxyphenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine

[0298]

[0299] Using 3-chloro-4-fluorobenzo[d]isothiazole 1,1-dioxide and 3-trifluoromethyl-5-methoxyaniline as starting materials, the synthesis method of Step 4 was used to obtain (3-trifluoromethyl-5-methoxyphenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine.

[0300] 1H NMR (400 MHz, DMSO-d6) δ 10.03 (s, 1H), 7.98 (q, J = 2.9, 2.3 Hz, 2H), 7.82 (s, 1H), 7.80 (dq, J = 9.4, 4.8 Hz, 1H), 7.73 (s, 1H), 7.19 (s, 1H), 3.88 (s, 3H).

[0301] Example 20 Synthesis of N-(2-(pyrrolidin-1-yl)-3-methylphenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine

[0302]

[0303] Using 3-chloro-4-fluorobenzo[d]isothiazole 1,1-dioxide and 2-(pyrrolidin-1-yl)-3-methylphenylamine as starting materials, the synthesis method of Step 4 was used to obtain N-(2-(pyrrolidin-1-yl)-3-methylphenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine.

[0304] 1H NMR (400 MHz, DMSO-d6) δ 10.78 (d, J = 8.7 Hz, 1H), 8.28 (d, J = 8.1 Hz, 1H), 8.01 - 7.91 (m, 2H), 7.85 - 7.76 (m, 1H), 7.29 (t, J = 7.9 Hz, 1H), 7.10 (d, J = 7.7 Hz, 1H), 3.33 (s, 3H), 3.20 (d, J = 12.4 Hz, 1H), 2.34 (s, 3H), 2.06 (d, J = 12.0 Hz, 1H), 2.06 (s, 3H).

[0305] Example 21 Synthesis of N-(2-(pyrrolidin-2-one)-6-fluorophenyl)-4-fluorobenzo[d]isothiazole- 1,1-dioxide-3-amine

[0306]

[0307] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1-dioxide and 2-(pyrrolidin-2-one)-6- fluorophenylamine as starting materials, N-(2-(pyrrolidin-2-one)-6-fluorophenyl)-4- fluorobenzo[d]isothiazole-1,1-dioxide-3-amine was obtained by the synthetic method of Step 4.

[0308] 1H NMR (400 MHz, DMSO-d6) δ 9.66 (d, J = 5.1 Hz, 1H), 8.03 - 7.91 (m, 2H), 7.82 - 7.73 (m, 1H), 7.54 (td, J = 8.2, 5.9 Hz, 1H), 7.39 (d, J = 8.4 Hz, 2H), 3.93 (t, J = 6.9 Hz, 2H), 2.46 (d, J = 7.9 Hz, 2H), 2.10 (p, J = 7.4 Hz, 2H).

[0309] Example 22 Synthesis of N-(2-(pyrrolidin-1-yl)-5-trifluoromethylphenyl)-4- fluorobenzo[d]isothiazole-1,1-dioxide-3-amine

[0310]

[0311] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1-dioxide and 2-(pyrrolidin-1-yl)-5- trifluoromethylphenylamine as starting materials, N-(2-(pyrrolidin-1-yl)-5- trifluoromethylphenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine was obtained by the synthetic method of Step 4.

[0312] 1H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 7.96 (td, J = 7.7, 4.1 Hz, 1H), 7.92 (dd, J = 7.5, 1.1 Hz, 1H), 7.76 (ddd, J = 10.1, 7.9, 1.2 Hz, 1H), 7.61 (d, J = 2.4 Hz, 1H), 7.51 (dd, J = 8.8, 2.4 Hz, 1H), 6.95 (d, J = 8.8 Hz, 1H), 3.38 - 3.30 (m, 4H), 1.88 - 1.80 (m, 4H).

[0313] Example 23 Synthesis of N-(2-(pyrrolidin-1-yl)-3,5-dimethylphenyl)-4- fluorobenzo[d]isothiazole-1,1-dioxide-3-amine

[0314]

[0315] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1-dioxide and 2-(pyrrolidin-1-yl)-3,5- dimethylphenylamine as starting materials, N-(2-(pyrrolidin-1-yl)-3,5-dimethylphenyl)-4- fluorobenzo[d]isothiazole-1,1-dioxide-3-amine was obtained by the synthetic procedure of Step 4.

[0316] 1H NMR (400 MHz, DMSO-d6) δ 10.77 (s, 1H), 8.11 (s, 1H), 7.95 (s, 2H), 7.78 (d, J = 9.9 Hz, 1H), 6.90 (s, 1H), 3.16 (q, J = 5.9, 4.9 Hz, 4H), 2.31 (d, J = 15.8 Hz, 6H), 2.03 (s, 4H).

[0317] Example 24 Synthesis of N-(2-(pyrrolidin-1-yl)-5-methylphenyl)-4-fluorobenzo[d]isothiazole- 1,1-dioxide-3-amine

[0318]

[0319] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1-dioxide and 2-(pyrrolidin-1-yl)-5- methylphenylamine as starting materials, N-(2-(pyrrolidin-1-yl)-5-methylphenyl)-4- fluorobenzo[d]isothiazole-1,1-dioxide-3-amine was obtained by the synthetic procedure of Step 4.

[0320] 1H NMR (400 MHz, DMSO-d6) δ 10.00 (d, J = 7.7 Hz, 1H), 7.92 (td, J = 3.5, 2.1 Hz, 2H), 7.80 (d, J = 2.1 Hz, 1H), 7.79 - 7.69 (m, 1H), 7.13 (d, J = 8.2 Hz, 1H), 7.02 (ddd, J = 8.1, 2.0, 0.8 Hz, 1H), 3.08 - 3.01 (m, 4H), 2.28 (s, 3H), 1.92 - 1.81 (m, 4H).

[0321] Example 25 Synthesis of N-(2-(methylisobutylamino)phenyl)-4-fluorobenzo[d]isothiazole- 1,1-dioxide-3-amine

[0322]

[0323] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1-dioxide and N-isobutyl-N-methyl-1,2- phenylenediamine as starting materials, N-(2-(methylisobutylamino)phenyl)-4- fluorobenzo[d]isothiazole-1,1-dioxide-3-amine was obtained by the synthetic procedure of Step 4.

[0324] 1H NMR (400 MHz, DMSO-d6) δ 10.10 (d, J = 7.4 Hz, 1H), 8.40 (d, J = 7.7 Hz, 1H), 8.03 - 7.92 (m, 2H), 7.87 - 7.78 (m, 1H), 7.46 (dd, J = 7.8, 1.6 Hz, 1H), 7.29 (pd, J = 7.5, 1.8 Hz, 2H), 2.81 (d, J = 7.1 Hz, 2H), 2.62 (s, 3H), 1.65 (hept, J = 6.8 Hz, 1H), 0.88 (d, J = 6.6 Hz, 6H).

[0325] Example 26 Synthesis of (S)-N-(3-(2-methylpyrrolidin-1-yl)pyridin-4-yl)-4- fluorobenzo[d]isothiazole-1,1-dioxide-3-amine

[0326]

[0327] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1-dioxide and 3-(2-methylpyrrolidin)-4- pyridine as starting materials, (S)-N-(3-(2-methylpyrrolidin-1-yl)pyridin-4-yl)-4- fluorobenzo[d]isothiazole-1,1-dioxide-3-amine was obtained by the synthetic procedure of Step 4.

[0328] 1H NMR (400 MHz, DMSO-d6) δ 8.19 (d, J = 6.3 Hz, 2H), 7.93 (d, J = 5.7 Hz, 1H), 7.90 - 7.78 (m, 2H), 7.63 (t, J = 7.7 Hz, 1H), 4.04 (s, 1H), 3.47 (ddd, J = 9.5, 7.5, 5.8 Hz, 1H), 2.99 (q, J = 7.9 Hz, 1H), 2.11 (dtd, J = 12.2, 7.6, 5.3 Hz, 1H), 1.96 - 1.74 (m, 2H), 1.50 (ddt, J = 12.1, 8.6, 7.0 Hz, 1H), 0.93 (d, J = 6.0 Hz, 3H).

[0329] Example 27 Synthesis of N-(3,5-diisopropylphenyl)-4-fluorobenzo[d]isothiazole- 1,1-dioxide-3-amine

[0330]

[0331] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1-dioxide and 3,5-diisopropylaniline as starting materials, N-(3,5-diisopropylphenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine was obtained by the synthetic method of Step 4.

[0332] 1H NMR (400 MHz, DMSO-d6) δ 9.75 (d, J = 5.0 Hz, 1H), 7.97 - 7.87 (m, 2H), 7.80 - 7.68 (m, 1H), 7.41 (d, J = 1.6 Hz, 2H), 7.03 (t, J = 1.6 Hz, 1H), 2.88 (hept, J = 6.9 Hz, 2H), 1.19 (d, J = 6.9 Hz, 12H).

[0333] Example 28 Synthesis of N-(2,6-diisopropylpyridin-4-yl)-4-fluorobenzo[d]isothiazole- 1,1-dioxide-3-amine

[0334]

[0335] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1-dioxide and 2,6 diisopropyl-4- aminopyridine as starting materials, N-(2,6-diisopropylpyridin-4-yl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine was obtained by the synthetic method of Step 4.

[0336] 1H NMR (400 MHz, DMSO-d6) δ 13.24 (s, 1H), 9.84 (s, 1H), 7.98-7.95 (d, 1H), 7.79 (s, 1H), 7.56 (s, 1H), 3.03 (s, 2H), 1.27 (d, J = 6.9 Hz, 12H)

[0337] Example 29 Synthesis of N-(3-(pyrrolidin-1-yl)-4-pyridin-4-yl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine

[0338]

[0339] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1-dioxide and 3-pyrrolidin-1-yl-4- aminopyridine as starting materials, N-(3-(pyrrolidin-1-yl)pyridin-4-yl)-4- fluorobenzo[d]isothiazole-1,1-dioxide-3-amine was obtained by the synthetic method of Step 4.

[0340] 1H NMR (400 MHz, DMSO-d6) δ 8.01 (s, 1H), 7.73 (d, J = 5.9 Hz, 4H), 7.56 (s, 1H), 3.42 (s, 4H), 1.90-1.82 (m, 4H).

[0341] Example 30 Synthesis of N-(3-(pyrrolidin-1-yl)pyridin-2-yl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine

[0342]

[0343] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1-dioxide and 3-(pyrrolidin-1-yl)pyridin- 2-amine as starting materials, N-(3-(pyrrolidin-1-yl)pyridin-2-yl)-4-fluorobenzo[d]isothiazole- 1,1-dioxide-3-amine was obtained by the synthetic method of Step 4.

[0344] 1H NMR (400 MHz, DMSO-d6) δ 14.57 (s, 1H), 7.83-7.72 (m, 3H), 7.57 (t, J = 8.9 Hz, 1H), 7.26-7.14 (m, 2H), 3.53 (s, 4H), 1.90-1.78 (m, 4H).

[0345] Example 31 Synthesis of N-(2-(pyrrolidin-1-yl)-3,5-ditrifluoromethylphenyl)-4- fluorobenzo[d]isothiazole-1,1-dioxide-3-amine

[0346]

[0347] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1 -dioxide and 2-(pyrrolidin-1 -yl)-3,5- dithfluoromethylaniline as starting materials, N-(2-(pyrrolidin-1 -yl)-3,5-dithfluoromethylphenyl)- 4-fluorobenzo[d]isothiazole-1,1 -dioxide-3-amine was obtained by the synthetic method of Step 4.

[0348] 1H NMR (400 MHz, DMSO-d6) δ 10.17 (d, J = 5.8 Hz, 1H), 8.81 (d, J = 2.2 Hz, 1H), 8.05 - 7.97 (m, 2H), 7.95 (d, J = 2.2 Hz, 1H), 7.84 (ddd, J = 10.6, 6.5, 2.5 Hz, 1H), 3.28 - 3.20 (m, 4H), 2.01 - 1.92 (m, 4H).

[0349] Example 32 Synthesis of N-(2-(cyclopropanecarboxamidomethyl)phenyl)-4-fluorobenzo[d]isothiazole-1,1 -dioxide-3-amine

[0350]

[0351] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1 -dioxide and 2-(cyclopropanecarboxamidomethyl)aniline as starting materials, N-(2-(cyclopropanecarboxamidomethyl)phenyl)-4- fluorobenzo[d]isothiazole-1,1 -dioxide-3-amine was obtained by the synthetic method of Step 4.

[0352] 1H NMR (400 MHz, DMSO-d6) δ 10.48 (d, J = 4.7 Hz, 1H), 8.77 (t, J = 5.9 Hz, 1H), 7.99 - 7.88 (m, 2H), 7.80 - 7.71 (m, 1H), 7.55 (dq, J = 7.2, 1.9, 1.4 Hz, 1H), 7.45 - 7.33 (m, 3H), 4.29 (d, J = 5.8 Hz, 2H), 1.54 (tt, J = 7.7, 4.7 Hz, 1H), 0.66 - 0.52 (m, 4H).

[0353] Example 33 Synthesis of N-(3-methoxyphenyl)-4-fluorobenzo[d]isothiazole-1,1 -dioxide-3-amine

[0354]

[0355] Using 3-chloro-4-fluorobenzo[d]isothiazol-1,1-dioxide and 3-methoxyaniline as raw materials, N-(3-methoxyphenyl)-4-fluorobenzo[d]isothiazol-1,1-dioxide-3-amine was obtained through the synthesis method in step 4.

[0356] 1H NMR (400MHz, DMSO-d6) δ9.82 (s, 1H), 8.02-7.92 (m, 2H), 7.78 (ddd, J=10.3, 6 .0, 2.9Hz, 1H), 7.43-7.34 (m, 3H), 6.89 (dt, J=6.0, 2.9Hz, 1H), 3.80 (s, 3H).

[0357] Example 34 Synthesis of N-(2-(piperidin-1-yl)phenyl)-4-fluorobenzo[d]isothiazol-1,1-dioxide-3-amine

[0358]

[0359] Using 3-chloro-4-fluorobenzo[d]isothiazol-1,1-dioxide and 2-(piperidin-1-yl)aniline as raw materials, N-(2-(piperidin-1-yl)phenyl)-4-fluorobenzo[d]isothiazol-1,1-dioxide-3-amine was obtained by the synthesis method in step 4.

[0360] 1H NMR (400MHz, DMSO-d6) δ10.21 (d, J=7.2Hz, 1H), 8.45-8.39 (m, 1H), 8.03-7.92 (m, 2H), 7.85 (ddd, J=10.9, 7.1, 2.0Hz, 1H), 7.45 (dd, J=7.7, 1.6Hz, 1H), 7.29 (dtd, J=22.4, 7.6, 1.6Hz, 2H), 2.84 (t, J=5.2Hz, 4H), 1.73 (p, J=5.5Hz, 4H), 1.58 (q, J=5.8Hz, 2H).

[0361] Example 35 Synthesis of N-(3-trifluoromethylphenyl)-4-fluorobenzo[d]isothiazol-1,1-dioxide-3-amine

[0362]

[0363] Using 3-chloro-4-fluorobenzo[d]isothiazol-1,1-dioxide and 3-trifluoromethylaniline as raw materials, N-(3-trifluoromethylphenyl)-4-fluorobenzo[d]isothiazol-1,1-dioxide-3-amine was obtained through the synthesis method in step 4.

[0364] 1H NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H), 8.16 (d, J = 2.1 Hz, 1H), 8.13 - 8.06 (m, 1H), 8.04 - 7.94 (m, 2H), 7.86 - 7.64 (m, 3H)

[0365] Example 36 Synthesis of N-(2-morpholinophenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine

[0366]

[0367] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1-dioxide and 2-morpholinoaniline as starting materials, N-(2-morpholinophenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine was obtained by the synthetic method of Step 4.

[0368] 1H NMR (400 MHz, DMSO-d6) δ 10.16 (d, J = 8.7 Hz, 1H), 8.43 (dd, J = 8.1, 1.6 Hz, 1H), 8.03 - 7.93 (m, 2H), 7.85 (ddd, J = 10.8, 7.1, 1.9 Hz, 1H), 7.51 (dd, J = 7.8, 1.6 Hz, 1H), 7.33 (dtd, J = 26.4, 7.6, 1.6 Hz, 2H), 3.83 - 3.77 (m, 4H), 2.92 (d, J = 9.1 Hz, 4H).

[0369] Example 37 Synthesis of (R)-N-(2-(3-fluoropyrrolidin-1-yl)phenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine

[0370]

[0371] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1-dioxide and (R)-2-(3-fluoropyrrolidin-1-yl)aniline as starting materials, (R)-N-(2-(3-fluoropyrrolidin-1-yl)phenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine was obtained by the synthetic method of Step 4.

[0372] 1H NMR (400 MHz, DMSO-d6) δ 10.02 (d, J = 6.5 Hz, 1H), 7.95 (dd, J = 6.4, 3.3 Hz, 2H), 7.78 (ddd, J = 10.2, 6.5, 2.5 Hz, 1H), 7.71 (dd, J = 7.9, 1.6 Hz, 1H), 7.26 (td, J = 7.8, 1.6 Hz, 1H), 7.12 (dd, J = 8.2, 1.4 Hz, 1H), 7.02 (td, J = 7.6, 1.3 Hz, 1H), 5.37 (dq, J = 54.8, 3.0 Hz, 1H), 3.64 - 3.31 (m, 3H), 3.20 (dt, J = 8.9, 6.0 Hz, 1H), 2.27 - 2.09 (m, 2H)

[0373] Example 38 Synthesis of (S)-N-(2-(3-fluoropyrrolidin-l-yl)phenyl)-4-fluorobenzo[d]isothiazole-l,l-dioxide-3-amine

[0374]

[0375] Using 3-chloro-4-fluorobenzo[d]isothiazole-l,l-dioxide and (S)-2-(3- fluoropyrrolidin-l-yl)aniline as starting materials, (S)-N-(2-(3-fluoropyrrolidin-l- yl)phenyl)-4-fluorobenzo[d]isothiazole-l,l-dioxide-3-amine was obtained by the synthetic procedure of Step 4.

[0376] 1H NMR (400 MHz, DMSO-d6) δ 10.02 (s, 1H), 7.95 (dd, J = 6.4, 3.3 Hz, 2H), 7.84 - 7.68 (m, 2H), 7.26 (ddd, J = 8.6, 7.4, 1.6 Hz, 1H), 7.12 (dd, J = 8.3, 1.4 Hz, 1H), 7.02 (td, J = 7.6, 1.4 Hz, 1H), 5.37 (dq, J = 54.7, 3.4, 2.9 Hz, 1H), 3.62 - 3.35 (m, 3H), 3.25 - 3.15 (m, 1H), 2.28 - 2.08 (m, 2H)

[0377] Example 39 Synthesis of N-(2-(pyrrolidin-l-yl)-6-fluorophenyl)-4-fluorobenzo[d]isothiazole-l,l-dioxide-3-amine

[0378]

[0379] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1 -dioxide and 2-(pyrrolidin-1 -yl)-6- fluorobenzeneamine as starting materials, N-(2-(pyrrolidin-1 -yl)-6-fluorophenyl)-4- fluorobenzo[d]isothiazole-1,1 -dioxide-3-amine was obtained by the synthetic method of Step 4.

[0380] 1H NMR (400 MHz, DMSO-d6) δ 9.97 (d, J = 4.2 Hz, 1H), 7.99 - 7.86 (m, 2H), 7.73 (ddd, J = 10.0, 8.0, 1.1 Hz, 1H), 7.19 (td, J = 8.3, 6.7 Hz, 1H), 6.56 (dddd, J = 8.2, 5.1, 3.9, 1.2 Hz, 2H), 3.33 - 3.25 (m, 4H), 1.83 - 1.75 (m, 4H).

[0381] Example 40 Synthesis of N-(2-(pyrrolidin-1-yl)-3-fluorophenyl)-4-fluorobenzo[d]isothiazole-1,1 - dioxide-3-amine

[0382]

[0383] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1 -dioxide and 2-(pyrrolidin-1 -yl)-3- fluorobenzeneamine as starting materials, N-(2-(pyrrolidin-1 -yl)-3-fluorophenyl)-4- fluorobenzo[d]isothiazole-1,1 -dioxide-3-amine was obtained by the synthetic method of Step 4.

[0384] 1H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H), 8.21 (d, J = 8.3 Hz, 1H), 8.02 - 7.92 (m, 2H), 7.81 (ddd, J = 10.8, 7.3, 1.8 Hz, 1H), 7.40 (td, J = 8.4, 5.9 Hz, 1H), 7.14 (dd, J = 11.8, 8.4 Hz, 1H), 3.19 (d, J = 12.2 Hz, 4H), 2.03 - 1.94 (m, 4H)

[0385] Example 41 Synthesis of N-(4-(pyrrolidin-1-yl)pyridin-3-yl)-4-fluorobenzo[d]isothiazole-1,1 - dioxide-3-amine

[0386]

[0387] Using 3-chloro-4-fluorobenzo[d]isothiazole 1,1-dioxide and 4-(pyrrolidin-1-yl)pyridin-3-amine as starting materials, N-(4-(pyrrolidin-1-yl)pyridin-3-yl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine was obtained by the synthetic method of Step 4.

[0388] 1H NMR (400 MHz, DMSO-d6) δ 8.01 (d, J = 5.9 Hz, 1H), 7.90 (s, 1H), 7.73 (t, J = 4.7 Hz, 3H), 7.56 (t, J = 9.0 Hz, 1H), 3.42 (d, J = 13.0 Hz, 4H), 1.90 - 1.82 (m, 4H)

[0389] Example 42 Synthesis of N-(2-(4H-[1,2,4]triazol-4-yl)phenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine

[0390]

[0391] Using 3-chloro-4-fluorobenzo[d]isothiazole 1,1-dioxide and 2-(4H-[1,2,4]-triazol-4-yl)aniline as starting materials, N-(2-(4H-[1,2,4]triazol-4-yl)phenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine was obtained by the synthetic method of Step 4.

[0392] 1H NMR (400 MHz, DMSO-d6) δ 10.00 (s, 1H), 8.79 (s, 2H), 7.96 (td, J = 7.7, 4.1 Hz, 1H), 7.91 (dd, J = 7.6, 1.0 Hz, 1H), 7.84 (dd, J = 7.8, 1.4 Hz, 1H), 7.75 (ddd, J = 9.2, 8.1, 1.0 Hz, 1H), 7.71 - 7.57 (m, 3H)

[0393] Example 43 Synthesis of N-(2-(1,3-oxazinan-2-one)phenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine

[0394]

[0395] Using 3-chloro-4-fluorobenzo[d]isothiazole 1,1-dioxide and 2-(1,3-oxazinan-2-one)aniline as starting materials, N-(2-(1,3-oxazinan-2-one)phenyl)-4-fluorobenzo[d]isothiazole-1,1-dioxide-3-amine was obtained by the synthetic method of Step 4.

[0396] 1H NMR (400 MHz, DMSO-d6) δ 9.31 (d, J = 4.3 Hz, 1H), 7.99 - 7.90 (m, 2H), 7.87 (dd, J = 7.9, 1.7 Hz, 1H), 7.82 - 7.70 (m, 1H), 7.57 (dd, J = 7.8, 1.7 Hz, 1H), 7.41 (dtd, J = 22.4, 7.5, 1.7 Hz, 2H), 4.31 (s, 2H), 3.84 (s, 2H), 2.05 (h, J = 5.4 Hz, 2H).

[0397] Example 44 Synthesis of N-(2-(pyrrolidine-2,5-dione)phenyl)-4-fluorobenzo[d]isothiazole- 1,1-dioxide-3-amine

[0398]

[0399] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1-dioxide and 2-(pyrrolidine-2,5-dione)aniline as starting materials, N-(2-(pyrrolidine-2,5-dione)phenyl)-4-fluorobenzo[d]isothiazole- 1,1-dioxide-3-amine was obtained by the synthetic method of Step 4.

[0400] 1H NMR (400 MHz, DMSO-d6) δ 9.35 (d, J = 5.6 Hz, 1H), 7.95 (dd, J = 7.0, 3.2 Hz, 2H), 7.83 (dd, J = 8.1, 1.5 Hz, 1H), 7.75 (ddd, J = 9.4, 7.0, 2.1 Hz, 1H), 7.59 (td, J = 7.7, 1.6 Hz, 1H), 7.49 (td, J = 7.7, 1.5 Hz, 1H), 7.39 (dd, J = 8.0, 1.6 Hz, 1H), 2.88 - 2.66 (m, 4H).

[0401] Example 45 Synthesis of N-(2-(3,3-difluoropyrrolidine-1-yl)phenyl)-4-fluorobenzo[d]isothiazole- 1,1-dioxide-3-amine

[0402]

[0403] Using 3-chloro-4-fluorobenzo[d]isothiazole-1,1-dioxide and (2-(3,3-difluoropyrrolidine-1- yl)aniline as starting materials, N-(2-(3,3-difluoropyrrolidine-1-yl)phenyl)-4-fluorobenzo[d]isothiazole- 1,1-dioxide-3-amine was obtained by the synthetic method of Step 4.

[0404] 1H NMR (400 MHz, DMSO-d6) δ 9.90 - 9.84 (m, 1H), 8.03 (dd, J = 8.0, 1.7 Hz, 1H), 7.92 (q, J = 3.6, 2.8 Hz, 2H), 7.82 - 7.72 (m, 1H), 7.34 (dd, J = 8.0, 1.6 Hz, 1H), 7.22 (dtd, J = 24.7, 7.4, 1.6 Hz, 2H), 3.55 (t, J = 13.0 Hz, 2H), 3.33 (t, J = 7.0 Hz, 2H), 2.41 (dt, J = 14.7, 7.0 Hz, 2H).

[0405] Example 46 Synthesis of N-(2-(pyrrolidin-1-yl)-3,5-difluorophenyl)-4- fluorobenzo[d]isothiazole-1,1-dioxide-3-amine

[0406]

[0407] N-(2-(pyrrolidin-1-yl)-3,5-difluorophenyl)-4-fluorobenzo[d]isothiazole-1,1- dioxide-3-amine was obtained by the synthetic method of Step 4 using 3-chloro-4- fluorobenzo[d]isothiazole-1,1-dioxide and 2-(pyrrolidin-1-yl)-3,5-difluoroaniline as starting materials.

[0408] 1H NMR (400 MHz, DMSO-d6) δ 10.46 (s, 1H), 8.04 (d, J = 8.2 Hz, 1H), 7.99 (s, 2H), 7.82 (d, J = 9.6 Hz, 1H), 7.21 (t, J = 10.4 Hz, 1H), 3.17 (d, J = 6.0 Hz, 4H), 1.99 (d, J = 5.9 Hz, 4H).

[0409] Example 47 Effect of the compound on transcription of HIF-2a downstream genes of 786-O cell line

[0410] 1. Experimental materials

[0411] Human renal clear cell carcinoma cell 786-O (Changsheng, CS0254), RPMI1640 medium (Keygen), trypsin-EDTA (0.25%, Gibco), fetal bovine serum (Cellmax, SA211.02); Trizol reagent (Takara), reverse transcription kit (Takara), SYBR GREEN fluorescent quantitative kit (Yixingbio).

[0412] 2. Experimental method

[0413] Fluorescent quantitative PCR

[0414] 2.1 Cell culture and dosing

[0415] 786-O cells were cultured in dishes using RPMI1640 medium containing 10% fetal bovine serum, and when they reached 90% confluence, they were seeded into 12-well plates. When the cell density was about 60%, 10 μM of PT2385 or 10 μM of compound (DMSO final concentration 1‰) was dosed, and samples were collected after incubation.

[0416] 2.2 RNA extraction

[0417] RNA was extracted using the Trizol method, and the specific steps were as follows:

[0418] a. Sample homogenization: for adherent cells cultured in 12-well plates, the culture medium was removed, and the cells were washed with PBS. The PBS was removed, and Trizol reagent was added to each well. After mixing by blowing and sucking, the sample was transferred to an EP tube, and after standing at room temperature for a while, the nucleoprotein complex was completely dissociated;

[0419] b. Phase separation: chloroform was added to the cell lysate, and after vortex mixing and standing at room temperature, the sample was centrifuged at 12,000 rpm for 10 min at 4°C. At this time, the sample was divided into three phases, the upper aqueous phase, the middle phase, and the lower phenol-chloroform phase. At this time, the RNA was completely in the upper aqueous phase, which was transferred to a new EP tube;

[0420] c. RNA isolation: isopropanol was added to the EP tube, vortexed, and centrifuged at 12,000 rpm for 10 min at 4°C to collect the RNA precipitate. The supernatant was discarded, and the precipitate was washed twice with 75% ethanol, centrifuged at 12,000 rpm for 8 min at 4°C, and the supernatant was discarded. The EP tube was opened and left at room temperature to evaporate the ethanol, and the dried RNA was colorless and transparent. Sterile water was added to dissolve the RNA, and the concentration was measured.

[0421] 2.3 Reverse transcription

[0422] Table 1 Reverse transcription system

[0423]

[0424] 2.4 Fluorescent quantitative PCR

[0425] Table 2 Fluorescent quantitative PCR system

[0426]

[0427]

[0428] The expression differences of target genes between different treatment groups were compared using the ΔΔC T method. The data were processed and expressed as mean ± SEM.

[0429] The effect of the compound on the transcription of HIF-2a downstream genes VEGFA and NDRG1 in 786-O cell line was detected by qPCR.

[0430] As shown in Figure 1 and 2 , the compound of the present application can significantly enhance the transcription of VEGFA and NDRG1, wherein the effect of compounds 1, 2, 4, 5, 7, 8, 10, 12, 16, 17, 18, 19, 20, 22, 23, 27, 34, 35, 36, 37, 38 is obvious, and the agonistic effect of compounds 1, 5, 8, 10, 16, 17, 18, 19, 34, 35, 36, 37, 38 is significantly better than that of the control compound.

[0431] Effect of the compound of Example 48 on the expression of HIF-2a downstream gene EPO

[0432] 1. Experimental materials

[0433] Human hepatocellular carcinoma Hep3B cells (China Shengsheng, CS0172), DMEM medium (Keygene Biological), trypsin-EDTA (0.25%, Gibco), fetal bovine serum (Cellmax, SA211.02); enzyme-linked immunosorbent assay kit (Hengyuan Biological).

[0434] 2. Experimental method

[0435] Enzyme-linked immunosorbent assay (ELISA);

[0436] a. Human hepatocellular carcinoma Hep3B cells were cultured in a dish using DMEM medium containing 10% fetal bovine serum, and when they grew to 90%, they were inoculated into a 12-well plate;

[0437] b. When the cell density was about 60%, 10 μM of PT2385 or 10 μM of other compounds (DMSO final concentration 1 ‰) was administered, and the cells were cultured under hypoxia;

[0438] c. The culture medium was centrifuged at 4°C, 12,000 rpm for 10 min, and the supernatant was placed on ice for use;

[0439] d. Gradient dilution of standard, respectively, add standard or sample to be tested on enzyme-labeled coated plate, incubate at 37°C and wash 5 times;

[0440] e. Add 50 μL of enzyme-labeled reagent to each well, incubate at 37°C and wash;

[0441] f. Each well was quantified according to the requirements of the ELISA color developing reagent kit manual;

[0442] g、Using Excel to draw standard curve regression equation, calculate the EPO concentration of each sample. Using GraphPad Prism to draw; data processing is expressed by mean ± SEM.

[0443] Using ELISA to detect the expression of EPO protein.

[0444] As shown in Figure 3 , the compounds of the present application can significantly improve the protein expression of EPO, wherein the agonistic activity of compounds 1, 2, 3, 5, 6, 7, 8, 9, 10, 12 and 14 is particularly obvious, and the agonistic effect of compounds 1, 2, 5, 8 and 10 is better than that of the control compounds.

[0445] Control compounds:

[0446] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details in accordance with all the teachings disclosed, and these changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A compound of Formula II: Formula II or a pharmaceutically acceptable salt thereof. Formula II wherein, X is N; each R1is independently selected from cyano, trifluoromethyl, and methoxy; R2is selected from H, D, bromo, and methoxy; R3is selected from H, D, methyl, and cyclopropyl; 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R3is selected from H, D, and cyclopropyl. n=2。 3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R3is H or D.

4. A compound selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

5. A compound of Formula III: Formula III or a pharmaceutically acceptable salt thereof. 、 、 、 、 、 、 、 、 、 and . Formula III wherein, X is N; wherein R1is selected from pyrrolidinyl, piperidinyl, (R)-3-fluoropyrrolidinyl, (S)-3- fluoropyrrolidinyl, and morpholinyl; R2is fluoro; n=1； R3is H or D; Z is C.

6. A compound selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

7. A method of preparing a compound of any one of claims 1-4, or a pharmaceutically acceptable salt thereof, comprising the steps of: , , , , , and . Step 1: reacting compound la with hydroxylamine hydrochloride to obtain compound lb; Step 2: halogenating compound lb to obtain compound lc; Step 3: reacting compound lc with compound B to obtain compound Id; Step 4: cyclizing compound Id to obtain the compound of Formula II’; 8. A method of preparing a compound of claim 5 or 6, or a pharmaceutically acceptable salt thereof, comprising the steps of: wherein R 1 , R 2 , R 3 and n are as defined in any one of claims 1 to 4. Step 1: reacting compound 2a with sodium nitrate, copper chloride, sulfur dioxide to obtain compound 2b; Step 2: aminating compound 2b to obtain compound 2c; Step 3: chlorinating compound 2c to obtain compound 2d; Step 4: reacting compound 2d with compound B’ to obtain the compound of Formula III’; 9. A pharmaceutical composition comprising a compound of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, and optionally a carrier and / or excipient. wherein R 1 , R 3 , Z and n are as defined in claim 5 or 6.

10. The pharmaceutical composition of claim 9, further comprising a prolyl hydroxylase inhibitor.

11. The pharmaceutical composition of claim 10, wherein the prolyl hydroxylase inhibitor is selected from the group consisting of Roxadustat, Vadadustat, Desidustat, Enasidenib, and Molidustat.

12. A vaccine adjuvant comprising a compound of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 9-11.

13. An immunogenic or immunostimulatory composition comprising the vaccine adjuvant of claim 12.

14. Use of a benzazolium compound of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 9-11, for the manufacture of a medicament for use as a hypoxia-inducible factor HIF-2 agonist, an immunomodulator, or for the treatment and / or prevention of a disease associated with abnormal HIF-2 signaling pathway. ​ 15. The use according to claim 14, wherein the disease associated with abnormal HIF-2 signaling pathway is selected from the group consisting of chronic kidney disease, dyslipidemia, high cholesterol, or a disease and / or condition associated with low EPO or EPO receptor activity, or characterized by EPO deficiency or red blood cell deficiency or defect.

16. The use according to claim 15, wherein the disease and / or condition associated with low EPO or EPO receptor activity, or characterized by EPO deficiency or red blood cell deficiency or defect is an ischemic disease.

17. The use according to claim 16, wherein the ischemic disease is selected from the group consisting of renal anemia, chronic kidney anemia, anemia of reduced erythropoiesis, stroke caused by ischemia, or myocardial ischemia.

18. Use of a benzazolium compound of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 9-11, in the manufacture of a vaccine adjuvant.

19. The use according to claim 18, wherein the compound, or a pharmaceutically acceptable salt thereof, stimulates an immune response in a subject.

20. The use according to claim 19, wherein the compound, or a pharmaceutically acceptable salt thereof, elicits or enhances an immune response in a subject.

21. The use according to claim 19 or 20, wherein the immune response is a non-specific immune response.

22. The use according to claim 19 or 20, wherein the immune response is an antigen-specific immune response.

23. The use according to claim 19 or 20, wherein the immune response comprises activation of B cells, activation of T cells, production of antibodies, and / or release of cytokines.

24. Use of an immunogenic or immunostimulatory composition of claim 13 in the manufacture of a vaccine.

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