Substituted angiotensin and endothelin receptor antagonist and use thereof

Abstract

The present invention relates to the technical field of chemical drugs, and provides a substituted angiotensin and endothelin receptor antagonist compound, and a preparation method therefor and a medical use thereof.

Description

Substituted angiotensin and endothelin receptor antagonists and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of chemical drugs, and provides a substituted angiotensin and endothelin receptor antagonist compound, a preparation method therefor, and medical use thereof. BACKGROUND

[0002] Saropsant (CAS: 254740-64-2, structural formula as follows) is a dual angiotensin and endothelin receptor antagonist in clinical development for the treatment of renal diseases or conditions, some of which do not have a specific treatment or are associated with symptoms that cannot be fully controlled by other therapies.

[0003] Atrasentan (CAS: 173937-91-2, structural formula as follows) is a potent and selective angiotensin and endothelin receptor antagonist that was previously evaluated in clinical trials for the treatment of prostate cancer and is now being evaluated in clinical trials for the treatment of chronic kidney disease associated with type II diabetes. It has also been shown to be able to reduce albuminuria in patients with diabetic nephropathy. SUMMARY

[0004] In view of the problems existing in the prior art, the present application provides a substituted angiotensin and endothelin receptor antagonist compound with a novel structure, a preparation method therefor, and medical use thereof.

[0005] Specifically, the present application provides a compound represented by general formula (I) or an isomer thereof, or a pharmaceutically acceptable salt thereof, wherein all the variables are as defined herein.

[0006] The present application is realized by the following technical scheme, a compound represented by general formula (I) or an isomer thereof, or a pharmaceutically acceptable salt thereof,

[0007] X is selected from hydrogen, alkylamine, alkyl, cycloalkyl, halogen, B1C(B2)=N-O-alkyl, alkenyl, alkyl-C=C-alkyl, cyano, alkylthio, alkylthioalkyl, alkoxy, alkoxyalkyl; B1, B2 are independently selected from H, alkyl;

[0008] The alkylamine, alkyl, alkylthio, alkylthioalkyl, alkoxy, alkoxyalkyl, alkyl-C=C-alkyl can be further substituted, and the substituent is selected from halogen, carboxylic acid, carboxylic acid alkyl ester, haloalkyl, cycloalkyl, cycloalkyloxy, heterocycloalkyl, cyano, hydroxyl, oxo, thio, alkenyl, alkynyl, amino, heteroaryl, aryl, aryl-O-, heteroaryl-O-, alkoxy, alkoxyalkyl, alkoxyalkoxy, haloalkyl, amido, or alkyl-substituted amido.

[0009] or X is selected from A1A2C(O)NA3-(CH2)n-, n = natural number from 1 to 3, A1, A2, A3 are independently selected from: H, alkyl, or A1, A2 are substituted or unsubstituted cycloalkyl, the substituents are selected from alkyl;

[0010] or X is selected from -(CH2)m-saturated or unsaturated heterocycloalkyl, m = natural number from 0 to 3, the heterocycloalkyl can be further substituted, the substituents are selected from alkyl, alkoxy, oxo, hydroxyl;

[0011] or X is selected from -(CH)p= saturated or unsaturated heterocycloalkyl, alkyl, p = natural number from 1 to 3, the heterocycloalkyl can be further substituted, the substituents are selected from alkyl, alkoxy, halogen;

[0012] T1, T2 are independently selected from: H or T1 and T2 are cycloalkyl, the cycloalkyl can be further substituted, the substituents are selected from halogen;

[0013] R1, R3, R5, R6, R7 are independently selected from: H, halogen, alkyl, alkoxy, cyano;

[0014] R2 is selected from: H, halogen, alkyl, alkoxy, cyano, when R2 is selected from hydrogen, X is selected from amino, heteroaryl-substituted alkyl or alkoxy, or X is selected from -(CH2)m-saturated or unsaturated heterocycloalkyl, m = natural number from 0 to 3, the heterocycloalkyl can be further substituted, the substituents are selected from alkyl, alkoxy, oxo, hydroxyl;

[0015] R4 is selected from H, alkyl, alkoxy;

[0016] R8 is selected from alkyl, halogen;

[0017] R 11 is selected from hydrogen, alkyl, -C(O)-alkyl.

[0018] As a preferred technical solution of the present application, the compound selected from the general formula (Ia) or its isomer, or its pharmaceutically acceptable salt is used:

[0019] wherein R1-R8, X are defined as described above.

[0020] As a preferred technical solution of the present application, the compound selected from the general formula (Ib) or its isomer, or its pharmaceutically acceptable salt is used:

[0021] R1-R8, X are defined as described above, R9 and R 10 are each independently selected from: hydrogen, halogen.

[0022] As a preferred technical solution of the present application, the compound is selected from the compounds represented by the general formula (Ic), (Id), (Ie) or isomers thereof, or pharmaceutically acceptable salts thereof:

[0023] X, T1, T2 are defined as above, R8 is selected from alkyl, halogen; R9 and R 10 are each independently selected from: hydrogen, halogen.

[0024] As a preferred technical solution of the present application, the alkyl is selected from C 1-4 alkyl, the C 1-4 alkyl is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl.

[0025] As a preferred technical solution of the present application, the cycloalkyl is selected from C 3-6 cycloalkyl, the C 3-6 cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; heterocycloalkyl is that one or more carbon atoms in cycloalkyl is replaced by heteroatom selected from O, N, S; unsaturated cycloalkyl is that there is unsaturated double bond or triple bond in the ring of cycloalkyl.

[0026] As a preferred technical solution of the present application, the alkoxy is selected from C 1-4 alkoxy, C 1-4 alkoxy is selected from methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, t-butoxy.

[0027] As a preferred technical solution of the present application, the halogen includes fluorine, chlorine, bromine, iodine.

[0028] As a preferred technical solution of the present application, the alkoxyalkyl is that one or more hydrogens in alkyl is replaced by alkoxy, for example, ethoxyethyl.

[0029] As a preferred technical solution of the present application, the alkenyl contains double bond unsaturated bond, including vinyl, propenyl, etc., the alkynyl contains triple bond unsaturated bond, including ethynyl, propynyl, etc.

[0030] As a preferred technical solution of the present application, X is selected from hydrogen, methyl, ethyl, n-propyl, methoxymethyl, methoxyethyl, methoxypropyl, ethoxymethyl, ethoxyethyl, ethoxypropyl, methylthiomethyl, methylthioethyl, methylthiopropyl, ethylthiomethyl, ethylthioethyl, ethylthiopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropyl, fluorine, bromine, isopropyl, cyano, Hydroxyethyl,

[0031] R1, R3, R5, R6, and R7 are independently selected from: H, fluorine, chlorine, methyl, methoxy, and cyano.

[0032] R2 is selected from fluorine, chlorine, methyl, methoxy, and cyano groups;

[0033] R4 is selected from H, methyl, and methoxy;

[0034] R8 is selected from methyl or chlorine.

[0035] As a preferred embodiment of the present invention, it is further preferred that R1, R3, R4, R5, R6, and R7 are independently selected from H.

[0036] As a preferred embodiment of the present invention, the compounds are selected from the following:

[0037] As a preferred embodiment of the present invention, the isomers of the compound represented by general formula (I) include, for example, those selected from:

[0038] As a preferred embodiment of the present invention, the compound represented by general formula (I) or its isomers, or its pharmaceutically usable salts, contains atomic isotopes in non-natural proportions, the isotopes being selected from deuterium (…). 2 H), Iodine-125 125 I) or C-14 14 C) etc., preferably one or more hydrogen atoms are coated with the isotope deuterium ( 2 H) substitution.

[0039] For example, deuterium ( 2 H)-substituted compounds are selected from:

[0040] Specifically, selected from:

[0041] As a preferred embodiment of the present invention, the isomers of the compound represented by general formula (I) include its transisomers, for example selected from:

[0042] The present application further provides the use of the compound or isomer thereof, or pharmaceutically acceptable salt thereof in the preparation of a medicament, in particular, preferably the use of the compound or pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing diseases associated with angiotensin and endothelin receptor antagonism.

[0043] Further, the diseases include treating chronic kidney disease, IgA, FSGS and hypertension and the like.

[0044] The present application further provides a pharmaceutical composition containing the aforementioned compound or isomer thereof, or pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

[0045] The compound of the present application is obtained by the following preparation route:

[0046] wherein R1, R2, R3, R4, R5, R6, R7 are as defined above.

[0047] or:

[0048] wherein R1, R2, R3, R4, R5, R6, R7 are as defined above, and X is selected from alkyl, alkoxy.

[0049] Unless otherwise indicated herein, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be construed as indeterminate or unclear in the absence of a specific definition, but should be interpreted according to its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding product or active ingredient thereof. The term "pharmaceutically acceptable" used herein is intended for those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0050] The "pharmaceutically acceptable salt" used herein is a derivative of the compound of the present application, wherein the parent compound is modified by salt formation with an acid or with a base.

[0051] including but not limited to as:

[0052] The prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to convert into the compounds of the present application. In addition, the prodrugs can be converted into the compounds of the present application in the in vivo environment by chemical or biochemical means.

[0053] Certain compounds of the application can exist in unsolvated as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present application.

[0054] The atoms of the molecules of the compounds of the application are isotopes, and derivatization with isotopes can generally afford enhanced specificity, reduced clearance, increased metabolic stability, and improved in vivo activity. Also, included is an embodiment wherein at least one atom is replaced by an atom having the same atomic number (number of protons) and different mass number (number of protons and neutrons). Examples of isotopes that are included in the compounds of the application include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, which respectively include 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl. In particular, radioactive isotopes such as3H or14C, which emit channelling radiation, can be used in the preparation of drugs or to trace compounds in vivo. Stable isotopes neither decay nor change with the amount, nor are they radioactive, and thus can be used safely. When the atoms of the molecules of the compounds of the application are isotopes, the isotopes can be converted according to the general methods by replacing the reagents used in the synthesis with reagents containing the corresponding isotope.

[0055] The term "heteroaryl" refers to a monocyclic or fused polycyclic aromatic ring structure containing one or more (preferably 1, 2, 3, or 4) heteroatoms independently selected from O, N, and S, and a specified number of carbon atoms. Specifically, the aromatic ring structure can have 5 to 12 ring members. The heteroaryl group is preferably 5 to 10 membered, more preferably 5 to 8 membered, and most preferably 5, 6, or 7 membered. The heteroaryl group can be, for example, a five- or six-membered monocyclic ring or a fused bicyclic structure formed by a fused five- and six-membered ring or two fused six-membered rings or, as another example, two fused five-membered rings. Each ring can contain up to four heteroatoms, typically selected from nitrogen, sulfur, and oxygen. The heteroaryl ring typically contains up to 4 heteroatoms, more typically up to 3 heteroatoms, more typically up to 2, for example a single heteroatom. In one embodiment, the heteroaryl ring contains at least one ring nitrogen atom. The nitrogen atoms in the heteroaryl ring can be basic, as in the case of imidazole or pyridine, or essentially non-basic, as in the case of indole or pyrrole nitrogen. In general, the number of basic nitrogen atoms present in the heteroaryl group (including any amino substituents of the ring) will be less than five.

[0056] Examples of five-membered monocyclic heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, furazanyl, oxazolyl, oxadiazolyl, oxatriazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl, and tetrazolyl. Examples of six-membered monocyclic heteroaryl groups include, but are not limited to, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, and triazinyl. Particular examples of bicyclic heteroaryl groups containing a five-membered ring fused to another five-membered ring include, but are not limited to, imidazothiazolyl and imidazimidazolyl. Particular examples of bicyclic heteroaryl groups containing a six-membered ring fused to a five-membered ring include, but are not limited to, benzofuranyl, benzothienyl, benzimidazolyl, benzoxazolyl, isobenzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, isobenzofuranyl, indolyl, isoindolyl, indolizinyl, purinyl (e.g., adenine, guanine), indazolyl, pyrazolopyrimidinyl, triazolopyrimidinyl, and pyrazolopyridinyl. Particular examples of bicyclic heteroaryl groups containing two fused six-membered rings include, but are not limited to, quinolinyl, isoquinolinyl, pyridopyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, and pteridinyl. Particular heteroaryl groups are those derived from thienyl, pyrrolyl, benzothienyl, benzofuranyl, indolyl, pyridyl, quinolinyl, imidazolyl, oxazolyl, and pyrazinyl.

[0057] The compounds of the present application can contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds can be radiolabeled with radioactive isotopes, such as deuterium ( 2 H), iodine-125 ( 125 I) or carbon-14 ( 14 C). All isotopic variations of the compounds of the present application, whether radioactive or not, are encompassed within the scope of the present application. Further, substitution with isotopes such as deuterium ( 2 H) affords certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life, reduced dosage requirements, and / or greater therapeutic efficacy. The isotope-labeled compounds of this application can be prepared by carrying out the procedures disclosed herein and / or known in the art using appropriate isotopically-labeled reagents in place of the non-isotopically labeled reagents previously employed.

[0058] The term "pharmaceutically acceptable carrier" means any formulation carrier or medium that does not interfere with the biological activity of the active substance and that is nontoxic to the host or patient in whom it is administered. Representative carriers include water, oil, vegetable and mineral, cream bases, lotion bases, ointment bases, and the like. These bases include suspending agents, viscosity increasing agents, penetration enhancers, and the like. Their formulation is well known to those skilled in the art of cosmetics or topical pharmaceuticals. Additional information on carriers can be found in Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.

[0059] The term "excipient" generally refers to a carrier, diluent, and / or vehicle needed to formulate an effective pharmaceutical composition.

[0060] The term "effective amount" or "therapeutically effective amount" with respect to a pharmaceutical or pharmacological agent means a sufficient amount of the agent to achieve the intended effect without being toxic to the subject. With respect to oral dosage forms of the present application, an "effective amount" of one active substance in a composition means the amount needed to achieve the intended effect in conjunction with another active substance in the composition. The determination of an effective amount will vary from subject to subject, depending on the age and general condition of the subject, as well as the particular active substance, and an appropriate effective amount in a given case can be determined by those skilled in the art using routine testing.

[0061] The term "active ingredient," "therapeutic agent," "active substance," or "active agent" means a chemical entity that is effective in treating a disorder, disease, or condition of interest.

[0062] The term "tautomer" or "tautomeric form" means structural isomers that differ in energy by a low energy barrier and can interconvert. If tautomerization is possible (as in solution), a chemical equilibrium of tautomers can be achieved. For example, protontautomer (also known as prototropic tautomer) includes interconversion by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomer includes interconversion by reorganization of some bonding electrons. Keto-enol tautomerization. Another example of tautomerization is phenol-keto tautomerization. Unless otherwise indicated, all tautomeric forms of the compounds of the present application are within the scope of the present application.

[0063] The compounds of this invention can exist in specific geometric or stereoisomeric forms. This invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)- isomers, (L)- isomers, transisomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this invention.

[0064] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain an enantiomer of a compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the desired enantiomer in pure form. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution using conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished by using chromatography employing a chiral stationary phase and optionally combined with chemical derivatization (e.g., from amines to carbamates).

[0065] "Optional" or "optionally" means that the event or condition described below may occur but is not required to occur, and the description includes both the scenario in which the event or condition occurs and the scenario in which the event or condition does not occur.

[0066] The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of the present invention. Attached Figure Description

[0067] Figure 1. Schematic diagram of AUC results of systolic blood pressure in rats of Example 147

[0068] Figure 2. Schematic diagram of 24-hour urinary albumin results in rats of Example 148

[0069] Figure 3. Schematic diagram of the urinary albumin / creatinine ratio (UACR) results in rats of Example 148.

[0070] Figure 4. Schematic diagram of PAS staining pathological scoring results of rat kidneys in Example 148 DETAILED DESCRIPTION

[0071] The application will be further described in conjunction with the examples below, but the embodiments of the application are not limited thereto.

[0072] The structure of the compound is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). The NMR shift (δ) is given in units of 10 -6 (ppm). The NMR is measured by a Bruker AVANCE-III nuclear magnetic instrument, and the measuring solvent is deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and the internal standard is tetramethylsilane (TMS).

[0073] The MS is measured by an ISQ EC mass spectrometer (manufacturer: Thermo, model: ISQ EC).

[0074] The high performance liquid chromatography (HPLC) analysis uses a Thermo U3000 HPLC DAD high performance liquid chromatograph.

[0075] The CombiFlash rapid preparation instrument uses a CombiFlash Rf+ LUMEN (TELEDYNE ISCO).

[0076] The thin layer chromatography silica gel plate uses Yantai Yinlong HSGF 254 or GF 254 The silica gel plate, the silica gel plate used in the thin layer chromatography (TLC) adopts a specification of 0.17mm-0.23mm, and the thin layer chromatography separation and purification product adopts a specification of 0.4mm-0.5mm.

[0077] The silica gel column chromatography generally uses Yushan Shangbang silica gel 100-200 mesh silica gel as the carrier.

[0078] DMF N,N-dimethylformamide, chloromethyl ethyl carbonate, potassium iodide, cesium carbonate, DCM dichloromethane, n-hexane, ethyl acetate.

[0079] General preparation method:

[0080] wherein, R1, R2, R3, R4, R5, R6, R7 are as defined above.

[0081] or:

[0082] wherein, R1, R2, R3, R4, R5, R6, R7 are as defined above, and X is selected from alkyl, alkoxy.

[0083] Example 1

[0084] Synthesis of 4'-(2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-2'-ethoxymethyl-5'-fluoro-[l,l-biphenyl]-2-sulfonamide

[0085] Step A: Synthesis of 3-(4-bromo-2-fluoro-5-(hydroxymethyl)benzyl)-2-butyl-l,3- diazaspiro[4.4]non-l-en-4-one

[0086] In a 50 mL reaction vial, 2-butyl-l,3-diazaspiro[4.4]non-l-en-4-one hydrochloride (170 mg, 0.74 mmol) was added under nitrogen protection, anhydrous DMF (5 mL) was added, stirred to dissolve, cooled to 0 °C, sodium hydride (59 mg, 1.47 mmol) was added, stirred at room temperature for 10 minutes, then cooled to 0 °C again, then 2-bromo-5-bromomethyl-4-fluorophenylmethanol (200 mg, 0.67 mmol, refer to patent WO2020 / 092383 for synthesis) was added, then continued to stir at room temperature for 50 minutes, saturated ammonium chloride was added to quench (50 mL), extracted with ethyl acetate (30 mL x 3), combined the organic phase, washed with saturated sodium chloride (60 mL), dried over anhydrous sodium sulfate, filtered, and rotary evaporated. The obtained residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1) to give 270 mg of colorless oily liquid product 3-(4-bromo-2-fluoro-5-(hydroxymethyl)benzyl)-2-butyl-l,3-diazaspiro[4.4]non-l-en-4-one (yield 95%). LC-MS: [M+H] = 411.2, 413.2. +

[0087] Step B: Synthesis of 3-(4-bromo-5-ethoxymethyl)-2-fluorobenzyl-2-butyl-l,3- diazaspiro[4.4]non-l-en-4-one

[0088] ​In a 50 mL reaction vial was placed 3-(4-bromo-2-fluoro-5-(hydroxymethyl)benzyl)-2-butyl-1,3-diazaspiro[4.4]non-1-en-4-one (270 mg, 0.66 mmol) under nitrogen protection, anhydrous DMF (5 mL) was added and stirred to dissolve, cooled to 0 °C, sodium hydride (32 mg, 0.79 mmol) was added, stirred at room temperature for 10 minutes, then cooled to 0 °C, ethyl iodide (120 mg, 0.79 mmol) was added, then stirred at room temperature for 50 minutes, quenched with saturated ammonium chloride (40 mL), extracted with ethyl acetate (20 mL x 3), combined the organic phase, washed with saturated sodium chloride (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography on silica gel (eluent: petroleum ether / ethyl acetate = 3 / 1) to give 140 mg of the product 3-(4-bromo-5-ethoxymethyl)-2-fluorobenzyl-2-butyl-1,3-diazaspiro[4.4]non-1-en-4-one as a yellow oil (yield 48%). LC-MS: [M+H] = 439.1, 441.1 + = 439.1, 441.1

[0089] Step C: Synthesis of 4'-(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl)-N-(4,5-dimethylisoxazol-3-yl)-2'-ethoxymethyl-5'-fluoro-N-methoxymethyl-[1,1'-biphenyl]-2-sulfonamide

[0090] In a 25 mL reaction vial was placed 3-(4-bromo-5-ethoxymethyl)-2-fluorobenzyl-2-butyl-1,3-diazaspiro[4.4]non-1-en-4-one (140 mg, 0.32 mmol), N-(4,5-dimethylisoxazol-3-yl)-N-(methoxymethyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide (160 mg, 0.38 mmol), potassium phosphate (200 mg, 0.96 mmol), tetrakis(triphenylphosphine)palladium (74 mg, 0.064 mmol), replaced with nitrogen, added ethylene glycol dimethyl ether (5 mL), water (0.5 mL), and heated in a 100 °C oil bath for 3 hours. The reaction was completed, quenched with water (10 mL), extracted with ethyl acetate (10 mL x 3), combined the organic phase, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography on silica gel (eluent: petroleum ether / ethyl acetate = 1 / 1) to give 170 mg of the product 4'-(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl)-N-(4,5-dimethylisoxazol-3-yl)-2'-ethoxymethyl-5'-fluoro-N-methoxymethyl-[1,1'-biphenyl]-2-sulfonamide as a colorless oil (yield 61%). LC-MS: [M+H]+ = 655.3.

[0091] Step D: Synthesis of 4'-(2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N- (4,5-dimethylisoxazol-3-yl)-2'-ethoxymethyl-5'-fluoro-[l,r-biphenyl]-2-sulfonamide

[0092] In a 25 mL reaction vial was placed 4'-(2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3- yl)methyl)-N-(4,5-dimethylisoxazol-3-yl)-2'-ethoxymethyl-5'-fluoro-N- methoxymethyl-[l,r-biphenyl]-2-sulfonamide (170 mg, 0.26 mmol), 4 M hydrochloric acid in dioxane (5 mL, 20 mmol), and stirred at 70 °C for 1 h. The reaction was complete, and the reaction was concentrated directly in vacuo to give a residue. The residue was purified by reverse phase column (C18, 120 g, acetonitrile: water = 4: 1, flow rate: 60 mL / min) to give 43 mg of white solid product 4'-(2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-2'-ethoxymethyl-5'-fluoro-[l,r-biphenyl]-2-sulfonamide (25.7% yield).

[0093] LC-MS: [M+H] + = 611.0.

[0094] 1 H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 8.07 (d, J = 7.2 Hz, 1H), 7.65 7.61 (m, 2H), 7.21 (d, J = 6.8 Hz, 1H), 7.07 (d, J = 7.7 Hz, 1H), 6.78 (d, J = 10.5 Hz, 1H), 4.78 (s, 2H), 3.92 (q, J = 13.0 Hz, 2H), 3.18 (q, J = 7.1 Hz, 2H), 2.38 (t, J = 7.5 Hz, 2H), 2.18 (s, 3H), 1.89 - 1.82 (m, 6H), 1.70 (d, J = 8.2 Hz, 2H), 1.64 (s, 3H), 1.54 (p, J = 7.6 Hz, 2H), 1.35 - 1.28 (m, 2H), 0.99 (t, J = 7.0 Hz, 3H), 0.84 (t, J = 7.3 Hz, 3H).

[0095] Example 9

[0096] Synthesis of 4'-(2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-2-methoxymethyl-5-fluoro-[l,l-biphenyl]-2-sulfonamide

[0097] Step A: Synthesis of 3-(4-bromo-5-methoxymethyl)-2-fluorobenzyl-2-butyl-l,3- diazaspiro[4.4]non-l-en-4-one

[0098] Into a 50 mL reaction vial was placed 3-(4-bromo-2-fluoro-5-hydroxymethyl)benzyl)- 2-butyl-l,3-diazaspiro[4.4]non-l-en-4-one (500 mg, 1.22 mmol) under nitrogen protection, anhydrous DMF (5 mL) was added and stirred to dissolve, cooled to 0 °C, sodium hydride (54 mg, 1.34 mmol) was added, stirred at room temperature for 10 minutes, then cooled to 0 °C again, followed by the addition of iodomethane (190 mg, 1.34 mmol), then stirred at room temperature for another 50 minutes, quenched by the addition of saturated ammonium chloride (40 mL), extracted with ethyl acetate (20 mL x 3), combined the organic phase, washed with saturated sodium chloride (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by column chromatography on silica gel (eluent: petroleum ether / ethyl acetate = 3 / 1) to give 220 mg of the product 3-(4-bromo-5-methoxymethyl)-2-fluorobenzyl-2-butyl-l,3- diazaspiro[4.4]non-l-en-4-one as a yellow oil (yield 43%). LC-MS: [M+H] = 427.1 +

[0099] Step B: Synthesis of 4-(2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-2-methoxymethyl-5-fluoro-N-methoxymethyl-[l,l'-biphenyl]-2- sulfonamide

[0100] ​In a 25ml reaction vial, 3-(4-bromo-5-methoxymethyl)-2-fluorobenzyl-2-butyl-1,3- diazaspiro[4.4]non-1-en-4-one (220mg, 0.52mmol), N-(bis-methyl-1,2-oxazol-3-yl)-N- (methoxymethyl)-2-(tetramethyl-1,3,2-dioxaborolan-2-yl)benzene-1-sulfonamide (260mg, 0.62mmol), potassium phosphate (330mg, 1.56mmol), palladium (triphenylphosphine) tetra (120mg, 0.1mmol), nitrogen purging, add ethylene glycol dimethyl ether (5ml), water (0.5ml), heat reaction in 100 degree oil bath for 3 hours. Reaction finished, quench with water (10ml), extract with ethyl acetate (10ml x 3), combine organic phase, dry over anhydrous sodium sulfate, filter, spin dry. The residue obtained was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1) to give 260mg of product 4-(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl)-N-(4,5-dimethylisoxazol-3-yl)-2- methoxymethyl-5-fluoro-N-methoxymethyl-[1,1'-biphenyl]-2-sulfonamide as colorless oily liquid (yield 78%). LC-MS: [M+H] = 641.3. + = 597.3.

[0101] Step c: synthesis of 4-(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-2-methoxymethyl-5-fluoro-[1,1'-biphenyl]-2-sulfonamide

[0102] In a 25ml reaction vial, 4-(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-2-methoxymethyl-5-fluoro-N-methoxymethyl-[1,1'-biphenyl]-2- sulfonamide (250mg, 0.39mmol), add 4M hydrochloric acid in dioxane (10ml, 40mmol), stir at 70 degree for 2 hours. Reaction finished, cool down the reaction mixture, directly concentrate under vacuum to give residue. The residue was purified by reverse phase column (C18, 120g, acetonitrile: water = 4:1, flow rate: 60ml / min) to give 58mg of product 4-(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl)-N-(4,5-dimethylisoxazol-3-yl)-2- methoxymethyl-5-fluoro-[1,1'-biphenyl]-2-sulfonamide as white solid (yield 23.8%). LC-MS: [M+H] = 597.3. + = 597.3.

[0103] 1 H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.12 - 8.04 (m, 1H), 7.67 (tt, J=7.5, 5.7 Hz, 2H), 7.28 - 7.21 (m, 1H), 7.09 (d, J=7.8 Hz, 1H), 6.79 (d, J=10.6 Hz, 1H), 4.78 (s, 2H), 3.95 - 3.83 (m, 2H), 3.05 (s, 3H), 2.38 (t, J=7.5 Hz, 2H), 2.21 (s, 3H), 1.90 - 1.82 (m, 6H), 1.76 - 1.68 (m, 2H), 1.68 (s, 3H), 1.54 (p, J=7.4 Hz, 2H), 1.35 - 1.27 (m, 2H), 0.84 (t, J=7.3 Hz, 3H).

[0104] Referring to the preparation method of Example 9, deuterated compounds 9A, 9B, 9C and 9D of compound 19 were prepared:

[0105] Example 33

[0106] Synthesis route

[0107] LC-MS: [M+H] + = 613.3.

[0108] 1 H NMR (400 MHz, DMSO-d6) δ 10.62 (s, 1H), 8.07 (dd, J=7.6, 1.8 Hz, 1H), 7.78 - 7.53 (m, 2H), 7.28 (dd, J=7.2, 1.8 Hz, 1H), 7.09 (d, J=7.7 Hz, 1H), 6.76 (d, J=10.5 Hz, 1H), 4.77 (s, 2H), 3.30 - 3.09 (m, 2H), 2.38 (td, J=7.3, 1.8 Hz, 2H), 2.21 (s, 3H), 1.92 - 1.81 (m, 6H), 1.79 (s, 3H), 1.69 (d, J=4.5 Hz, 3H), 1.57 - 1.47 (m, 2H), 1.36 - 1.23 (m, 4H), 0.83 (t, J=7.4 Hz, 3H).

[0109] Example 34

[0110] Synthesis of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4- chloro-5-methyloxazol-3-yl)-5'-fluoro-2'-(methoxymethyl)-[l,l'-biphenyl]-2- sulfonamide

[0111] Synthesis route:

[0112] Step A: Synthesis of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N- (4-chloro-5-methyloxazol-3-yl)-5'-fluoro-2'-(methoxymethyl)-N-[(2- (trimethylsilyl)ethoxy)methyl]-[l,l'-biphenyl]-2-sulfonamide

[0113] To 2-butyl-3-[(2-fluoro-5-(methoxymethyl)-4-(tetramethyl-l,3,2-dioxaborolan-2-yl)phenyl)methyl]- 1,3-diazaspiro[4.4]non-l-en-4-one (100 mg, 0.21 mmol), 2-bromo-N-(4-chloro-5-methyl- 1,2-oxazol-3-yl)-N-[(2-(trimethylsilyl)ethoxy)methyl]benzene-l-sulfonamide (0.13 g, 0.27 mmol) were dissolved in dioxane (5 mL) and water (0.5 mL), potassium carbonate (0.15 g, 1.05 mmol) and [l,l'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex (17 mg, 0.021 mmol) were added. The reaction mixture was heated to 80 °C under nitrogen and stirred overnight. The reaction was complete and the reaction was stopped. The reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 2, 33%) to give 53 mg of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4-chloro-5- methyloxazol-3-yl)-5'-fluoro-2'-(methoxymethyl)-N-[(2-(trimethylsilyl)ethoxy)methyl]- [l,l'-biphenyl]-2-sulfonamide (yield 33%) as a yellow liquid. LC-MS: [M+H] = 747.4. +

[0114] Step B: Synthesis of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4- chloro-5-methyloxazol-3-yl)-5'-fluoro-2'-(methoxymethyl)-[l,l'-biphenyl]-2-sulfonamide

[0115] ​To a solution of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4- chloro-5-methylisoxazol-3-yl)-5'-fluoro-2'-(methoxymethyl)-N-[(2- (trimethylsilyl)ethoxy)methyl]-[l,l'-biphenyl]-2-sulfonamide (32 mg) in 1,4- dioxane (4 mL) was added TBAF (1 M in THF, 0.1 mL) at room temperature. The reaction mixture was heated to 70 °C for 1 h. The reaction was complete. The reaction was quenched with saturated aqueous NH4C1 solution. The mixture was extracted with DCM (15 mL x 3). The combined organic phase was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate) to give 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4-chloro-5- methylisoxazol-3-yl)-5'-fluoro-2'-(methoxymethyl)-[l,l'-biphenyl]-2-sulfonamide (32 mg, 83% yield) as a white solid.

[0116] LC-MS: [M+H] = 617.2. +

[0117] 1 H NMR (400 MHz, DMSO-d6) δ 12.09 (s, 1H), 8.06 - 7.99 (m, 1H), 7.54 (dd, J = 5.8, 3.4 Hz, 2H), 7.17 (d, J = 7.8 Hz, 1H), 7.14 - 7.08 (m, 1H), 6.83 (d, J = 10.8 Hz, 1H), 4.86 (s, 2H), 3.99 - 3.84 (m, 2H), 3.06 (s, 3H), 2.00 (s, 3H), 1.94 - 1.86 (m, 8H), 1.55 (p, J = 7.5 Hz, 2H), 1.38 - 1.30 (m, 2H), 1.24 (s, 2H), 0.86 (t, J = 7.3 Hz, 3H).

[0118] Examples 2-8, 10-32, 35-140

[0119] Referring to the aforementioned preparation method, the compounds 2-8, 10-32, 35-140 were prepared: ​

[0120] wherein, for example:

[0121] Synthesis of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-methoxyethyl)-[l,l'-biphenyl]-2-sulfonamide

[0122] Synthesis route:

[0123] Step A: Synthesis of 3-(4-bromo-2-fluoro-5-(hydroxymethyl)benzyl)-2-butyl-l,3- diazaspiro[4.4]non-l-en-4-one

[0124] In a 25 ml reaction vial, 2-butyl-l,3-diazaspiro[4.4]non-l-en-4-one hydrochloride (186 mg, 0.81 mmol) was added under nitrogen protection, anhydrous DMF (5 ml) was added, stirred and dissolved, cooled to 0 °C, sodium hydride (87 mg, 2.17 mmol) was added, stirred at room temperature for 30 minutes, then cooled to 0 °C, then (2-bromo-5-(bromomethyl)-4-fluorophenyl)methanol (184 mg, 0.62 mmol, refer to patent WO2020 / 092383 for synthesis) was added, then continued to stir at room temperature for 2 hours, saturated ammonium chloride was added to quench (5 ml), extracted with ethyl acetate (5 ml x 3), the organic phases were combined, washed with saturated sodium chloride (5 ml x 3), dried over anhydrous sodium sulfate, filtered, and rotary evaporated. The obtained residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1) to obtain 225 mg of yellow oily liquid product 3-(4-bromo-2-fluoro-5-(hydroxymethyl)benzyl)-2-butyl-l,3-diazaspiro[4.4]non-l-en-4-one (yield 88%). LC-MS: [M+H] + = 413.1

[0125] Step B: Synthesis of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-5'-fluoro-2'-(hydroxymethyl)-N-(methoxymethyl)-[l,l'-biphenyl]-2- sulfonamide

[0126] In a 25 ml reaction vial was placed 3-(4-bromo-2-fluoro-5-(hydroxymethyl)benzyl)-2-butyl-1,3-diazaspiro[4.4]non-1-en-4-one (200 mg, 0.49 mmol), N-(4,5-dimethylisoxazol-3-yl)-N-(methoxymethyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide (270 mg, 0.64 mmol), potassium carbonate (130 mg, 0.98 mmol), 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride dichloromethane complex (40 mg, 0.049 mmol), nitrogen was purged, 1,4-dioxane (5 ml), water (2 ml), heated to reaction in 100 degree oil bath for 3 hours. Additional 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride dichloromethane complex (40 mg, 0.049 mmol) was added, the reaction was continued to stir for 3 hours, water was added to quench (10 ml), extracted with ethyl acetate (20 ml x 3), the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1) to give 272 mg of colorless oily liquid product 4'-((2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl)-N-(4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-(hydroxymethyl)-N-(methoxymethyl)-[1,1'-biphenyl]-2-sulfonamide (yield 87%). LC-MS: [M+H] + = 627.5

[0127] Step C: Synthesis of 4'-((2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl)-N-(4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-formyl-N-(methoxymethyl)-[1,1'-biphenyl]-2-sulfonamide

[0128] In a 25ml reaction vial, 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)- N-(4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-(hydroxymethyl)-N-(methoxymethyl)- [l,l'-biphenyl]-2-sulfonamide (680mg, 1.09mmol), manganese dioxide (376mg, 4.3mmol) were replaced with nitrogen, DCE (10ml) was added, and the reaction was heated in a 60°C oil bath for 10h. The reaction was filtered directly, and the residue was dried in vacuo. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1) to give 330mg of the product 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-5'-fluoro-2'-(formyl)-N-(methoxymethyl)-[l,l'-biphenyl]-2- sulfonamide (yield 50%) as a colorless oily liquid. LC-MS: [M+H] = 625.3 + = 625.3

[0129] Step D: (E)-4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-5'-fluoro-N-(methoxymethyl)-2'-(2-methoxyvinyl)-[l,l'- biphenyl]-2-sulfonamide

[0130] In a 25ml reaction vial, (methoxymethyl)triphenylphosphonium bromide (550mg, 1.6mmol) was replaced with nitrogen, THF (10ml) was added, and potassium tert-butoxide (180mg, 1.6mmol) was added. After 0.5h at room temperature, a THF (5ml) solution of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-5'-fluoro-2'-(formyl)-N-(methoxymethyl)-[l,l'-biphenyl]-2- sulfonamide (200mg, 0.32mmol) was added at 0°C. The reaction was stirred at room temperature for 4h, and then quenched with saturated ammonium chloride solution (30ml). The organic phase was extracted with ethyl acetate (20ml x 3), dried over anhydrous sodium sulfate, filtered, and dried in vacuo. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1) to give 210mg of the product (E)-4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-5'-fluoro-N-(methoxymethyl)-2'-(2-methoxyvinyl)-[l,l'- biphenyl]-2-sulfonamide (yield 47%) as a colorless oily liquid. LC-MS: [M+H]+ = 653.1

[0131] Step E: Synthesis of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N- (4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-methoxyethyl)-N-(methoxymethyl)- [l,l'-biphenyl]-2-sulfonamide

[0132] In a 25 ml reaction flask was added (E)-4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l- en-3-yl)methyl)-N-(4,5-dimethylisoxazol-3-yl)-5'-fluoro-N-(methoxymethyl)-2'-(2- methoxyethenyl)-[l,l'-biphenyl]-2-sulfonamide (80 mg, 0.12 mmol), palladium on carbon (5%, 130 mg, 0.06 mmol), nitrogen was purged, added ethyl acetate (5 ml), hydrogen was purged, and reacted at room temperature for 10 hours. The reaction system was directly filtered and dried. The obtained residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1) to obtain 64 mg of colorless oily liquid product 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-methoxyethyl)-N-(methoxymethyl)-[l,l'-biphenyl]- 2-sulfonamide (yield 80%). LC-MS: [M+H] + = 655.0

[0133] Step F: Synthesis of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N- (4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-methoxyethyl)-[l,l'-biphenyl]-2-sulfonamide

[0134] In a 25 mL reaction vial was placed 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3- yl)methyl)-N-(4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-methoxyethyl)-N- (methoxymethyl)-[l,l'-biphenyl]-2-sulfonamide (80 mg, 0.13 mmol), 4 M hydrochloric acid in dioxane (4 mL, 16 mmol), water (0.4 mL, 22 mmol), stirred at room temperature overnight. The reaction was cooled to 0 °C, adjusted to pH 5-6 with concentrated ammonia, extracted with ethyl acetate (20 mL x 3), combined organic phase, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1) to give 50 mg of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-methoxyethyl)-[l,l'-biphenyl]-2-sulfonamide. LC-MS: [M+H] = 611.3 + = 611.3

[0135] 1 H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.08 (dd, J = 7.7, 1.7 Hz, 1H), 7.72-7.59 (m, 2H), 7.25 (dd, J = 7.2, 1.7 Hz, 1H), 6.98 (d, J = 7.7 Hz, 1H), 6.71 (d, J = 10.7 Hz, 1H), 4.75 (s, 2H), 3.26 (td, J = 6.8, 3.2 Hz, 2H), 3.06 (s, 3H), 2.41-2.27 (m, 4H), 2.21 (s, 3H), 1.91-1.80 (m, 6H), 1.73-1.67 (m, 5H), 1.59-1.49 (m, 2H), 1.35-1.26 (m, 2H), 0.84 (t, J = 7.3 Hz, 3H).

[0136] Synthesis of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-(methoxy-d3)ethyl)-[l,l'-biphenyl]-2- sulfonamide

[0137] Synthesis route

[0138] Step A: Synthesis of (2-bromo-4-fluoro-5-methylphenyl)methanol

[0139] The substrate 2-bromo-4-fluoro-5-methylbenzoic acid (10 g, 42.91 mmol) was dissolved in THF (100 mL) at room temperature, and borane tetrahydrofuran complex (11.06 g, 128.73 mmol) was added dropwise slowly at 0 °C. After the addition, the reaction was allowed to warm up to room temperature and stirred overnight. TLC showed that the starting material was consumed and a new spot of product was formed. Dilute hydrochloric acid (1 N, 30 mL) was added slowly to the reaction at 0 °C and stirred for 5 min, then water (30 mL) and ethyl acetate (250 mL) were added to the reaction. The aqueous phase was extracted with ethyl acetate (60 mL). The combined organic phase was washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and then purified by column chromatography on silica gel (eluent: ethyl acetate / n-hexane = 1 / 2, 33%) to give the product (2-bromo-4-fluoro-5-methylphenyl)methanol 9.4 g (yield 100%) as a white solid.

[0140] Step B: Synthesis of 1-bromo-2-(bromomethyl)-5-fluoro-4-methylbenzene

[0141] 1-bromo-2-(bromomethyl)-5-fluoro-4-methylbenzene (8.9 g, 40.63 mmol) was added at room temperature, and stirred to dissolve under nitrogen protection. Triethylamine (12.33 g, 121.89 mmol) and 4-dimethylaminopyridine (0.25 g, 2.03 mmol) were added, and the mixture was cooled to 0 °C in an ice water bath. Methylsulfonic anhydride (10.62 g, 60.95 mmol) was added dropwise in 1 mL of THF, and the mixture was stirred at room temperature for 1 h. TLC showed that the starting material was completely consumed and a new spot of product was formed. Lithium bromide (17.64 g, 203.15 mmol) was added under an ice water bath, and the mixture was stirred at room temperature for 2 h. TLC showed that the intermediate (OMs) from the previous step was completely consumed and a new spot of product was formed. Water (30 mL) was added, and the mixture was extracted with EA (200 mL*2). The organic phase was washed with saturated sodium chloride solution (30 mL), dried, filtered, concentrated, and then purified. Purification: After drying, the product 1-bromo-2-(bromomethyl)-5-fluoro-4-methylbenzene was obtained as a yellow liquid by column chromatography 6.6 g (yield 57.61%).

[0142] Step C: Synthesis of 2-(2-bromo-4-fluoro-5-methylphenyl)acetonitrile

[0143] The substrate, 1-bromo-2-(bromomethyl)-5-fluoro-4-methylbenzene (6.6 g, 23.41 mmol) was dissolved in N,N-dimethylformamide 46 mL at room temperature, and a solution of tetrabutylammonium cyanide (8.64 g, 25.75 mmol,) in N,N-dimethylformamide 20 mL was added dropwise at 0 °C. After the addition, the reaction was stirred at room temperature for 1 h. TLC showed that the starting material was consumed and the product was formed. To the reaction, saturated sodium bicarbonate (20 mL) and ethyl acetate (200 mL*2) were added at 0 °C. The organic phase was washed with water (20 mL*2), saturated sodium chloride solution (20 mL*2) successively, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a residue, which was separated by column chromatography to give the product 2-(2-bromo-4-fluoro-5-methylphenyl)acetonitrile 4.33 g (yield 81.11%) as a colorless liquid.

[0144] Step D: Synthesis of methyl 2-(2-bromo-4-fluoro-5-methylphenyl)acetate

[0145] The substrate, 2-(2-bromo-4-fluoro-5-methylphenyl)acetonitrile (3.56 g, 15.61 mmol) was dissolved in methanol 78 mL at room temperature, and a solution of hydrochloric acid in dioxane (4 M, 78 mL) was added dropwise at 0 °C. After the addition, the reaction was stirred at room temperature overnight. LCMS showed that the starting material was completely reacted and the product was formed obviously. The reaction was concentrated and filtered to give the product methyl 2-(2-bromo-4-fluoro-5-methylphenyl)acetate 3.5 g (yield 85.88%) as a light yellow liquid.

[0146] LC-MS: [M+H] + = 262.9

[0147] Step E: Synthesis of methyl 2-(2-bromo-5-(bromomethyl)-4-fluorophenyl)acetate

[0148] The substrate, methyl 2-(2-bromo-4-fluoro-5-methylphenyl)acetate (3.5 g, 13.51 mmol), N-bromosuccinimide (2.64 g, 14.86 mmol), and azobisisobutyronitrile (2.22 g, 13.51 mmol) were dissolved in dichloroethane 80 mL at room temperature, and the reaction was stirred at 80 °C overnight. LCMS showed that the starting material was completely reacted, and the reaction was extracted with water (30 mL) and dichloromethane (200 mL). The organic phase was washed with saturated brine 30 mL, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the product methyl 2-(2-bromo-5-(bromomethyl)-4-fluorophenyl)acetate 2.34 g (yield 50.95%) as a light yellow liquid.

[0149] Step F: Synthesis of 2-(2-bromo-5-(bromomethyl)-4-fluorophenyl)ethan-1-ol

[0150] The substrate, methyl 2-(2-bromo-5-(bromomethyl)-4-fluorophenyl)acetate (2.34 g, 6.88 mmol) was dissolved in tetrahydrofuran 46 mL at room temperature, diisobutylaluminum hydride (2.94 g, 20.64 mmol) was added slowly dropwise at 0 °C (about 10 min). The reaction was allowed to warm to room temperature and stirred for 3 h. TLC showed the starting material was consumed completely. To the reaction was added 1 M hydrochloric acid (30 mL) and ethyl acetate (200 mL) at 0 °C. The aqueous phase was extracted with ethyl acetate 200 mL. The combined organic phase was washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to give the product, 2-(2-bromo-5-(bromomethyl)-4-fluorophenyl)ethan-1-ol 1.87 g (yield 87.09%) as a yellow oil.

[0151] Step G: Synthesis of 3-(4-bromo-2-fluoro-5-(2-hydroxyethyl)benzyl)-2-butyl-1,3- diazaspiro[4.4]non-1-en-4-one

[0152] The substrate, methyl 2-(2-bromo-5-(bromomethyl)-4-fluorophenyl)acetate (2.34 g, 6.88 mmol) was dissolved in tetrahydrofuran 46 mL at room temperature, diisobutylaluminum hydride (2.94 g, 20.64 mmol) was added slowly dropwise at 0 °C (about 10 min). The reaction was allowed to warm to room temperature and stirred for 3 h. TLC showed the starting material was consumed completely. To the reaction was added 1 M hydrochloric acid (30 mL) and ethyl acetate (200 mL) at 0 °C. The aqueous phase was extracted with ethyl acetate 200 mL. The combined organic phase was washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to give the product, 2-(2-bromo-5-(bromomethyl)-4-fluorophenyl)ethan-1-ol 1.87 g (yield 87.09%) as a yellow oil.

[0153] LC-MS: [M+H] + = 427.0.

[0154] Step H: Synthesis of 3-(4-bromo-2-fluoro-5-(2-(methoxy-D3)ethyl)benzyl)-2-butyl-1,3- diazaspiro[4.4]non-1-en-4-one

[0155] Deuterated iodomethane (356.60 mg, 2.46 mmol) was added at room temperature under nitrogen protection, anhydrous N,N-dimethylformamide 5 mL was added, stirred and dissolved, then 3-(4-bromo-5-(bromomethyl)-2-fluorobenzyl)-2-butyl-1,3-diazaspiro[4.4]non-1-en-4-one (500 mg, 0.82 mmol) was added, cooled to 0 °C, sodium hydride (98.4 mg, 2.46 mmol) was added, and the mixture was stirred at 0 °C to room temperature for 2 h. TLC monitoring showed that the raw material was completely reacted and obvious product was generated. 10 mL of saturated ammonium chloride was added for quenching, 100 mL of ethyl acetate was added for extraction, the organic phase was washed with water (10*3 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The sample was purified after stirring. Silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 2, 25%) to obtain the product 3-(4-bromo-2-fluoro-5-(2-(methoxy-D3)ethyl)benzyl)-2-butyl-1,3-diazaspiro[4.4]non-1-en-4-one 300 mg (82.41% yield).

[0156] LC-MS: [M+H] + = 444.1.

[0157] Step I: Synthesis of 4'-((2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl)-N-(4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-(methoxy-d3)ethyl)-N-((2-(trimethylsilyl)ethoxy)methyl)-[1,1'-biphenyl]-2-sulfonamide

[0158] To a solution of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-(methyloxy-d3)ethyl)-[l,l'-biphenyl]-2- sulfonamide (380 mg, 0.51 mmol) in tetrahydrofuran (5 mL) was added tetrabutylammonium fluoride (2.67 g, 10.2 mmol) at room temperature. The reaction mixture was stirred at 65 °C for 2 h. The reaction was monitored by LCMS. The reaction mixture was cooled to room temperature and diluted with water (5 mL). The mixture was extracted with ethyl acetate (2 x 10 mL). The combined organic extracts were washed with water (2 x 5 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (eluting with ethyl acetate / hexanes = 1 / 3) to give 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-(methyloxy-d3)ethyl)-[l,l'-biphenyl]-2- sulfonamide as a colorless oil (400 mg, 79.07% yield).

[0159] LC-MS: [M+H] = 744.2. +

[0160] Step J: Synthesis of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-(methyloxy-d3)ethyl)-[l,l'-biphenyl]-2- sulfonamide

[0161] To a solution of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-(methyloxy-d3)ethyl)-[l,l'-biphenyl]-2- sulfonamide (380 mg, 0.51 mmol) in tetrahydrofuran (5 mL) was added tetrabutylammonium fluoride (2.67 g, 10.2 mmol) at room temperature. The reaction mixture was stirred at 65 °C for 2 h. The reaction was monitored by LCMS. The reaction mixture was cooled to room temperature and diluted with water (5 mL). The mixture was extracted with ethyl acetate (2 x 10 mL). The combined organic extracts were washed with water (2 x 5 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (eluting with ethyl acetate / hexanes = 1 / 3) to give 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-(methyloxy-d3)ethyl)-[l,l'-biphenyl]-2- sulfonamide as a colorless oil (400 mg, 79.07% yield).

[0162] ​The reaction mixture was cooled in an ice-water bath, quenched with 5 mL of saturated citric acid solution and 5 mL of water, extracted with 100 mL of ethyl acetate, washed with water (10 mL), washed with saturated sodium chloride (10 mL*2), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then purified by trituration (eluent: ethyl acetate / n-hexane = 1 / 1, 50%). The collected product was dried under vacuum to give 220 mg of 4'-((2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl)-N-(4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-(methoxy-d3)ethyl)-[1,1'-biphenyl]-2-sulfonamide.

[0163] LC-MS: [M+H] + = 614.1.

[0164] 1 H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.08 (dd, J = 7.7, 1.7 Hz, 1H), 7.73-7.59 (m, 2H), 7.24 (dd, J = 7.3, 1.7 Hz, 1H), 6.98 (d, J = 7.7 Hz, 1H), 6.70 (d, J = 10.7 Hz, 1H), 4.74 (s, 2H), 3.25 (td, J = 6.8, 3.1 Hz, 2H), 2.41-2.27 (m, 4H), 2.21 (s, 3H), 1.88-1.67 (m, 11H), 1.53 (p, J = 7.4 Hz, 2H), 1.34-1.26 (m, 2H), 0.83 (t, J = 7.3 Hz, 3H).

[0165] Synthesis of 4'-((2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl)-N-(4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-(2-methoxyethoxy)ethyl)-[1,1'-biphenyl]-2-sulfonamide

[0166] Synthesis route:

[0167] Step A: Synthesis of 3-(4-bromo-2-fluoro-5-(2-hydroxyethyl)benzyl)-2-butyl-1,3-diazaspiro[4.4]non-1-en-4-one

[0168] In a 50 mL reaction vial was placed 3-(4-bromo-2-fluoro-5-(2- hydroxyethyl)benzyl)-2-butyl-1,3-diazaspiro[4.4]non-1 -en-4-one (300 mg, 0.71 mmol), anhydrous DMF (5 mL) was added, stirred to dissolve, sodium hydride (43.0 mg, 1.06 mmol) was added at 0 degree, stirred at room temperature for 30 minutes, then 1 -iodo-2- ethyl methyl ether (200 mg, 1.06 mmol) was added, stirred at room temperature for 16 hours. Saturated ammonium chloride was added to quench (50 mL), extracted with ethyl acetate (20 mL x 3), combined the organic phase, washed with saturated sodium chloride (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 5 / 1 ) to give 100 mg of yellow oily liquid product 3-(4-bromo-2-fluoro-5-(2-(2-methoxyethoxy)ethyl)benzyl)-2-butyl-1,3-diazaspiro[4.4]non-1 -en-4-one (yield 29%). LC-MS: [M+H] + = 427.0

[0169] Step B: Synthesis of 3-(4-bromo-2-fluoro-5-(2-(2-methoxyethoxy)ethyl)benzyl)-2-butyl-1,3-diazaspiro[4.4]non-1 -en-4-one

[0170] In a 50 mL reaction vial was placed 3-(4-bromo-2-fluoro-5-(2- hydroxyethyl)benzyl)-2-butyl-1,3-diazaspiro[4.4]non-1 -en-4-one (300 mg, 0.71 mmol), anhydrous DMF (5 mL) was added, stirred to dissolve, sodium hydride (43.0 mg, 1.06 mmol) was added at 0 degree, stirred at room temperature for 30 minutes, then 1 -iodo-2- ethyl methyl ether (200 mg, 1.06 mmol) was added, stirred at room temperature for 16 hours. Saturated ammonium chloride was added to quench (50 mL), extracted with ethyl acetate (20 mL x 3), combined the organic phase, washed with saturated sodium chloride (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 5 / 1 ) to give 100 mg of yellow oily liquid product 3-(4-bromo-2-fluoro-5-(2-(2-methoxyethoxy)ethyl)benzyl)-2-butyl-1,3-diazaspiro[4.4]non-1 -en-4-one (yield 29%). LC-MS: [M+H] + = 485.0.

[0171] Step C: Synthesis of 4'-((2-Tert-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)- N-(4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-(2-methoxyethoxy)ethyl)-N- (methoxymethyl)-[l,l'-biphenyl]-2-sulfonamide

[0172] In a 50 mL reaction vial was placed 3-(4-bromo-2-fluoro-5-(2-(2- methoxyethoxy)ethyl)benzyl)-2-butyl-l,3-diazaspiro[4.4]non-l-en-4-one (100 mg, 0.21 mmol), N-(bis-methyl-l,2-oxazol-3-yl)-N-(methoxymethyl)-2-(tetramethyl- 1,3,2-dioxaborolan-2-yl)benzene-l-sulfonamide (120 mg, 0.27 mmol), potassium carbonate (150 mg, 1.05 mmol), [l,l'-bis(diphenylphosphino)ferrocene] palladium dichloride dichloromethane complex (17 mg, 0.021 mmol), nitrogen was purged, dioxane (10 mL), water (1 mL) was added, and the reaction was heated in a 100 degree oil bath for 3 hours. The reaction was quenched with water (10 mL) and extracted with ethyl acetate (10 mL x 3). The organic phase was combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1) to give 110 mg of the product 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5- dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-(2-methoxyethoxy)ethyl)-N-(methoxymethyl)- [l,l'-biphenyl]-2-sulfonamide (75% yield) as a colorless oil. LC-MS: [M+H] = 699.1. +

[0173] Step D: Synthesis of 4'-((2-Butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N- (4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-(2-methoxyethoxy)ethyl)-[l,l'-biphenyl]- 2-sulfonamide

[0174] ​In a 50 mL reaction vial, 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-(2-methoxyethoxy)ethyl)-N-(methoxymethyl)-[l,l'-biphenyl]-2-sulfonamide (100 mg, 0.14 mmol) was added 4M hydrochloric acid in dioxane (10 mL, 40 mmol) and stirred at 60 °C for 2 hours. The reaction was complete. The reaction mixture was concentrated directly under vacuum to give a residue. The residue was purified by reverse phase column (C18, 80 g, acetonitrile: water = 4: 1, flow rate: 60 mL / min) to give 31 mg of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl)-N-(4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-(2-methoxyethoxy)ethyl)-[l,l'-biphenyl]-2-sulfonamide.

[0175] LCMS = [M+H] + = 655.1

[0176] 1H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H), 8.08 (dd, J = 7.5, 1.9 Hz, 1H), 7.66 (pd, J = 7.4, 1.6 Hz, 2H), 7.29 - 7.22 (m, 1H), 6.98 (d, J = 7.7 Hz, 1H), 6.70 (d, J = 10.7 Hz, 1H), 4.74 (s, 2H), 3.32 - 3.27 (m, 6H), 3.18 (s, 3H), 2.40 - 2.26 (m, 4H), 2.21 (s, 3H), 1.90 - 1.80 (m, 6H), 1.76 - 1.62 (m, 5H), 1.59 - 1.47 (m, 2H), 1.34 - 1.27 (m, 2H), 0.84 (t, J = 7.3 Hz, 3H).

[0177] Synthesis of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl-d2)-N-(4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-methoxyethyl)-[l, l'-biphenyl]-2-sulfonamide

[0178] Synthesis route:

[0179] Step A: Synthesis of ethyl 4-bromo-2-fluoro-5-methylbenzoate

[0180] In a 500 ml flask, 4-bromo-2-fluoro-5-methylbenzoic acid (50 g, 214.6 mmol) was taken in a 500 ml flask, anhydrous ethanol (400 ml) was added under nitrogen atmosphere, then hydrochloric acid in 1,4-dioxane (4 M, 100 ml, 400 mmol) was added, and stirred at 80 °C overnight. Filtered, dried, dried, ethyl acetate (500 ml) was added, washed with saturated sodium bicarbonate (100 ml x 3), washed with saturated sodium chloride (100 ml), dried to give the product ethyl 4-bromo-2-fluoro-5-methylbenzoate (52.8 g, yield 94.25%).

[0181] Step B: Synthesis of ethyl 4-bromo-5-(bromomethyl)-2-fluorobenzoate

[0182] In a 1000 ml round bottom flask, ethyl 4-bromo-2-fluoro-5-methylbenzoate (25 g, 95.75 mmol) was taken, anhydrous acetonitrile (500 ml) was added under nitrogen atmosphere, N-bromosuccinimide (22.15 g, 124.48 mmol) was added, benzoyl peroxide (2.32 g, 9.58 mmol) was added, stirred overnight at 80 °C in an oil bath under nitrogen atmosphere. The reaction was completed and dried, purified by silica gel column to give 23 g of product ethyl 4-bromo-5-(bromomethyl)-2-fluorobenzoate. (Yield: 70.6%).

[0183] Step C: Synthesis of (4-bromo-5-(bromomethyl)-2-fluorophenyl)methanol

[0184] In a 500 ml three necked flask, ethyl 4-bromo-5-(bromomethyl)-2-fluorobenzoate (15 g, 44.12 mmol) was taken, tetrahydrofuran (300 ml) was added, cooled to 0 °C. Diisobutylaluminum hydride (1.5 M) (73.53 ml, 110.3 mmol) was added, stirred for 2 h at 0 °C, quenched the reaction by careful dropwise addition of 1 M aqueous hydrochloric acid at 0 °C until no gas was evolved, 200 ml of 1 M aqueous hydrochloric acid was added, extracted with 300 ml of ethyl acetate twice, the organic phase was combined, washed with 200 ml of water, washed with 200 ml of saturated sodium chloride, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated to give 11.9 g of white solid product (4-bromo-5-(bromomethyl)-2-fluorophenyl)methanol. (Yield: 90.5%).

[0185] Step D: Synthesis of 2-bromo-4-fluoro-5-(hydroxymethyl)benzaldehyde

[0186] Into a 500 mL reaction flask was placed (4-bromo-5-(bromomethyl)-2- fluorophenyl)methanol (12 g, 40.28 mmol), under nitrogen protection, anhydrous tetrahydrofuran (250 mL) was added, N-methylmorpholine oxide (18.88 g, 161.12 mmol) was added, the reaction was heated at 75 °C overnight, after the reaction was completed, 250 mL of ethyl acetate was added, washed with 300 mL of water, 300 mL of saturated sodium chloride, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum, the crude product was purified by silica gel column to give 7.2 g of product 2-bromo-4-fluoro-5-(hydroxymethyl)benzaldehyde. (Yield: 76.7%).

[0187] Step E: Synthesis of (4-bromo-2-fluoro-5-(2-methoxyethenyl)phenyl)methanol

[0188] Into a 100 mL reaction flask was placed (methoxymethyl)triphenylphosphonium bromide (9.97 g, 25.7 mmol), anhydrous tetrahydrofuran (30 mL) was added, mixed well, a solution of potassium tert-butoxide in tetrahydrofuran (30 mL, 1 mol / L) was slowly added at 0 °C, stirred at 0 °C for 30 min, then 2-bromo-4-fluoro-5-(hydroxymethyl)benzaldehyde (2.00 g, 8.58 mmol) was added, then stirred at room temperature for 1 h. After the reaction was completed, saturated ammonium chloride was added to quench (100 mL), extracted with ethyl acetate (60 mL x 3), the organic phases were combined, washed with saturated sodium chloride (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 5 / 1) to give 2.2 g of yellow liquid product (4-bromo-2-fluoro-5-(2-methoxyethenyl)phenyl)methanol (yield 98%). LC-MS: [M+H] + = 263.0

[0189] Step F: Synthesis of (4-bromo-2-fluoro-5-(2-methoxyethyl)phenyl)methanol

[0190] Into a 100 mL reaction flask was placed (4-bromo-2-fluoro-5-(2-methoxyethenyl)phenyl)methanol (2.20 g, 8.43 mmol), anhydrous ethyl acetate (50 mL) was added, stirred to dissolve, platinum dioxide (190 mg, 0.84 mmol) was added at 25 °C, then replaced with hydrogen three times, stirred at room temperature under normal pressure for 2 h. After the reaction was completed, the reaction liquid was filtered through diatomite, and the filter cake was washed with ethyl acetate several times. The resulting filtrate was concentrated under vacuum to give a residue. The resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1) to give 1.2 g of yellow liquid product (4-bromo-2-fluoro-5-(2-methoxyethyl)phenyl)methanol (yield 54%). LC-MS: [M+H] + = 265.0

[0191] Step G: Synthesis of 4-bromo-2-fluoro-5-(2-methoxyethyl)benzoic acid

[0192] Into a 100 mL reaction vial was placed 4-bromo-2-fluoro-5-(2- methoxyethyl)phenyl)methanol (1.00 g, 3.80 mmol) and cuprous chloride (38.0 mg, 0.380 mmol), followed by anhydrous acetonitrile (15 mL), stirred to dissolve, and a solution of tert-butyl hydroperoxide in n-decane (3.8 mL, 5 M) was added dropwise at 0 °C. The reaction was stirred at room temperature for 16 h. Upon completion of the reaction, saturated sodium sulfite (50 mL) was added, and the mixture was extracted with ethyl acetate (20 mL x 3). The combined organic phase was washed with saturated sodium chloride (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by column chromatography on silica gel (eluent: petroleum ether / ethyl acetate = 2 / 1) to give 500 mg of 4-bromo-2-fluoro-5-(2-methoxyethyl)benzoic acid as a white solid (47.5% yield). LC-MS: [M-H] + = 277.0

[0193] Step H: Synthesis of (4-bromo-2-fluoro-5-(2-methoxyethyl)phenyl)methane-d2-ol

[0194] Into a 50 mL reaction vial was placed 4-bromo-2-fluoro-5-(2- methoxyethyl)benzoic acid (500 mg, 1.80 mmol), followed by anhydrous tetrahydrofuran (15 mL), stirred to dissolve, and deuterated borane in tetrahydrofuran solution (5.4 mL, 5.4 mmol, 1 M) was added slowly under nitrogen atmosphere at room temperature (25 °C). After the addition was completed, the mixture was stirred at 25 °C for 3 h. Upon completion of the reaction, 5 mL of deuterated hydrochloric acid was added slowly at 0 °C, and the mixture was stirred for 1 h. The resulting residue was concentrated in vacuo. The resulting residue was purified by column chromatography on silica gel (eluent: petroleum ether / ethyl acetate = 3 / 1) to give 400 mg of (4-bromo-2-fluoro-5-(2-methoxyethyl)phenyl)methane-d2-ol as a light yellow oil (83.6% yield). LC-MS: [M+H] + = 267.0

[0195] Step I: Synthesis of 1-bromo-4-(bromomethyl-d2)-5-fluoro-2-(2- methoxyethyl)benzene

[0196] In a 50 mL reaction vial, add (4-bromo-2-fluoro-5-(2-methoxyethyl)phenyl)methane-d2-ol (400 mg, 1.51 mmol), carbon tetrabromide (1.00 g, 3.02 mmol) and anhydrous dichloromethane (10 mL), stir to dissolve, slowly add triphenylphosphine (710 mg, 2.72 mmol) at 0 °C under nitrogen atmosphere. After addition, stir at 0 °C for 1 h. After completion of the reaction, slowly pour it into 30 mL of water, then extract with dichloromethane (10 mL x 3) three times. Combine the organic phase, dry, filter, concentrate to get a residue. Purify the residue by normal phase column (n-hexane: ethyl acetate = 1:0 to 50:1) to get 400 mg of 1-bromo-4-(bromomethyl-d2)-5-fluoro-2-(2-methoxyethyl)benzene as a light yellow oil (yield 80.8%).

[0197] Step J: Synthesis of 3-((4-bromo-2-fluoro-5-(2-methoxyethyl)phenyl)methyl-d2)-2- butyl-1,3-diazaspiro[4.4]non-1-en-4-one

[0198] In a 50 mL reaction vial, add 2-butyl-1,3-diazaspiro[4.4]non-1-en-4-one hydrochloride (280 mg, 1.22 mmol), add anhydrous DMF (5 mL), stir to dissolve, add sodium hydride (110 mg, 2.68 mmol) at 0 °C, stir at room temperature for 30 min, then add 1-bromo-4-(bromomethyl-d2)-5-fluoro-2-(2-methoxyethyl)benzene (400 mg, 1.22 mmol), stir at room temperature for 2 h. Add saturated ammonium chloride to quench (50 mL), extract with ethyl acetate (20 mL x 3), combine the organic phase, wash with saturated sodium chloride (50 mL), dry over anhydrous sodium sulfate, filter, and spin dry. Purify the resulting residue by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1) to get 500 mg of 3-((4-bromo-2-fluoro-5-(2-methoxyethyl)phenyl)methyl-d2)-2-butyl-1,3-diazaspiro[4.4]non-1-en-4-one as a yellow oily liquid product (yield 92%). LC-MS: [M+H] = 443.0. + = 443.0.

[0199] Step K: Synthesis of 4'-((2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl-d2)-N-(4,5- dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-methoxyethyl)-N-((2-(trimethylsilyl)ethoxy)methyl)-

[0200] In a 50 mL reaction vial was placed 3-((4-bromo-2-fluoro-5-(2-methoxyethyl)phenyl)methyl-d2)-2-butyl-1,3-diazaspiro[4.4]non-1-en-4-one (500 mg, 1.13 mmol), (2-(N-(4,5-dimethylisoxazol-3-yl)-N-((2-(trimethylsilyl)ethoxy)methyl)sulfamoyl)phenyl)boronic acid (120 mg, 0.27 mmol), potassium carbonate (780 mg, 5.65 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (92.0 mg, 0.110 mmol), nitrogen was purged, dioxane (10 mL), heavy water (1 mL), was placed in a 100 degree oil bath to heat the reaction for 16 hours. The reaction was complete, the reaction was allowed to cool to room temperature, ice water was added to quench (30 mL), ethyl acetate was extracted (30 mL x 4), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1) to give 700 mg of the product 4'-((2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl-d2)-N-(4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-methoxyethyl)-N-((2-(trimethylsilyl)ethoxy)methyl)-[1,1'-biphenyl]-2-sulfonamide as a colorless oily liquid (83% yield). LC-MS: [M+H] + = 744.1.

[0201] Step L: 4'-((2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl-d2)-N-(4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-methoxyethyl)-[1,1'-biphenyl]-2-sulfonamide

[0202] In a 50 mL reaction vial, 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl- d2)-N-(4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-methoxyethyl)-N-((2- (trimethylsilyl)ethoxy)methyl)-[l,l'-biphenyl]-2-sulfonamide (700 mg, 0.940 mmol), deuterated trifluoroacetic acid (3 mL) and dichloromethane (10 mL) were added and stirred at 25 °C for 2 h. The reaction was completed, concentrated directly under vacuum, then deuterated water (20 mL) and dichloromethane (10 mL) were added, the aqueous phase was adjusted to near neutral with concentrated aqueous ammonia, then extracted with dichloromethane (10 mL x 4). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography to obtain 412 mg of 4'-((2-butyl-4-oxo-l,3-diazaspiro[4.4]non-l-en-3-yl)methyl-d2)-N-(4,5-dimethylisoxazol-3-yl)-5'-fluoro-2'-(2-methoxyethyl)-[l,l'-biphenyl]-2-sulfonamide. LCMS: [M+H] + = 613.1

[0203] 1 H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 8.08 (dd, J = 7.7, 1.7 Hz, 1H), 7.72 - 7.60 (m, 2H), 7.28 - 7.20 (m, 1H), 6.99 (d, J = 7.7 Hz, 1H), 6.70 (d, J = 10.6 Hz, 1H), 3.29 - 3.20 (m, 2H), 3.06 (s, 3H), 2.42 - 2.26 (m, 4H), 2.21 (s, 3H), 1.92 - 1.78 (m, 6H), 1.75 - 1.62 (m, 5H), 1.58 - 1.48 (m, 2H), 1.35 - 1.22 (m, 2H), 0.83 (t, J = 7.3 Hz, 3H).

[0204] Preparation of Compound 117 hydrochloride salt

[0205] The foregoing method was repeated to prepare Compound 117, and 3 g of the free base was dissolved in 15 mL of dichloromethane, 1.25 mL of hydrogen chloride dioxane (4 M / L) was added, and after stirring for 1 h, it was concentrated to a solid at 50 °C, and then dried at 50 °C overnight to obtain Compound 117 hydrochloride salt.

[0206] LCMS = [M-HCl+H] + = 613.1

[0207] 1H NMR (400 MHz, DMSO-d6) δ 10.68 (s, 1H), 8.08 (dd, J = 7.7, 1.7 Hz, 1H), 7.73 - 7.61 (m, 2H), 7.28 (d, J = 7.8 Hz, 1H), 7.23 (dd, J = 7.2, 1.7 Hz, 1H), 6.76 (d, J = 10.8 Hz, 1H), 3.29 (t, J = 7.0 Hz, 2H), 3.07 (s, 3H), 2.83 (t, J = 7.8 Hz, 2H), 2.39 (ddd, J = 32.3, 14.2, 7.2 Hz, 2H), 2.22 (s, 3H), 2.05 - 1.79 (m, 6H), 1.87 (d, J = 6.2 Hz, 2H), 1.71 (s, 3H), 1.62 (q, J = 7.5 Hz, 2H), 1.32 (q, J = 7.4 Hz, 2H), 0.86 (t, J = 7.3 Hz, 3H).

[0208] Example 141 Endothelin Type A Receptor (ETAR) Inhibitor Activity Assay

[0209] This experiment used ETA-CHO stable cell line and FLIPR calcium flux detection method to determine the antagonistic effect of the test compound on endothelin type A receptor ETA.

[0210] Experimental method: digest and collect ETA-CHO cells, count after resuspension, then inoculate into 384-well cell plates, then place the cell plates in a 37°C, 5% CO2 incubator for about 12h;

[0211] The next day, prepare the Assay Buffer according to the instructions of the FLIPR Calcium 6 Assay Kit kit. Thaw 20x Component A to room temperature, dilute it to 1x loading buffer with Assay buffer, and place it at room temperature for standby; remove the culture medium in the cell plate, quickly add 35μL 1x loading buffer to each well, centrifuge and then place the cell plate in a 37°C incubator for 120min in the dark;

[0212] Prepare the positive compound and the test compound working solution, and transfer 5μL to the corresponding cell wells, and incubate at 37°C for 30min in the dark;

[0213] Prepare the agonist working solution, and transfer 20μL / well to the 384-well compound source plate;

[0214] Put the cell plate, agonist source plate and tip into the FLIPR instrument, add 10 μL of diluted agonist into each well using FLIPR Tetra, and collect data at 515 nm-575 nm wavelength.

[0215] Plot the signal value against the compound concentration, and use the GraphPad Prism software to perform nonlinear regression to fit the curve and calculate the IC 50 .

[0216] Example 14 Determination of angiotensin II type 1 receptor (AT1R) inhibitor activity

[0217] This experiment uses AT1-CHO stable cell line and HTRF detection method to determine the antagonistic effect of the test compound on angiotensin II type 1 receptor (AT1R)

[0218] Experimental method:

[0219] 1. According to the experimental procedure of the IP-One-Gq Kit, dilute the 5x Stimulation buffer (SB) provided in the kit with ddH2O to 1x SB. Prepare the positive compound and test compound stock solution, and perform gradient dilution, then take 2 μL and add to 38 μL of 1x SB, dilute again to prepare the working solution.

[0220] 2. Digest and collect AT1-CHO cells, count the cell density after resuspension to 1.2x10^6 / mL, then inoculate 8.4 μL per well into a 384-well cell plate, so that the number of cells per well is 10000.

[0221] 3. Add different concentrations of compounds to the corresponding cell wells according to the amount of 2.8 μL per well, and incubate in a 37°C incubator for 30 mins.

[0222] 4. Prepare the agonist AngII using 1x SB, then add the agonist to the 384-well plate according to the amount of 2.8 μL, and incubate in a 37°C incubator for 30 mins.

[0223] 5. Dilute the d2 Reagent and Tb Cryptate Antibody provided in the kit to 1x using lysis&Detection Buffer, add 3 μL of d2 and Tb to the 384-well plate, respectively, and incubate at room temperature for 1 h, then detect using the enzyme label instrument HTRF module (665 / 620 nm) to collect experimental data.

[0224] 6. Curve fitting and IC50values were determined by non-linear regression method using GraphPad Prism software by plotting signal values versus compound concentrations. 50 Calculations.

[0225] The compounds of the present application exhibit dual activity as inhibitors of endothelin type A receptor (ETAR) and angiotensin type 1 receptor (AT1R) as determined by Examples 141 and 142.

[0226] wherein, preferably, the endothelin type A receptor (ETAR) inhibitor activity IC50of the compound is < 100 nm, further preferably < 50 nm, for example, compounds 9, 15 and deuterated compounds thereof, etc., which is superior to saralasin and WO2023025277A1 compounds 44, 45. 50 < 100 nm, further preferably < 50 nm, for example, compounds 9, 15 and deuterated compounds thereof, etc., which is superior to saralasin and WO2023025277A1 compounds 44, 45.

[0227] wherein, preferably, the angiotensin type 1 receptor (AT1R) inhibitor activity IC50of the compound is < 100 nm, further preferably < 50 nm, for example, compounds 9, 15 and deuterated compounds thereof, etc., which is superior to saralasin and WO2023025277A1 compounds 44, 45. 50 < 100 nm, further preferably < 50 nm, for example, compounds 9, 15 and deuterated compounds thereof, etc., which is superior to saralasin and WO2023025277A1 compounds 44, 45.

[0228] According to Examples 141 and 142, the following are the data for some of the compounds:

[0229] Example 143 Determination of the effect of plasma protein binding on AT1 receptor antagonist activity

[0230] Experimental method:

[0231] 1. According to the experimental procedure of the IP-One-Gq Kit, dilute the 5x Stimulation buffer (SB) provided in the kit to 1x SB with ddH2O. Prepare the positive compound and the test compound stock solution, and perform gradient dilution to prepare the working solution.

[0232] 2. Digest the collected AT1-CHO cells, count after resuspension, and add 1x Stimulation Buffer provided in the IP-1 detection reagent or 1x Stimulation Buffer containing 10.42% human, rat, monkey plasma to resuspend again, so that the final cell density is 1.2x10 6 Then inoculate in the 384-well cell plate at an amount of 8.4 μL per well, so that the number of cells per well is 10000. Set up the plasma-free positive control well (containing 1 μM positive drug) and the negative control well (containing 1% DMSO)

[0233] 3. Different concentrations of compounds were added into the corresponding wells in an amount of 2.8 μL per well, and incubated in a 37 °C incubator for 30 min.

[0234] 4. Agonist Ang II was prepared using lx SB, diluted to 5 nM, and then added into the 384-well in an amount of 2.8 μL per well, and incubated in a 37 °C incubator for 30 min.

[0235] 5. The d2 Reagent and Tb Cryptate Antibody in the kit were diluted to lx using lysis & Detection Buffer, and d2 and Tb were added into the 384-well in an amount of 3 μL per well, respectively, and incubated at room temperature for 1 h, and then detected using an Enzyme Labeling Meter HTRF module (665 / 620 nm) to collect experimental data.

[0236] 6. The curve fitting and IC 50 calculated by plotting the signal value against the compound concentration using the non-linear regression method of GraphPad Prism software.

[0237] The results show that the IC 50 of the compound on AT1 receptor is larger in the presence of plasma, but the inhibitory activity of the preferred compound (such as compound 15) on AT1 is better than that of sparsentan, whether in the presence or absence of plasma.

[0238] Example 14 In vitro liver microsomal stability experiment of compound 4 in different animal species

[0239] 1.1 Preparation of stock solution and working solution

[0240] The compound and positive drug were dissolved in DMSO to obtain a 10 mM stock solution, and the stock solution was diluted with acetonitrile-water (1:1, v / v) to obtain a 100 μM solution

[0241] 1.2 Liver microsomal stability determination

[0242] The compound was incubated in duplicate with rat and human liver microsomal dilutions (0.5 mg / mL), respectively. The final concentration of the drug in the incubation system was 5 μM, and the total incubation time was 60 minutes. During the incubation process, samples were taken at time points 0.0, 20, and 60 minutes, respectively, and the reaction was terminated by adding an acetonitrile-methanol (v / v = 1:1) solution containing an internal standard (150 μg / mL propranolol). Verapamil (1 μM) was used as a positive control drug for the incubation system. The test samples were analyzed using UPLC-MS / MS, and the remaining percentage, the in vitro intrinsic clearance rate, and the half-life were calculated.

[0243] 1.3 Data analysis

[0244] The peak area ratio of the analyte to the internal standard was used to calculate the relative percentage content (residual rate %) after incubation of the compound and to perform an exponential function fitting. The calculation formula is as follows:

[0245] t 1 / 2 = 0.693 / k, k = rate constant (-slope value);

[0246] ER (extraction rate) = CL in vivo / liver blood flow

[0247] Physiological parameters

[0248] Classification criteria: slow metabolism (ER < 0.3), moderate metabolism (0.3 < ER < 0.7), fast metabolism (ER > 0.7).

[0249] The test results are as follows:

[0250] Stability of the compound in human and rat liver microsomes

[0251] The preferred compound of the present application has good stability.

[0252] Caco2 cell permeability experiment of the compound of Example 145

[0253] 1. Preparation of the test substance administration solution: prepare a 10 mM test compound stock solution with DMSO. Prepare a 10 mM positive control stock solution with DMSO. In this test, digoxin, minoxidil and atenolol are used as control compounds.

[0254] 2. Evaluation of cell monolayer integrity

[0255] Before the start of the permeability test, measure the transmembrane electrical resistance value (TEER) of the cells with an ohmmeter, and calculate the TEER value of the monolayer cells. After the TEER value meets the standard, replace the culture medium, and continue to culture the cells until the test is carried out.

[0256] 3. Permeability test

[0257] (1) First, the Caco-2 cells need to be cultured on a multi-well transparent culture membrane to form a cell monolayer, and cultured for 14-28 days to ensure that the Caco-2 cells reach a certain degree of differentiation, so as to better simulate the intestinal epithelial cell layer.

[0258] (2) Add the compound to be tested to HBSS, and let it contact the Caco-2 cells.

[0259] (3) Incubate the cells in an incubator at a constant temperature, humidity and CO2 concentration for 2 hours.

[0260] (4) After the incubation is completed, the upper solution (above the cells) and the lower solution (below the cells layer into the pores of the membrane) are collected separately. The concentration of the compound in the samples is measured using liquid chromatography-mass spectrometry (LC-MS / MS).

[0261] 4 Data analysis

[0262] (1) The apparent permeability coefficient (P app ) is calculated as follows: P app = (VA x [drug] acceptor ) / (Area x Time x [drug] initial, donor)

[0263] Where VA is the volume in the receptor well (in mL), Area is the surface area of the membrane (0.143 cm 2 for permeable supports for 96-well plates), and time is the total transport time (in seconds).

[0264] (2) The efflux ratio (ER) is calculated as follows: Efflux Ratio = P app(B-A) / P app(A-B)

[0265] Where P app(B-A) represents the apparent permeability coefficient from the basolateral side to the apical direction, and P app(A-B) represents the apparent permeability coefficient from the apical to the basolateral side.

[0266] 5 Data results

[0267] The preferred compounds of the present application have good permeability.

[0268] Example 1 46 Enzyme inhibition test study of the compound

[0269] 1 Preparation of the test substance administration solution:

[0270] Prepare a 10 mM test compound stock solution with DMSO. Prepare a 10 mM positive control stock solution with DMSO. The present test uses ketoconazole as the control compound.

[0271] 2 Test design

[0272] The inhibitory potential on cytochrome P450 enzyme CYP3A was evaluated using a phosphate buffer saline (100 mM, pH 7.4) system containing 0.2 mg / mL human liver microsomes. The test concentrations were 0.0137, 0.0412, 0.123, 1.11, 3.33, 10, 30, 50 μM. All samples were incubated in duplicate. The positive substrate in the incubation system was 5 μM midazolam (CYP3A). The concentration of ketoconazole (CYP3A) in the incubation system was 0.001, 0.005, 0.01, 0.05, 0.25, 1, 10 and 50 μM. The incubation was carried out at 37 °C, and the reaction was initiated by adding NADPH solution to a final concentration of 1 mM. The incubation time for CYP3A (midazolam as substrate) was 10 min, and after the incubation, 400 μL of internal standard (containing 150 μg / mL propranolol) acetonitrile-methanol (v:v = 1:1) solution was added to precipitate the protein, and the amount of metabolite product of the labeled substrate was detected by UPLC-MS / MS to calculate the IC 50 value of the inhibition.

[0273] 3 Test results

[0274] Example IC 50 values of CYP3A inhibition are as follows:

[0275] Test results

[0276] The preferred compounds of the present application have weaker enzyme inhibition and less risk of drug interaction.

[0277] Example 147 Hypertensive rat antihypertensive test

[0278] Method: The antihypertensive effect of the compound was evaluated using spontaneously hypertensive rats (SHR), and the rat blood pressure was determined using the tail cuff method. After the adaptation period, the animals were grouped based on systolic blood pressure (SBP). The control compound, compound 117 hydrochloride was administered at a dose of 30 mg / kg once a day for 7 consecutive days. The rat blood pressure was measured at 1, 3, 7 and 24 h after administration on the 7th day, and the systolic blood pressure (SBP) AUC within 24 h was calculated.

[0279] Results: The test results are shown in Figure 1, and after 7 days of continuous administration, the control compound (saralasin) and compound 117 hydrochloride can significantly reduce the systolic blood pressure of spontaneously hypertensive rats, and the antihypertensive effect of compound 117 is better.

[0280] Example 148 Pharmacodynamics of rat IgA nephropathy model

[0281] Methods: Male SD rats were used in the experiment. After the adaptation period, the rats were unilaterally nephrectomized and recovered for one week. Then the rats were induced by BSA / LPS / CCL4 for 12 weeks. The rats were grouped according to the urine albumin level at the fourth week. The rats were dosed from the fifth week for 8 weeks. The rats in the sham group and model control group were given vehicle once a day. The rats in the control compound (sparsentan) group and the compound 117 hydrochloride group were given sparsentan and compound 117 hydrochloride at a dose of 30 mg / kg once a day. At the eighth week, the rats were put in metabolic cages to collect 24h urine. The urine albumin and urine creatinine were detected. The serum urea nitrogen and serum creatinine were detected. The rats were euthanized at the end of the experiment. The kidneys were collected and weighed. The left kidney was divided into two parts. One part was fixed in 10% formalin and the other part was embedded in OCT. The PAS staining was used for histopathological examination.

[0282] The results are shown in Figures 2-4. After 8 weeks of continuous administration, compound 117 hydrochloride at a dose of 30 mg / kg significantly reduced the 24h urine albumin level of rats and improved the urine albumin / creatinine ratio (UACR). The pathological results showed that compared with the model group, the glomerular sclerosis of the dosed groups was significantly improved, indicating that compound 117 hydrochloride at a dose of 30 mg / kg can reduce the urine albumin level of IgA nephropathy and delay the progression of the disease. The therapeutic effect is better than that of the control compound at the same dose.

[0283] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples. Any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods and shall be within the scope of protection of the present application.

Claims

1. A compound of general formula (I) or an isomer thereof, or a pharmaceutically usable salt thereof, characterized in that, X is selected from the group consisting of hydrogen, alkylamino, alkyl, cycloalkyl, halogen, B1C(B2)=N-O-alkyl, alkenyl, alkyl-C=C-alkyl, cyano, alkylthio, alkylthioalkyl, alkoxy, alkoxyalkyl; B1, B2are independently selected from the group consisting of H, alkyl; said alkylamino, alkyl, alkylthio, alkylthioalkyl, alkoxy, alkoxyalkyl, alkyl-C=C-alkyl can be further substituted with substituents selected from the group consisting of halogen, carboxylic acid, carboxylic acid alkyl ester, haloalkyl, cycloalkyl, cycloalkyloxy, heterocycloalkyl, cyano, hydroxy, oxo, thioxo, alkenyl, alkynyl, amino, heteroaryl, aryl, aryl-O-, heteroaryl-O-, alkoxy, alkoxyalkyl, alkoxyalkoxy, haloalkyl, amido or alkyl substituted amido; or X is selected from the group consisting of A1A2C(O)NA3-(CH2)n-, n = a natural number from 1 to 3, A1, A2, A3are independently selected from the group consisting of H, alkyl, or A1, A2form a substituted or unsubstituted cycloalkyl, the substituents being selected from the group consisting of alkyl; or X is selected from the group consisting of -(CH2)m-saturated or unsaturated heterocycloalkyl, m = a natural number from 0 to 3, the heterocycloalkyl can be further substituted with substituents selected from the group consisting of alkyl, alkoxy, oxo, hydroxy; or X is selected from the group consisting of -(CH)p=saturated or unsaturated heterocycloalkyl, alkyl, p = a natural number from 1 to 3, the heterocycloalkyl can be further substituted with substituents selected from the group consisting of alkyl, alkoxy, halogen; T1, T2are independently selected from the group consisting of H or T1and T2form a cycloalkyl, the cycloalkyl can be further substituted with substituents selected from the group consisting of halogen; R1, R3, R5, R6, R7are independently selected from the group consisting of H, halogen, alkyl, alkoxy, cyano; R2is selected from the group consisting of H, halogen, alkyl, alkoxy, cyano, when R2is selected from hydrogen, X is selected from the group consisting of amino, heteroaryl substituted alkyl or alkoxy, or X is selected from the group consisting of -(CH2)m-saturated or unsaturated heterocycloalkyl, m = a natural number from 0 to 3, the heterocycloalkyl can be further substituted with substituents selected from the group consisting of alkyl, alkoxy, oxo, hydroxy; R4is selected from the group consisting of H, alkyl, alkoxy; R8is selected from the group consisting of alkyl, halogen; R 11 selected from hydrogen, alkyl, -C(O)-alkyl.

2. The compound according to claim 1 or its isomers, or its pharmaceutically acceptable salts, characterized in that, a compound selected from the group consisting of compounds of general formula (Ia) or its isomers, or pharmaceutically acceptable salts thereof: R1to R8, X are as defined in claim 1.

3. The compound according to claim 1, or its isomer, or its pharmaceutically acceptable salt, wherein a compound selected from the group consisting of compounds of general formula (Ib) or its isomers, or pharmaceutically acceptable salts thereof: R1-R8, X are as defined in claim 1, R9and R 10 are each independently selected from the group consisting of: hydrogen, halogen.

4. The compound according to claim 1, or its isomer, or its pharmaceutically acceptable salt, wherein compounds selected from the group consisting of compounds of the general formula (Ic), (Id), (Ie) or an isomer thereof, or a pharmaceutically acceptable salt thereof: X, T1, T2 are as defined in claim 1, R8 is selected from alkyl, halogen; R9 and R 10 are each independently selected from the group consisting of: hydrogen, halogen.

5. The compound according to any one of claims 1 to 4, or an isomer thereof, or a pharmaceutically acceptable salt thereof, wherein, said alkyl is selected from the group consisting of C 1-4 alkyl, said C 1-4 said alkyl is selected from the group consisting of methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl; said cycloalkyl is selected from the group consisting of C 3-6 cycloalkyl, said C 3-6 said cycloalkyl is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; heterocycloalkyl is a cycloalkyl wherein one or more carbon atoms of the ring are replaced by a heteroatom selected from the group consisting of O, N, S; unsaturated cycloalkyl means that the ring of the cycloalkyl group contains one or more unsaturated double or triple bonds.

6. The compound according to any one of claims 1 to 4, or an isomer thereof, or a pharmaceutically acceptable salt thereof, wherein, said alkoxy is selected from the group consisting of C 1-4 alkoxy, C 1-4 said alkoxy is selected from the group consisting of methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec-butoxy, t-butoxy.

7. The compound according to any one of claims 1 to 4, or an isomer thereof, or a pharmaceutically acceptable salt thereof, wherein, The halogen includes fluorine, chlorine, bromine, iodine.

8. The compound according to any one of claims 1-4, or an isomer thereof, or a pharmaceutically acceptable salt thereof, wherein, Alkoxyalkyl means that one or more hydrogens of an alkyl group are replaced by alkoxy.

9. The compound according to any one of claims 1 to 4, or an isomer thereof, or a pharmaceutically acceptable salt thereof, wherein, X is selected from hydrogen, methyl, ethyl, n-propyl, methoxymethyl, methoxyethyl, methoxypropyl, ethoxymethyl, ethoxyethyl, ethoxypropyl, methylthiomethyl, methylthioethyl, methylthiopropyl, ethylthiomethyl, ethylthioethyl, ethylthiopropyl, methoxy, ethoxy, n-propoxy, isopropoxy. cyclopropyl, fluoro, bromo, isopropyl, cyano, hydroxyethyl, R1, R3, R5, R6, R7are independently selected from the group consisting of H, fluorine, chlorine, methyl, methoxy, cyano; R2is selected from the group consisting of fluorine, chlorine, methyl, methoxy, cyano; R4is selected from the group consisting of H, methyl, methoxy; R8is selected from the group consisting of methyl, chlorine.

10. The compound according to claim 1, or isomers thereof, or pharmaceutically acceptable salts thereof, wherein, selected from the following compounds:

11. The compound according to claim 1, or isomers thereof, or pharmaceutically acceptable salts thereof, wherein, said compound is substituted with deuterium ( 2 H) for one or more hydrogen atoms.

12. The compound according to claim 11, or isomers thereof, or pharmaceutically acceptable salts thereof, wherein, the isotope deuterium ( 2 H) substituted compounds are selected from:

13. A pharmaceutical composition comprising a compound according to any one of the preceding claims 1 to 12, or an isomer thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

14. Use of a compound according to any one of claims 1 to 12, or an isomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 13, for the manufacture of a medicament for the treatment and / or prevention of diseases associated with angiotensin and endothelin receptor antagonism.

15. Use according to claim 14, characterized in that, The diseases include chronic kidney disease, IgA, FSGS and hypertension.

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